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List of Contributors

A. Arenas, Department of Rehabilitation Medicine, University of Miami School of Medicine, P.O. Box
016960 (D-461), Miami, FL 33101, USA
K. Anderson, Reeve-Irvine Research Center, 1216 GNRF, University of California, Irvine, Irvine, CA
92697-4292, USA
H.W.G. Baker, University of Melbourne Department of Obstetrics and Gynaecology Melbourne IVF
Reproductive Services, Royal Women’s Hospital, Carlton, Vic. 3058, Australia
M.S. Beattie, Department of Neuroscience, laboratory of CNS Repair and Spinal Trauma and Repair
Laboratories, The Ohio State University College of Medicine and Pulic Health, Columbus, OH, USA
L.A. Birder, Departments of Medicine and Pharmacology, University of Pittsburgh School of Medicine,
Pittsburgh, PA 15261, USA
A.F. Brading, Oxford Continence Group, University Department of Pharmacology, Mansfield Road,
Oxford OX1 3QT, UK
J.C. Bresnahan, Department of Neuroscience, Laboratory of CNS Repair and Spinal Trauma and Repair
Laboratories, The Ohio State University College of Medicine and Public Health, Columbus, OH, USA
J.A. Brock, Spinal Injuries Research Centre, Prince of Wales Medical Research Institute, Gate 1, Barker
Street, Randwick, NSW 2031, Australia
A. Brown, Biotherapeutics Research Group, The Spinal Cord Injury Team, Robarts Research Institute and
The Graduate Program in Neuroscience, The University of Western Ontario, P.O. Box 5015, 100 Perth
Drive, London, ON N6A 5K8, Canada
D.J. Brown, Victorian Spinal Cord Service, Austin Health, Heidelberg, Vic., Australia
E.A.L. Chung, St Mark’s Hospital, Northwick Park, Watford Road, Harrow, Middlesex, HA1 3UJ, UK
V.E. Claydon, International Collaboration on Repair Discoveries (ICORD), University of British Co-
lumbia, Vancouver, BC V6 T 1Z4, Canada
H.L. Collins, Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201,
USA
M.D. Craggs, Centre for Spinal Research, Functional Assessment and Restoration, London Spinal Cord
Injuries Unit, Royal National Orthopaedic Hospital NHS Trust, Brockley Hill, Stanmore, Middlesex
HA7 4LP, UK
W.C. de Groat, Departments of Pharmacology and Urology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
G.A. Dekaban, Spinal Cord Injury Team, BioTherapeutics Research Group, Robarts Research Institute,
100 Perth Drive, P.O. Box 5015, London, ON N6A 5K8, Canada
S.E. DiCarlo, Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201,
USA
J.W. Downie, Department of Urology and Pharmacology, Faculty of Medicine, Dalhousie University,
5850 College St., Halifax, NS B3 H 1X5, Canada
S.L. Elliott, Departments of Psychiatry and Urology, University of British Columbia, British Columbia
Center for Sexual Medicine, Echelon-5, 855 West 12th Avenue, Vancouver, Vancouver Sperm Retrieval
Clinic, Vancouver Hospital and G.F. Strong Rehabilitation Centre, Vancouver, BC, Canada

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G.S. Emch, Department of Neuroscience, Georgetown University Medical Center, TRB EP04, Washing-
ton, DC 20057, USA
A.V. Emmanuel, St Mark’s Hospital, Northwick Park, Watford Road, Harrow, Middlesex, HA1 3UJ, UK
P. Enck, Department of Psychosomatic Medicine, University Hospitals Tuingen, Schaffhausenstr 113,
72072 Tubingen, Germany
F.A. Frizelle, Colorectal Unit, Department of Surgery, Christchurch Hospital and Burwood Spinal Unit,
Christchurch, New Zealand
R.A. Gaunt, Department of Biomedical Engineering and Center for Neuroscience, University of Alberta,
507 HMRC, Edmonton, AB T6G 2S2, Canada
I. Greving, Department of Internal Medicine, Elisabeth Hospital, Gelsenkirchen, Germany
D. Gris, Spinal Cord Injury Team, BioTherapeutics Research Group, Robarts Research Institute, 100
Perth Drive, P.O. Box 5015, London, ON N6A 5K8, Canada
L.A. Havton, Department of Neurology, David Geffen School of Medicine at University of California Los
Angeles, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA
S.T. Hill, Victorian Spinal Cord Service, Austin Health, Heidelberg and Melbourne IVF Reproductive
Services, Royal Women’s Hospital, Melbourne, Vic., Australia
T.X. Hoang, Department of Neurology, David Geffen School of Medicine at University of California Los
Angeles, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA
C.H. Hubscher, Department of Anatomical Sciences and Neurobiology, University of Louisville School of
Medicine, Louisville, KY 40292, USA
J.E. Jacob, Biotherapeutics Research Group, The Spinal Cord Injury Team, Robarts Research Institute
and The Graduate Program in Neuroscience, The University of Western Ontario, P.O. Box 5015, 100
Perth Drive, London, ON N6A 5K8, Canada
R.D. Johnson, Department of Physiological Sciences, College of Veterinary Medicine and the McKnight
Brain Institute, University of Florida, Gainesville, FL 32610-0144, USA
A.K. Karlsson, Spinal Injuries Unit, Sahlgrenska University Hospital, Institute of Clinical Neuroscience,
Sahlgrenska Academy, S 413 45 Goteborg, Sweden
J.R. Keast, Pain Management Research Institute, University of Sydney at Royal North Shore Hospital, St
Leonards, NSW, Australia
S. Klosterhalfen, Institute of Medical Psychology, University Hospitals Dusseldorf, Germany
A. Krassioukov, International Collaboration on Repair Discoveries (ICORD), Division of Physical Med-
icine, School of Rehabilitation and Department of Medicine, University of British Columbia, Vancou-
ver, BC V6 T 1Z4, Canada and Department of Physical Medicine and Rehabilitation, University of
Western Ontario, London, ON, Canada
G.M. Leedy, Division of Social Work, University of Wyoming, Laramie, WY, USA
I.J. Llewellyn-Smith, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford
Park, SA 5042, Australia
A.C. Lynch, Colorectal Unit, Department of Surgery, Christchurch Hospital and Burwood Spinal Unit,
Christchurch, New Zealand
D.R. Marsh, Department of Anatomy and Cell Biology, Dalhousie University, Halifax, NS, Canada
C.J. Mathias, Neurovascular Medicine Unit, Faculty of Medicine, Imperial College London at St. Mary’s
Hospital, London W2 1NY, UK, Autonomic Unit, National Hospital for Neurology and Neurosurgery,
Queen Square, and Institute of Neurology, University College London, London, UK
E.M. McLachlan, Spinal Injuries Research Centre, Prince of Wales Medical Research Institute, Gate 1,
Barker Street, Randwick, NSW 2031, Australia
Y.S. Nout, Department of Neuroscience, Laboratory of CNS Repair and Spinal Trauma and Repair
Laboratories, The Ohio State University College of Medicine and Public Health, Columbus, OH, USA
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P.J. Potter, Regional Spinal Cord Injury Rehabilitation Program, and Physical Medicine and Rehabil-
itation, St. Joseph’s Health Center, The University of Western Ontario, London, ON, Canada
A. Prochazka, Department of Biomedical Engineering and Center for Neuroscience, University of Alberta,
507 HMRC, Edmonton, AB T6G 2S2, Canada
A.G. Rabchevsky, University of Kentucky, Spinal Cord & Brain Injury Research Center and Department
of Physiology, 741 South Limestone Street, B 371 BBSRB, Lexington, KY 40536-0509, USA
T. Ramalingam, Specialist Registrar in Colorectal Surgery, Headquarters Army Medical Directorate,
Former Army Staff College, Camberley, Surrey GU16 4NP, UK
D.W. Rodenbaugh, Department of Molecular and Integrative Physiology, University of Michigan, Ann
Arbor, MI 48109, USA
L.P. Schramm, Departments of Biomedical Engineering and Neuroscience, The Johns Hopkins University
School of Medicine, 606 Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205, USA
S.J. Shefchyk, Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB
R3E 3J7, Canada
M.L. Sipski, Veterans Administration Rehabilitation Research and Development, Center of Excellence in
Functional Recovery and Spinal Cord Injury, Miami, FL 33101, USA
M.A. Vizzard, Departments of Neurology and Anatomy and Neurobiology, University of Vermont College
of Medicine, Burlington, VT 05405, USA
L.C. Weaver, Spinal Cord Injury Laboratory, BioTherapeutics Research Group, Robarts Research In-
stitute, 100 Perth Drive, P.O. Box 5015, London, ON N6A 5K8, Canada
B. Wietek, Department of Radiology, University Hospitals Tubingen, Germany
J.R. Wrathall, Department of Neuroscience, Georgetown University Medical Center, TRB EP04, Wash-
ington, DC 20057, USA
N. Yoshimura, Departments of Pharmacology and Urology, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
N.D.T. Zinck, Department of Pharmacology, Faculty of Medicine, Dalhousie University, 5850 College St.,
Halifax, NS B3 H 1X5, Canada
Dedication

‘‘Spinal cord injury is a ferocious assault on the body that leaves havoc in its wake. Paralysis is
certainly part of its legacy, but there are other equally devastating consequences including
autonomic dysfunction: compromised cardiovascular, bowel, bladder, and sexual function.
Treatments and cures for these losses would greatly improve the quality of life for all of us living
with spinal cord injury. I am hopeful that the multi-faceted and collaborative approach to spinal
cord repair evidenced by this book and its contributors means that there will be effective
therapies for autonomic dysfunction in the not too distant future.’’
Christopher Reeve, September 30, 2004

Christopher Reeve sent this endorsement to us only 10 days before his death. His passionate advocacy for
research that would better the lives of all who have suffered spinal cord injury has affected all of us who
work in this field. We dedicate this book to Christopher Reeve and to all, who like him, strive to overcome
the tragedy of spinal cord injury. As Canadian editors, we particularly would like to thank and
acknowledge the efforts of our advocates, Mr. Rick Hansen and Ms. Barbara Turnbull for their tireless
efforts to bring awareness, expertise and funding to the field of spinal cord injury research, in all of its
dimensions. Finally, we dedicate this book to everyone who has sustained a spinal cord injury and lives
courageously, hoping that the efforts of science will bring timely rewards.

Lynne Weaver and Canio Polosa


on behalf of the contributors,
March 1, 2005

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Foreword

Autonomic dysfunction after spinal cord injury: the perspective of a person with a spinal cord injury.

‘‘Something’s wrong. I’m hot. No, wait, now I’m cold. But why am I sweating? I never sweat. My
legs won’t stop jumping. Now my hands are in tight fists and my torso is tight as well. Why does
my scalp itch? Oh no, the headache is starting. I’m getting dysreflexic. What is causing it? My
catheter seems okay, I don’t feel any kinks. But why isn’t there any urine in the leg bag? Oh no, is
the catheter clogged up? The headache is getting worse. It feels like a nail is being hammered into
my head. What am I going to do? It’s going to get worse. I know it, I know what’s going to
happen. But I’m all alone. I can’t reach anyone to ask for help. How am I going to fix this? I’m
going to have to drive myself to the doctor. I’m trying to adjust everything, but it’s not working.
My head, it hurts so bad. I can’t think straight. Everything looks a little bit blurry. It’s hard to
breathe now, the spasms are so severe. I feel like I’m going to throw up. Why is this happening to
me? Why? I’m crying now, I’m so afraid. I know what can happen if I don’t stop this. My heart is
beating wildly, my head is hurting more and more. It feels like it’s going to explode. I have to
hurry to the doctor before it’s too late. I could die from this y’’

This is what happened to me a few years ago during one of my worst attacks of autonomic dysreflexia.
Fortunately, I was able to make it to the doctor’s office and convince him of the urgency of the situation. He
changed the catheter, which was indeed clogged with sediment, and within a matter of seconds after the
volume of my bladder was reduced, the grossly obvious symptoms vanished. There are after effects from
such an episode, however. The biggest being extreme exhaustion, which is not trivial when you’re paralyzed.
For a little background on my spinal cord injury, I’m classified as a C5 ASIA grade B and was injured in
a motor vehicle accident in December of 1988. The most prominent source of my autonomic dysreflexia
stems from my bladder. While in the hospital, I tried various methods of managing my bladder and finally
settled on using a suprapubic catheter. This provides me with the greatest level of independence, comfort,
and reliability. I do use an anti-cholinergic medication to reduce spasticity in my bladder. This is a very
important point to stress. If I miss my medication or am delayed in taking it, I experience a continuous state
of mild dysreflexia. That consists of increased spasticity in my body, hot/cold flashes, and an itchy scalp. I
do not develop the severe headache, however, but I also do not know what fluctuations are occurring with
my blood pressure when in that state. These symptoms disappear shortly after I resume the medication.
Just as every spinal cord injury is different, the primary stimulus and pattern of symptoms of autonomic
dysreflexia experienced by each individual are different. The two most common stimuli appear to be
bladder or bowel distension. One anecdotal observation is that the onset and intensity of the symptoms
seem to occur faster and more severely with increasing time post-injury. I have experienced this first-hand
and have been told the same thing by many other people with spinal cord injury. Is this truly common
among the majority of people who develop autonomic dysreflexia? If so, what is the underlying biological
mechanism? And, what long-term damage develops in people with chronic spinal cord injury who repeat-
edly experience episodes of dysreflexia? These are but a few of the problems that need to be addressed in the
scientific and clinical settings.

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Aside from acute autonomic dysreflexia episodes, there are many autonomic dysfunctions that present
difficulties that people with any level of spinal cord injury have to deal with on a daily basis. Some of the
most prominent problems include impairments in bladder and bowel control, sexual function, body tem-
perature regulation, cardiovascular control, and metabolism. Any of these dysfunctions can significantly
reduce a person’s quality of life.
Now, in addition to being a quadriplegic, I am also a scientist and, when I first entered the field of spinal
cord injury science in 2000, I observed that research regarding autonomic dysfunctions resulting from
spinal cord injury was not very prevalent. Yet these are problems that everybody with cord injury ex-
periences to some degree. This is a perplexing paradox. In an effort to address this issue, I conducted a
study to determine what areas of functional recovery were most important to people living with spinal cord
injury. Regaining bladder/bowel function and eliminating autonomic dysreflexia was the first or second
highest priority for approximately 40% of quadriplegics and paraplegics and, similarly, regaining sexual
function was the first or second highest priority to 28.3% of quadriplegics and 45.5% of paraplegics
(Anderson, 2004). These results demonstrate that research regarding autonomic dysfunctions is extremely
important. To that end, this book has been written about what is already known and to serve as a platform
for fueling future research.
After all, it is all of these autonomic functions that we take for granted when we have them and that
dominate our lives when we lose them.

References

Anderson, K.D. (2004) Targeting recovery: priorities of the spinal cord injured population. J Neurotrauma, 21: 1371–1383.

Kim Anderson
Contents

List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Foreword by Kim Anderson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Overview: Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and
signs
A-.K. Karlsson (Goteborg, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Section I. Anatomical Changes Mediating Autonomic Dysfunction After Cord Injury

1. Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons


I.J. Llewellyn-Smith, L.C. Weaver and J.R. Keast (Bedford Park, SA, London, ON,
Canada and St. Leonards, NSW, Australia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2. Spinal sympathetic interneurons: Their identification and roles after spinal cord injury
L.P. Schramm (Baltimore, MD, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3. Which pathways must be spared in the injured human spinal cord to retain cardiovascular
control?
A. Krassioukov (Vancouver, BC, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Section II. Urinary Bladder Dysfunction

4. Disordered control of the urinary bladder after human spinal cord injury: what are the
problems?
P.J. Potter (London, ON, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

5. Mechanisms underlying the recovery of lower urinary tract function following spinal cord
injury
W.C. de Groat and N.Yoshimura (Pittsburgh, PA, USA). . . . . . . . . . . . . . . . . . . . . 59

6. Spinal mechanisms contributing to urethral striated sphincter control during continence and
micturition: ‘‘How good things might go bad’’
S.J. Shefchyk (Winnipeg, MB, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

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7. Neurochemical plasticity and the role of neurotrophic factors in bladder reflex pathways
after spinal cord injury
M.A. Vizzard (Burlington, VT, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

8. Effect of injury severity on lower urinary tract function after experimental spinal
cord injury
J.R. Wrathall and G.S. Emch (Washington, DC, USA) . . . . . . . . . . . . . . . . . . . . . . 117

9. Role of the urothelium in urinary bladder dysfunction following spinal cord injury
L.A. Birder (Pittsburgh, PA, USA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

10. Plasticity in the injured spinal cord: can we use it to advantage to reestablish effective
bladder voiding and continence?
N.D.T. Zinck and J.W. Downie (Halifax, NS, Canada) . . . . . . . . . . . . . . . . . . . . . . 147

11. Control of urinary bladder function with devices: successes and failures
R.A. Gaunt and A. Prochazka (Edmonton, AB, Canada). . . . . . . . . . . . . . . . . . . . . 163

12. Novel repair strategies to restore bladder function following cauda equina/ conus medullaris
injuries
T.X. Hoang and L.A. Havton (Los Angeles, CA, USA) . . . . . . . . . . . . . . . . . . . . . . 195

13. Pelvic somato-visceral reflexes after spinal cord injury: measures of functional loss and
partial preservation
M.D. Craggs (Stanmore, UK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Section III. Cardiovascular Dysfunction

14. The clinical problems in cardiovascular control following spinal cord injury: an overview
A. Krassioukov and V.E. Claydon (Vancouver, BC, Canada) . . . . . . . . . . . . . . . . . . 223

15. Orthostatic hypotension and paroxysmal hypertension in humans with high spinal cord
injury
C.J. Mathias (London, UK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

16. Autonomic dysreflexia after spinal cord injury: central mechanisms and strategies for
prevention
L.C. Weaver, D.R. Marsh, D. Gris, A. Brown and G.A. Dekaban (London, ON,
Canada and Halifax, NS, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

17. Segmental organization of spinal reflexes mediating autonomic dysreflexia after spinal cord
injury
A.G. Rabchevsky (Lexington, KY, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

18. Spinal cord injury alters cardiac electrophysiology and increases the susceptibility to
ventricular arrhythmias
H.L. Collins, D.W. Rodenbaugh and S.E. DiCarlo (Detroit and Ann Arbor,
MI, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
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19. Adaptations of peripheral vasoconstrictor pathways after spinal cord injury


E.M. McLachlan and J.A. Brock (Randwick, NSW, Australia). . . . . . . . . . . . . . . . . 289

20. Genetic approaches to autonomic dysreflexia


A. Brown and J.E. Jacob (London, ON, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . 299

Section IV. Bowel Dysfunction

21. Gastrointestinal symptoms related to autonomic dysfunction following spinal cord injury
E.A.L. Chung and A.V. Emmanuel (Harrow, UK) . . . . . . . . . . . . . . . . . . . . . . . . . 317

22. Colorectal motility and defecation after spinal cord injury in humans
A.C. Lynch and F.A. Frizelle (Christchurch, New Zealand) . . . . . . . . . . . . . . . . . . . 335

23. Mechanisms controlling normal defecation and the potential effects of spinal cord injury
A.F. Brading and T. Ramalingam (Oxford and Camberley, UK) . . . . . . . . . . . . . . . 345

24. Alterations in eliminative and sexual reflexes after spinal cord injury: defecatory function
and development of spasticity in pelvic floor musculature
Y.S. Nout, G.M. Leedy, M.S. Beattie and J.C. Bresnahan (Columbus, OH and
Laramie, WY, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

25. Upper and lower gastrointestinal motor and sensory dysfunction after human spinal cord
injury
P. Enck, I. Greving, S. Klosterhalfen and B. Wietek (Tubingen, Gelsenkirchen and
Dusseldorf, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

Section V. Sexual Dysfunction

26. Problems of sexual function after spinal cord injury


S.L. Elliott (Vancouver, BC, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

27. Ascending spinal pathways from sexual organs: effects of chronic spinal lesions
C.H. Hubscher (Louisville, KY, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

28. Descending pathways modulating the spinal circuitry for ejaculation: effects of chronic
spinal cord injury
R.D. Johnson (Gainesville, FL, USA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415

29. Male fertility and sexual function after spinal cord injury
D.J. Brown, S.T. Hill and H.W.G. Baker (Heidelberg, Melbourne and Carlton,
Australia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

30. Female sexual function after spinal cord injury


M.L. Sipski and A. Arenas (Miami, FL, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449


Introduction

This book was inspired by a gathering of basic and clinical scientists, and healthcare providers, for a
workshop on autonomic dysfunction after spinal cord injury held in Banff, Alberta, Canada in July, 2003.
The discussions in this meeting highlighted the contrast between the high priority assigned by people with
spinal cord injury to finding a cure for their autonomic dysfunctions, and the limited awareness of these
issues or attention given to them by the scientific and medical community, other than care providers who
interact with cord-injured people regularly. Research is needed to gain greater understanding of the
mechanisms of these problems and to develop treatments and prevention strategies for them. To provide a
foundation for such endeavours, this book contains a compilation of what is known about bladder,
cardiovascular, bowel and sexual dysfunction after spinal cord injury, as it relates to the changes within the
autonomic nervous system control of these functions.
The book is organized into sections that focus on each of the affected visceral functions: urinary bladder,
cardiovascular, gastrointestinal and sexual. The book begins with a description of the time course of
autonomic dysfunctions and their ramifications from the first hours after a spinal cord injury to the more
stable chronic states. The next section contains three chapters that address anatomical findings that may
provide some of the foundation for autonomic dysfunctions in many of the systems. The system-specific
chapters then follow in four sections. Each section begins with a chapter or two defining the clinical
problems experienced by people with cord injury. The following chapters present research, basic and
clinical, that address the autonomic dysfunctions.
We have noted themes that transcend the different sections and can pertain to bladder, bowel, cardio-
vascular and sexual functions. For example, sprouting of axons, including the central processes of sensory
neurons, within the injured spinal cord can be advantageous or detrimental, depending on the amount,
location and potential for new contacts of this sprouting. This may also pertain to changes in the
autonomic ganglia outside the central nervous system. Another theme is loss of coordination and balance
of parasympathetic, sympathetic and somatic systems in the absence of modulatory influences from
supraspinal neuronal systems. Bladder dyssynergia and autonomic dysreflexia, with its episodic hyperten-
sion, have much in common. Lack of coordinated control of pelvic neurons leads to failure of defaecation
and ejaculation. Spinal cord injury affects more than spinal neurons; it impacts on peripheral ganglia and
target tissues such as blood vessels and the wall of the urinary bladder. Growth factors that one would
think should be advantageous to repair of the injured spinal cord, may actually promote development of
circuits that impair rather than support recovered function. Many other themes also thread through this
book.
Finally, we would like to acknowledge several people who have helped us during the preparation of this
book. Ms. Bibi Pettypiece organized the meeting in Banff that started this project and was in commu-
nication with all of the contributing authors to coordinate the details that can so easily become a burden.
Her assistance was invaluable. Ms. Eilis Hamilton applied her considerable skill with graphic presentation
to many of the illustrations in this book, adding to their clarity. The authors who have benefited from her
assistance are very appreciative. Mr. Tom Merriweather and Ms. Maureen Twaig from Progress in Brain
Research, Elsevier, have been encouraging and helpful throughout our effort. Lastly, we realize that we
have not included all of the work that has been done on the subject of autonomic dysfunction after spinal

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cord injury. Excellent work is being done in addition to that described in these chapters, and we apologize
to those who did not have the opportunity to contribute to this book.

Lynne Weaver
Canio Polosa
Editors
L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

Overview

Autonomic dysfunction in spinal cord injury: clinical


presentation of symptoms and signs

Ann-Katrin Karlsson

Spinal Injuries Unit, Sahlgrenska University Hospital, Institute of Clinical Neuroscience, Sahlgrenska Academy, S 413 45
Goteborg, Sweden

Abstract: Spinal cord injury and especially cervical spinal cord injury implies serious disturbances in
autonomic nervous system function. The clinical effects of these disturbances are striking. In the acute
phase, the autonomic imbalance and its effect on cardiovascular, respiratory system and temperature
regulation may be life threatening. Serious complications such as over-hydration with the risk of pulmo-
nary edema or hyponatremia are seen. The cord-injured person suffers from autonomic nervous system
dysfunction also affecting bladder and bowel control, renal and sexual function. Paralytic ileus may cause
vomiting and aspiration, which in turn interferes with respiratory function in those with cervical spinal cord
injury. The cord-injured person is at risk to develop pressure sores from the moment of the accident. Two to
three months post-injury the cord-injured person with a lesion level above the fifth thoracic segment may
develop autonomic dysreflexia, characterised by sympathetically mediated vasoconstriction in muscular,
skin, renal and presumably gastrointestinal vascular beds induced by an afferent peripheral stimulation
below lesion level. The reaction might cause cerebrovascular complications and has effects on metabolism.
Some of the autonomic disturbances are transient and a new balance is reached months post-injury, while
others persist for life.

Spinal cord injury, in a moment, dramatically with spinal cord injury consider the disturbances in
changes the life of the affected person. The loss of autonomic nervous system function even more
control of skeletal muscle, as well as of sensations devastating than the loss of motor and sensory
from below the injury, together with the impair- function.
ment of thermoregulation, urinary bladder and
bowel function produce a profound deterioration
in the quality of life for people after spinal cord The moment of the accident
injury. Tetraplegic subjects rank improvement in
hand function as the most important factor to en- A cervical spinal cord injury may be life threaten-
hance quality of life (Anderson, 2004). However, ing. When the level of lesion is above the third
paraplegic subjects give normal sexual function the cervical segment (C3), the injured person needs
highest priority and, when the first and second immediate assistance of respiration due to the loss
choice was combined, recovery of normal bladder of the supraspinal excitatory drive of the phrenic
and bowel function were given the highest priority motor neurons located at C3–C4. Even when the
in both groups of subjects. This shows that people level of the lesion is below C3, the cord-injured
person may suffer from life-threatening conditions
Corresponding author. Tel.:+46 31 3421000; due to autonomic nervous system dysfunction.
Fax: +46 31 415835; E-mail: ann-katrin.karlsson@neuro.gu.se Cardiac arrest may be one of the causes of death in

DOI: 10.1016/S0079-6123(05)52034-X 1
2

the first few minutes following a cervical spinal if the injured person lies more than 2 h in the same
cord injury due to the disruption of central sym- position on a hard spine board, he/she is at risk of
pathetic control and the concomitant unopposed developing pressure sores. This risk is attributed to
vagal outflow. The incidence of this complication the loss of sensory inputs to the brain from below
is difficult to estimate, although some data indicate the level of the lesion, but this may not be the
a decreasing incidence during the last decades. The whole explanation since unconscious patients
number of patients who reach hospital alive has without spinal cord injury do not seem to be at
increased more than twofold from the 1940s to the as high risk of developing pressure sore as are the
late 1970s. This probably reflects improved skill in cord injury patients, conscious or unconscious.
treatment of a potential spinal cord injury by the Regulation of blood flow in the skin exposed to
first aid team, since unskilled handling of an un- pressure seems to be deranged in the decentralized
stable neck might result in an ascending neurolog- areas of the body, even though subcutaneous
ical level of the lesion, making it life threatening. adipose tissue blood flow during resting conditions
shows no difference when compared to that in
able-bodied people (Karlsson et al., 1997a).
In the emergency room

When arriving at the hospital the person with a Blood pressure control and the syndrome of
cervical spinal cord injury presents with the fol- inappropriate anti-diuretic hormone secretion
lowing symptoms: flaccid paresis, exclusively dia-
phragmatic respiration, low blood pressure and After the extent of the injury is visualized radio-
mostly bradycardia. It is well recognized that spi- graphically, the injured patient is transported ei-
nal cord injury implies inability to empty the blad- ther to an operating theatre, an intensive care unit
der voluntary and, accordingly, an indwelling or to a spinal cord injury unit. Careful monitoring
catheter is always placed in the urinary bladder of blood pressure, heart rate, respiratory rate and
in the emergency room when a spinal cord injury is body temperature begins. A mean arterial blood
suspected. At this stage, new risks appear. Because pressure above 80 mmHg is recommended (Hadley,
the spinal cord injury is often part of a multitrau- 2002), and this level is sometimes maintained
ma the low blood pressure after cervical spinal by intravenous fluid supply and/or by the use of
cord injury may be misinterpreted as consequent pressor agents. Urine output in the cord-injured
to extensive blood loss. A treatment with rapid patient usually is low during the first days post-
infusion of intravenous fluids might lead to pul- injury, probably due to an inappropriate secretion
monary edema. On the other hand, the cord-in- of anti-diuretic hormone. Three to six days post-
jured person may be bleeding in the abdomen and injury urine output reaches 5–6 l/day; the accu-
this may be difficult to diagnose because of the mulated water is excreted. If urine output is strictly
pre-existing low blood pressure, absence of tonic monitored daily, this polyuria may be misinter-
contraction of the abdominal muscles (guarding preted as a sign of inability to concentrate the
reaction) and absence of pain. For this and other urine, and if the loss of water is fully substituted by
reasons, the cord-injured person needs to be care- intravenous fluids, or vasopressin is given, hypo-
fully investigated by computerized tomography natremia may develop. Some years ago a 28-year-
scanning and magnetic resonance imaging. An un- old man sustained a C7 spinal cord injury resulting
stable fracture in the spinal column with the risk of in tetraplegia and developed a serious hypo-
deterioration of the neurological outcome some- natremia with concomitant loss of vision (Karlsson
times results in placing the patient on a hard table, and Krassioukov, 2004). His cervical fracture was
a so-called ‘‘spine board.’’ Then the patient can be stabilized surgically and during the day of surgery
moved from the emergency room to X-ray or to he received 8.6 l of fluid intravenously. When his
the intensive care unit without having to be moved urine output some days later increased to 6 l, it was
from bed to examination tables and back. However, misinterpreted as inability to concentrate the urine
3

and vasopressin was given. Serum sodium concen- such as simultaneous injuries in the chest. How-
tration decreased to 121 mmol/l (at the lowest lev- ever, the 40% loss of vital capacity, due to the
el), and he was treated by restriction of fluid intake paralysis of the inter-costal muscles, is also of im-
and by mineral corticoids. His sodium level nor- portance. The loss of sympathetically mediated
malized slowly. As soon as his water intake ex- bronchial dilatation may add further to the risk of
ceeded 0.7 l/day his serum level of sodium developing respiratory failure. Inhalation of
decreased. As a result of these problems, he had bronchodilators is usually used in the acute phase
a partial loss of vision that was permanent. Urine following spinal cord injury. In spite of this treat-
output must be calculated for a longer period than ment, excessive mucus production and stagnation
the previous day to avoid this risk of overcom- of secretion is seen. The autonomic nervous system
pensation. imbalance might be life threatening during this
condition, since a person with cervical injury who
has a tendency to hypoxia might sustain a severe
Bradycardia
bradycardia or heart arrest during tracheal suc-
tioning. Irritation of the trachea is a heavy stim-
Another effect of the loss of supraspinal control of
ulus of vagal outflow even in able-bodied people
the sympathetic nervous system is the bradycardia
and the reaction in cord-injured patients might be
that is seen sometimes during the first 2–3 weeks
an exaggeration of this reaction due to the loss of
post-injury. An example of this is a man, who at
supraspinal control of the sympathetic nervous
the age of 39 years sustained a spinal cord injury at
system. Pretreatment with anti-cholinergic drugs is
the C5 level, an ASIA C type injury. He was
sometimes needed before tracheal suctioning.
treated in the intensive care unit for 2 days and his
condition was uncomplicated except for brad-
ycardia. At the spinal cord injury unit, he showed
Temperature regulation
signs of a decreased arterial oxygen tension a tra-
cheal suction induced a cardiac arrest. He was
The respiratory problems might lead to pneumo-
transferred to the intensive care unit, where he
nia with high fever. Then the cord-injured patient
stayed for 12 days. He had a prolonged period of
is faced with another effect of autonomic distur-
bradycardia with a mean heart rate of 48 bpm that
bances: that is the inability to lose excess heat by
lasted for 2–3 weeks. The cardiologist was con-
sweating. This inability might be life threatening
sulted and he prescribed a 24 h electrocardiogram
during high fever or in an extremely warm climate.
recording. The recording showed sinus brad-
Several years ago a young man was treated at Sa-
ycardia with a mean rate of 48 bpm and the in-
hlgrenska University Hospital. He had sustained a
stallation of a pacemaker was suggested. However,
C4 spinal cord injury and had an aspiration of
a week later the bradycardia resolved spontane-
fluid into his lungs during the transport to hospi-
ously and during the next 2 weeks the heart rate
tal. He developed pneumonia and his temperature
increased to a mean of 57 bpm. This imbalance in
increased from 41.0 to 42.41 C and, at this high
the acute phase seems to be replaced by a new
temperature, he died of a cardiac arrest.
balance later since, when measuring heart rate
Even though central temperature control is un-
variability in the chronic phase, no difference is
affected by cervical spinal cord injury, we some-
found when comparing cord-injured subjects to
times see a prolonged period of increased
able-bodied subjects (Gao et al., 2002).
temperature in newly injured patient. Careful ex-
amination reveals no signs of infection or inflam-
Respiratory system mation and 3–6 weeks post-injury, the temperature
normalizes. Some people with cervical spinal cord
In the first weeks post-injury there is a risk of res- injury complain of feeling very cold. This is very
piratory failure in the cervical spinal cord injury marked after a shower, when the patient some-
patient. This is sometimes due to obvious reasons times needs a heating quilt or a heater to feel
4

comfortable. The ability to increase temperature needs to be turned every second hour in order to
by shivering is lost below the lesion level and may avoid skin problems. What makes the skin more
explain some of the sensation. However, some vulnerable to pressure during fever is not known.
patients suffer from coldness all the time. Another problem with the skin, seen during the
first months post-injury, is acne vulgaris that
sometimes flares up in the person with cervical in-
Blood pressure and mobilization
jury. This condition is not life threatening and oc-
curs above as well as below lesion level. Whether
When the fracture is stabilized and the neurological
this is due solely to hormonal disturbances elicited
level of lesion is stable, the person with spinal cord
by a stress reaction or to a combination with au-
injury needs to be mobilized. The low blood pres-
tonomic dysfunction is not known.
sure and the inability to increase blood pressure by
vasoconstriction below lesion level make mobiliza-
tion of the person with cervical injury difficult. Urinary system, bladder control
It has to be done gradually by tilting the patient
101/day while blood pressure and neurological sta- The autonomic nervous system dysfunction in-
tus are continuously monitored. When the patient volves the urinary system during the initial post-
tolerates a 401 tilt, they are usually able to sit in a injury stage of ‘‘spinal shock’’ and for the lifetime
wheelchair. Age at injury seems to affect the ability of the person. The dysfunction entails loss of con-
to mobilize the patients, since the elderly usually trol of the urinary outlet and, during spinal shock,
need more time to become mobilized. Whether this loss of sensation from the bladder wall making the
is due to a lower tolerance to low blood pressure or patient at risk of over-distension of the bladder.
to greater decreases in blood pressure during mo- During spinal shock the bladder is atonic irrespec-
bilization is not known. The renin–angiotensin sys- tive of level of lesion. When the stage of spinal
tem plays a role in blood pressure control in shock is past, which may take 3–4 months, people
cervical spinal cord injury (Johnson et al., 1971; with cervical or thoracic lesions develop a spinal
Sutters et al., 1992). This was clearly demonstrated reflex bladder that expels urine under high intra-
when we treated a man in his 40 s with a cervical vesical pressure at a certain amount of bladder
injury who had suffered from renal failure prior to filling, a condition categorized as upper motor
his injury. His mobilization was prolonged and he neuron lesion. People with lower lumbar and/or
suffered from symptoms of low blood pressure for sacral levels of lesion retain an atonic bladder, a
several months after his injury. lower motor neuron lesion. Regulation of bladder
emptying appears rather robust and might be nor-
malized even if the person suffers from some de-
Skin and sensation
gree of paresis and loss of sensation. In a
retrospective chart review of 249 patients with up-
Very few patients are able to lie or sit more than 4 h
per motor neuron lesions who had been treated at
in the same position without getting redness in the
the Spinal Cord Injury Unit in Göteborg,
skin of areas used for body support; this is the first
we found that almost 30% of the individuals
sign of pressure sores. The patient needs to be
recovered normal micturition and most of them
turned every fourth hour even during a skull trac-
had injuries classified as ASIA C and D (Karin
tion period. Four to five caregivers are needed to do
Pettersson, personal communication).
a safe log-roll of the patient. When the patient is
mobilized to a wheel chair sitting position, selection
of cushions is of great importance in order to avoid Urinary system, renal function
pressure sores. The risk of developing pressure sores
persists in the cord-injured person and is increased Renal dysfunction has previously been the major
during severe infections with increased body tem- cause of death following spinal cord injury. The
perature. Under these circumstances the patient mortality rate due to renal failure has decreased
5

from about 40% in the late 1940s (Whiteneck aspiration and the influence on respiration are
et al., 1992) to 3–5% during the last decade (Webb controlled. The pathogenesis of this temporary
et al., 1984). This dramatic change is probably due paralytic ileus is unclear and the time frame for
to improvements in bladder emptying regime as return of bowel activity does not correspond to
well as to the introduction of antibiotics to treat return of reflex activity in the bladder or return of
urinary tract infections. Even better, it seems that tonus and reflexes in the skeleto-muscular system.
renal function has a capacity to improve during Even though a program for bowel emptying is in-
the first years post-injury. In a retrospective chart troduced, the evacuation of stool may be protract-
review, we found that glomerular filtration rate ed. This might be due to the new balance of
was low in those with cervical spinal cord injury in parasympathetic control, with an intact innerva-
the first months after injury. However, at a follow- tion of the ascending colon via the vagus nerve and
up 2–3 years later we found that the glomerular the loss of supraspinal control of the sacral para-
filtration rate had increased, at least in the group sympathetic supply to the colon.
who emptied the bladder by clean intermittent When the bowel program has started the cord-
catheterization (Karin Pettersson, personal com- injured person may face new problems. There is a
munication). risk of developing anal incontinence, and the risk
is highest with lesions in the lower lumbar level
due to a low tonus in the external anal sphincter.
Gastrointestinal system This is perhaps one of the most devastating effects
of the injury to the spinal cord and may be one
The gastrointestinal system is also affected by the important reason why cord-injured people do not
spinal cord injury. Newly injured patients are at return to work after their injury.
increased risk of developing stress related gastric The higher tonus and the uninhibited activity in
ulcers and are regularly offered anti-acid treat- the anal sphincter after cervical and thoracic le-
ment. It might be that the unopposed vagal out- sions might give rise to severe pain that seems to
flow plays a role and increases the risk of ulcer originate from the anal sphincter. The pain is
formation. Other problems from the intestinal sys- made worse by anal fissures and hemorrhoids, and
tem are obvious to the patient soon after injury. this pain, as well as anal incontinence and consti-
The bowel is silent and the voluntary control of pation, might later lead to colostomy. However,
bowel emptying is lost. This paralytic ileus ceases the pain problem is not always resolved even
within 1–2 weeks, but if liquids or solid food is though the rectum and anal region are bypassed.
given prior to this there is a clear risk of a pro-
longed period of paralytic ileus, with the concom-
itant risk of nausea and vomiting. A patient who is Sexual function
placed in skull traction is hard to manage properly
during vomiting and there is a great risk of aspi- Some cord-injured men already in the intensive
ration. Furthermore, a paralytic ileus could give care unit ask about their ability to have an active
rise to a meteoristic abdomen, which might inter- sex life and become fathers. The erectile dysfunc-
fere with respiration by interference with the tion in men after spinal cord injury has different
breathing movement of the diaphragm. characteristics depending on level of lesion, and
Programs for bowel emptying must be intro- mainly follows the pattern of bladder dysfunction.
duced and we choose a rather conservative way of The person with an upper motor neuron lesion
treatment initially: no ingestion of food or drink usually has the capacity for reflex erection by tac-
until the bowel has been emptied. In people with tile stimulation. The person with a lower motor
lesions at the cervical and thoracic level this takes neuron lesion has loss of all erectile function. The
about 3–6 days, but in the low lumbar lesion level capacity of psychogenic erection is lost in all cord-
the emptying might be further delayed by several injured men with a complete lesion. Retrograde
days. By this regime, the risk of vomiting and ejaculation is the rule when there is an ejaculation
6

at all. Today, we can offer drugs and different sympathetic neurons may be asymptomatic and
stimulations in order to improve erectile function much more frequent than previously known. Con-
and to produce an anterograde ejaculation. Vibra- tinuous measurement of noradrenalin in blood
tion and electro-stimulation need careful monitor- samples collected every 30 min in 24 h revealed
ing of blood pressure since these methods readily several peaks even in subjects who were asympto-
evoke autonomic dysreflexia. By the means of vi- matic (Karlsson et al., 1997b).
bration and electro-stimulation in combination The reaction is triggered by distension of hollow
with insemination or in vitro fertilization, men af- organs below lesion level such as the urinary blad-
ter spinal cord injury can become fathers. At our der, the gall bladder, the renal pelvis and ureters or
Spinal Cord Injury Unit, with 35–50 newly injured the gastrointestinal system. A bone fracture below
patients admitted every year, we have about 75 lesion level may also give rise to the reaction.
children who have cord-injured fathers (Agneta When the triggering factor is withdrawn, the blood
Siösteen, personal communication). pressure returns to normal. However, if the cord-
Sexual function is impaired also in women. The injured person or the treating staff is unfamiliar
female analogue to erectile dysfunction, that is, with the reaction, there is a serious risk of com-
loss of lubrication, needs treatment. Fertility is plications as intracranial hemorrhage or cerebral
unaffected, but the autonomic disturbances make infarction. In our clinical practice there have been
the woman with spinal cord injury at increased two cases in the last 2 years. The first was a woman
risk of urinary incontinence, urinary tract infection in her 50s who sustained a C4–C5 level spinal cord
and pressure sores during child bearing. They are injury and tetraplegia a few years ago. She was
also at risk of developing severe autonomic discharged to home and some months later be-
dysreflexia during labor. came severely constipated and developed an auto-
nomic dysreflexia reaction. She was treated at a
local hospital where there was inadequate knowl-
Autonomic dysreflexia edge of autonomic dysreflexia. The blood pressure
stayed very high and the patient developed a cer-
Two to four months post-injury the person with ebral infarction. Another case was seen recently. A
cervical spinal cord injury may suddenly experi- person with cervical spinal cord injury who had
ence a flushing in the face and complain of severe been injured for more than 30 years was treated
headache. The blood pressure increases from 100/60 for a serious infection. He developed an abscess in
to 240/120 mmHg. When looking for triggering the abdomen and suffered from a prolonged ep-
factors, the staff might find an obstruction of the isode of dysreflexia. During this period we meas-
urinary outlet. When urine passes again the blood ured his cerebral blood flow and found signs of
pressure returns to normal. This reaction, the so- decreased perfusion in parts of the brain, indicat-
called autonomic dysreflexia reaction, is seen in ing vasoconstriction. We speculated that this vaso-
cord-injured people with a lesion level above T6. constriction might have been an autoregulatory
The clinical reaction is not an all-or-none reaction response of the cerebral vessels in the presence of
but graded; in mild cases the person just feels a greatly increased systemic arterial pressure.
small chill. Investigations have shown that the re- The autonomic dysreflexia reaction has other
action is caused by a severe vasoconstriction below side effects and can influence metabolism. Region-
lesion level in skin, muscular (Karlsson et al., al investigation of adipose tissue metabolism
1998) and renal vascular beds (Gao et al., 2002). above and below lesion level showed an increase
Presumably also the splanchnic/gastrointestinal in glycerol release — activation of lipolysis —
vascular bed is involved. The reaction is mediat- during induced dysreflexia, that is, during sympa-
ed by the sympathetic nervous system as shown by thetic activation below lesion level (Karlsson et al.,
a profound increase in noradrenalin spillover be- 1998). Whether this is of importance for the insulin
low lesion level (Karlsson et al., 1998; Gao et al., resistance sometimes seen after spinal cord injury
2002). The peripheral afferent stimulation of the (Karlsson, 1999) is not known.
7

Another associated risk with the autonomic Long-term effects


dysreflexia reaction is the vasoconstriction in the
renal vascular bed (Gao et al., 2002). This sympa- Gradually after spinal cord injury some risks seem
thetic activation below lesion level might contrib- to diminish, others persist and new ones develop.
ute to the development of renal failure. Previously, By 15–20 years post-injury new complications
many tetraplegic patients used bladder tapping to from the circulatory system are sometimes seen.
condom drainage as a method of bladder empty- Following large meals the cord-injured person with
ing. Bladder tapping is known to induce auto- a cervical lesion is sometimes unable to sit upright
nomic dysreflexia every time it is performed. The due to rapid fall in blood pressure. Whether this is
introduction of intermittent catheterization may merely an effect of duration of injury or a marker
thus have the advantage of maintaining renal of concomitant disease is not known. The muscle
function both by the intermittent total emptying of and skin vascular beds are unable to vasoconstrict
the bladder, with markedly lower risk of urinary during the post-prandial increase in blood flow in
tract infections, and by the absence of triggering the intestinal vascular bed, but this limitation is
factors for renal vasoconstriction. present since the onset of injury and therefore,
does not solely explain the new complication.
People with cervical spinal cord injury sometimes
experience low urine output during the daytime
Visceral sensation
when they are in sitting position. During the night
there is a huge urine flow, making the patient at risk
The loss of sensation includes not only skin and
of incontinence or over-distension of the bladder.
joints but also loss of visceral sensation. This is of
importance during pathological processes in the
internal organs, and also seems to include loss of
Time course of autonomic nervous system changes
normal sensation of hunger and satiety, a condi-
— transitional stage?
tion that might contribute to disturbances in body
weight sometimes seen in tetraplegic people. A
Bradycardia lasts for few weeks. Paralytic ileus
weight gain implies the risk of diabetes, impaired
lasts for 1–2 weeks. Signs of the inappropriate an-
glucose control and increased levels of serum lip-
ti-diuretic hormone secretion syndrome appear
ids. A higher risk in cord-injured than in able-
during the first week. The risk of respiratory fail-
bodied subjects has been reported (Duckworth
ure is great during the first weeks after injury. The
et al., 1983; Bauman and Spungen, 2001), although
bladder is flaccid for 3–4 months. Body tempera-
a study that controlled for inheritance by compar-
ture is increased for 3–4 weeks. The risk of deep
ing cord-injured subjects to their siblings showed
vein thrombosis is increased for weeks or months.
no difference in glucose tolerance evaluated by the
There are many signs of a transitional stage in
hyper-insulinemic, normo-glycemic clamp method
autonomic nervous system dysfunction following
(Karlsson, 1999).
spinal cord injury. However, as described above,
the time frame is highly divergent. Some of the
changes are attributed to the spinal shock, or more
Thrombo-emboli precisely to the recovery from spinal shock. How-
ever, our knowledge of the pathology behind spi-
During the first months post-injury the cord-in- nal shock is sparse, and it seems that the return of
jured person is at increased risk of developing deep reflex activity in the spinal cord follows a different
vein thrombosis. Treatment with anti-coagulants time course. Muscular tone and tendon reflexes
is given for 3–6 months. The role of the autonomic appear within 6–8 weeks, whereas reflexes for
nervous system in this increased risk is unclear. bladder emptying may return much later, up to
Later in life, cord-injured people appear not to 3–4 months post-injury. The imbalance in heart
have increased risk of deep vein thrombosis. rate regulation has a time course of its own. We
8

have some knowledge from animal and human Karlsson, A.K. (1999) Insulin resistance and sympathetic func-
research regarding the plasticity of spinal neural tion in high spinal cord injury. Spinal Cord, 37: 494–500.
Krassioukov, A.V., Bunge, R.P., Pucket, W.R. and Bygrave,
circuits after injury (Krassioukov and Weaver,
M.A. (1999) The changes in human spinal sympathetic pre-
1995; Krassioukov et al., 1999) but the clinical ganglionic neurons after spinal cord injury. Spinal Cord, 37:
implications of these findings are unclear. 6–13.
After spinal cord injury, new balances are cre- Karlsson, A.K., Elam, M., Friberg, P., Biering-Sorensen, F.,
ated in the autonomic nervous system and we need Sullivan, L. and Lonnroth, P. (1997a) Regulation of lipolysis
to create more knowledge about how this is done. by the sympathetic nervous system: a microdialysis study in
normal and spinal cord-injured subjects. Metabolism, 46:
We also have to develop more understanding of 388–394.
the overall disturbances in the autonomic nervous Karlsson, A.K., Elam, M., Friberg, P., Sullivan, L., Attvall, S.
system of cord-injured people since these distur- and Lonnroth, P. (1997b) Peripheral afferent stimulation of
bances have a profound impact on their life. decentralized sympathetic neurons activates lipolysis in spinal
cord-injured subjects. Metabolism, 46: 1465–1469.
Karlsson, A.K., Friberg, P., Lonnroth, P., Sullivan, L. and
Elam, M. (1998) Regional sympathetic function in high spi-
References nal cord injury during mental stress and autonomic dysre-
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Anderson, K.D. (2004) Targeting recovery: Priorities of the spi- Karlsson, A.K. and Krassioukov, A.V. (2004) Hyponatremia-
nal cord-injured population. J. Neurotrauma, 21: 1371–1383. induced transient visual disturbances in acute spinal cord in-
Bauman, W.A. and Spungen, A.M. (2001) Carbohydrate and jury. Spinal Cord, 42(3): 204–207.
lipid metabolism in chronic spinal cord injury. J. Spinal Cord Krassioukov, A.V. and Weaver, L.C. (1995) Reflex and mor-
Med., 24: 266–277. phological changes in spinal preganglionic neurons after cord
Duckworth, W.C., Jallepalli, P. and Solomon, S.S. (1983) Glu- injury in rats. Clin. Exp. Hypertens., 17: 361–373.
cose intolerance in spinal cord injury. Arch. Phys. Med. Re- Sutters, M., Wakefield, C., O’Neil, K., Appleyard, M., Frankel,
habil., 64: 107–110. H., Mathias, C.J. and Peart, W.S. (1992) The cardiovascular,
Gao, S.A., Ambring, A., Lambert, G. and Karlsson, A.K. endocrine and renal response of tetraplegic and paraplegic
(2002) Autonomic control of the heart and renal vascular bed subjects to dietary sodium restriction. J. Physiol., 457:
during autonomic dysreflexia in high spinal cord injury. Clin. 515–523.
Auton. Res., 12: 457–464. Webb, D.R., Fitzpatrick, J.M. and O’Flynn, J.D. (1984) A 15-
Hadley, M.N., Walters, B.C., Grabb, P.A., Oyesiku, N.M., year follow-up of 406 consecutive spinal cord injuries. Br. J.
Przbylski, G.J., Resnick, D.K. and Ryken, T.C. (2002) Man- Urol., 56: 614–617.
agement of acute spinal cord injuries in an intensive care unit Whiteneck, G.G., Charlifue, S.W., Frankel, H.L., Fraser, M.H.,
or other monitored settings. Neurosurgery, 50: S51–S57. Gardner, B.P., Gerhart, K.A., Krishnan, K.R., Menter, R.R.,
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 1

Effects of spinal cord injury on synaptic inputs to


sympathetic preganglionic neurons

Ida J. Llewellyn-Smith1,, Lynne C. Weaver2 and Janet R. Keast3

1
Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, SA 5042, Australia
2
Spinal Cord Injury Laboratory, BioTherapeutics Research Group, Robarts Research Institute, London, ON, Canada
3
Pain Management Research Institute, University of Sydney at Royal North Shore Hospital, St Leonards,
NSW, Australia

Abstract: Spinal cord injuries often lead to disorders in the control of autonomic function, including
problems with blood pressure regulation, voiding, defecation and reproduction. The root cause of all these
problems is the destruction of brain pathways that control spinal autonomic neurons lying caudal to the
lesion. Changes induced by spinal cord injuries have been most extensively studied in sympathetic pre-
ganglionic neurons, cholinergic autonomic neurons with cell bodies in the lateral horn of thoracic and
upper lumbar spinal cord that are the sources of sympathetic outflow. After an injury, sympathetic pre-
ganglionic neurons in mid-thoracic cord show plastic changes in their morphology. There is also extensive
loss of synaptic input from the brain, leaving these neurons profoundly denervated in the acute phase of
injury. Our recent studies on sympathetic preganglionic neurons in lower thoracic and upper lumbar cord
that regulate the pelvic viscera suggest that these neurons are not so severely affected by spinal cord injury.
Spinal interneurons appear to contribute most of the synaptic input to these neurons so that injury does not
result in extensive denervation. Since intraspinal circuitry remains intact after injury, drug treatments
targeting these neurons should help to normalize sympathetically mediated pelvic visceral reflexes. Fur-
thermore, sympathetic pelvic visceral control may be more easily restored after an injury because it is less
dependent on the re-establishment of direct synaptic input from regrowing brain axons.

Every year, tens of thousands of people worldwide and severity of these problems is dependent
suffer a spinal cord injury, often with devastating on the level and completeness of the injury. Two
consequences. Injured people can lose mobility cardiovascular consequences of spinal cord injury
because they become partially or totally paralyzed are resting or postural hypotension and autonomic
and experience persistent pain and spasticity. As dysreflexia, a condition in which strokes or death
this volume documents, their quality of life can can occur when noxious or innocuous sensory
also be significantly damaged by injury-induced stimuli entering the cord below the injury reflexly
disorders in the control of autonomic function, induce episodes of hypertension. Spinal cord
including problems with blood pressure regulation, injury can also produce a variety of difficulties
voiding, defecation and reproduction. The presence that impair the voiding of urine, including
detrusor hyperreflexia, detrusor sphincter dyssy-
Corresponding author. Tel.: +61-8-8204-4456; nergia and detrusor areflexia. Fecal incontinence
Fax: +61-8-8204-5268;
and constipation are also outcomes of spinal cord
E-mail: Ida.Llewellyn-Smith@flinders.edu.au injury; and inability to achieve psychogenic and

DOI: 10.1016/S0079-6123(05)52001-6 11
12

reflexogenic erections and ejaculatory dysfunction dorsolateral funiculus in the white matter; sympa-
in men and failure of vaginal lubrication in women thetic preganglionic neurons in the dorsolateral
have a significant impact on sexual life after injury. funiculus are more common in the rostral than the
The root cause of all these autonomic sequelae caudal thoracic cord (Strack et al., 1988). A small
of spinal cord injury is the disruption of the brain proportion of sympathetic preganglionic neurons
pathways that control spinal autonomic neurons. are associated with the bundles of dendrites that
Subsequently, when axons conveying supraspinal course mediolaterally between the intermediolateral
drive are dying or dead, neuronal circuits caudal to cell column and central canal. These sympathetic
the injury are able to reorganize. Primary afferent preganglionic neurons have spindle-shaped cell
neurons sprout (Christensen and Hulsebosch, bodies and comprise the intercalated nucleus. The
1997; Krenz and Weaver, 1998b; Krenz et al., somata and dendrites of the sympathetic pregangl-
1999; Wong et al., 2000; Weaver et al., 2001; On- ionic neurons in the intercalated nucleus are ori-
darza et al., 2003) and spinal autonomic interneu- ented parallel to the dendritic bundles with which
rons can become key regulators of some of the they are associated. A final concentration of sym-
information that is conveyed from the spinal cord pathetic preganglionic cell bodies, which are usually
to the periphery (see Schramm, this volume). fusiform in shape, occurs in the central autonomic
Changes also occur in the synaptic circuitry con- area dorsal to the central canal. Somata occupying
trolling sympathetic preganglionic neurons that this position are most frequently encountered at the
have axons providing central drive to all sympa- caudal end of the sympathetic preganglionic neuron
thetic postganglionic neuron in paravertebral and distribution, i.e., in the lowest thoracic and upper
prevertebral ganglia and to chromaffin cells in the lumbar segments.
adrenal medulla.

Morphological changes after spinal cord injury


Location and morphology of sympathetic
preganglionic neurons Spinal cord injury can evoke significant changes in
sympathetic preganglionic neurons, as well as de-
Sympathetic preganglionic neurons are small- to priving them of input from the brain. While neu-
medium-sized cholinergic neurons and their cell rons at the site of a cord injury can be killed,
bodies are located in the thoracic and upper lumbar sympathetic preganglionic neurons that lie distant
spinal cord in four distinct subnuclei within the from the lesion site may also be affected, at least in
lateral horn (Cabot, 1990). The majority of sym- the acute phase of injury. Within 3 days of a com-
pathetic preganglionic somata occur in the inter- plete spinal cord transection at thoracic segment 4/5,
mediolateral cell column, which lies at the border the dendrites of sympathoadrenal preganglionic
between the grey and white matter of the spinal neurons in the intermediolateral cell column of
cord. In the intermediolateral cell column, the cell mid-thoracic cord have retracted to about one-
bodies of sympathetic preganglionic neurons are third of their original length and the diameter of
spindle-shaped or fusiform and occur in groups or their cell bodies has decreased to about 60% of
‘‘nests’’ that are spaced at short intervals along the that in intact cord (Llewellyn-Smith and Weaver,
grey–white boundary. Most of the dendrites of the 2001). This reduction in soma size and dendrite
neurons in the intermediolateral cell column run length is less pronounced 7 days after transection;
rostrally or caudally and can be hundreds of and, by 14 days post-operatively, the sympathetic
micrometers long. In addition, some sympathetic preganglionic neurons and their dendrites are not
preganglionic neurons in the intermediolateral significantly different in size from those in intact
cell column have dendrites that are oriented med- cord (Krassioukov and Weaver, 1996; Krenz and
iolaterally, traveling either into the dorsolateral Weaver, 1998a). The shrinkage and regrowth of
funiculus or toward the central canal. Other sym- sympathetic preganglionic somata and dendrites
pathetic preganglionic somata are situated in the correlate with the degeneration and clearance from
13

the intermediolateral cell column of axons de- the intermediolateral cell column, around it in
tached from their cell bodies by the transection. spinal cord laminae V and VII and in lamina X
Many degenerating profiles of severed axons can dorsal to the central canal (Cabot et al., 1994; Joshi
be seen ultrastructurally at 3 days after injury, but et al., 1995; Clarke et al., 1998; Cano et al., 2001;
virtually none are present at 14 days (Weaver et al., Deuchars et al., 2001; Tang et al., 2004). The axons
1997). Although new synapses form on sympatho- of presympathetic supraspinal and intraspinal neu-
adrenal preganglionic neurons after a spinal cord rons contain a diverse array of neurotransmitters,
injury (Weaver et al., 1997), reinnervation by ax- many of which have been shown to have direct
ons of spinal neurons is unlikely to be the spur for effects on sympathetic preganglionic neurons.
regrowth of dendrites. Even at 14 days after a Neurons in the brain are probably the exclusive
complete transection, sympathetic preganglionic source of monoamine innervation, whereas both
neurons in the mid-thoracic cord are profoundly supraspinal and intraspinal neurons contribute ax-
denervated. The density of synapses on their cell ons containing amino acids and neuropeptides.
bodies has been cut to half of that in intact cord
and their axodendritic input is reduced by 70%
(Llewellyn-Smith and Weaver, 2001). Whether or Amino acids
not sympathetic preganglionic neurons continue to
be reinnervated after 2 weeks of injury has not Glutamate, g-aminobutyric acid (GABA) and
been studied ultrastructurally. However, anastomo- glycine, all produce fast synaptic responses in sym-
sing networks of axons immunoreactive for growth- pathetic preganglionic neurons (e.g., Mo and Dun,
associated protein 43, a marker for developing and 1987a, b; Inokuchi et al., 1992a, b; Krupp and Feltz,
regenerating axons, are present at least as long as 6 1995; Krupp et al., 1997) and are considered the
weeks after spinal cord injury (Cassam et al., 1999). main fast-acting transmitters regulating their activ-
Continuing reorganization of synaptic circuitry ity (Dampney, 1994). Axons immunoreactive for
controlling the activity of sympathetic preganglion- these amino acids synapse on sympathetic pregangl-
ic neurons may underlie the increasing severity of ionic neurons (Bacon and Smith, 1988; Bogan et al.,
attacks of autonomic dysreflexia (Maiorov et al., 1989; Cabot et al., 1992) and quantitative ultra-
1997a, b; Marsh and Weaver, 2004). structural studies have demonstrated that synaptic
vesicles containing at least one type of amino acid
are present in virtually all of the axons that provide
Innervation of sympathetic preganglionic neurons in input to these neurons (Llewellyn-Smith et al., 1992,
intact and injured cord 1995b, 1998). Brainstem neurons innervate sympa-
thetic preganglionic neurons monosynaptically
In intact cord, sympathetic preganglionic neurons (Zagon and Smith, 1993; Deuchars et al., 1995,
are innervated by both supraspinal and intraspinal 1997); and these spinally projecting neurons contain
neurons. Virus tracing studies have been particu- markers for glutamate and GABA axons, including
larly useful for revealing the locations of presym- phosphate activated glutaminase, vesicular gluta-
pathetic neurons, i.e., those that are directly mate transporter 2 and glutamic acid decarboxylase
antecedent to sympathetic preganglionic neurons (Minson et al., 1991; Stornetta et al., 2002, 2004).
and are likely to be involved in regulating their Furthermore, immunoreactivity for glutamate or
activity. Supraspinal inputs to sympathetic pre- GABA occurs in boutons in the intermediolateral
ganglionic neurons come from five main brain re- cell column that have been anterogradely labeled
gions, including the rostral ventrolateral medulla, from the medulla (Llewellyn-Smith et al., 1995b).
the rostral ventromedial medulla, the caudal raphe Although neurons in the brain provide many of the
nuclei, the A5 region and the paraventricular nu- glutamate- and GABA-immunoreactive axons in
cleus of the hypothalamus (Strack et al., 1989; the intermediolateral cell column, spinal cord injury
Sved et al., 2001). Spinal neurons that project to does not deprive sympathetic preganglionic neurons
sympathetic preganglionic neurons occur within of amino acid-containing inputs. Ultrastructural
14

studies at times when severed supraspinal axons are immunoreactive for dopamine b-hydroxylase
degenerating or have just been removed from below (Cassam et al., 1997), the enzyme that produces
a lesion show that glutamate- and GABA-immuno- noradrenaline from dopamine; and we have de-
reactive synaptic contacts persist on sympathetic scribed axons caudal to a 2-week transection that
preganglionic neurons caudal to either 3- or 7-day contain tyrosine hydroxylase and form synapses
complete spinal cord transections (Llewellyn-Smith in the intermediolateral cell column (Fig. 2B;
et al., 1997; Llewellyn-Smith and Weaver, 2001). Llewellyn-Smith et al., 1995a). Hence, the cat-
Hence, intraspinal neurons as well as supraspinal echolamine enzyme-immunoreactive fibers present
neurons provide amino acid-containing inputs to at 2 weeks may arise from these neurons. Some
sympathetic preganglionic neurons. immunoreactivity may also be present in the non-
terminal portions of severed axons since, at 2
weeks, degenerating terminals cannot be found in
Monoamines the intermediolateral cell column at the ultrastruc-
tural level (Weaver et al., 1997). Determining
Adrenaline and noradrenaline evoke both whether the spinal neurons that express catechola-
excitatory and inhibitory responses in sympathet- mine enzymes at 2 weeks after transection synthe-
ic preganglionic neurons (Coote et al., 1981; size dopamine, adrenaline or noradrenaline will
Kadzielawa, 1983; Ma and Dun, 1985a; Miyazaki require different experimental strategies, such as
et al., 1989; Lewis and Coote, 1990a). At the light fluorescence histochemistry, or multiple-label
microscope level, sympathetic preganglionic neu- immunofluorescence for investigating coexistence
rons at all levels of intact cord are surrounded by of relevant synthetic enzymes or amine transport-
networks of nerve fibers immunoreactive for en- ers. A more detailed anatomical analysis of
zymes of catecholamine synthesis, such as tyrosine 11-week transected cord will also be needed to as-
hydroxylase (Fig. 1A) and phenylethanolamine certain whether there are any enzyme-immunore-
N-methyltransferase (Fig. 1B). In intact cord, phe- active fibers present at the chronic stage of injury.
nylethanolamine N-methyltransferase-immunore- Pharmacological and physiological studies indi-
active axons have been confirmed to synapse on cate that, in general, serotonin (5-hydroxytrypta-
sympathetic preganglionic neurons at the electron mine (5-HT)) has a sympathoexcitatory action on
microscope level (Milner et al., 1988; Bernstein- sympathetic nerve activity and directly on sympa-
Goral and Bohn, 1989) and we have found thetic preganglionic neurons (e.g., Ma and Dun,
that sympathetic preganglionic neurons receive 1986; Yusof and Coote, 1988; Lewis and Coote,
synapses from axons with immunoreactivity for 1990b; Pickering et al., 1994). Serotonergic axons,
tyrosine hydroxylase (Fig. 2A). All of this cat- marked by either immunoreactivity for 5-HT or
echolamine input to sympathetic preganglionic the serotonin transporter, also form a dense plexus
neurons probably originates in the brain (see be- of axons around sympathetic preganglionic neu-
low). Major sources are the C1 adrenergic neurons rons (Figs. 3A and C) and synapses by 5-HT-
of the rostral ventrolateral medulla and the nor- immunoreactive axons have been demonstrated on
adrenergic neurons of the A5 group (Jansen et al., sympathetic preganglionic neurons that project to
1995). The supraspinal origin of catecholamine in- the superior cervical ganglion and adrenal medulla
put is supported by the disappearance of immuno- (Bacon and Smith, 1988; Vera et al., 1990; Jensen
reactivity for catecholamine-synthesizing enzymes et al., 1995). Retrograde and viral tracing studies
caudal to a complete spinal cord transection. indicate that the serotonergic axons surrounding
Staining for tyrosine hydroxylase and phenyl- sympathetic preganglionic neurons arise from rap-
ethanolamine N-methyltransferase is substantially he neurons, mainly those in the medullary nuclei
reduced at 2 weeks post-operatively (Figs. 1C and (Loewy and McKellar, 1981; Bowker et al., 1982;
D) and is absent by 11 weeks (Figs. 1E and F). Jansen et al., 1995). Very rare 5-HT-immunoreac-
Interestingly, after spinal cord injury, some neurons tive neurons have been detected in the spinal cord
in the intermediate grey of the spinal cord become (Newton et al., 1986). However, these are unlikely
15

Fig. 1. Axons containing immunoreactivity for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenyl-
ethanolamine N-methyltransferase (PNMT), disappear from the intermediolateral cell column caudal to a complete spinal cord
transection (TX). (A, B) Intact cord. (C, D) 2-week transected cord. (E, F) 11-week transected cord. (A, C, E) Stained for tyrosine
hydroxylase and (B, D, F) stained for phenylethanolamine N-methyltransferase (PNMT). Transections were located in caudal thoracic
segment 4/rostral thoracic segment 5. All micrographs show the intermediolateral cell column in thoracic segment 6. Bars, 100 mm.

to nnervate sympathetic preganglionic neurons be- Neuropeptides


cause 5-HT-immunoreactive and serotonin trans-
porter-immunoreactive axons are absent from Axons containing a substantial array of neuro-
autonomic areas caudal to a 2-week transection peptides have been demonstrated by light micros-
(Figs. 3B and D). Hence, severed serotonergic ax- copy in regions of the cord where the cell bodies
ons disappear from the cord before axons that are and dendrites of sympathetic preganglionic neu-
immunoreactive for catecholamine enzymes. rons are located (Table 1) and a number of these
16

Fig. 2. Axons immunoreactive for tyrosine hydroxylase form synapses in intact and transected cord. (A) In the intermediolateral cell
column of intact cord, a tyrosine hydroxylase-immunoreactive varicosity (TH) forms a synapse (arrowhead) on a dendrite that
contains cholera toxin B subunit (CTB) retrogradely transported from the adrenal medulla. An adjacent non-immunoreactive var-
icosity (asterisk) synapses (arrowheads) on the same dendrite. (B) In the intermediolateral cell column of thoracic segment 8, a tyrosine
hydroxylase-immunoreactive varicosity (TH) forms a synapse (arrowheads) on a dendrite 2 weeks after a complete spinal cord
transection (TX) in caudal thoracic segment 4/rostral thoracic segment 5. An adjacent non-immunoreactive varicosity (asterisk)
synapses (arrowhead) on the same dendrite. Bars, 500 nm.

Fig. 3. Axons containing the serotonergic markers, 5-HT or the serotonin transporter, disappear from the intermediolateral cell
column caudal to a complete spinal cord transection (TX). (A, C) Intact cord. (B, D) 2-week transected cord. (A, B) Stained for 5-HT
and (C, D) stained for the serotonin transporter (SERT). Transections were located in caudal thoracic segment 4/rostral thoracic
segment 5. All micrographs show the intermediolateral cell column in thoracic segment 6. Bar in (D) applies to (A—D), 100 mm.
17

Table 1. Neuropeptide immunoreactivity identified in axons in autonomic regions of the thoracic and upper lumbar spinal cord

Angiotensin II Neurotensin
Avian pancreatic polypeptide (APP) Nociceptin
Calcitonin gene-related peptide (CGRP) Orexina
Cholecystokinin (CCK) Oxytocin
Cocaine and amphetamine regulated transcript (CART)a Pituitary adenylate cyclase activating polypeptide (PACAP)a
Corticotropin releasing factor (CRF) Somatostatin
Enkephalina Substance Pa
Galanin Thyrotropin releasing hormone
Neuropeptide Y (NPY)a Vasoactive intestinal peptide (VIP)
Neurophysin Vasopressin
a
Axons containing these neuropeptides have been shown to form synapses either in the intermediolateral cell column or on identified
sympathetic preganglionic neurons.

have been shown to synapse on sympathetic pre- acetyltransferase-immunoreactive (i.e., choliner-


ganglionic neurons (Bacon and Smith, 1988; Vera gic) neurons in the intermediolateral cell column
et al., 1990; Llewellyn-Smith et al., 1991; Pilowsky (Llewellyn-Smith and Weaver, 2004). Hence, some
et al., 1992). When applied to sympathetic pre- of the axons containing these neuropeptides come
ganglionic neurons, many of these neuropeptides from spinal interneurons to innervate the choli-
evoke synaptic responses (e.g., Ma and Dun, nergic sympathetic preganglionic neurons. The
1985b; Dun and Mo, 1988; Kolaj et al., 1997; source of the neuropeptide Y-immunoreactive ax-
Lai et al., 1997; Antunes et al., 2001; van den Top ons that persist in the lateral horn may be the
et al., 2003). Some of the neuropeptide-immuno- neurons in laminae V and VII in intact cord that
reactive axons supplying sympathetic preganglion- express neuropeptide Y mRNA (Minson et al.,
ic neurons arise exclusively from neurons in the 2001). The intraspinal enkephalin innervation of
brain, including those containing oxytocin, vaso- autonomic regions may arise from small enkepha-
pressin and orexin. However, other neuropeptides, lin-immunoreactive neurons in lamina X that
such as substance P, enkephalin and neuropeptide we have detected in sections from intact cord
Y, occur in autonomic areas of intact and fixed with high concentrations of glutaraldehyde
transected cord (Figs. 4–6), suggesting that both (Llewellyn-Smith and Keast, unpublished obser-
supraspinal and intraspinal neurons supply the vations), as neurons in this location are known to
lateral horn. Substance P is co-localized with se- communicate with sympathetic preganglionic neu-
rotonin in brainstem neurons that innervate the rons (Cano et al., 2001; Tang et al., 2004). The cell
spinal cord (Sasek et al., 1990). A large subset bodies of origin of the intraspinal substance P in-
of spinally projecting cardiovascular neurons in put have yet to be defined.
the medulla contain preproenkephalin mRNA
(Stornetta et al., 1999) and immunoreactivity for
neuropeptide Y and mRNA for preproneuropep- Rostrocaudal differences in sympathetic
tide Y have been identified in medullospinal neu- preganglionic neurons and their innervation
rons (Minson et al., 1994; Stornetta et al., 1999).
Nevertheless, complete spinal cord transection The sympathetic nervous system was original-
does not destroy all varicose axons in the inter- ly thought to act in an undifferentiated way to
mediolateral cell column immunoreactive for sub- allow an animal to respond appropriately to life-
stance P, enkephalin or neuropeptide Y (Davis threatening situations. However, over the past two
and Cabot, 1984; Romagnano et al., 1987; Cassam decades, it has become increasingly clear that cen-
et al., 1997) and we have shown that, caudal to a 7- tral control of sympathetic outflow is differential,
day complete transection, axons containing each permitting specific functional groups of sympathetic
of these neuropeptides form synapses on choline preganglionic neurons to respond in different ways
18

Fig. 4. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and ne-
uropeptide Y, are still present in the intermediolateral cell column of mid-thoracic spinal cord segments. (A, C, E) Intact cord. (B, D,
F) 2-week transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (E, F) stained for
neuropeptide Y (NPY). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. Micrographs show the
intermediolateral cell column from thoracic segments 7, 8 or 9. Bars, 100 mm.

to the same homeostatic challenge (reviewed by the outcomes of spinal cord injury will vary de-
Morrison, 2001). Differences in the spatial ar- pending on the functional group of sympathetic
rangement of sympathetic preganglionic neurons preganglionic neurons that are deprived of their
and in their innervation are likely to be the ana- supraspinal input.
tomical basis for these differentiated physiological Sympathetic preganglionic neurons are topo-
responses. The differences in the innervation of graphically organized along the rostrocaudal axis
sympathetic preganglionic neurons suggest that of the spinal cord (Strack et al., 1988). Preganglionic
19

Fig. 5. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and ne-
uropeptide Y, are still present in the intermediolateral cell column of lower thoracic and upper lumbar segments. (A, C, E) Intact cord.
(B, D, F) 2-week transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (E, F) stained for
neuropeptide Y (NPY). Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. Micrographs show the
intermediolateral cell column from thoracic segment 13 or lumbar segment 1. Bars, 100 mm.

neurons in the rostral thoracic cord supply rostral and adrenaline from chromaffin cells. The caudal
sympathetic ganglia (e.g., the superior cervical end of the range includes sympathetic pregangl-
ganglion and the stellate ganglion) and participate ionic neurons involved in regulating the activity of
in the regulation of targets in the upper body, like organs in the lower body, like the urinary bladder,
pupils, salivary glands and the heart. The mid- lower bowel and reproductive organs. Despite this
thoracic cord contains sympathetic preganglionic general topographical organization, the distribu-
neurons that project to the celiac ganglion as part tions of sympathetic preganglionic neurons pro-
of the circuitry controlling mesenteric vasculature, jecting to different target ganglia or adrenal
gut motility and gut secretion as well as sympa- chromaffin cells overlap so that sympathetic pre-
thetic preganglionic neurons projecting to the ad- ganglionic neurons of different functions are in-
renal medulla to regulate release of noradrenaline termixed within each spinal segment. For example,
20

Fig. 6. Caudal to a complete spinal cord transection (TX), axons containing the neuropeptides, substance P, enkephalin and ne-
uropeptide Y, are still present in the central autonomic area of upper lumbar segments. (A, C, E) Intact cord. (B, D, F) 2-week
transected cord. (A, B) Stained for substance P (SP), (C, D) stained for enkephalin (ENK) and (F) stained for neuropeptide Y (NPY).
Transections were located in caudal thoracic segment 4/rostral thoracic segment 5. All micrographs show the central autonomic area in
lumbar segment 2. cc, central canal. Bars, 100 mm.

in the rat, thoracic segment 6 contains a mixture of rostrocaudal arrangement of their cell bodies. In
sympathetic preganglionic neurons that send ax- autonomic areas of cat thoracolumbar cord, nerve
ons to the superior cervical ganglion, stellate gan- fibers immunoreactive for 5-HT, substance P,
glion, celiac ganglion or adrenal medulla. somatostatin, oxytocin, neurotensin or neurophy-
Although not very well studied, rostrocaudal sin show a non-uniform rostrocaudal distribution
differences in the innervation of sympathetic pre- (Krukoff et al., 1985) as do 5-HT-immunoreactive
ganglionic neurons parallel the target-based axons in rabbit intermediolateral cell column
21

(Jensen et al., 1995). In rats, oxytocin-immunore- axons at the light microscopic level (Figs. 5C,
active axons closely appose sympathetic pregangl- and 6C). Similarly, dense baskets were present
ionic neurons retrogradely labeled from the around retrogradely labeled neurons in cords at
cervical sympathetic trunk; but sympathoadrenal 2 and 11 weeks after transection (e.g., Figs. 7 and 8).
preganglionic neurons are not innervated by ox- These observations imply that most of the
ytocin fibers (Holets and Elde, 1982; Appel and enkephalin input to pelvic visceral sympathetic
Elde, 1988). Inputs to choline acetyltransferase- preganglionic neurons comes from spinal neurons
immunoreactive sympathetic preganglionic neu- below the transection. Furthermore, the density of
rons that express Fos in response to hypotension the enkephalin innervation of neurons projecting
also show a heterogenous pattern of innervation to the major pelvic ganglion suggests that inter-
by some types of axons (Minson et al., 2002). neurons are likely to provide the predominant in-
Although apposing almost all Fos-positive sym- put to sympathetic preganglionic neurons that
pathetic preganglionic neurons in upper and control the pelvic viscera.
middle thoracic cord, neuropeptide Y or phenyl- At the electron microscope level, we observed
ethanolamine N-methyltransferase-immunoreac- that the density of synapses on sympathetic pre-
tive axons avoid significant proportions of these ganglionic neurons projecting to the major pelvic
neurons in lower thoracic segments. More than ganglion did not appear to differ in intact and
half of the hypotension-sensitive sympathetic transected cord, although this observation was not
preganglionic neurons in the middle and lower quantified. This conclusion was supported by
thoracic cord lacked appositions from galanin- quantification of enkephalin-immunoreactive in-
immunoreactive axons, whereas some choline ace- put to these neurons. In intact cord, sympathetic
tyltransferase-positive, Fos-negative neurons in preganglionic neurons that projected to the major
the lumbar cord lay in dense baskets of galanin- pelvic ganglion received many synapses from
positive fibers. enkephalin-immunoreactive axon terminals. In
Recently, we have examined enkephalin- the intermediolateral cell column, 52% of the
immunoreactive inputs to sympathetic pregangl- synaptic input to retrogradely labeled cell bodies
ionic neurons retrogradely labeled with cholera was enkephalin-immunoreactive. Furthermore,
toxin B subunit from the major pelvic ganglion this enkephalin innervation was targeted to cell
(Llewellyn-Smith et al., 2005), which contains bodies in preference to dendrites since only 29% of
sympathetic and parasympathetic preganglionic the input to retrogradely labeled dendrites was
neurons innervating the urinary bladder, lower enkephalin positive. In the 2-week transected cord,
bowel and reproductive organs. This work has re- enkephalin occurred in 65% of the varicosities that
vealed a striking difference between the reaction to synapsed on sympathetic preganglionic somata
spinal cord injury of these neurons, which have that projected to the major pelvic ganglion from
somata in thoracic segment 12 to lumbar segment the intermediolateral cell column. The proportion-
2, and more rostral sympathetic preganglionic al change in input between cell bodies in intact and
neurons. In contrast to choline acetyltransferase- transected cord was not statistically significant.
immunoreactive sympathetic preganglionic neu- However, the increase in enkephalin input from
rons in thoracic segment 8 (Llewellyn-Smith and 52% to 65% suggests a small loss of synapses due
Weaver, 2001), sympathetic preganglionic neurons to transection. This loss might have been revealed
that are in circuits controlling pelvic viscera appear if data had been collected from a larger number of
to retain most of their innervation after a complete rats. These data indicate that the enkephalin input
spinal cord transection. In intact cord, we found to pelvic visceral sympathetic preganglionic neu-
that sympathetic preganglionic neurons projecting rons is not significantly affected by transection,
to the major pelvic ganglion from the intermedio- due to the fact that it is predominantly intraspinal.
lateral cell column, the intercalated nucleus and Since pelvic visceral sympathetic preganglionic
central autonomic area were surrounded by very neurons are not substantially denervated after spi-
dense baskets of enkephalin-immunoreactive nal cord injury, their somata and dendrites may
22

Fig. 7. In transected cord, enkephalin-immunoreactive axons closely appose sympathetic preganglionic neurons projecting to the
major pelvic ganglion. Transections (TX) were located in caudal thoracic segment 4/rostral thoracic segment 5. (A) A sympathetic
preganglionic neuron (asterisk) that has retrogradely transported cholera toxin B subunit (CTB) from the major pelvic ganglion
(MPG) lies at the lateral edge of the intermediolateral cell column (IML) in 11-week transected cord. A host of enkephalin (ENK)-
immunoreactive varicosities form close appositions on the sympathetic preganglionic neuron. A retrogradely labeled dendrite in the
white matter (WM) also receives many close appositions from enkephalin-containing terminals. Some appositions are indicated by
arrowheads. Bar, 20 mm. (B) Retrogradely labeled sympathetic preganglionic neurons (asterisks) in the central autonomic area lie
within a very dense network of enkephalin (ENK)-immunoreactive axons. Bar, 50 mm.

not undergo the shrinkage and regrowth that we affected, they should be more easily restored after
have previously documented in more rostral sym- an injury because there will be less dependence on
pathetic preganglionic neurons immediately after the re-establishment of direct synaptic input from
injury. However, further studies are needed to ex- regrowing supraspinal axons. It will be interesting to
plore this possibility. see whether spinal interneurons are equally impor-
The dominance of intraspinal pathways in the tant in the regulation of parasympathetic pregangl-
control of sympathetic preganglionic neurons sup- ionic neurons, which project to the major pelvic
plying the major pelvic ganglion has important im- ganglion from the lower lumbar and upper sacral
plications for the restoration of pelvic visceral cord and are also critical for pelvic visceral function.
function after spinal cord injury. Since intraspinal
circuits controlling pelvic visceral sympathetic pre- Acknowledgments
ganglionic neurons are relatively unaffected by spi-
nal cord injury, drug treatments that target this Project Grants (#229907 to ILS and #000044 to
persistent circuitry should help to normalize sym- JRK) and Research Fellowships (#229921 to ILS
pathetically mediated pelvic visceral reflexes. Fur- and #358709 to JRK) from the National Health
thermore, since sympathetic components are less and Medical Research Council of Australia, grants
23

Fig. 8. In transected cord, enkephalin-immunoreactive axons synapse on sympathetic preganglionic neurons projecting to the major
pelvic ganglion. In the central autonomic area of 2-week transected cord, an enkephalin-immunoreactive varicosity (ENK) forms a
synapse (arrowheads) on a dendrite that contains a crystal due to retrograde transport of cholera toxin B subunit (CTB) from the
major pelvic ganglion (MPG). The transection was located in caudal thoracic segment 4/rostral thoracic segment 5. Bar, 500 nm.

from the National Heart Foundation of Australia Bowker, R.M., Westlund, K.N., Sullivan, M.C. and Coulter,
(#G98A0097 and #G00A0512 to ILS), a Visiting J.D. (1982) Organization of descending serotonergic projec-
tions to the spinal cord. Prog. Brain Res., 57: 239–265.
Scientist Award from the Heart and Stroke Foun-
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dation of Canada (ILS), Ontario Heart and Stroke architecture, ultrastructure and biophysical properties. In:
Foundation (LCW) and the Canadian Institutes of Loewy A.D. and Spyer K.M. (Eds.), Central Regulation of
Health Research (LCW) supported this work. Autonomic Functions. Oxford University Press, pp. 44–67.
Carolyn Martin, Natalie Fenwick and Lee Travis Cabot, J.B., Alessi, V. and Bushnell, A. (1992) Glycine-like
provided expert technical assistance. immunoreactive input to sympathetic preganglionic neurons.
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Cabot, J.B., Alessi, V., Carroll, J. and Ligorio, M. (1994) Spinal
cord lamina V and lamina VII interneuronal projections to
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 2

Spinal sympathetic interneurons: Their identification


and roles after spinal cord injury

Lawrence P. Schramm

Departments of Biomedical Engineering and Neuroscience, The Johns Hopkins University School of Medicine, 606 Traylor
Building, 720 Rutland Avenue, Baltimore, MD 21205, USA

Abstract: Primary afferent neurons rarely, if ever, synapse on the sympathetic preganglionic neurons that
regulate the cardiovascular system, nor do sympathetic preganglionic neurons normally exhibit sponta-
neous activity in the absence of excitatory inputs. Therefore, after serious spinal cord injury ‘‘spinal
sympathetic interneurons’’ provide the sole excitatory and inhibitory inputs to sympathetic preganglionic
neurons. Few studies have addressed the anatomy and physiology of spinal sympathetic interneurons, to a
great extent because they are difficult to identify. Therefore, this chapter begins with descriptions of both
neurophysiological and neuroanatomical criteria for identifying spinal sympathetic interneurons, and it
discusses the advantages and disadvantages of each. Spinal sympathetic interneurons also have been little
studied because their importance in intact animals has been unknown, whereas the roles of direct pro-
jections from the brain to sympathetic preganglionic neurons are better known. This chapter presents
evidence that spinal sympathetic interneurons play only a minor role in sympathetic regulation when the
spinal cord is intact. However, they play an important role after spinal cord injury, both in generating
ongoing activity in sympathetic nerves and in mediating segmental and intersegmental sympathetic reflexes.
The spinal sympathetic interneurons that most directly influence the activity of sympathetic preganglionic
neurons after spinal cord injury are located close to their associated sympathetic preganglionic neurons,
and the inputs from distant segments that mediate multisegmental reflexes are relayed to sympathetic
preganglionic neurons multisynaptically via spinal sympathetic interneurons. Finally, spinal sympathetic
interneurons are more likely to be excited and less likely to be inhibited by both noxious and innocuous
somatic stimuli after chronic spinal transection. The onset of this hyperexcitability corresponds to mor-
phological changes in both sympathetic preganglionic neurons and primary afferents, and it may reflect the
pathophysiological processes that lead to autonomic dysreflexia and the hypertensive crises that may occur
with it in people after chronic spinal injury.

Introduction maker potentials, and under ordinary


circumstances their activity is determined by
Sympathetic preganglionic neurons are the final synaptic inputs from the brain and spinal cord
neurons within the central nervous system that (see Laskey and Polosa, 1988, for review). The
regulate sympathetic output to nearly every tissue regulation of the activity of sympathetic pregangl-
and organ. Like somatic motoneurons, sympa- ionic neurons by brainstem systems has been ex-
thetic preganglionic neurons do not exhibit pace- tensively investigated (Laskey and Polosa, 1988;
Cabot, 1996; Blessing, 1997). However, less is
Corresponding author. Tel.: +410-955-3026; known about the regulation of sympathetic activ-
Fax: +410-955-9826; E-mail: LSCHRAMM@bme.jhu.edu ity after spinal cord injury, when inputs from the

DOI: 10.1016/S0079-6123(05)52002-8 27
28

brainstem are lost. Sympathetic activity after after spinal cord injury and because they may play
spinal cord injury is enigmatic because it ranges positive roles in the recovery of autonomic function
from abnormally low, leading to bouts of hypo- or pathological roles in mediating autonomic dys-
tension, to abnormally high, leading to hyperten- function during spinal cord repair and regeneration.
sive crises (Mathias and Frankel, 1992). One The anatomy and physiology of spinal sympa-
characteristic upon which there appears to be lit- thetic systems have been comprehensively re-
tle disagreement is that few, if any, spinal primary viewed (Laskey and Polosa, 1988; Cabot, 1996;
afferents synapse directly upon sympathetic pre- Weaver and Polosa, 1997). Therefore, this chapter
ganglionic neurons (Laskey and Polosa, 1988). will concentrate on recent studies of the anatomy
Therefore, by definition, after a complete spinal and physiology of spinal sympathetic interneurons
cord transection spinal interneurons convey all in rats with intact, acutely transected, and chron-
spinal inputs to sympathetic preganglionic neu- ically transected spinal cords. I begin by reviewing
rons, whether these inputs are derived from pri- the methods used to identify and characterize spi-
mary afferents or from intraspinal sources of nal sympathetic interneurons.
ongoing sympathetic activity.
For the purposes of this chapter, I define spinal Spinal sympathetic interneurons are identified both
interneurons as all spinal neurons other than (1) physiologically and anatomically
somatic motoneurons and (2) autonomic pregangl-
ionic neurons. I define spinal sympathetic inter- Ideally, we could identify each spinal sympathetic
neurons as spinal interneurons with connections interneuron, whether characterized physiologically
that can directly or indirectly affect sympathetic or anatomically, by tracing its axon to synapses
activity. Some spinal neurons that play no role in upon sympathetic preganglionic neurons. However,
sources of sympathetic activity in intact spinal this is possible under only special conditions, usu-
cords may participate in those sources after spinal ally in vitro (see, for example, Deuchars et al.,
cord injury. These neurons may, themselves, be 2001). In all other cases, the ‘‘sympathetic’’ nature
under tonic descending inhibition when the spinal of spinal sympathetic interneurons must either be
cord is intact, or their connections to sympathetic inferred from their neurophysiological properties
preganglionic neurons may be via other interneu- or by tracing their connections to sympathetic
rons that are tonically inhibited. Such neurons preganglionic neurons using specialized, trans-
would be classified as spinal sympathetic interneu- synaptic, retrograde labeling methods.
rons after, but not before, spinal cord injury. Gebber and colleagues pioneered neurophysio-
That spinal sympathetic interneurons have been logical identification of spinal sympathetic inter-
little studied is understandable for two reasons. neurons by identifying spinal neurons with
First, as discussed below, they are unique neither discharge patterns that were correlated with the
in their neurotransmitters nor their morphology. discharge patterns in either pre- or postganglionic
Thus, they are not readily identified. Second, Miller sympathetic axons (Gebber and McCall, 1976;
et al. (2001) found very few spinal interneurons Barman and Gebber, 1984). These investigators
with activities correlated with ongoing renal sym- cross-correlated the ongoing activity of single spi-
pathetic nerve activity in rats with intact spinal nal interneurons and the ongoing activity in sym-
cords. This observation suggests that spinal sym- pathetic nerves. Neurons with activities either
pathetic interneurons play a minor role in the reg- positively or negatively correlated with sympathet-
ulation of sympathetic activity in animals with ic nerve activity were defined as spinal sympathetic
intact spinal cords. Therefore, these interneurons interneurons. This remains the only neurophysio-
have not attracted attention in studies of normal, logical method for identifying spinal sympathetic
autonomic regulation of the circulation. In recent interneurons (Chau et al., 1997, 2000; Miller et al.,
years, however, spinal sympathetic interneurons 2001; Tang et al., 2003).
have attracted more attention because they may Neurophysiological identification of spinal sym-
play important roles in autonomic dysfunction pathetic interneurons has two drawbacks. First,
29

how does one distinguish between spinal sympa-


thetic interneurons and sympathetic preganglionic
neurons? The activities of both types of neurons
could be correlated with sympathetic nerve activ-
ity. Second, how does one distinguish between
spinal sympathetic interneurons and other inter-
neurons which share inputs with sympathetic pre-
ganglionic neurons but which are not involved in
sympathetic processing?
Distinguishing between spinal sympathetic in-
terneurons and sympathetic preganglionic neurons
is the easier of these problems. As shown by
Gebber and McCall (1976), sympathetic pregangl-
ionic neurons rarely discharge at rates exceeding
20 Hz. Therefore, the minimum interspike interval
for sympathetic preganglionic neurons is approx-
imately 50 ms. The discharge patterns of spinal
interneurons, on the other hand, usually include
bursts of action potentials with interspike intervals
of 20 ms or less. Therefore, with the relatively mi-
nor risk of misidentifying some spinal sympathetic
interneurons with low discharge rates, spinal sym-
pathetic interneurons can be distinguished from
sympathetic preganglionic neurons by the presence
of short interspike intervals in their discharge pat-
terns. An additional criterion for some spinal sym- Fig. 1. Neurophysiological identification of spinal sympathetic
pathetic interneurons is their dorsal location in the interneurons by cross-correlation. Upper panel: renal sympa-
spinal cord. Sympathetic preganglionic neurons thetic nerve activity (RSNA, upper trace) and simultaneously
are never located within spinal laminae I–V. recorded occurrences of action potentials in a spinal interneu-
ron (lower trace). Lower panel: cross-correlation between a
Therefore, sympathetically correlated neurons lo- 10 min recording of spinal neuronal action potentials and si-
cated in the dorsal laminae of the spinal cord are multaneously recorded RSNA. From Krassioukov et al. (2002),
very likely to be spinal sympathetic interneurons. with permission.
Figure 1 illustrates the neurophysiological iden-
tification of a putative spinal sympathetic inter- these activities was calculated (Fig. 1, lower panel,
neuron. The ongoing activity of a spinal neuron dark trace). Zero time on the correlogram was the
was recorded at a depth of 300 mm from the dorsal instant at which the interneuron began a burst of
surface of the 10th thoracic (T10) spinal segment of activity. The sharp positive peak in the correlo-
an anesthetized rat, acutely spinally transected at gram, approximately 75 ms after the onset of the
the 3rd cervical segment (C3). The neuron was burst, indicated that bursts of renal sympathetic
identified as an interneuron both by its dorsal po- nerve activity regularly lagged the onset of bursts
sition and by the presence of bursts of activity with of activity of the interneuron by 75 ms. To gauge
interspike intervals of 10 ms. At the temporal res- the significance of the correlation, the interdis-
olution of Fig. 1, these bursts are visible as darker charge intervals of the interneuron’s ongoing
action potential indicators (upper panel, lower activity were shuffled 10 times to generate 10 ‘‘dum-
trace). Ongoing renal sympathetic nerve activity my’’ cross-correlations with renal sympathetic nerve
(Fig. 1, upper panel, upper trace) was recorded activity (Fig. 1, lower panel, lighter traces). The
simultaneously with the ongoing activity of the positive correlation between the interneuron’s actu-
interneuron, and the cross-correlation between al ongoing activity and ongoing renal sympathetic
30

nerve activity was so much larger than the enve- activity. The polarities of somatically evoked re-
lope of the 10 dummy correlations that the prob- sponses of uncorrelated interneurons were much
ability that this relationship could have occurred less likely to match those of simultaneous respons-
by chance was very small. es in renal sympathetic nerve activity. Further-
A more difficult problem than distinguishing more, the excitatory fields of uncorrelated neurons
spinal sympathetic interneurons from sympathetic were significantly larger than those of correlated
preganglionic neurons is distinguishing spinal neurons, and they were often larger than the ex-
sympathetic interneurons from interneurons that citatory fields for renal sympathetic nerve activity.
are only coincidently correlated with sympathetic Excitatory fields are defined as the area of body
nerve activity. An example of such a coincidence surface from which stimulation of sensory recep-
would be the case in which both sympathetic pre- tors evoked excitation of the neuron.
ganglionic neurons and interneurons were driven Once identified neurophysiologically, spinal
by a common synaptic input. Indeed, this distinc- sympathetic interneurons can be anatomically lo-
tion cannot be made unambiguously using neuro- cated and morphologically characterized either by
physiological techniques. Nevertheless, confidence intracellular labeling (Deuchars et al., 2001) or by
in identifying spinal sympathetic interneurons is the juxtacellular labeling method (Pinault, 1996;
possible when (1) bursts of ongoing or evoked ac- Schreihofer and Guyenet, 1997; Tang et al., 2003).
tivity of a sympathetically correlated interneuron The juxtacellular method involves approaching the
usually lead ongoing or evoked bursts of sympa- soma or proximal dendrites of a spinal sympa-
thetic nerve activity by an interval consistent with thetic interneuron very closely and passing positive
the calculated conduction time from the interneu- current pulses into it through a biocytin-filled elec-
ron to the recording site on the sympathetic nerve trode. The current apparently electroporates (gen-
and (2) evoked excitatory and inhibitory responses erates temporary pores in) the neuron’s membrane
of interneurons to applied stimuli are correlated and carries biocytin into the cell. Biocytin rapidly
with responses in sympathetic nerve activity to the diffuses throughout the neuron’s soma and dend-
same stimuli. rites. Labeled neurons are identified and recon-
Figure 1 illustrates a case in which the first of structed histologically after treatment with a
these criteria was met. The 75 ms lag between streptavidin-conjugated chromogen. Although this
bursts of ongoing activity of the interneuron and method has been used to visualize spinal sympa-
bursts of ongoing sympathetic nerve activity rep- thetic interneurons (Tang et al., 2003), it suffers
resents an aggregate conduction velocity of ap- from two drawbacks. First, respiratory and vas-
proximately 0.5 m/s, which is consistent with the cular movements of the spinal cord often prevent
expected conduction velocity of the largely un- one from approaching interneurons closely enough
myelinated axons of the renal sympathetic nerve. to label them without injuring them. Second, al-
Subsequently, this neuron also met the second cri- though the somas and dendrites of labeled spinal
terion. Pinch of the left flank, within the region of sympathetic interneurons are well demonstrated
the T10 dermatome, excited both the activity of by juxtacellular labeling, axons are never observed.
this neuron and renal sympathetic nerve activity, Axons of spinal sympathetic interneurons can be
whereas pinch of the left hip and left shoulder demonstrated by intracellular labeling. To date,
caused decreases in both this neuron’s activity and however, intracellular labeling of spinal sympa-
renal sympathetic nerve activity (data not shown). thetic interneurons has been accomplished only in
Chau et al. (1997) found that the polarities vitro (Deuchars et al., 2001).
(direction in which firing frequency changed, up Although neurophysiological studies permit
or down) of somatically evoked responses of the functional characterization of spinal sympathetic
majority of interneurons with ongoing activities interneurons, correlation methods, alone, cannot
positively correlated with renal sympathetic nerve unequivocally identify spinal sympathetic interneu-
activity matched the polarities of simultaneously rons. Spinal sympathetic interneurons can be more
evoked responses in renal sympathetic nerve definitively identified by retrograde, trans-synaptic
31

tracing from sympathetic preganglionic neurons. infection and the perfusion of the animal. This
In an ingenious series of experiments, Cabot and interval usually ranges between 3 and 6 days. For
colleagues (1994) simultaneously injected the beta identification of spinal sympathetic interneurons,
subunit of cholera toxin (cholera toxin B) and infected rats are kept for approximately 72 h before
wheat germ agglutinin into the superior cervical perfusion. Rats kept for this time manifest no
ganglion of rats. Both the cholera toxin B and the visible symptoms of the infection.
wheat germ agglutinin were transported from the When virus is injected into the adrenal gland,
ganglion to the somas and dendrites of sympa- both preganglionic neurons projecting directly to
thetic preganglionic neurons with synapses in that adrenal chromaffin cells and postganglionic neu-
ganglion. However, only the wheat germ aggluti- rons projecting to both adrenal medullary and ad-
nin was transported further in the retrograde di- renal cortical blood vessels are infected. Therefore,
rection, across the synapses made by spinal the spinal sympathetic interneurons infected by
sympathetic interneurons on sympathetic pre- injection of virus into the adrenal gland may be-
ganglionic neurons, thereby labeling the spinal long to at least two classes of interneurons, neu-
sympathetic interneurons. Thus, sympathetic pre- rons involved in overall metabolic regulation and
ganglionic neurons were identified by their com- neurons involved in the regulation of the adrenal
bined labeling with cholera toxin B and wheat circulation. As discussed below, the distinction
germ agglutinin. Spinal sympathetic interneurons between these classes of interneurons may be of
were identified by their labeling with wheat germ limited importance because it is likely that neither
agglutinin but not cholera toxin B. Although these play an important role in animals with intact spi-
were landmark experiments, they were hampered nal cords. After spinal cord lesions, most stimuli
by faint labeling of spinal sympathetic interneu- that activate one class of adrenal spinal sympa-
rons, due in large part to restricted, retrograde, thetic interneurons are likely to activate both.
trans-synaptic transport of wheat germ agglutinin As in the case of the cholera toxin B and wheat
from sympathetic preganglionic neurons. germ agglutinin experiments described above, viral
More recently, spinal sympathetic interneurons methods also require distinguishing between sym-
have been identified by the retrograde, trans-synap- pathetic preganglionic neurons and spinal sympa-
tic transport of herpes viruses (Strack et al., 1989a, thetic interneurons. Sympathetic preganglionic
b; Schramm et al., 1993; Clarke et al., 1998; Tang neurons can be identified because, in addition to
et al., 2004). Herpes simplex and pseudorabies vi- being immunohistochemically labeled for the virus,
rus are two herpes viruses that are rapidly taken up they also label positively for choline acetyl transf-
by the axons of sympathetic postganglionic neu- erase, a synthetic enzyme for acetyl choline found
rons and by the axons of preganglionic neurons in relatively few spinal neurons other than sympa-
projecting to the adrenal medulla. Virus is trans- thetic preganglionic neurons and somatic motoneu-
ported back to the somas of these neurons where it rons. Thus, spinal neurons that co-label for virus
replicates and moves trans-synaptically to the neu- and choline acetyl transferase can be identified as
rons’ synaptic antecedents. Thus, virus taken up sympathetic preganglionic neurons, and neurons
from a peripheral organ or tissue by sympathetic that are infected but do not co-label for choline
postganglionic neurons infects the sympathetic pre- acetyl transferase can be identified as spinal sym-
ganglionic neurons that synapse on those neurons. pathetic interneurons. Sympathetic preganglionic
Virus replicates in the sympathetic preganglionic neurons and somatic motoneurons can be distin-
neurons, and spinal and brainstem interneurons guished by their differential, dorsoventral locations.
that synapse on infected sympathetic preganglionic An alternative method for identifying sympathetic
neurons are infected by further retrograde, trans- preganglionic neurons depends on their propensity
synaptic movement of virus. Antibodies to the vi- for transporting retrograde tracers from the circu-
ruses are used to label infected neurons. The ap- lation to their somas and dendrites (Fig. 2). In this
proximate number of synapses traversed by the method, a large quantity (8–12 mg/kg) of a con-
virus can be controlled by the interval between the ventional retrograde tracer such as Fluorogolds
32

Fig. 2. Anatomical identification of interneurons using pseudorabies virus and Fluorogolds. Left panel: ultraviolet illumination.
Sympathetic preganglionic neuron (white arrow) identified by fluorescence of intraperitoneally injected Fluorogolds. Right panel:
under illumination for the chromogen used to identify pseudorabies virus, both the sympathetic preganglionic neuron (white arrow)
and a spinal sympathetic interneuron (gray arrow) are visible as gray neurons. The spinal sympathetic interneuron is definitively
identified by its absence under ultraviolet illumination. After Tang et al. (2004), with permission.

is injected either intraperitoneally or subcutaneously Spinal interneurons play a more important role in
(Anderson and Edwards, 1994). Approximately 1 generating sympathetic activity after spinal cord
week post-injection, most peripherally projecting lesions in rats
neurons (such as autonomic preganglionic neurons
and somatic motoneurons) are labeled with the Although most investigators have found that ac-
tracer and can be detected under ultraviolet illu- tivity is reduced in sympathetic nerves of unanest-
mination. Although the labeling of somatic moto- hetized people (Wallin, 1986) and rats
neurons is highly variable by tracers administered (Krassioukov and Weaver, 1995; Randall et al.,
intraperitoneally, the labeling of autonomic pre- 2005) after spinal cord transection, many investi-
ganglionic neurons is more uniform. A potential gators report that detectable levels of ongoing ac-
drawback of this method is that freshly adminis- tivity remain in some nerves. Therefore, spinal
tered Fluorogolds appears to interfere with some sympathetic interneurons must provide ongoing
viral tracing methods (Strack and Loewy; excitatory input to sympathetic preganglionic neu-
Schramm, unpublished data). In our hands, how- rons in the absence of pathways from the brain-
ever, pseudorabies virus can be safely injected 1 stem sympatho-excitatory systems. Although in
week after treatment with this retrograde tracer. anesthetized, surgically prepared rats with acute
The major drawback of the viral tracing meth- spinal transections, sympathetic activity is sub-
ods is that infection by the virus may be capri- stantially reduced in some nerves, it is maintained
cious. Within a population of identically treated, or even increased in others (Meckler and Weaver,
virus-injected animals, some may not exhibit any 1985; Taylor and Schramm, 1987).
infection, some may exhibit infections that appear The observations of decreased activity in some
highly specific (infecting only sympathetic pre- nerves are easily explained by the decrease in sup-
ganglionic neurons and spinal sympathetic inter- raspinal drive to some sympathetic preganglionic
neurons) and some may exhibit infections that neurons after spinal cord injury. Maintenance —
destroy many neurons. A second drawback is that and even increases — in sympathetic activity after
the number of synapses retrogradely traversed by spinal cord injury are less easily explained. Very
the virus can only be estimated from the survival likely, sympathetic preganglionic neurons whose
time. Finally, the viral infection often initiates an activity was either not diminished or was increased
immune response that, itself, could alter the fur- after spinal transection received little drive from
ther transport of the virus. brainstem circuits before transection. Alternatively,
33

brainstem sources of activity for these neurons ongoing activities of almost 50% of the interneu-
were replaced by even more powerful intraspinal rons recorded at T10 were correlated to ongoing
sources after transection. In either case, it also is renal sympathetic nerve activity, the activities of
likely that potentially excitatory spinal inputs to only 16% of interneurons at T8 were correlated
these sympathetic preganglionic neurons were with renal sympathetic nerve activity. The activi-
under tonic inhibition from supraspinal systems. ties of no interneurons at T2, T13 or L2 were cor-
Thus, I propose that spinal transection abolishes related with renal sympathetic nerve activity. In
descending excitation, either directly to sympa- unpublished studies (Chau and Schramm), this
thetic preganglionic neurons or indirectly to spinal exploration extended to C2 and L5 without detect-
sympathetic interneurons. However, it also abol- ing additional interneurons correlated with renal
ishes descending inhibition of spinal systems with sympathetic nerve activity. Because anatomical
excitatory inputs to sympathetic preganglionic data indicate that the sympathetic preganglionic
neurons. Ruggiero et al. (1997a, b) provided clear neurons that are most likely to generate renal
evidence that acute spinal transection releases the sympathetic nerve activity lie in 8th through 12th
activities of many dorsal horn and intermediate thoracic segments (Tang et al., 2004), these data
zone neurons from inhibition. They found that show that circuits in distant spinal segments play
acute cervical spinal cord transection in anestheti- little role in generating ongoing renal sympathetic
zed rats and pigs significantly increased the nerve activity. Whether a similar degree of longi-
number of neurons expressing the c-fos gene in tudinal specificity exists for cardiac and pelvic
many dorsal horn laminae and in lamina VII of the sympathetic nerves remains to be determined.
thoracic spinal cord.
Based on these observations, Miller et al. (2001) Long propriospinal pathways affecting sympathetic
predicted that spinal neurons with ongoing activ- activity are multisynaptic
ities correlated with renal sympathetic nerve ac-
tivity would be relatively rare in rats with intact Although distant spinal segments appear to play
spinal cords because spinal circuits that might ex- little or no role in generating ongoing sympathetic
cite sympathetic preganglionic neurons would be activity in a given segment after spinal transection,
under tonic, supraspinal inhibition. As noted sympathetic reflexes can be evoked by stimulating
above, this prediction was confirmed by their ob- afferents to distant segments (see Weaver and
servation that the activities of only one-fifth as Polosa, 1997, for review). To what extent are the
many spinal interneurons were correlated with re- sympathetic reflexes elicited from distant segments
nal sympathetic nerve activity in rats with intact mediated by monosynaptic projections to sympa-
spinal cords as were correlated in rats with acutely thetic preganglionic neurons? Cabot et al. (1994)
transected spinal cords. noted that spinal sympathetic interneurons retro-
gradely labeled by injecting wheat germ agglutinin
The generation of ongoing sympathetic activity after into the superior cervical ganglion exhibited ‘‘a
spinal transection is localized to a restricted number strict segmental organization’’ with respect to their
of spinal segments associated sympathetic preganglionic neurons. In
other words, wheat germ agglutinin-labeled neu-
As described above, after acute spinal transection, rons (spinal sympathetic interneurons) were not
activity persists in some sympathetic nerves. found in segments that did not contain cholera
To what extent is this ongoing activity generated toxin B-labeled neurons (sympathetic preganglion-
locally, and to what extent does it represent activ- ic neurons).
ity common to the entire spinal cord? Chau et al. These observations were confirmed in the renal
(1997) searched the spinal cord from T2 to the 2nd sympathetic system using the retrograde transport
lumbar (L2) segment for interneurons with activ- of pseudorabies virus. Tang et al. (2004) found
ities correlated to renal sympathetic nerve activity that between caudal cervical and caudal lumbar
in rats with acute spinal transections. Although the segments, infected spinal sympathetic interneurons
34

were located only in segments of caudal thoracic adrenal sympathetic preganglionic neurons were
and rostral lumbar segments, the segments in located across the entire mediolateral span of lamina
which infected sympathetic preganglionic neurons VII, and spinal sympathetic interneurons were
were also located. The first infected thoracic similarly distributed, usually intercalated among
interneurons appear 68–72 h after injection of the sympathetic preganglionic neurons. Pseudorabies
pseudorabies virus into the kidney. This delay is virus injected into the kidney of the rat also
identical to that required to infect brainstem neu- infected sympathetic preganglionic neurons locat-
rons that have known, monosynaptic projections ed across the entire intermediate zone of the spinal
to sympathetic preganglionic neurons. Apparently cord between the lateral funiculus and lamina X
in this model, the time required for retrograde (Tang et al., 2004). The majority of spinal sympa-
transport of pseudorabies virus from its uptake thetic interneurons labeled in those experiments
at a synapse to the soma of the next neuron is brief were similarly distributed.
compared to the time necessary for enough Although most anatomically identified spinal
replication to occur for the virus to be visible sympathetic interneurons have been detected
immunohistochemically in that newly infected among, or just dorsal to, populations of sympa-
neuron. Similarly, the transport time is brief com- thetic preganglionic neurons, small numbers iden-
pared to the time required for replication to in- tified after renal injections of pseudorabies virus
crease the intracellular concentration of virus for were located (in descending order of density) in
retrograde infection of a neuron’s synaptic ante- lamina IV, II, and I (Tang et al., 2004). Interest-
cedents. Because neurons as far rostral as the ingly, spinal sympathetic interneurons identified
paraventricular nucleus of the hypothalamus can by their positive cross-correlations with renal sym-
be infected in as little as 72 h, the absence of spinal pathetic nerve activity were distributed somewhat
sympathetic interneurons in caudal cervical, ros- more widely than anatomically identified spinal
tral thoracic and caudal lumbar spinal cord that sympathetic interneurons, for instance in the me-
lack infected sympathetic preganglionic neurons dial portions of laminae I, II, and III (Chau et al.,
strongly suggests that long propriospinal inputs to 2000; Tang et al., 2003). The wider distribution of
sympathetic preganglionic neurons infected from neurophysiologically identified spinal sympathetic
renal injections are multisynaptic. interneurons was not surprising. Anatomically
The majority of spinal sympathetic interneurons identified spinal sympathetic interneurons were
projecting monosynaptically to sympathetic pre- visualized using a relatively short, post-infection
ganglionic neurons are located either among or survival time (72 h). As discussed above, during
just dorsal to their functionally related popula- that time, pseudorabies virus would have been un-
tions of sympathetic preganglionic neurons. Not likely to have traversed more than the two
only are the longitudinal distributions of spinal synapses between the renal sympathetic postgangl-
sympathetic interneurons and their related sympa- ionic neurons and the first spinal sympathetic in-
thetic preganglionic neurons similar, but the den- terneurons presynaptic to infected sympathetic
sities of spinal sympathetic interneurons are preganglionic neurons. Spinal sympathetic inter-
greatest in or near the spinal laminae that contain neurons identified by cross-correlation, on the
their associated sympathetic preganglionic neu- other hand, could have been located in spinal cir-
rons. Thus, Cabot et al. (1994) localized spinal cuits many synapses removed from sympathetic
sympathetic interneurons to the sympathetic pre- preganglionic neurons and could, therefore, be ex-
ganglionic neuron-rich lateral portion of lamina pected to be located more remotely.
VII and the reticulated (lateral) portion of lamina The locations of spinal sympathetic interneu-
V, just dorsal to the intermediolateral column. rons with respect to sympathetic preganglionic
Clarke et al. (1998) used the retrograde transport neurons may be more important than their loca-
of modified Herpes simplex virus to identify spinal tions with respect to their inputs. Histological re-
sympathetic interneurons that were presynaptic to construction of spinal sympathetic interneurons
adrenal sympathetic preganglionic neurons. Infected (Deuchars et al., 2001; Tang et al., 2003) indicated
35

that the dendritic trees of these neurons often ex-


tended hundreds of microns in two, and sometimes
three, dimensions. Tang et al. (2004) concluded
that the dendrites of some individual spinal sym-
pathetic interneurons were so extensive that they
could receive not only primary afferent inputs but
inputs from a variety of descending or propriospi-
nal pathways as well.

Spinal sympathetic interneurons in rats are more


likely to be excited and less likely to be inhibited by
somatic stimuli after chronic spinal cord transection Fig. 3. Responses of spinal sympathetic interneurons to so-
matic stimulation 1 month after T3 spinal cord transection.
Because the severity of autonomic dysreflexia Upper panel: schematic drawing of the cutaneous regions from
increases with time after spinal cord injury which responses of spinal sympathetically correlated interneu-
(Krassioukov and Weaver, 1995; Krassioukov rons were elicited. Lower panel: representative rate meter re-
sponses of a sympathetically correlated neuron to noxious (left)
et al., 2003), we have supplemented the studies of and innocuous (right) stimulation of cutaneous regions 1–5 in a
rats with acutely transected spinal cords described rat chronically transected at T3. From Krassioukov et al.
above with studies after chronic, T3, spinal tran- (2002), with permission.
section. In both chronically and acutely spinally
transected rats, Krassioukov et al. (2002) identified
spinal sympathetic interneurons in the T10 segment of somatic regions that project to caudal lumbar
by cross-correlation with renal sympathetic nerve spinal cord (Fig. 3, regions 4 and 5) decreased on-
activity. They compared the responses in activity going renal sympathetic nerve activity. The ongo-
of spinal sympathetic interneurons to somatic ing activities of a majority of T10 spinal
stimulation in those two populations. To stand- sympathetic interneurons were inhibited by stim-
ardize stimulation sites, the left body wall was di- ulation of these regions. One month after spinal
vided into five regions, beginning at approximately cord transection, both noxious and innocuous
the T8 dermatome and ending at the left hip and stimulation of regions 1, 3, and 5 were significantly
hindlimb (Fig. 3). more likely to increase the activities of spinal sym-
Two types of stimuli were delivered to these re- pathetic interneurons than in the acutely trans-
gions, a 10-s pinch with toothed forceps (noxious) ected state, and innocuous stimulation of regions 1
and 10 s of brushing with a cotton applicator (in- and 5 was less likely to decrease their activities.
nocuous). Responses in the activities of spinal Although autonomic dysreflexia may occur in
sympathetic interneurons and renal sympathetic the acute stage of spinal cord injury (Krassioukov
nerve activity observed in acutely spinally trans- et al., 2003), it is far more common in the chronic
ected rats by Krassioukov et al. (2002) corre- stage in both humans (Mathias and Frankel, 1992)
sponded closely to those reported previously in and rats (Krassioukov and Weaver, 1995). In rats,
rats with acutely transected spinal cord (Chau the onset of autonomic dysreflexia correlated well
et al., 1997, 2000). Both noxious and innocuous with morphological changes in sympathetic pre-
stimulation of somatic regions projecting to caudal ganglionic neurons (Krenz and Weaver, 1998b)
thoracic spinal cord (Fig. 3, regions 1–3), increased and with increases in sprouting of primary afferent
the magnitudes of bursts in ongoing renal sympa- axons (Krenz and Weaver, 1998a; Wong et al.,
thetic nerve activity. Responses in the activities of 2000). Some of these axons appeared to synapse
spinal sympathetic interneurons were more varia- on neurons appropriately positioned to be spinal
ble. Nevertheless, the majority of T10 spinal sym- sympathetic interneurons (Wong et al., 2000). The
pathetic interneurons were excited by stimulation electrophysiological experiments described above
of regions 1–3. Noxious and innocuous stimulation provided a neurophysiological correlation to both
36

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37

Mathias, C.J. and Frankel, H.L. (1992) Autonomic disturbanc- Schreihofer, A.M. and Guyenet, P.G. (1997) Identification of
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Published by Elsevier B.V.

CHAPTER 3

Which pathways must be spared in the injured human


spinal cord to retain cardiovascular control?

Andrei Krassioukov

International Collaboration on Repair Discoveries (ICORD) and School of Rehabilitation, University of British Columbia,
Vancouver, BC, Canada and Department of Physical Medicine and Rehabilitation, University of Western Ontario,
London, ON, Canada

Abstract: Cardiovascular abnormalities following spinal cord injury are attributed to autonomic instability
caused by a combination of changes occurring within the spinal cord, including loss of descending au-
tonomic control and plastic changes within spinal and peripheral circuits. Previous animal studies have
shown that localized disruption of the descending vasomotor pathways results in cardiovascular changes
similar to those observed following cord injury. However, the location of these pathways in humans is
uncertain. This chapter presents clinical and histopathological findings from individuals with spinal cord
injury that associates a common area of white matter destruction with severe cardiovascular symptoms.
These data provide evidence that descending vasomotor pathways in the human spinal cord project through
the dorsal aspects of the lateral funiculus.

Introduction cord-injured individuals (Piepmeier et al., 1985;


Lehmann et al., 1987; Atkinson and Atkinson, 1996).
People with cervical or high thoracic spinal cord Likewise, the severity of autonomic dysreflexia
injury face life-long abnormalities in systemic ar- correlates with completeness of spinal injury
terial pressure control (Mathias and Frankel, as assessed by the American Spinal Injury
1992; Karlsson, 1999; Teasell et al., 2000). In gen- Association (ASIA) score: only 27% of incom-
eral, their basal systemic arterial pressure is lower plete quadriplegics present signs of dysreflexia in
than normal and is complicated by orthostatic comparison with 91% of complete quadriplegics
intolerance (Mathias and Frankel, 1992; Cariga (Curt et al., 1997).
et al., 2002). In addition, these cord-injured people Determinations of plasma catecholamine levels
experience transient episodes of hypertension, in cord-injured individuals, and other evidence,
known as ‘‘autonomic dysreflexia’’ that are often suggest that a decrease in sympathetic neuronal
associated with disturbances in heart rate and activity is the main cause of the hypotension and
rhythm (Krassioukov et al., 2003; Clydon et al., postural intolerance (Figoni, 1984; Mathias, 1995;
2005). The severity of spinal cord injury varies Karlsson et al., 1998; Gao et al., 2002). The de-
between individuals and impacts greatly upon creased sympathetic activity, in turn, presumably
cardiovascular control. For example, hypotension results from damage to the spinal pathways that
affects between 20% and 30% of all spinal carry facilitatory input from the lower brainstem
to the sympathetic preganglionic neurons. The de-
Corresponding author. Tel.: +603 822 2673 (off.); struction of these descending vasomotor pathways,
resulting in the loss of excitatory supraspinal input
+604 822 9305 (lab); Fax: +604 822 2924;
E-mail: krassioukov@icord.org to the spinal sympathetic preganglionic neurons,

DOI: 10.1016/S0079-6123(05)52003-X 39
40

is currently considered the major factor for the


persistent lack of sympathetic tone after spinal
cord injury (Mathias and Frankel, 1992; Atkinson
and Atkinson, 1996). Damage to the spinal path-
ways that carry inhibitory input from the lower
brainstem to the sympathetic preganglionic neu-
rons may have a role in autonomic dysreflexia, by
allowing exaggerated activity in the spinal reflex
circuits, caudal to the lesion, that connect spinal
afferent projections to preganglionic neurons. Fig. 1. Schematic diagrams of the possible localization of de-
scending vasomotor pathways within the spinal cord as previ-
Damage to the pathways from the lower brain- ously reported in experimental animals (A) and human
stem to sympathetic preganglionic neurons has a investigations (B). These pathways were localized within dif-
central role in generating the abnormal systemic ferent regions of the white matter in primates (Kerr and
arterial pressure control, typical of cervical or high Alexander, 1964) (arrow 1), in cats and rodents (Lebedev et al.,
thoracic spinal cord injury. Therefore, these path- 1986; Reis et al., 1988; Ruggiero et al., 1989) (arrow 2), and in
humans (Nathan and Smith, 1987) (arrow 3). (B) The boxed
ways are a high priority target for repair, regen- areas indicate the two potential localizations of descending
erative and neuroprotective treatment. Knowledge vasomotor pathways in man, which we examined in the present
of the localization of these pathways in the human investigation: the dorsal aspect of lateral funiculus (Area I), and
spinal cord is therefore essential. This chapter de- white matter adjacent to dorsolateral aspects of the intermedio-
scribes an approach to obtaining such knowledge. lateral cell column (Area II). (From Furlan et al. (2003), with
permission from J. Neurotrauma.)
Previous work in experimental animals, using
electrical stimulation or lesions, led to the conclu-
symptoms had the greatest damage in Area I. The
sion that the pathways for cardiovascular control
remainder of the cases, who had only minor car-
run in the dorsal aspect of the lateral funiculus of
diovascular symptoms, or no symptoms at all, had
the spinal cord white matter (Kerr and Alexander,
significantly less damage in this area. The degree
1964; Illert and Gabriel, 1972; Foreman and
of damage to Area II did not correlate well with
Wurster, 1973; Lebedev et al., 1986). Henceforth,
the extent of cardiovascular dysfunction in these
this area of the white matter will be referred to as
individuals.
Area I (Fig. 1). By contrast, a study in patients
On the basis of these data it may be concluded
undergoing limited cordotomy for the relief of
that, in humans, the pathways from the lower
chronic pain resistant to medical treatment, has
brainstem to the sympathetic preganglionic neu-
suggested that these pathways run in the white
rons run in the dorsal aspect of the lateral fun-
matter adjacent to the dorsolateral aspect of the
iculus of the white matter. Since this is the general
intermediolateral cell column (Nathan and Smith,
region where previous work on experimental an-
1987). Henceforth, this area of the white matter
imals (rat, cat, dog, others) had localized similar
will be referred to as Area II (Fig. 1).
pathways, this study further confirms the relevance
This chapter describes a retrospective study of
to humans of animal model studies of autonomic
cases of spinal cord injury from which detailed
dysfunctions after spinal cord injury.
clinical records and spinal cord specimens were
available. The cases with the most severe cardio-
vascular symptoms were identified. It was hypoth-
esized that this case subset would also have the Study groups
most severe damage to the pathways from lower
brainstem to the sympathetic preganglionic neu- We retrospectively reviewed the charts of the spi-
rons. The extent and severity of white matter nal cord injury cases included in this study and
damage was estimated using stains for myelin and collected data on age and gender, causes of spinal
for an axoplasmic marker. This study has shown cord injury, neurological assessment (including
that the group with the most severe cardiovascular severity and level of injury), cardiovascular
41

parameters, and clinical history predating the A total of seven cases with spinal cord injury
spinal cord injury, for example, pre-existing (two females and five males, aged 31–82 years with
cardiovascular disease. Detailed information on a mean of 60.0 years), and five individuals with
cardiovascular parameters was collected during intact central nervous system (two females and
the acute stage of injury in all individuals. We also three males, aged 30–73 years with a mean of 51.4
searched for evidence of episodes of autonomic years) were analyzed. Individuals from the control
dysreflexia in these individuals. Heart rate and group were comparable to the cord injury group
blood pressure had been evaluated in each patient with regard to age (P ¼ 0.42) and gender
hourly for the first 2 weeks and then every 2 h until (P ¼ 1.0). All spinal cord-injured individuals had
discharge. Daily averages were calculated from a cervical injury. Neurological evaluation using the
all measurements available for each day in the ASIA scale showed that individuals in Group 1
patients’ charts during a 5-week post-injury period. had a more severe cord injury (Table 1).
These cases of cord injury all had a cervical injury
and were assigned to one of the two groups: cases Cardiovascular parameters
that developed severe cardiovascular dysfunction
during the acute post-injury period (Group 1), and There were significant differences in the cardio-
cases with no or minor cardiovascular dysfunction vascular parameters between individuals in
in this period (Group 2). The control group Groups 1 and 2. Severe hypotension, bradycardia,
(Group 3) included five cases with intact central and episodes of autonomic dysreflexia, which are
nervous system. Neurological evaluation of the signs of disrupted supraspinal cardiovascular con-
severity of cord injury was conducted through as- trol, were prominent among the cases in Group 1.
sessment of motor and sensory impairments ac- Severe hypotension (neurogenic shock) in the early
cording to the ASIA scoring system (Maynard Jr. post-injury period required the administration of
et al., 1997). The ASIA Grade A represents the vasopressive agents to all individuals in Group 1.
most severe, complete injury with complete motor Intravenous dopamine was administered, on aver-
and sensory impairment, and ASIA Grade D age, for 774.1 days (1–19 days) in this group. In
characterizes minor, incomplete cord injury (mild contrast, only one individual in Group 2 required
motor dysfunction and no sensory loss). infusion of dopamine for a period of 11 h. After

Table 1. Clinical and neurological data in spinal cord-injured individuals with severe (Group 1) or minor cardiovascular dysfunction
(Group 2) and in control cases (Group 3)

Groups Cases Gender Age (years) ASIA grade Level of SCI Cause of SCI Time from SCI to death

1 1 Female 31 A C 2,3 Diving accident 9 months


2 Male 66 B C 4,5 Fall 6 months
3 Female 43 A C 5,6 Motor vehicle accident 3.5 months
4 Male 66 A C 6,7 Spontaneous epidural hemorrhage 5 months
2 5 Male 65 C C 1,2 Motor vehicle accident 36 months
6 Male 67 A C 2,3 Fall 3.5 months
7 Male 82 B C 5,6 Spontaneous epidural hemorrhage 5 weeks
3 8 Male 30 N/A N/A N/A N/A
9 Male 53 N/A N/A N/A N/A
10 Female 37 N/A N/A N/A N/A
11 Male 73 N/A N/A N/A N/A
12 Female 64 N/A N/A N/A N/A

N/A, not applicable.


Adapted from Furlan et al. (2003), with permission from J. Neurotrauma.
42

the first 2 days, the cases in Group 2 had normal for examination. In Group 3, the third thoracic
arterial pressures, no bradycardia and no episodes segment was examined. Two sets of alternate spi-
of autonomic dysreflexia. Although individuals in nal cord sections (5–8 mm) were obtained and
Group 1 were treated with vasopressor agents in stained for: (1) general histology and myelin pres-
the early stage of cord injury, their systolic and ervation using hematoxylin and eosin and luxol
diastolic blood pressures were significantly lower fast blue; and (2) axonal preservation using
than those of Group 2 during the first 4 weeks post immunocytochemical staining for neurofilament
injury. Individuals in Group 1, and some of Group 200 (Sigma, 1:200).
2 also had bradycardia during the first 2 days after Sections were viewed with bright field illumina-
injury. Then, the mean daily heart rate in Group 1 tion (Axioscope, Zeiss), and the extent of injury
remained significantly lower than the heart rate in and axonal degeneration was examined using the
Group 2 for the following 4 weeks after injury. By Northern Eclipse imaging software (Version 6.0,
the end of the 4th week, the cases in Group 1 had Empix Imaging Inc). In sections stained for myelin
recovered cardiovascular function and the two with luxol fast blue, the total area of demyelina-
groups did not differ with respect to heart rate and tion (Fig. 2, pink areas within the white matter)
blood pressure. was measured and presented as a fraction (percent
Three individuals in Group 1 (Cases 1, 3, and 4 and standard error of the mean) of the total sur-
of Table 1) developed episodes of autonomic face area of the spinal cord section (including
dysreflexia during their stay at the hospital. A white and gray matter). In sections stained for ne-
typical episode of dysreflexia occurred in Case 1 at urofilament 200, axonal counts were conducted in
day 4. This was her first episode of dysreflexia. selected areas of white matter (see below).
During this episode her systolic and diastolic blood Axonal preservation was examined within the
pressures reached 180 and 100 mmHg, respectively, two areas that have been previously suggested to
from a resting mean pressure of 90 mmHg. This contain descending vasomotor pathways: the dor-
episode was accompanied by an increased heart sal aspects of the lateral funiculus (Area I in Fig.
rate to 80 beats per minute (from a resting rate of 1B), and the white matter adjacent to the dorso-
55 beats per minute). Pounding headache, double lateral portions of the intermediolateral column
vision and anxiety were the major complaints (Area II in Fig. 1B) (Kerr and Alexander, 1964;
during this episode. Elevation of the head and Foreman and Wurster, 1973; Lebedev et al., 1986;
analgesic medication were effective in managing Nathan and Smith, 1987). We also examined the
this episode. No episodes of autonomic dysreflexia extent of axonal preservation within the dorsal
were reported in individuals in Group 2. Pre- columns and the corticospinal tracts in each case
existing systemic hypertension was established in (Fig. 3). Sections stained for neurofilament 200
Case 4 (Group 1) and Case 5 (Group 2) prior to were examined under low magnification (  1.25)
spinal cord injury. Although daily recording of and areas of interests were identified (Fig. 3). For
cardiovascular parameters was not performed in each section, using high magnification (  20) im-
individuals from Group 3, we found no clinical ages, at least three representative fields from the
data to suggest cardiovascular abnormalities. chosen areas were examined (Fig. 3a–3d). Finally,
the axonal counts were conducted using the
Northern Eclipse software. Axonal preservation
Histopathological findings was expressed as the mean number of preserved
axons per 10,000 mm27SEM.
The spinal cord tissue from the cases included in Cross-sectional analysis of spinal cord sections
this study was fixed with 10% buffered formalin from the high thoracic cord, caudal to the injury
for 2 weeks and paraffin embedded. In no case did site, revealed that the extent of white matter de-
the postmortem interval exceed 24 h. In each cord generation was 24.6572.1% (range: 19.29–29.66%)
injury case, at least one segment caudal to the level in Group 1, and 8.271.2% (range: 6.06–10.15%)
of injury (upper thoracic segments) was selected in Group 2 (Fig. 2). This showed that spinal cords
43

Fig. 2. Myelin staining with Luxol Fast Blue of spinal cord sections from the high thoracic spinal cord. (A) Spinal cord injury case who
developed severe cardiovascular complications (Group 1); (B) spinal cord injury case with no significant cardiovascular dysfunction
(Group 2); and (C) individual with intact CNS (control case, Group 3). Calibration bar is 2 mm. A well-defined butterfly shaped area of
the gray matter is present in all sections. Myelin-containing white matter is stained blue. Areas of axonal degeneration and myelin loss
(pink areas within the white matter) are present in sections from Cases 3 and 7. (D) Average values of white matter degeneration
(expressed as a percent of total spinal cord area) in spinal cord sections from individuals in Group 1 were significantly greater than
those of individuals from Group 2. (From Furlan et al. (2003), with permission from J. Neurotrauma.)

from cases of Group 1 with severe cardiovascular similar (P ¼ 0.167). There were fewer preserved
dysfunction after cord injury had more extensive axons per 10,000 mm2 in Area I (dorsal aspects of
areas of white matter degeneration than spinal the lateral funiculus) in individuals from Group 1
cords from cases who had no or minor cardiovas- (2075) than in individuals from Group 2 (52715;
cular dysfunction after injury (Po0.002). P ¼ 0.029) and Group 3 (6573; Po0.001). Also,
Axons within the spinal cord were unequiv- the number of preserved axons within the Area I in
ocally identified in spinal cord sections using Group 2 was significantly less than in Group 3
immunohistochemistry for neurofilament 200 and (P ¼ 0.034).
bright field microscopy (Fig. 3B). In control cases, The number of preserved axons per 10,000 mm2
axons were evenly distributed throughout the white within Area II (white matter adjacent to the
matter. However, there was a striking difference in dorsolateral aspects of the intermediolateral cell
axonal preservation in different regions of the spi- column) in Group 1 (5977) was significantly re-
nal cord from individuals with spinal cord injury. duced in comparison with Group 2 (93713;
The number of preserved axons per 10,000 mm2 P ¼ 0.028) and Group 3 (109715; P ¼ 0.013).
within the corticospinal tract in Group 1 (2175) There were no significant differences in axonal
was significantly lower than in Group 2 (88712; counts within Area II between Groups 2 and 3
Po0.001) and Group 3 (117714; Po0.001). (P ¼ 0.357).
There was no significant difference between the
number of axons within the corticospinal tract of Discussion
individuals from Groups 2 and 3 (P ¼ 0.184).
The number of preserved axons per 10,000 mm2 Previous investigations have demonstrated that
within the dorsal column in Group 1 (100713), hypotension, bradycardia, and autonomic dysre-
Group 2 (131714), and Group 3 (136714) was flexia occur more frequently in individuals with
44

Fig. 3. (A) Staining of axons by immunocytochemistry for neurofilament 200 (NF200) in low power (  1.25) photomicrograph of a
spinal cord from a cord-injured individual with severe cardiovascular dysfunction (Case 4). Calibration bar is 1 mm. The squares
indicate the areas of the spinal cord in which preserved axons were counted. The four areas examined in this study for axonal
preservation were the following: dorsal column (DC), Area I, lateral corticospinal tracts (CST), and Area II. (B) High magnification
(  20) of different areas of the spinal cord stained with NF200 from three representative cases, one from each of the groups. Brown-
stained dots represent cross-sections of spinal axons immunocytochemically identified with NF200. There was a significant axonal loss
within Area 1 (panel b-1) and the CST (panel c-1) in all individuals from Group 1. (From Furlan et al. (2003), with permission from
J. Neurotrauma.)

severe cervical spinal cord injury (Lehmann et al., and the difference in incidence and severity of car-
1987; Mathias and Frankel, 1992; Noreau et al., diovascular symptoms in the two groups. In addi-
2000; Silver, 2000). This study has demonstrated a tion, the histology shows that the axon loss was
relationship between the location and severity of not homogeneously distributed across the area of
pathology in the spinal cord and cardiovascular the sections. The dorsal columns had very little
dysfunction in human cases of spinal cord injury. axon loss. Area I and the corticospinal tract
The histological analysis demonstrates that Group showed the greatest loss. Area II had an interme-
1, with significant cardiovascular dysfunction, had diate amount of loss.
greater myelin and axon loss than the cases in We expected that the severe cardiovascular
Group 2, who had insignificant cardiovascular symptoms of Group 1 would be associated with
dysfunction. By these criteria, injury was more se- a very large axon loss in the area traversed by the
vere in Group 1 than in Group 2. This matches the descending vasomotor pathways. Area I had a loss
higher ASIA grade for Group 1 than for Group 2, of 70% of axons in Group 1 cases, whereas this
45

area in Group 2 was decreased by 20%. In con- within the human spinal cord (Bunge et al., 1993;
trast, Group 1 lost 20% of axons in Area II vs. the Hayes and Kakulas, 1997; Puckett et al., 1997;
loss of 15% by Group 2, changes that were rela- Kakulas, 1999). Moreover, some histopathological
tively similar. Accordingly, the cardiovascular dys- findings in humans are significantly different from
function in Group 1 and the lack of dysfunction in those observed in animal models of cord injury
Group 2 correlate best with the axonal losses in (Puckett et al., 1997). Therefore, to extrapolate
Area I. Thus, Area I, the dorsal area of the dorso- information from animal models to human disor-
lateral funiculus, seems a more likely candidate ders, it is essential to compare findings from an-
than the more ventral Area II as the site of the imal and human studies.
cardiovascular pathways. This view is consistent Using cervical electrical stimulation and selec-
with previous work by Fehlings and Tator (1995) tive lesions, numerous investigators have reported
demonstrating a relationship between loss of func- that the descending vasomotor pathways are lo-
tion and axonal loss after spinal cord injury (SCI). calized within extensive areas from ventral to dor-
They showed that inclined plane score varied log- sal in the peripheral aspects of the lateral funiculus
arithmically with number of axons at the injury in cats and monkeys (Kerr and Alexander, 1964;
site, such that loss of greater than 50% of axons is Illert and Gabriel, 1972; Foreman and Wurster,
required for a significant drop in neurological 1973). Barman and Wurster (1975) demonstrated
function. that the descending sympathetic pathways are
These observations contradict the conclusions situated on the surface of the dorsolateral
reached by Nathan and Smith (1987) that, in funiculus and are organized in a dorsal-to-ventral
humans, the descending vasomotor pathways are manner based on electrical stimulation in dogs.
localized to the white matter adjacent to the Lebedev et al. (1986) carried out an electrophys-
dorsolateral aspect of the intermediolateral cell iological study before and after the dorsolateral
column (Area II of the present study). These funiculus transection showing that descending
conclusions were based on the analysis of vasomotor pathways are situated within the
postmortem spinal cord sections from patients dorsal parts of the lateral funiculus in cats, the
who underwent antero-lateral cordotomies for area which corresponds to Area I in our
control of intractable pain, and subsequently, investigation.
developed cardiovascular symptoms such as The knowledge of the area greatly responsible
hypotension and orthostatic intolerance. However, for vasomotor control has significant relevance to
careful analysis of their data showed that the recovery from spinal cord injury. Area I is su-
antero-lateral cordotomy resulted also in partial perficial and easily accessible from the dorsolat-
destruction of the lateral funiculus (Area I of the eral surface of the spinal cord. It is conceivable
present study). In other words, the cases in the that topical treatments could be applied to this
report by Nathan and Smith (1987) had damage in region. Thus, an avenue for future studies is an
both areas examined in the present study. There- exploration of techniques for delivering treatment
fore, it cannot be excluded that the relevant to this region. An additional question, raised by
damage was to Area I. this study, is whether it is possible to distinguish
Much of our present understanding of the anatomically, within Area I, the region of faci-
pathophysiology of central nervous system litatory pathways responsible for the maintenance
(CNS) disorders, including spinal cord injury, is of blood pressure and the orthostatic tolerance,
based on extrapolations from animal models from the location of inhibitory pathways that
(Krassioukov and Weaver, 1996; Krenz and limit the spinal reflexes responsible for dysreflexia.
Weaver, 1998; Maiorov et al., 1998; Osborn This investigation documents that anatomical
et al., 1989). Although considerable clinical data studies of cases of spinal cord injury can provide
are available on neurological function after human crucial information that may assist with treatment
spinal cord injury, only a limited number of stud- of the disabling cardiovascular consequences of
ies have been directed to histopathological changes spinal cord injury.
46

Acknowledgments Furlan, J.C., Fehlings, M.G., Shannon, P., Norenberg, M.D.


and Krassioukov, A.V. (2003) Descending vasomotor path-
This study was conducted with the support of a ways in humans: correlation between axonal preservation
and cardiovascular dysfunction after spinal cord injury.
Christopher Reeve Paralysis Foundation grant J. Neurotrauma, 20(12): 1351–1363.
(KB2-0003-1), a Cervical Spine Research Society Gao, S.A., Ambring, A., Lambert, G. and Karlsson, A.K.
grant, support from the Canadian Syringomyelia (2002) Autonomic control of the heart and renal vascular bed
Network, and a grant from the Heart and Stroke during autonomic dysreflexia in high spinal cord injury. Clin.
Foundation of Ontario (NA4951) awarded to Auton. Res., 12(6): 457–464.
Hayes, K.C. and Kakulas, B.A. (1997) Neuropathology of hu-
Dr. A. Krassioukov. Dr. J. Furlan (Toronto, man spinal cord injury sustained in sports-related activities.
ON) was a postdoctoral fellow who conducted a J. Neurotrauma, 14(4): 235–248.
major part of the histopathological analysis. The Illert, M. and Gabriel, M. (1972) Descending pathways in the
author also would like to acknowledge Dr. A. cervical cord of cats affecting blood pressure and sympathetic
Marcillo (Miami, FL), Mrs. Lorraine Yamamoto activity. Pflugers Arch., 335: 109–124.
Kakulas, B.A. (1999) A review of the neuropathology of human
(Burlington, ON), and Mrs. Lynda Rickards, R.N.
spinal cord injury with emphasis on special features. J. Spinal
(Toronto, ON) for their assistance and support Cord Med., 22: 119–124.
during the project. Karlsson, A.K. (1999) Autonomic dysreflexia. Spinal Cord, 37:
383–391.
Karlsson, A.K., Friberg, P., Lonnroth, P., Sullivan, L. and
Elam, M. (1998) Regional sympathetic function in high
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Published by Elsevier B.V.

CHAPTER 4

Disordered control of the urinary bladder after


human spinal cord injury: what are the problems?

Patrick J. Potter

Regional Spinal Cord Injury Rehabilitation Program, and Physical Medicine and Rehabilitation,
St. Joseph’s Health Center, The University of Western Ontario, London, ON, Canada

Abstract: Spinal cord injury has a profound impact on the storage and voiding functions of the urinary
bladder. Loss of autonomic and somatic control mechanisms leads to hypo- or hyperactivity of the bladder
wall and sphincters causing problems that range from incontinence to complete loss of the capacity to
empty the bladder. This chapter outlines the types of bladder dysfunction that occur after spinal cord
injury, their relative prevalence and current practices used to manage the problems. With all the inter-
ventions that are available, management of bladder function often still remains a compromise, as the
medications and physical interventions available may stimulate or block components of the voiding reflex,
but are often not fully restorative in this effort.

Introduction and autonomic functions usually do not recover


completely (Menter and Hudson, 1995; Wolfe
Urinary bladder control is dependent upon coor- et al., 2002; Potter et al., 2004). When only
dinated interaction between the somatic and au- partial resolution of the impairment in bladder
tonomic nervous systems (Burns et al., 2001; de control occurs, then accommodation to the altered
Groat and Yoshimura, 2001). When the supraspi- physiological condition requires interventions that
nal, coordinated control of these systems is lost, range from pharmacological treatments to appli-
the resulting bladder dysfunction is termed ‘‘ne- ances and mechanical or electrical devices, as pre-
urogenic bladder impairment’’ or a neurogenic sented in the following chapters. For previous
bladder. The presence of a neurogenic urinary reviews of the neurogenic bladder after spinal cord
bladder is extremely common after spinal cord in- injury see Perkash (2004) and Burns et al. (2001).
jury (Waites et al., 1993; Cardenas and Hooton,
1995; Vines, 1996; Sapounzi-Krepia et al., 1998;
Stover et al., 1989; Chen et al., 1999; Wolfe et al., Brief overview of normal bladder function
2002). In general, this dysfunction is not unique to
spinal cord injury as it can arise from disrupted The urinary bladder is a fluid reservoir that nor-
peripheral or central nervous system control. For mally empties completely in a well-controlled man-
example, a neurogenic bladder can occur in dis- ner. All of the clinical problems encountered after
eases such as multiple sclerosis (Anderson et al., spinal cord injury are manifestations of impairment
1976). Although some neurological recovery may in these two basic functions of the bladder: storage
occur following a spinal cord injury, motor, sensory and emptying. The lower urinary tract is made up
of the bladder, internal sphincter, external sphinc-
Corresponding author. Tel.:+519 685 4080; ter and urethra. The bladder wall is composed of
Fax: +519 685 4081; E-mail: patrick.potter@sjhc.london.on.ca smooth muscle, termed the detrusor muscle, and

DOI: 10.1016/S0079-6123(05)52004-1 51
52

has a base or trigone, a body and a neck. Para- spinal cord and in the bladder lead to malfunctions
sympathetic control of the detrusor muscle origi- in these reflexes and in the storage and voiding
nates from preganglionic axons in the pelvic nerve functions of the urinary bladder.
with cell bodies in the 2nd to 4th sacral (S) spinal
cord segments. Detrusor contraction is mediated
Clinical presentations of bladder dysfunction after
primarily by parasympathetic stimulation. Sympa-
spinal cord injury
thetic control of this muscle comes from the hypo-
gastric nerve that contains axons of preganglionic
Neurogenic bladder impairments depend greatly
neurons that are located in the 10th thoracic (T10)
upon the level and extent of central nervous system
to 2nd lumbar (L2) spinal cord segments. Sensory
injury. The lower levels of injury, such as at the level
innervation of the bladder wall travels to the S2–S4
of the conus medullaris, are more likely to result in
spinal segments via the pelvic nerve. The internal
a flaccid bladder. Thoracic and cervical level inju-
bladder sphincter is made of smooth muscle and is
ries commonly generate mixed pictures of detrusor
located at the junction of the bladder neck and
hyperactivity, sphincter spasticity and lack of coor-
urethra. This sphincter receives parasympathetic
dination between the detrusor and sphincters that is
and sympathetic innervation like that of the de-
termed dyssynergia. Because the urinary bladder is
trusor. However, in this muscle, sympathetic stim-
innervated bilaterally, hemi-cord impairments such
ulation causes contraction. The external sphincter
as the Brown Sequard Syndrome often do not result
is striated muscle that surrounds the urethra and is
in significant bladder dysfunction. Furthermore, in-
controlled by somatic innervation from the S2–S4
juries that spare the central portion of the cord re-
spinal segments that reach this sphincter via the
sult in relative sparing of bladder function.
pudendal nerve. Details of the anatomy and phar-
Issues of bladder dysfunction relate to four major
macology of these pathways are presented in later
problems: (1) inadequate or excessive detrusor
chapters and in reviews by de Groat and
function, (2) inadequate or excessive sphincter func-
Yoshimura (2001) and Burns et al. (2001).
tion, (3) dyssynergy between detrusor and sphincter
To promote the storage function of the urinary
actions and (4) impaired ability to sense the bladder
bladder, sympathetic innervation plays two key
(Lisenemayer and Oakley, 2003). Approaches to
roles. First, through a-adrenergic receptors, the
treatment can therefore be based on manipulation
neck and internal sphincter of the bladder are con-
of these functions. Often combinations of ap-
tracted to close the bladder outlet (Ek et al., 1977).
proaches are required and the type of bladder man-
Next, via b-adrenergic receptors, the body of the
agement may change through a cord-injured
bladder relaxes. Normally, filling of the bladder
person’s life. For example, during acute care im-
occurs with minimal increases in pressure. Voiding
mediately after injury, a Foley catheter is often in-
the bladder is a coordinated process that involves
serted to drain the flaccid bladder. As some degree
contraction of the detrusor muscle with concomi-
of continence develops with time after injury, this
tant relaxation of the striated muscle of the urethra
approach would likely change to intermittent cath-
and pelvic floor and relaxation of internal and ex-
eterization during rehabilitation. After discharge
ternal sphincters. This requires integrated control
from the hospital, some people who are able to re-
from pontine centers in the brain and sacral spinal
gain an active lifestyle, even including participation
neurons (see de Groat and Yoshimura, 2001).
in sports, rely on condom drainage into a leg bag
When the bladder volume increases, sensory input
that obviates the need for strictly timed procedures
from the bladder wall to the sacral spinal neurons
such as intermittent catheterization.
increases until the threshold for the micturition re-
flex is reached and reflex voiding can be initiated.
This process in the able-bodied person is under Inadequate detrusor function
voluntary control and is accomplished by well-
regulated and integrated autonomic and somatic Inadequate contraction of the detrusor muscle
reflexes. After spinal cord injury, changes in the is often associated with spinal cord injuries that
53

impair the distal conus medullaris region of the of the detrusor muscle is extremely variable be-
spinal cord. These very low injuries mimic lower tween individuals. Clinical approaches to treat this
motor neuron impairment such as found in problem include anticholinergic (anti-muscarinic)
peripheral neuropathies, resulting in absent or sig- medication if excessive intravesical pressures pre-
nificantly decreased detrusor contraction. There- vail. If the detrusor muscle cannot be relaxed ad-
fore the approach to remedy this problem is to equately with such medication to provide
augment emptying. Such augmentation can be in continence between intermittent catherizations,
the form of cholinergic muscarinic receptor stim- an indwelling catheter or attached device such as
ulation such as the oral administration of bethane- a condom catheter is necessary. If, due to lack of
col. Bethanecol must be taken every 4–6 h, and a sensation, voiding cannot be managed effectively
bladder response occurs 30 min to 1 h after taking or conveniently, an external appliance (condom
the drug. Therefore, drug use has to be timed drainage) may be used in males. However similar
to coordinate with bladder fullness. Mechanical devices are notoriously difficult to maintain in fe-
techniques used by some include increasing intra- males, resulting, instead, in the use of an indwell-
abdominal pressure with external mechanical pres- ing Foley or suprapubic (inserted through the
sure such as the Credé maneuver that utilizes for- lower abdominal wall) catheter. Decreasing de-
ward flexion over the subject’s hand as it presses trusor contractions may also be accomplished by
into the abdomen to facilitate voiding. Often emp- chemically blocking C-fiber bladder afferent ne-
tying with this procedure is incomplete, and to urotransmission with intravesical vanilloids such
prevent urinary tract infections from occurring as as capsaicin or resiniferatoxin or by intravesical
a consequence of the residual urine in the bladder, administration of anticholinergics. Another in-
the Credé maneuver may be combined with one travesical approach under investigation is injec-
catheterization per day to empty the bladder com- tion of botulinum toxin into the detrusor muscle to
pletely. This procedure is not successful in people cause relaxation (Reitz et al., 2004). Intravesical
with detrusor–sphincter dyssynergia as it also can administration of medication is more invasive than
cause contraction of the sphincters, blocking the oral medications but does offer options when oral
outflow of urine (Chancellor et al., 1990). The anticholinergic drugs are not effective. When an
most common approach to the management of an intravesical route is used for treatment, effects are
inadequate detrusor response is intermittent cath- temporary and repeated treatments are necessary.
eterization. In some cases of significantly de- The ideal time frames for repeated intravesical
creased detrusor function, spontaneous detrusor drug administration are not well established. More
contractions may occur that fail to empty the invasive approaches for reducing detrusor hyper-
bladder but are a cause of incontinence. In such activity include denervation procedures such as
cases, an anticholinergic (anti-muscarinic) drug sacral rhizotomy, a procedure that must be viewed
such as oxybutin and intermittent catheterization cautiously as it is irreversible. To address reduced
may be combined. bladder capacity due to detrusor hyperreflexia, the
bladder size and capacity may be increased by a
surgical augmentation cystoplasty using a piece
Excessive detrusor function of bowel.

Increased detrusor tone or spasticity (detrusor


hyperreflexia) is part of the upper motor neuron Inadequate sphincter function
syndrome. In this situation, the detrusor muscle is
considered to be ‘‘unstable,’’ contracting at lower Inadequate sphincter function, whether associated
bladder volumes and often producing excessive with inadequate or excessive detrusor function,
intravesical pressures. Detrusor hyperreflexia of- results in incontinence. Sphincter tone can be en-
ten, but not always, occurs with thoracic and cer- hanced by blocking muscarinic cholinergic recep-
vical cord injuries, and the extent of hyperactivity tors or by stimulating b-adrenergic receptors. Of
54

these two possible approaches, blocking mu- adrenergic antagonist as discussed above, and ap-
scarinic cholinergic receptors is usually the supe- plying a device for collection of urine such as a
rior, although combination therapy may be condom catheter. Alternatively, detrusor contrac-
utilized. In this condition, the goal is to restore tion may be blocked pharmacologically and blad-
the storage function of the bladder. Once this is der emptying accomplished by intermittent
accomplished, if the person cannot initiate void- catheterization. Often these approaches are only
ing, then emptying is done by intermittent cathe- partially successful and a compromise in bladder
terization. If continence cannot be maintained management is reached within the tolerance limits
with drugs, then voiding into an appliance such as for side effects of the medication. Either the person
a condom catheter is possible, and cholinergic voids more frequently, and experiences urgency, or
muscarinic receptor agonists such as bethanecol catheterizes more frequently. The most common
can be utilized to facilitate voiding. Operative ap- approach in males is to reduce sphincter tone and
proaches to enhance the usefulness of the bladder apply an external device.
neck in maintaining continence include surgically
modifying the bladder neck or implantation of an
Impaired ability to sense the bladder
artificial sphincter. The most common approach
for dealing with inadequate sphincter tone is to
At this time, 4-aminopyridine is the only pharma-
enhance the contraction pharmacologically and
cological agent that has been demonstrated to en-
utilize intermittent catheterization for bladder
hance electrical conduction in the spinal cord,
emptying.
enhancing sensation of bladder contraction and
fullness in some individuals (Potter et al., 1998). In
Excessive sphincter tone the absence of such sensation, management must
be accomplished by systems that continuously
a-2 receptor sympathetic adrenergic blockade is drain the urine such as condom drainage, indwell-
the mainstay of pharmacological management of ing catheters (suprapubic or Foley) or diapers or
the contracted bladder neck, to allow emptying in methods that employ timed, regular emptying such
the presence of excessive sphincter tone. Originally as intermittent catheterization.
developed to treat hypertension, this group of
medications has evolved, through several genera-
Infection can be a consequence of all
tions, to a family of drugs that can be taken once
management systems
per day and that have infrequent and less severe
side effects such as hypotension. Other approaches
Infection is a problem, secondary to almost all
to the spastic sphincter include sphincterotomy
methods of managing the neurogenic bladder after
and pudendal nerve section.
spinal cord injury (Bennett et al., 1995; Stover
et al., 1989; Esclarin De Ruz et al., 2000). As the
Detrusor-sphincter dyssynergia prevalence of resistant bacteria increases (Waites
et al., 2000; Siroky, 2002) commonly used, inex-
After spinal cord injury, the clinical presentation pensive antibiotics become ineffective. The fre-
of a person can be an inability to empty the blad- quency of urinary tract infections may necessitate
der either spontaneously or by self-initiated void- antibiotic prophylaxis (Galloway, 1997; Waites
ing. With these symptoms, urodynamic studies are et al., 2001; Morton et al., 2002). Alternative prep-
required to ascertain whether the impairment arations such as cranberry juice that contribute to
stems from inadequate detrusor function, exces- maintenance of the integrity of the bladder urot-
sive sphincter activity or dyssynergy between the helium as a barrier to bacteria then become more
two muscle groups. Approaches to treating dyssy- important considerations for long-term prophy-
nergia usually involve decreasing bladder neck re- laxis (Reid et al., 2003). For the cord-injured per-
sistance with a drug such as an a-2 receptor son, often the most sought after management
55

strategy is that which, in their mind, most mimics strictures, bladder diverticuli, chronic cystitis and
‘‘normal function’’ (Jamil, 2001). increased incidence of bladder cancer. These prev-
alence studies reveal that, although we are well
aware of the high incidence of neurogenic bladder,
Incidence and prevalence of urinary bladder we are still limited in our ability to manage its
dysfunction after cord injury consequences. For example, incomplete emptying
is associated with high residual urine volume,
Most people, during the first days after spinal cord which is a risk factor for incontinence and
injury, have evidence of a neurogenic bladder. In infection (Shekelle et al., 1999; Trautner and
people with incomplete injury, the majority of re- Darouiche, 2002). Recognizing that continence is
covery of bladder function is evident in the first the first issue associated with a neurogenic bladder,
6–9 months and improvement can continue for pain and infection are equally important long-term
2 years after injury. The negative consequences of sequelae (Post et al., 1998). Although continence
the neurogenic bladder to the health and quality of may be controlled with devices, sepsis, pain and
life for cord-injured people are decreasing with incontinence may result from recurrent urinary
current improvements in management and under- tract infections.
standing of the causes of the problems. In one of
the early papers on urological aspects of rehabil-
itation, Bors (1951), described up to 80% mortal- Conclusion
ity of spinal cord injured soldiers in World War I,
before they were able to return to the United The consequences of spinal cord injury to the
States. By the time of World War II the survival function of the urinary bladder are severe and play
rate had increased to 88%. Bors attributed this a serious role in the health and well-being of the
improvement to greater understanding of the cord-injured person for life. For the bladder to be
pathophysiology of the neurogenic bladder and an effective reservoir, we fully utilize reflex con-
the advent of antibiotics. Mortality due to uro- traction of the sphincter and reflex detrusor relax-
logical causes is now estimated to be o3% (Jamil, ation. For the bladder to empty, these processes
2001). The current focus of modern rehabilitation must be reversed. After minor impairments void-
medicine and research is directed toward issues of ing may still be possible but with greater effort,
morbidity and not mortality. The prevalence of a incontinence, incomplete emptying, increased fre-
neurogenic bladder after spinal cord injury is high quency, urgency or hesitancy. Given that we often
(Anson and Shepard, 1996; Noreau et al., 2000). A cannot fully reverse the effects of impaired neuro-
study by the Model Spinal Cord Injury Systems of logical control of bladder function, even with the
Care determined that 81% of persons with spinal extensive array of available medication, the most
cord injury reported some degree of impaired effective approach to the management of ne-
bladder function (McKinley et al., 1999). Even urogenic bladder remains to find the best balance
more significant are the secondary sequelae, in- between a person’s need for emptying their blad-
cluding frequent urinary tract infections, pain sec- der, their tolerance for medication, assistive de-
ondary to urinary tract infection and pain vices and appliances, and the social consequences
secondary to indwelling devices such as Foley of maintaining continence (Stover et al., 1989;
catheters. Cardenas and Hooton, 1995, Liguori et al., 1997;
In a survey of Spinal Cord Injured persons Yavuzer et al., 2000; Boschen et al., 2003).
(Wolfe et al., 2002; Potter et al., 2004) regarding Although restoration of normal function is the
the long-term sequelae of spinal cord injury, uro- ultimate goal of research, development of superior
logical problems had a high prevalence. The ‘‘neu- management methods is a high priority as well.
rological impairment’’ of bladder function does Newer generations of pharmacological agents are
not appear to change with time, but time and ag- being developed to provide better therapeutic re-
ing result in secondary problems such as urethral sponses with less side effects. Using strategies such
56

as administering medications directly into the Esclarin De Ruz, A., Garcia Leoni, E. and Herruzo Cabrera, R.
bladder can minimize side effects of drugs. Meth- (2000) Epidemiology and risk factors for urinary tract infec-
ods for intermittent catheterization have been im- tion in patients with spinal cord injury. J. Urol., 164:
1285–1289.
proved by devices such as hydrophilic catheters Galloway, A. (1997) Prevention of urinary tract infection in
that can be inserted with much less friction than patients with spinal cord injury—a microbiological review.
conventional catheters (Hedlund et al., 2001; Spinal Cord, 35: 198–204.
Vapnek et al., 2003). Probiotic treatment, the in- Hedlund, H., Hjelmais, K., Jonsson, O., Klarslov, P. and
travesicular administration of healthy bacteria to Talja, M. (2001) Hydrophilic versus non-coated catheters for
prevent infection, may reduce the need for antibi- intermittent catheterization. Scand. J. Urol. Nephrol., 35:
49–53.
otics. Research must be directed toward finding a Jamil, F. (2001) Towards a catheter free status in neurogenic
cure for the bladder dysfunction after cord injury, bladder dysfunction: a review of bladder management op-
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 5

Mechanisms underlying the recovery of lower urinary


tract function following spinal cord injury

William C. de Groat and Naoki Yoshimura

Departments of Pharmacology and Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Abstract: The lower urinary tract has two main functions, the storage and periodic expulsion of urine,
which are regulated by a complex neural control system in the brain and lumbosacral spinal cord. This
neural system coordinates the activity of two functional units in the lower urinary tract: (1) a reservoir (the
urinary bladder) and (2) an outlet (consisting of bladder neck, urethra and striated muscles of the pelvic
floor). During urine storage the outlet is closed and the bladder is quiescent, thereby maintaining a low
intravesical pressure over a wide range of bladder volumes. During micturition the outlet relaxes and the
bladder contracts to promote the release of urine. This reciprocal relationship between bladder and outlet is
generated by visceral reflex circuits, some of which are under voluntary control. Experimental studies in
animals indicate that the micturition reflex is mediated by a spinobulbospinal pathway passing through a
coordination center (the pontine micturition center) located in the rostral brainstem. This reflex pathway is
in turn modulated by higher centers in the cerebral cortex that are presumably involved in the voluntary
control of micturition. Spinal cord injury at cervical or thoracic levels disrupts voluntary control of voiding
as well as the normal reflex pathways that coordinate bladder and sphincter functions. Following spinal cord
injury, the bladder is initially areflexic but then becomes hyperreflexic due to the emergence of a spinal
micturition reflex pathway. Studies in animals indicate that the recovery of bladder function after spinal
cord injury is dependent in part on plasticity of bladder afferent pathways and the unmasking of reflexes
triggered by capsaicin-sensitive C-fiber bladder afferent neurons. The plasticity is associated with changes in
the properties of ion channels and electrical excitability of afferent neurons, and appears to be mediated in
part by neurotrophic factors released in the spinal cord and the peripheral target organs.

Introduction certain level of function even after elimination of


extrinsic neural input.
The functions of the lower urinary tract to store The lower urinary tract is also unusual with
and periodically release urine are dependent upon regard to its pattern of activity and the complexity
neural circuits located in the brain, spinal cord and of its neural regulation. For example, the urinary
peripheral ganglia (Barrington, 1925; Kuru, 1965; bladder has two principal modes of operation:
de Groat et al., 1993; Morrison et al., 2002). storage and elimination. Thus many of the neural
This dependence on central nervous control circuits exhibit switch-like or phasic patterns of
distinguishes the lower urinary tract from many activity (de Groat, 1975) in contrast to tonic
other visceral structures (e.g., the gastrointestinal patterns occurring in autonomic pathways to
tract and cardiovascular system) that maintain a cardiovascular organs. In addition, micturition is
under voluntary control and depends upon learned
Corresponding author. Tel.: +412 648 9357; behavior that develops during maturation of the
Fax: +412 648 1945; E-mail: degroat@server.pharm.pitt.edu nervous system, whereas many other visceral

DOI: 10.1016/S0079-6123(05)52005-3 59
60

functions are regulated involuntarily. Micturition sympathetic (hypogastric nerves and sympathetic
also depends on the integration of autonomic and chain) (Fig. 1) (de Groat et al., 1993).
somatic efferent mechanisms within the lumbosacral
spinal cord (Chancellor and Yoshimura, 2002;
Morrison et al., 2002). This is necessary during Parasympathetic pathways
urine storage and elimination to coordinate the
activity of visceral organs (the bladder and The sacral, parasympathetic, efferent pathway
urethra) with that of urethral striated muscles. provides the major excitatory input to the blad-
The dependence of lower urinary tract functions der and consists of spinal preganglionic neurons
on complex central neural networks renders these (Morgan et al., 1979) with cell bodies, in most of
functions susceptible to a variety of neurological the species studied, situated in the gray matter of
disorders (Torrens and Morrison, 1987; Chancellor the sacral spinal segments. In some species, for
and Yoshimura, 2002). This chapter will review example, the rat, the cell bodies span the caudal
studies in animals and humans that have provided lumbar and rostral sacral segments. Regardless of
insights into the neural control of the lower the actual location of the cell bodies, this group of
urinary tract and the disruption of this control neurons will be called the ‘‘sacral parasympathetic
by spinal cord injury. nucleus’’ in this chapter. These neurons send axons
to peripheral ganglion cells that, in turn, innervate
the bladder and urethral smooth muscle (Fig. 1).
Anatomy and innervation Transmission in bladder ganglia is mediated by
acetylcholine that excites the ganglion cells by act-
The storage and periodic elimination of urine are ing on nicotinic cholinergic receptors, whereas
regulated by the activity of two functional units in parasympathetic neuroeffector transmission in the
the lower urinary tract: (1) a reservoir (the bladder) bladder is mediated by acetylcholine acting on
and (2) an outlet (consisting of bladder neck, muscarinic receptors (de Groat and Yoshimura,
urethra and striated muscles of the pelvic floor). 2001; Andersson and Arner, 2004). Both M2 and
Under normal conditions, the urinary bladder and M3 muscarinic receptor subtypes are expressed in
outlet exhibit a reciprocal relationship. During bladder smooth muscle; however, examination of
urine storage, the bladder neck and proximal subtype-selective muscarinic receptor antagonists
urethra are closed; and the bladder smooth muscle and studies of muscarinic receptor knockout mice
is quiescent, allowing intravesical pressure to have revealed that the M3 subtype is the principal
remain low over a wide range of bladder volumes. receptor involved in excitatory transmission.
During voluntary micturition, the initial event is a In bladders of various animals, stimulation
reduction of intraurethral pressure, which reflects of parasympathetic nerves also produces a
a relaxation of the pelvic floor and the periurethral non-cholinergic contraction that is resistant to
striated muscles, and an opening of the bladder atropine and other muscarinic receptor blocking
neck (Chancellor and Yoshimura, 2002). The agents. Adenosine triphosphate (ATP) has been
changes in the urethra are followed in a few identified as the excitatory transmitter mediating
seconds by a detrusor contraction and a rise in the non-cholinergic contraction (Ralevic and
intravesical pressure that is maintained until the Burnstock, 1998; Burnstock, 2001). ATP excites
bladder empties. Reflex inhibition of the smooth the bladder smooth muscle by acting on P2X
and striated muscles of the urethra also contrib- purinergic receptors that are ligand-gated ion
utes to the reduction of outlet resistance during channels. Among the seven types of P2X recep-
micturition. These changes are coordinated by three tors that have been identified, P2X1 is the major
sets of nerves emerging from the thoracolumbar subtype expressed in the rat and also in the human
and sacral levels of the spinal cord: (1) sacral bladder smooth muscle. Although purinergic
parasympathetic (pelvic nerves), (2) sacral somatic excitatory transmission is not important in the
(pudendal nerves) and (3) thoracolumbar normal human bladder, it appears to be involved
61

Fig. 1. Diagram showing the sympathetic, parasympathetic and somatic innervation of the urogenital tract of the male cat. Sym-
pathetic preganglionic pathways emerge from the lumbar spinal cord and pass to the sympathetic chain ganglia (SCG) and then via the
inferior splanchnic nerves (ISN) to the inferior mesenteric ganglia (IMG). Preganglionic and postganglionic sympathetic axons then
travel in the hypogastric nerve (HGN) to the pelvic plexus and the urogenital organs. Parasympathetic preganglionic axons that
originate in the sacral spinal cord pass in the pelvic nerve to ganglion cells in the pelvic plexus and to distal ganglia in the organs. Sacral
somatic pathways are contained in the pudendal nerve, which provides an innervation to the penis, the ischiocavernosus (IC),
bulbocavernosus (BC) and external urethral sphincter (EUS) muscles. The pudendal and pelvic nerves also receive postganglionic
axons from the caudal SCG. These three sets of nerves contain afferent axons from the lumbosacral dorsal root ganglia. Abbreviations:
U, ureter; PG, prostate gland; VD, vas deferens.

in bladders in patients with pathological condi- receptor antagonists or by desensitization of P2X


tions such as chronic urethral outlet obstruction or purinergic receptors, indicating that acetylcholine
interstitial cystitis (Palea et al., 1993; O’Reilly or ATP is involved in excitatory transmission to
et al., 2002). urethral smooth muscle (Zoubek et al., 1993).
Parasympathetic pathways to the urethra induce
relaxation during voiding. In various species the Sympathetic pathways
relaxation is not affected by muscarinic antago-
nists and therefore is not mediated by acetylcho- Sympathetic preganglionic pathways that arise
line. However inhibitors of nitric oxide synthase from the 11th thoracic (T11) to 2nd lumbar (L2)
block the relaxation in vivo during reflex voiding spinal segments pass to the sympathetic chain
or block the relaxation of urethral smooth muscle ganglia (SCG) and then to prevertebral ganglia in
strips induced in vitro by electrical stimulation of the superior hypogastric and pelvic plexuses
intramural nerves indicating that nitric oxide is the (Fig. 1) and also to short adrenergic neurons in
inhibitory transmitter involved in relaxation the bladder and urethra (de Groat et al., 1993;
(Burnett et al., 1997; Ho et al., 1999; Morrison Delancey et al., 2002). Sympathetic postganglionic
et al., 2002). In some species, neurally evoked con- nerves that release norepinephrine provide an
tractions of the urethra are reduced by muscarinic excitatory input to smooth muscle of the urethra
62

and bladder base, an inhibitory input to smooth et al., 2003, 2004). Studies of the biomechanical
muscle in the bladder body as well as inhibitory properties of the intact female rat urethra in vitro
and facilitatory inputs to vesical parasympathetic have confirmed the large contribution of striated
ganglia (Andersson, 1993; de Groat and Booth, muscle activity and nicotinic receptor mechanisms
1993). Radioligand receptor binding studies to the contractions of the mid-urethra (Jankowski
showed that a-adrenergic receptors are concen- et al., 2004).
trated in the bladder base and proximal urethra,
whereas b-adrenergic receptors are most promi-
nent in the bladder body (Andersson, 1993). These Afferent pathways
observations are consistent with pharmacological
studies showing that sympathetic nerve stimula- Afferent axons innervating the urinary tract are
tion or exogenous catecholamines produce b- present in the three sets of nerves that innervate
adrenergic receptor-mediated inhibition of the the lower urinary tract (Janig and Morrison, 1986;
body and a-adrenergic receptor-mediated contrac- de Groat et al., 1993; Bahns et al., 1998; Morrison
tion of the base, dome and urethra. Molecular and et al., 2002). The most important afferents for in-
contractility studies have shown that b3-adrenergic itiating micturition are those passing through the
receptors elicit inhibition and a1-adrenergic recep- pelvic nerve to the sacral spinal cord. These affer-
tors elicit contractions. The a1A-adrenergic recep- ents are small myelinated (Ad) and unmyelinated
tor subtype is most prominent in the normal (C) fibers that convey information from receptors
bladders but the a1D-subtype is upregulated in in the bladder wall to second-order neurons in the
bladders from patients with outlet obstruction, spinal cord. Ad bladder afferents in the cat re-
raising the possibility that a1-adrenergic receptor spond in a graded manner to passive distension as
excitatory mechanisms in the bladder might con- well as active contraction of the bladder and ex-
tribute to irritative lower urinary tract symptoms hibit intravesical pressure thresholds in the range
in patients with obstruction (de Groat and of 5–15 mmHg, similar to the pressures at which
Yoshimura, 2001; Morrison et al., 2002). humans report the first sensation of bladder filling
(Chancellor and Yoshimura, 2002; Morrison et al.,
2002). These fibers also code for noxious stimuli in
Somatic pathways the bladder. On the other hand, C-fiber bladder
afferents in the cat have high thresholds and com-
The external urethral sphincter, which is composed monly do not respond to even high levels of in-
of striated muscle, receives a somatic cholinergic travesical pressure (Habler et al., 1990). However,
innervation via the pudendal nerve from anterior activity in some of these afferents is unmasked or
horn cells in the third and fourth sacral segments enhanced by chemical irritation of the bladder
(Fig. 1). Branches of the pudendal nerve and other mucosa. These findings indicate that C-fiber affer-
sacral somatic nerves also carry efferent impulses ents in the cat have specialized functions, such as
to muscles of the pelvic floor and proprioceptive the signaling of inflammatory or noxious events in
afferent signals from these muscles as well as sen- the lower urinary tract. Nociceptive and me-
sory information from the urethra. Analysis of chanoceptive information is also carried in the
urethral closure mechanisms in the female rat dur- hypogastric nerves to the thoracolumbar segments
ing bladder distension-evoked and sneeze-induced of the spinal cord (Bahns et al., 1998).
stress conditions revealed that the major rise in In rats, A- and C-fiber bladder afferents are not
urethral pressure occurred in the mid-urethra and distinguishable on the basis of stimulus modality;
was mediated by efferent pathways in the puden- thus both types of afferents consist of mechano-
dal nerve to the external urethral sphincter as well and chemo-sensitive populations (Sengupta and
as pathways in nerves to the iliococcygeus and Gebhart, 1994; Morrison et al., 1999; Shea et al.,
pubococcygeus muscles, but not by pathways in 2000; Rong et al., 2002). C-fiber afferents that
the sympathetic or parasympathetic nerves (Kamo respond only to bladder filling have also been
63

identified in the rat bladder and appear to be contain peptides: calcitonin gene-related peptide,
volume receptors possibly sensitive to stretch of vasoactive intestinal polypeptide, pituitary-adenyl
the mucosa. C-fiber afferents are sensitive to the cyclase activating polypeptide (PACAP), tachy-
neurotoxins, capsaicin and resiniferatoxin as well kinins, galanin and opioid peptides (de Groat,
as to other substances such as tachykinins, nitric 1987; Keast and de Groat, 1992; Maggi, 1993;
oxide, ATP, prostaglandins and neurotrophic fac- Morrison et al., 2002). Nerves containing these
tors released into the bladder by afferent nerves, peptides are common in the bladder, in the sub-
urothelial cells and inflammatory cells (Vizzard mucosal and epithelial layers, and around blood
et al., 1995; Lee et al., 2000; Chuang et al., 2001; vessels (de Groat and Yoshimura, 2001). In the
Yoshimura et al., 2001a; Morrison et al., 2002). spinal cord, peptidergic nerve terminals have a
These substances can sensitize the afferent nerves distribution very similar to the distribution of
and change their response to mechanical stimuli. pelvic nerve afferents labeled with horseradish
The properties of lumbosacral dorsal root gan- peroxidase (Kawatani et al., 1985; de Groat, 1987).
glion cells innervating the bladder, urethra and Peptidergic bladder afferent neurons in the rat also
external urethral sphincter in the rat have been express TrkA, a high-affinity receptor for nerve
studied with patch clamp recording techniques in growth factor (NGF) (McMahon et al., 1994) and
combination with axonal tracing methods to receptors for capsaicin (TRPV1) (Yoshimura
identify the different populations of neurons et al., 1996, 1998a, 2003) and tachykinins (NK-2
(Yoshimura et al., 1996, 2001b, 2003; Yoshimura and NK-3 receptors) (Morrison et al., 2002;
and de Groat, 1997, 1999; Black et al., 2003). Sculptoreanu and de Groat, 2003). Capsaicin, a
Based on responsiveness to capsaicin it is estimated neurotoxin that can release peptides from afferent
that approximately 70% of bladder afferent terminals, produces inflammatory responses, in-
neurons in the rat are of the C-fiber type. These cluding plasma extravasation and vasodilatation,
neurons exhibit high threshold, tetrodotoxin- when applied locally to the bladder in experimen-
resistant sodium channels and action potentials. tal animals (Maggi, 1993). These findings suggest
They show phasic firing (one to two spikes) in re- that the neuropeptides may be important trans-
sponse to prolonged depolarizing current pulses. mitters in the afferent pathways from the lower
Approximately 90% of the bladder C-fiber afferent urinary tract. Tachykinins may also act back on
neurons also are excited by ATP, which induces a afferent terminals in an auto-feedback manner
depolarization and firing by activating P2X3 or to modulate the excitability of the terminals
P2X2/3 receptors (Zhong et al., 2003). These (Morrison et al., 2002; Sculptoreanu and de Groat,
neurons express isolectin-B4 binding, which is 2003).
commonly used as a marker for ATP responsive
sensory neurons. A-fiber afferent neurons are Urothelial– afferent interactions
resistant to capsaicin and ATP, and exhibit low
threshold tetrodotoxin-sensitive sodium channels Recent studies have revealed that the urothelium,
and action potentials and tonic firing (multiple which has been traditionally viewed as a passive
spikes) to depolarizing current pulses. C-fiber barrier at the bladder luminal surface (Lavelle
bladder afferent neurons also express a slowly et al., 2000; Lewis, 2000), also has specialized
decaying A-type K+ current that controls spike sensory and signaling properties that allow
threshold and firing frequency (Yoshimura et al., urothelial cells to respond to their chemical and
1996, 2003). Suppression of this K+ current induces physical environment and to engage in reciprocal
hyperexcitability of bladder afferent neurons. These chemical communication with neighboring nerves
properties of dorsal root ganglion cells are in the bladder wall (Ferguson et al., 1997; Birder
consistent with the different properties of A- and et al., 1998, 2001, 2002, 2003; Cockayne et al.,
C-fiber afferent receptors in the bladder. 2000). These properties include: (1) expression of
Immunohistochemical studies have shown that a nicotinic, muscarinic, tachykinin, adrenergic and
large percentage of bladder afferent neurons capsaicin (TRPV1) receptors, (2) responsiveness to
64

transmitters released from sensory nerves, (3) close


physical association with afferent nerves and (4)
ability to release chemical mediators such as ATP
and nitric oxide that can regulate the activity of
adjacent nerves and thereby trigger local vascular
changes and/or reflex bladder contractions. The
role of ATP in urothelial–afferent communication
has attracted considerable attention because blad-
der distension releases ATP from the urothelium
and intravesical administration of ATP induces
bladder hyperactivity, an effect blocked by admin-
istration of P2X purinergic receptor antagonists
that suppress the excitatory action of ATP on
bladder afferent neurons (Morrison et al., 2002).
Mice in which the P2X3 receptor was knocked out
exhibited hypoactive bladder activity and ineffi-
cient voiding (Cockayne et al., 2000), suggesting
that activation of P2X3 receptors on bladder
afferent nerves by ATP released from the urothe-
lium is essential for normal bladder function. The
chapter by Birder (this volume) presents a more
detailed discussion of urothelial–afferent interac-
tions and changes in the urothelium after spinal
Fig. 2. Combined cystometrograms and sphincter electromyo-
cord injury. grams (EMG) comparing reflex voiding responses in an infant
(A) and in a paraplegic subject (C) with a voluntary voiding
Reflex mechanisms controlling the lower response in an able-bodied adult (B). The abscissa in all records
urinary tract represents bladder volume in milliliters and the ordinates rep-
resent bladder pressure in cm H2O and electrical activity of the
EMG recording. On the left side of each trace the arrows in-
The neural pathways controlling lower urinary dicate the start of a slow infusion of fluid into the bladder
tract function are organized as simple on–off (bladder filling). Vertical dashed lines indicate the start of
switching circuits that maintain a reciprocal rela- sphincter relaxation that precedes by a few seconds the bladder
tionship between the urinary bladder and urethral contraction in A and B. (B) Note that a voluntary cessation of
voiding (stop) is associated with an initial increase in sphincter
outlet (de Groat et al., 1981, 1993) (Fig. 2). The
EMG followed by a reciprocal relaxation of the bladder. A
principal reflex components of these switching resumption of voiding is again associated with sphincter relax-
circuits are listed in Table 1 and illustrated in ation and a delayed increase in bladder pressure. On the other
Fig. 3. Intravesical pressure measurements during hand, in the paraplegic subject (C) the reciprocal relationship
bladder filling in both humans and animals reveal between bladder and sphincter is abolished. During bladder
filling, transient uninhibited bladder contractions occur in as-
low and slowly increasing bladder pressures when
sociation with sphincter activity. Further filling leads to more
bladder volume is below the threshold for inducing prolonged and simultaneous contractions of the bladder and
voiding (Fig. 2). The accommodation of the blad- sphincter (bladder-sphincter dyssynergia). Loss of the recipro-
der to increasing volumes of urine is primarily a cal relationship between bladder and sphincter in paraplegic
passive phenomenon dependent upon the intrinsic people interferes with bladder emptying.
properties of the vesical smooth muscle and qui-
escence of the parasympathetic efferent pathway. urethra (Table 1, Fig. 3) (de Groat and Lalley,
In addition, in some species urine storage is also 1972; de Groat et al., 1981). During bladder filling
facilitated by sympathetic reflexes that mediate the activity of the sphincter electromyogram
an inhibition of bladder activity, closure of the (EMG) increases (Fig. 2), reflecting an increase
bladder neck and contraction of the proximal in efferent firing in the pudendal nerve and an
65

Table 1. Reflexes to the lower urinary tract

Afferent Pathway Efferent Pathway Central Pathway

Urine storage

Low-level vesical afferent activity (pelvic nerve) 1. External sphincter contraction (somatic nerves) Spinal reflexes
2. Internal sphincter contraction (sympathetic nerves)
3. Detrusor inhibition (sympathetic nerves)
4. Ganglionic inhibition (sympathetic nerves)
5. Sacral parasympathetic outflow inactive

Micturition

High-level vesical afferent activity (pelvic nerve) 1. Inhibition of external sphincter activity S-B-S reflexa
2. Inhibition of sympathetic outflow S-B-S reflex
3. Activation of parasympathetic outflow to the bladder S-B-S reflex
4. Activation of parasympathetic outflow to the urethra Spinal reflex

a
S-B-S reflex, spinobulbospinal reflex

increase in outlet resistance that contributes to the tracing, measurements of gene expression and
maintenance of urinary continence. patch-clamp recording in spinal cord slice prepa-
The storage phase of the urinary bladder can be rations have recently provided many new insights
switched to the voiding phase either involuntarily into the morphological and electrophysiological
(reflexly) or voluntarily (Fig. 2). The reflex switch properties of these reflex components. Neurotropic
is readily demonstrated in the human infant viruses, such as pseudorabies virus, have been
(Fig. 2A) or in the anesthetized animal when the particularly useful since they can be injected into a
volume of urine exceeds the micturition threshold. target organ (urinary bladder, urethra and urethral
At this point, increased afferent firing from tension sphincter) and then move intraaxonally from the
receptors in the bladder reverses the pattern of periphery to the central nervous system, where
efferent outflow, producing firing in the sacral they replicate and then pass retrogradely across
parasympathetic pathways and inhibition of synapses to infect second- and third-order neurons
sympathetic and somatic pathways. The expulsion in the neural pathways (Vizzard et al., 1995;
phase consists of an initial relaxation of the Nadelhaft and Vera, 1996; Sugaya et al., 1997).
urethral sphincter (Fig. 2) followed in a few Since pseudorabies virus can be transported across
seconds by a contraction of the bladder, and an many synapses, it could sequentially infect all of
increase in bladder pressure and flow of urine. the neurons that connect directly or indirectly to
Relaxation of the urethral outlet is mediated by the lower urinary tract (Fig. 4).
activation of a parasympathetic reflex pathway to
the urethra that triggers the release of nitric oxide, Anatomy of the spinal cord
an inhibitory transmitter, as well as by removal of
adrenergic and somatic cholinergic excitatory The spinal cord gray matter is divided into three
inputs to the urethra. Secondary reflexes elicited general regions: (1) the dorsal horn that contains
by flow of urine through the urethra facilitate interneurons that process sensory input, (2) the
bladder emptying. ventral horn that contains motoneurons and (3)
The reflex circuitry controlling micturition the intermediate region, located between the dorsal
consists of four basic components: spinal efferent and ventral horns, that contains interneurons and
neurons, spinal interneurons, primary afferent autonomic preganglionic neurons. These regions
neurons and neurons in the brain that modulate are further subdivided into layers or laminae that
spinal reflex pathways. Transneuronal virus are numbered, starting with the superficial layer of
66

Fig. 3. Diagram showing neural circuits controlling continence and micturition. (A) Urine storage reflexes. During the storage of
urine, distention of the bladder produces low level vesical afferent firing, which in turn stimulates (1) the sympathetic outflow to the
bladder outlet (base and urethra) and (2) pudendal outflow to the external urethral sphincter. These responses occur by spinal reflex
pathways and represent guarding reflexes, which promote continence. Sympathetic firing also inhibits the detrusor muscle and mod-
ulates transmission in bladder ganglia. A region in the rostral pons (the pontine storage center) increases external urethral sphincter
activity. (B) Voiding reflexes. During elimination of urine, intense bladder afferent firing activates spinobulbospinal reflex pathways
passing through the pontine micturition center, which stimulate the parasympathetic outflow to the bladder and internal sphincter
smooth muscle and inhibit the sympathetic and pudendal outflow to the urethral outlet. Ascending afferent input from the spinal cord
may pass through relay neurons in the periaqueductal gray (PAG) before reaching the pontine micturition center.

the dorsal horn (lamina I) and extending to the intermediolateral gray matter (laminae V–VII) in
ventral horn (lamina IX), and the commissure the sacral (in the rat, also caudal lumbar) segments
connecting the two sides of the spinal cord (lamina of the spinal cord (Fig. 4) (Morgan et al., 1981; de
X) (Fig. 5D). Groat et al., 1982; Araki and de Groat, 1997;
Miura et al., 2001a), whereas sympathetic pre-
Efferent pathways ganglionic neurons are located in both medial
(lamina X) and lateral sites (laminae V–VII) in the
Parasympathetic preganglionic neurons innervat- intermediate gray matter of the rostral lumbar
ing the lower urinary tract are located in the spinal cord. Parasympathetic preganglionic
67

Fig. 4. Transneuronal virus tracing of the central pathways controlling the urinary bladder of the rat. Injection of pseudorabies virus
into the wall of the urinary bladder leads to retrograde transport of virus (dashed arrows) and sequential infection of postganglionic
neurons, preganglionic neurons and then various central neural circuits synaptically linked to the preganglionic neurons. Normal
synaptic connections are indicated by solid arrows. At long survival times virus can be detected with immunocytochemical techniques
in neurons at specific sites throughout the spinal cord and brain extending to the pontine micturition center in the pons (i.e.,
Barrington’s nucleus) and to the cerebral cortex. Other sites in the brain labeled by virus are: (1) the paraventricular nucleus (PVN),
medial preoptic area (MPOA) and periventricular nucleus (Peri V.N.) of the hypothalamus, (2) periaqueductal gray (PAG), (3) locus
coeruleus (LC) and subcoeruleus, (4) red nucleus, (5) medullary raphe nuclei and (6) the noradrenergic cell group designated A5. Sixth
lumbar (L6) spinal cord section showing on the left side the distribution of virus labeled parasympathetic preganglionic neurons (&)
and interneurons (K) in the region of the parasympathetic nucleus, the dorsal commissure (DCM) and the superficial laminae of the
dorsal horn (DH), 72 h after injection of the virus into the bladder. The right side shows the entire population of preganglionic neurons
(PGN)(&) labeled by axonal tracing with the fluorescent dye (Fluorogold), injected into the pelvic ganglia and the distribution of
virus-labeled bladder PGN (’). Composite diagram of neurons in 12 spinal sections (42 mm each).

neurons send dendrites to discrete regions of the 1990; Sasaki, 1994). This dendritic distribution of
spinal cord including: (1) the lateral and dorsal sphincter motoneurons is similar to that of sacral
lateral funiculus, (2) lamina I on the lateral edge of preganglionic neurons indicating that these two
the dorsal horn, (3) the dorsal gray commissure populations of neurons may receive synaptic
(lamina X) and (4) gray matter and lateral fun- inputs from the same interneuronal sites and
iculus ventral to the autonomic nucleus (Morgan fiber tracts in the spinal cord.
et al., 1993). As discussed below, this dendritic
structure very likely indicates the origin of impor- Afferent projections in the spinal cord
tant synaptic inputs to these cells. Pudendal moto-
neurons innervating the external urethral sphincter Afferent pathways from the lower urinary tract
in the cat are located in the ventrolateral division project to discrete regions of the dorsal horn that
of Onuf’s nucleus and send dendritic projections contain the interneurons as well as the soma and/
into (1) the lateral funiculus, (2) lamina X (3) in- or dendrites of efferent neurons innervating the
termediolateral gray matter and (4) rostrocaudally lower urinary tract. Pelvic nerve afferent pathways
within the nucleus (Thor et al., 1989; Beattie et al., from the urinary bladder of the cat and rat project
68

Fig. 5. Comparison of the distribution of bladder afferent projections to the 6th lumbar (L6) spinal cord of the rat (A) with the
distribution of c-fos positive cells in the L6 spinal segment following chemical irritation of the lower urinary tract of the rat (B) and the
distribution of interneurons in the L6 spinal cord labeled by transneuronal transport of pseudorabies virus injected into the urinary
bladder (C). Afferents labeled by wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injected into the urinary bladder. C-fos
immunoreactivity is present in the nuclei of cells. DH, dorsal horn; SPN, sacral parasympathetic nucleus; CC central canal. (D)
Drawing shows the laminar organization of the cat spinal cord.

into Lissauer’s tract at the apex of the dorsal horn Spinal interneurons
and then pass rostrocaudally giving off collaterals
that extend laterally and medially through the su- As shown in Fig. 5C, interneurons retrogradely
perficial layer of the dorsal horn (lamina I) into the labeled by injection of pseudorabies virus into the
deeper layers (laminae V–VII and X) at the base of urinary bladder of the rat are located in the regions
the dorsal horn (Fig. 5A) (Morgan et al., 1981; of the spinal cord receiving afferent input from the
Steers et al., 1991). The lateral pathway, which is bladder (Nadelhaft and Vera, 1995; Sugaya et al.,
the most prominent projection, terminates in the 1997). Interneuronal locations also overlap in
region of the sacral parasympathetic nucleus many sites with the dendritic distribution of the
(SPN) and also sends some axons to the dorsal efferent neurons. A similar distribution of labeled
commissure (Fig. 5A). Pudendal afferent pathways interneurons has been noted following injections
from the urethra and urethral sphincter exhibit a of virus into the urethra (Vizzard et al., 1995) or
similar pattern of termination in the sacral spinal the external urethral sphincter, indicating a prom-
cord (Thor et al., 1989). The overlap of bladder inent overlap of the interneuronal pathways con-
and urethral afferents in the lateral dorsal horn trolling the various target organs of the lower
and dorsal commissure indicates these regions are urinary tract.
likely to be important sites of viscerosomatic in- The spinal neurons involved in processing af-
tegration and be involved in coordinating bladder ferent input from the lower urinary tract have been
and sphincter activity. identified by the expression of the immediate early
69

gene, c-fos (Fig. 5B). In the rat, noxious or non- group (Fig. 4). Several regions in the hypothalamus
noxious stimulation of the bladder and urethra and the cerebral cortex also exhibited virus-infected
increases the levels of Fos protein, primarily in the cells. Neurons in the cortex were located primarily
dorsal commissure, the superficial dorsal horn and in the medial frontal cortex. Other anatomical
in the area of the sacral parasympathetic nucleus studies in which anterograde tracer substances
(Fig. 5B) (Birder and de Groat, 1993; Birder et al., were injected into brain areas and then identified
1999). Some of these interneurons send long pro- in terminals in the spinal cord are consistent with
jections to the brain, whereas others make local the virus tracing data (Morrison et al., 2002).
connections in the spinal cord and participate in
segmental spinal reflexes.
Patch clamp recordings from parasympathetic Organization of urine storage and voiding reflexes
preganglionic neurons in the neonatal rat spinal
slice preparation have revealed that interneurons Sympathetic storage pathway
located immediately dorsal and medial to the
parasympathetic nucleus make direct monosynap- The integrity of the sympathetic input to the lower
tic connections with the preganglionic neurons urinary tract is not essential for the performance of
(Araki, 1994; Araki and de Groat, 1996, 1997). micturition (Torrens and Morrison, 1986; de
Microstimulation of interneurons in both loca- Groat et al., 1993). However, physiologic experi-
tions elicits glutamatergic, N-methyl-D-aspartic ments in animals indicate that during bladder
acid (NMDA) and non-NMDA excitatory postsy- filling, the sympathetic system does provide a tonic
naptic currents in the preganglionic neurons. Stim- inhibitory input to the bladder as well as an
ulation of a subpopulation of medial interneurons excitatory input to the urethra. This sympathetic
elicits gamma-amino butyric acid (GABA)ergic input is physiologically significant since surgical
and glycinergic inhibitory postsynaptic currents. interruption or pharmacologic blockade of the
Thus local interneurons are likely to play an im- sympathetic innervation can reduce urethral
portant role in both excitatory and inhibitory re- outflow resistance, reduce bladder capacity and
flex pathways controlling the preganglionic increase the frequency and amplitude of bladder
outflow to the lower urinary tract. Glutamatergic contractions recorded under constant volume
excitatory currents have also been elicited in pre- conditions.
ganglionic neurons by stimulation of the projec- Sympathetic reflex activity is elicited by a
tions from lamina X and the lateral funiculus sacrolumbar, intersegmental spinal reflex pathway
(Miura et al., 2001a, 2003). that is triggered by vesical afferent activity in the
pelvic nerves (Fig. 3A, Table 1) (de Groat and
Lalley, 1972). The reflex pathway is inhibited when
Pathways in the brain bladder pressure is raised to the threshold for
producing micturition. This inhibitory response is
The neurons in the brain that control the lower abolished by transection of the spinal cord at the
urinary tract have been studied with a variety of lower thoracic level, indicating that it originates at
anatomical tracing techniques in several species a supraspinal site, possibly the pontine micturition
(Morrison et al., 2002). In the rat, transneuronal center. Thus, the vesicosympathetic reflex repre-
virus tracing methods have identified many popu- sents a negative feedback mechanism, whereby an
lations of neurons that are involved in the control increase in bladder pressure tends to increase in-
of bladder, urethra and the urethral sphincter hibitory input to vesical ganglia and smooth mus-
including: Barrington’s nucleus (the pontine mi- cle, thus allowing the bladder to accommodate
cturition center), medullary raphe nucleus which large volumes (Fig. 3A). Increased sympathetic
contains serotonergic neurons, the locus coeruleus excitatory input to the bladder base and urethra
which contains noradrenergic neurons, per- would complement these mechanisms by increas-
iaqueductal gray and the A5 noradrenergic cell ing outflow resistance.
70

Urethral sphincter storage pathway through the pontine micturition center (Fig. 3B).
The pathway functions as on–off switch (de Groat,
Motoneurons innervating the striated muscles of 1975) that is activated by a critical level of afferent
the urethral sphincter exhibit a tonic discharge activity arising from tension receptors in the blad-
that increases during bladder filling. This activity der, and is in turn modulated by inhibitory and ex-
is mediated in part by low-level afferent input from citatory influences from areas of the brain rostral to
the bladder (Fig. 3A, Table 1). During micturition the pons (e.g., diencephalon and cerebral cortex)
the firing of sphincter motoneurons is inhibited. (Torrens and Morrison, 1986; de Groat et al., 1993).
This inhibition is dependent in part on supraspinal In contrast to the reflex control of the bladder,
mechanisms since it is not as prominent in chronic the parasympathetic control of the urethra in the
spinal animals. Electrical stimulation of the pon- rat appears to be dependent on pathways organ-
tine micturition center induces sphincter relaxation ized in the spinal cord (Table 1) that are modulated
suggesting that bulbospinal pathways from the by input from the brain. Nitric oxide-mediated
pons may be responsible for maintaining the nor- relaxation of the urethra that occurs in response to
mal reciprocal relationship between bladder and bladder distension is reduced but not eliminated by
sphincter (Holstege et al., 1986; Shefchyk, 1989; acute transection of the spinal cord (Kakizaki
Mallory et al., 1991). et al., 1997, Cheng et al., 1997). The reflex
relaxation of the urethral smooth muscle is very
prominent in chronic spinal-cord-transected rats.
Spinobulbospinal parasympathetic micturition Electrophysiological studies in cats and rats
pathway have confirmed that the parasympathetic efferent
outflow to the urinary bladder is activated by a
Micturition is mediated by activation of the sacral long latency supraspinal reflex pathway (de Groat
parasympathetic efferent pathway to the bladder et al., 1981, 1982, 1993; Mallory et al., 1989;
and the urethra as well as reciprocal inhibition of Cheng et al., 1999). In cats, recordings from sacral
the somatic pathway to the urethral sphincter parasympathetic preganglionic neurons innervat-
(Table 1, Fig. 3B). Studies in animals using brain ing the urinary bladder show that reflex firing oc-
lesioning techniques revealed that neurons in the curs with a long latency (65–100 ms) following
brainstem at the level of the inferior colliculus (i.e., stimulation of myelinated (Ad) vesical afferents in
the pontine micturition center) have an essential the pelvic nerve (Fig. 6). Afferent stimulation also
role in the control of the parasympathetic compo- evokes negative field potentials in the rostral pons
nent of micturition (Barrington, 1925; Kuru, 1965; at latencies of 30–40 ms, whereas electrical stimu-
Torrens and Morrison, 1986; Mallory et al., 1991; lation in the pons excites sacral preganglionic neu-
de Groat et al., 1993). Removal of areas of the rons at latencies of 45–60 ms. The sum of the
brain above the inferior colliculus by inter- latencies for the spinobulbar and bulbospinal
collicular decerebration usually facilitates micturit- components of the reflex pathway approximates
ion by elimination of inhibitory inputs from more the latency for the entire reflex. In cats, it is be-
rostral centers (Yokoyama et al., 2000). However, lieved that the ascending afferent pathways from
transections at any point below the colliculi abol- the spinal cord project to a relay station in the
ish micturition. Bilateral lesions in the rostral periaqueductal gray (PAG), which then connects
pons, in the region of the pontine micturition cen- to the pontine micturition center (Fig. 3B) (Blok
ter in cats, also abolish micturition (Barrington, and Holstege, 1994; Blok et al., 1995; Blok, 2002).
1925), whereas electrical or chemical stimulation at
these sites triggers bladder contractions and mi-
cturition (Kuru, 1965; Sugaya et al., 1987; Kruse Pontine micturition center
et al., 1990; Mallory et al., 1991; Noto et al., 1991b).
These observations led to the concept of a spino- Physiological and anatomical experiments have
bulbospinal micturition reflex pathway that passes provided substantial support for the concept that
71

Fig. 6. Diagram showing the organization of the parasympathetic excitatory reflex pathway to the detrusor muscle. Scheme is based on
electrophysiological studies in cats. In animals with an intact spinal cord, micturition is initiated by a supraspinal reflex pathway
passing through a center in the brain stem. The pathway is triggered by myelinated afferents (A-d fibers), which are connected to the
tension receptors in the bladder wall. Injury to the spinal cord above the sacral segments interrupts the connections between the brain
and spinal autonomic centers and initially blocks micturition. However, over a period of several weeks following cord injury, a spinal
reflex mechanism emerges, which is triggered by unmyelinated vesical afferents (C-fibers); the A-fiber-afferent inputs are ineffective.
The C-fiber reflex pathway is usually weak or undetectable in animals with an intact nervous system. Stimulation of the C-fiber bladder
afferents by instillation of ice water into the bladder (cold stimulation) activates voiding responses in people with spinal cord injury.
Capsaicin (20–30 mg, subcutaneously) blocks the C-fiber reflex in chronic spinal cats, but does not block micturition reflexes in intact
cats. Intravesical capsaicin also suppresses detrusor hyper-reflexia and cold-evoked reflexes in people with neurogenic bladder dys-
function.

neuronal circuitry in the pontine micturition cen- putative inhibitory transmitters into the pontine
ter functions as a switch in the micturition reflex micturition center of the cat can increase the vol-
pathway. The switch seems to regulate bladder ume threshold for inducing micturition and in
capacity and also coordinate the activity of the high doses completely block reflex voiding, indi-
bladder and external urethral sphincter. Electrical cating that synapses in this region are important
or chemical stimulation in the pontine micturition for regulating the set point for reflex voiding and
center of the rat, cat and dog induces: (1) a sup- also are an essential link in the reflex pathway
pression of urethral EMG, (2) firing of sacral (Mallory et al., 1991). Brain imaging studies in
preganglionic neurons, (3) bladder contractions humans using positron emission tomography
and (4) release of urine (Torrens and Morrison, (PET) or functional magnetic resonance imaging
1986; Mallory et al., 1989; de Groat et al., 1993; have identified increased neuronal activity in the
Blok, 2002). On the other hand, microinjections of pontine micturition center and PAG during
72

voiding (Blok et al., 1997, 1998; Athwal et al., Yoshimura, 2001). Glutamic acid, which is the
1999, 2001). major excitatory transmitter in the central nervous
system, has an important role in the control of the
micturition reflex. Experiments in rats indicate
Suprapontine control of micturition
that glutamatergic transmission in the spinal cord
is essential for bladder and urethral reflexes and
The organization of suprapontine pathways con-
for the spinal processing of afferent input from the
trolling micturition is less well defined, despite the
bladder. Both NMDA and a-amino-3-hydroxy-
fact that there is a large body of literature dealing
5-methyl-4-isoxazoleproprionic acid (AMPA)/
with the responses of the lower urinary tract to
kainate receptors are involved in glutamatergic
lesions or electrical stimulation of the brain (de
transmission in the micturition reflex pathway
Groat et al., 1993). In brief, it appears that the
(Yoshiyama et al., 1993, 1994, 1995, 1997; Sugaya
voluntary control of micturition is dependent
and de Groat, 1994; Matsumoto et al., 1995a, b).
upon (1) connections between the frontal cortex
A study using spinal slice preparations from neo-
and the septal and the preoptic regions of the
natal rats also revealed that sacral preganglionic
hypothalamus and (2) connections between the
neurons directly receive glutamatergic excitatory
paracentral lobule and the brain stem and spinal
inputs through NMDA and AMPA/kainate re-
cord (Torrens and Morrison, 1986; de Groat et al.,
ceptors from spinal interneurons in the region of
1993). Lesions to these areas of cortex resulting
the sacral parasympathetic nucleus (Araki and de
from tumors, aneurysms or cerebrovascular
Groat, 1996). Thus, it is likely that glutamatergic
disease, appear to remove inhibitory control over
transmission is important at various sites in the
the anterior hypothalamic area that normally
micturition reflex pathway.
provides an excitatory input to micturition
The spinobulbospinal micturition reflex path-
centers in the brainstem (Yokoyama et al., 2000).
way controlling bladder and urethral reflexes is
Human PET scan studies have revealed that two
also modulated by various neurotransmitters such
cortical areas (the right dorsolateral prefrontal
as norepinephrine (Yoshimura et al., 1990a, b;
cortex and the anterior cingulate gyrus) were
Ishizuka et al., 1996), dopamine (Yoshimura et al.,
active during voiding (Blok et al., 1997, 1998;
1993, 1998b; Seki et al., 2001), 5-hydroxytrypta-
Blok, 2002). The hypothalamus including the
mine (Thor et al., 1990; Steers et al., 1992a; Espey
preoptic area as well as the pons and the PAG
and Downie, 1995; Danuser and Thor, 1996),
also showed activity in concert with voluntary
gamma aminobutyric acid (Steers et al., 1992b;
micturition. Other PET studies that examined the
Igawa et al., 1993; Araki, 1994), acetylcholine
changes in brain activity during filling of the
(Sugaya et al., 1987; Ishiura et al., 2001) and
bladder revealed that increased activity occurred in
neuropeptides, tachykinins, enkephalins (Booth
the PAG, the midline pons, the mid-cingulate
et al., 1985; Noto et al., 1991a), vasoactive intesti-
gyrus and bilaterally in the frontal lobes (Athwal
nal polypeptide (de Groat et al., 1990) and PACAP
et al., 1999, 2001; Matsuura et al., 2002). These
(Ishizuka et al., 1995), acting through different re-
results are consistent with the notion that the PAG
ceptor subtypes (de Groat and Yoshimura, 2001).
receives information about bladder fullness and
then relays this information to other brain areas
involved in the control of bladder storage.
Neurogenic dysfunction of the lower urinary tract

Supraspinal and spinal neurotransmitters Neurogenic disturbances of micturition can be


controlling micturition classified into two general categories: failure to
store and failure to eliminate urine (Wein, 2002).
Various neurotransmitters at the spinal and Problems with storage occur with differing degrees
supraspinal level are involved in regulation of of severity, ranging from reduced bladder capacity
micturition and continence (de Groat and and frequency of urination to urgency and
73

incontinence. A common finding is that disorders powerful and cause urinary incontinence. In addi-
affecting the brain, particularly suprapontine areas, tion, the bladder is usually only emptied partially
produce hyperactive or uninhibited bladders. owing to development of simultaneous contrac-
Cerebrovascular accidents, Parkinson’s disease, tions of the bladder and the striated urethral
tumors or demyelinating diseases are common sphincter (detrusor–sphincter dyssynergia, Fig. 2C).
causes of this problem (Betts, 1999; Sakakibara Inefficient voiding may also be due to unsustained
and Fowler, 1999; Wein, 2002). bladder contractions.
Failure to eliminate urine occurs under various In normal micturition, activation of the pontine
conditions that interrupt the detrusor to detrusor micturition center simultaneously induces bladder
excitatory reflex pathway or that interfere with the contractions and a suppression of sphincter activ-
coordination between detrusor and sphincters ity. A complete suprasacral lesion interrupts this
(Chancellor and Yoshimura, 2002; Wein, 2002). coordination between the bladder and the striated
Areflexic bladders can occur with (1) damage to sphincter (Fig. 2C). Thus, cord-injured people with
the pelvic nerve or the sacral spinal cord (lower chronic upper motoneuron lesions exhibit (1)
motor neuron lesions), (2) lesions of the afferent detrusor hyperreflexia, (2) coordinated relaxation
pathways (e.g., diabetes, tabes dorsalis, pernicious of urethral sphincter smooth muscle and (3)
anemia, herniated intervertebral disc) or (3) the dyssynergic contraction of urethral sphincter
acute stage of spinal cord injury rostral to the striated muscle (detrusor–sphincter dyssynergia).
sacral segments (an upper motor neuron lesion) All patterns of detrusor hyperreflexia associated
(Fam and Yalla, 1988; Chancellor and Blaivas, with detrusor-sphincter dyssynergia lead to high
1996; Yoshimura, 1999). intravesical pressure and/or severe bladder
trabeculation with the formation of diverticula,
which often induce vesicoureteral reflux and dete-
Spinal cord injury rostral to the lumbosacral level rioration of the upper urinary tract (Fam and
Yalla, 1988; Chancellor and Blaivas, 1996).
The upper motoneuron type of spinal cord injury People with spinal cord injury at the level of T6
initially leads to a phase of spinal shock that is or higher, often exhibit autonomic dysreflexia,
followed by a recovery phase during which neu- which is characterized by arterial pressor responses
rological changes emerge. During the period of induced by stimuli below the level of spinal cord
spinal shock immediately after spinal cord injury, lesion such as bladder distension, fecal impaction
there is a flaccid paralysis and absence of reflex or bladder inflammation (Fam and Yalla, 1988).
activity below the level of lesion; thus the urinary These stimuli cause excitation of sympathetic
bladder becomes areflexic. However, activity of pathways and induce arteriolar vasoconstriction
striated and smooth muscle sphincters rapidly re- and hypertension as well as piloerection and
covers after suprasacral injuries. Because sphincter sweating below the level of injury.
tone is present, urinary retention develops and
cord-injured patients have to be treated with in-
termittent or continuous catheterization to elimi- Spinal cord injury at or below the sacral level
nate urine from the urinary bladder. Following the
spinal shock phase, reflex detrusor activity reap- In the lower motoneuron type of spinal cord
pears after 2–12 weeks in most cases (Fam and injury, complete lesions of the sacral cord or the
Yalla, 1988; Chancellor and Blaivas, 1996). cauda equina usually result in flaccidity of the
During the recovery phase, the detrusor devel- bladder and its outlet. The bladder becomes
ops involuntary reflex contractions (neurogenic areflexic, thereby bladder compliance and bladder
detrusor overactivity) in response to visceral stim- capacity are increased. Pressures in the striated
uli such as bladder filling or suprapubic manual urethral sphincter are decreased. When the lesion
compression. After the recovery period, these extends to the thoracolumbar spinal cord, the
involuntary bladder contractions become more sympathetic outflow to the internal smooth muscle
74

sphincter of the urethra is also damaged, and the although voiding contractions in spinal cord-
bladder neck becomes incompetent in association injured rats were still triggered by capsaicin-
with hypoactive detrusor and striated sphincter resistant Ad-fiber afferents (Cheng et al., 1995;
(Fam and Yalla, 1988; Chancellor and Blaivas, Cheng and de Groat, 2004). Capsaicin treatment
1996; Yoshimura, 1999). also eliminated detrusor-sphincter dyssynergia in
anesthetized chronic spinal rats. Clinical studies
demonstrated that C-fiber afferents innervating
Changes in functions of bladder afferent pathways the bladder are involved in detrusor hyperreflexia
after spinal cord injury and autonomic dysreflexia in spinal cord-injured
people (Geirsson et al., 1995; Igawa et al., 1996,
A slow recovery of lower urinary tract function 2003; Cruz et al., 1997) and detrusor hyperreflexia
following spinal cord injury is also observed in in people with multiple sclerosis (Fowler et al.,
animals such as cats and rats (de Groat et al., 1992; Szallasi and Fowler, 2002). Taken together,
1990; Kruse et al., 1993; de Groat, 1995). Com- it is clear that the functional properties of C-fiber
plete transection of the thoracic spinal cord ini- afferents in the bladder are altered following spinal
tially produces detrusor areflexia in both species, cord injury, thereby inducing hyperreflexic bladder
followed by the emergence of detrusor hype- activity in both humans and animals (Fig. 6).
rreflexia with detrusor-sphincter dyssynergia. Other evidence of a reorganization of C-
Electrophysiological and pharmacological studies fiber-mediated reflex pathways in subjects with
have shown that the reflex pathways controlling suprasacral spinal cord injury was obtained in
the lower urinary tract are markedly different be- studies of the cold stimulation-evoked voiding re-
tween spinal intact and spinal-injured animals. In flex. Instillation of cold water into the bladder in
spinal intact cats and rats, the micturition reflex is these subjects induces reflex voiding (the Bors Ice
mediated by a long-latency supraspinal reflex Water Test) (Bors and Comarr, 1971; Geirsson
pathway, passing through the pons, that is acti- et al., 1993, 1995). This reflex does not occur in
vated by myelinated Ad-fiber bladder afferents normal subjects, except for infants (Geirsson et al.,
(de Groat et al., 1981, 1993, 1998; Mallory et al., 1994). It has been shown in the cat that C-fiber
1989; Yoshimura, 1999). However, in chronic spi- bladder afferents are responsible for cold-induced
nal cats the afferent limb of the micturition reflex bladder reflexes (Fall et al., 1990). Intravesical ad-
consists of unmyelinated C-fiber afferents (Fig. 6). ministration of capsaicin to paraplegic people
It has also been demonstrated that in chronic spi- blocks the cold-induced bladder reflexes, indicat-
nal cats (3–6 weeks after injury), subcutaneously ing that they are mediated by C-fiber afferents
administered capsaicin, a C-fiber neurotoxin, com- (Geirsson et al., 1995, Igawa et al., 1996, 2003).
pletely blocked reflex bladder contractions induced Studies in rats revealed that cold stimulation in-
by bladder distention, whereas capsaicin had no duced detrusor-sphincter dyssynergia and that
inhibitory effects on reflex bladder contractions in capsaicin treatment prevented it (Cheng et al.,
spinal intact cats (de Groat et al., 1990; Cheng 1997). Thus, cold- and capsaicin-sensitive C-fiber
et al., 1999). Thus, it is plausible that C-fiber bladder afferents can evoke detrusor hyperreflexia
bladder afferents that usually do not respond to and detrusor-sphincter dyssynergia. These re-
bladder distention (i.e., silent C-fibers) (Habler sponses are facilitated after the elimination of
et al., 1990) become mechano-sensitive and initiate supraspinal controls by spinal cord injury (Fig. 6).
automatic micturition after spinal cord injury.
Increased excitability of C-fiber afferents after
spinal cord injury has also been demonstrated in Changes in the firing properties of bladder afferent
rats in which detrusor hyperreflexia was identified neurons following spinal cord injury
during cystometrograms as non-voiding bladder
contractions prior to micturition. The non-voiding The mechanisms for inducing hyperexcitability of
contractions were suppressed by capsaicin, C-fiber bladder afferents were investigated by
75

whole-cell patch-clamp recording in dissociated channels from the tetrodotoxin-resistant type to


dorsal root ganglion neurons innervating the rat the tetrodotoxin-sensitive type. Since tetrodotoxin-
urinary bladder (Yoshimura and de Groat, 1997). sensitive Na+ currents have a lower threshold for
Chronic spinal cord injury in rats produced hyper- activation than tetrodotoxin-resistant currents, it
trophy of bladder afferent neurons as reflected by is reasonable to assume that these changes in ex-
an increase in cell diameter and cell input capac- pression of Na+ channels in bladder afferent neu-
itance. This confirmed earlier findings that bladder rons after spinal cord injury contribute to a low
afferent neurons in the L6-S1 (1st sacral) dorsal threshold for spike activation in these neurons.
root ganglia undergo somal hypertrophy (45–50% In chronic spinal cord-transected rats, bladder
increase in cross-sectional area) in chronic spinal afferent neurons with tetrodotoxin-sensitive spikes
cord-transected rats (Kruse et al., 1995). In addi- exhibited no apparent membrane potential relax-
tion to neuronal hypertrophy, bladder afferent ation when the neurons were gradually depolar-
neurons in chronic spinal rats increased their ex- ized by injecting inward currents. In these neurons,
citability. In contrast to the majority (approxi- voltage responses induced by current injections
mately 70%) of bladder afferent neurons from were not altered by application of 4-aminopyri-
spinal cord intact rats that exhibited high thresh- dine, a K+ channel blocker, although the neurons
old tetrodotoxin-resistant humped action poten- with tetrodotoxin-resistant spikes still had 4-ami-
tials (Yoshimura et al., 1996), 60% of bladder nopyridine-sensitive membrane potential relaxa-
afferent neurons in chronic spinal cord-transected tion during depolarization as found in spinal cord
rats exhibited tetrodotoxin-sensitive low-threshold intact rats. The phenomenon of membrane poten-
action potentials. The mean threshold for spike tial relaxation is due to slowly inactivating IA cur-
activation in cord-transected rats ( 25.5 mV) was rents that can be elicited by depolarization from
21% lower than in intact animals ( 20 mV). the resting membrane potential (Yoshimura et al.,
1996; Yoshimura, 1999). Therefore it is likely that
Plasticity in Na+ and K+ channels of bladder following spinal cord injury A-type K+ channels
afferent neurons following spinal cord injury are suppressed in parallel with an increased ex-
pression of tetrodotoxin-sensitive Na+ currents,
The alteration of electrophysiological properties in thereby increasing excitability of bladder afferent
bladder afferent neurons after spinal cord injury neurons. Since tetrodotoxin-resistant Na+ cur-
was also reflected in changes in density of different rents and IA currents are preferentially expressed
types of Na+ currents (Yoshimura and de Groat, in small-sized C-fiber afferent neurons in spinal
1997; Black et al., 2003). Consistent with the in- cord intact rats (Gold et al., 1996a; Yoshimura
crement in the proportion of neurons with tetrodo- et al., 1996), the changes in these channels after
toxin-sensitive spikes, the number of bladder spinal cord injury must occur primarily in C-fiber
afferent neurons that predominantly expressed bladder afferent neurons and contribute to in-
tetrodotoxin-sensitive Na+ currents (60–100% of creased cell excitability of these neurons.
total Na+ currents) also increased. The density of Immunohistochemical studies using antibodies to
tetrodotoxin-sensitive Na+ currents in bladder af- tetrodotoxin-resistant Na+ channel protein (Nav
ferent neurons significantly increased from 32.1 to 1.8) revealed that tetrodotoxin-resistant Na+
80.6 pA/pF, while tetrodotoxin-resistant current channels are located not only in small-sized dor-
density decreased from 60.5 to 17.9 pA/pF fol- sal root ganglion cell bodies, but also in superficial
lowing spinal cord injury. In addition, an increase laminae of the dorsal horn of the spinal cord
in tetrodotoxin-sensitive Na+ currents was detect- (Novakovic et al., 1998; Yoshimura et al., 2001).
ed in some bladder afferent neurons that still re- Thus changes occurring in afferent cell bodies fol-
tained a predominance of tetrodotoxin-resistant lowing spinal cord injury may also occur at affer-
currents (450% of total Na+ currents) after spi- ent axons and/or receptors in the bladder and thus
nal cord injury. These data indicate that spinal contribute to the emergence of the C-fiber-
cord injury induces a switch in expression of Na+ mediated spinal micturition reflex.
76

and detrusor-sphincter dyssynergia after spinal


cord injury (Fig. 7).
In addition, we have recently found that in-
creased NGF in the spinal cord after spinal cord
injury is also responsible for inducing hyperexcit-
ability of C-fiber bladder afferent pathways, and
that intrathecal application of NGF antibodies,
which neutralized NGF in the spinal cord, sup-
pressed detrusor hyperreflexia and detrusor-
sphincter dyssynergia in spinal cord injured rats
(Seki et al., 2002, 2004a, b). Intrathecal adminis-
Fig. 7. Diagram of hypothetical mechanisms inducing lower
urinary tract dysfunction following spinal cord injury (SCI).
tration of NGF antibodies also reportedly blocked
The subsequent events occurring after SCI are indicated by the autonomic dysreflexia in paraplegic rats (Krenz
numbers 1–7. Injury to the spinal cord (1) causes detrusor- et al., 1999). Thus, NGF and its receptors in the
sphincter dyssynergia (DSD) (2) leading to functional obstruc- bladder and/or the spinal cord are potential targets
tion of urethra. Increased urethral resistance induces bladder for new therapies to suppress detrusor hype-
hypertrophy (3), resulting in increased levels of nerve growth
factor (NGF, 4) in the bladder smooth muscle. The nerve
rreflexia and detrusor-sphincter dyssynergia after
growth factor (NGF) level in the spinal cord (5) is also in- spinal cord injury (Fig. 7).
creased after SCI. Increased NGF in the bladder and spinal
cord is transported to bladder afferent pathways (6), followed
by hyperexcitability of bladder afferent pathways (7). Hyper- Spinal mechanisms involving vasoactive intestinal
excitability of bladder afferent pathways causes or enhances
neurogenic detrusor overactivity (8) and DSD (2).
polypeptide (VIP) and pituitary adenylate cyclase
activating polypeptide (PACAP)

Role of neurotrophic factors Vasoactive intestinal polypeptide (VIP) and


pituitary adenylate cyclase activating polypeptide
Nerve growth factor (NGF) has been implicated as (PACAP) are contained in afferent neurons inner-
a chemical mediator of pathology-induced changes vating the urinary bladder of the cat and rat
in C-fiber afferent nerve excitability and reflex (Kawatani et al., 1985; see Vizzard, this volume,
bladder activity (Yoshimura, 1999; Vizzard, 2000). for references). In the cat sacral spinal cord, VIP
It has been demonstrated that chronic administra- is present exclusively in C-fiber afferent axons
tion of NGF into the bladder of rats induced (Morgan et al., 1999), and is located in afferent ter-
bladder hyperactivity and increased the firing fre- minals projecting to the sacral parasympathetic nu-
quency of dissociated bladder afferent neurons cleus (Kawatani et al., 1985). In chronic spinal cats,
(Yoshimura et al., 1999), and that the production VIP-immunoreactivity is distributed over a wider
of neurotrophic factors including NGF increased area of the lateral dorsal horn in the sacral spinal
in the bladder after spinal cord injury (Vizzard, cord, suggesting C-fiber afferent axonal sprouting
2000, this volume). It has also been shown that the after spinal injury (Thor et al., 1986). In addition,
bladder hyperactivity and hypertrophy of afferent the effects of intrathecal administration of VIP are
and efferent neurons innervating the hypertrophic changed. In normal cats, VIP inhibits the micturit-
bladder in rats with partial urethral obstruction ion reflex, whereas in paraplegic cats VIP facilitates
was antagonized in part by systemic autoimmuni- the micturition reflex (de Groat et al., 1990). These
zation against NGF (Steers et al., 1996). Thus it findings suggest that the action of a putative C-fiber
seems that target organ–neural interactions medi- afferent transmitter may underlie the emergence of
ated by neurotrophic factors such as NGF pro- C-fiber bladder reflexes in the paraplegic cat.
duced in the hypertrophied bladder muscle may In normal and spinal cord-injured rats, VIP and
contribute to changes in C-fiber bladder afferent PACAP, another member of the secretin/glucagon/
pathways that underlie the detrusor hyperreflexia VIP peptide family, facilitated the micturition
77

reflex by actions on the spinal cord (Ishizuka et al., mRNAs levels of GluR-A and GluR-B AMPA
1995; Yoshiyama and de Groat, 1997; Vizzard, receptor subunits and NR1, but not NR2 NMDA
this volume). Patch-clamp studies in the neonatal receptor subunits (Shibata et al., 1999). On the
rat spinal slice preparation revealed that PACAP other hand, motoneurons in the urethral sphincter
has a direct excitatory action on parasympathetic nucleus express all four AMPA receptor subunits
preganglionic neurons due in part to blockade of (GluR-A, -B, -C and -D) in conjunction with
K+ channels and also has an indirect action med- moderate amounts of NR2A and NR2B as well as
itated by activation of excitatory interneurons high levels of NR1 receptor subunits. It seems
(Miura et al., 2001b). PACAP increased the fre- likely that this difference in expression accounts
quency of spontaneous excitatory postsynaptic for the different sensitivity of bladder and sphinc-
potentials as well as spontaneous firing and de- ter reflexes to glutamatergic antagonists.
creased the threshold for action potential genera-
tion. PACAP also increased the number and Spinal tachykinin mechanisms
frequency of action potentials elicited by depolar-
izing current pulses. PACAP levels in bladder Tachykinins, such as substance P and neurokinin
afferent neurons and in spinal cord projections are A, that are released at C-fiber afferent terminals
upregulated after spinal cord injury (Zvarova can act in the bladder wall to modulate the excit-
et al., 2005; Vizzard, this volume). These findings ability of afferent nerves and induce bladder con-
suggest that putative C-fiber afferent transmitters, tractions (de Groat et al., 1993; Morrison et al.,
such as VIP in the cat or PACAP in the rat, may 2002), and are also thought to be involved in the
underlie the emergence of C-fiber afferent evoked spinal cord as mediators of excitatory transmission
bladder reflexes after spinal cord injury. between primary afferent nerves and second-order
spinal neurons that express NK1 receptors. Al-
Spinal glutamatergic mechanisms though some studies have reported an increase in
bladder capacity and a decrease in maximal void-
Glutamic acid plays an essential role as an exci- ing pressure after intrathecal administration of
tatory transmitter in the spinal reflex pathways NK1 receptor antagonists in normal conscious or
controlling bladder and external urethral sphincter anesthetized rats, other experiments have failed to
activity in both normal and spinal cord-injured detect a change in bladder function after admin-
rats. A study of the effect of selective antagonists istration of these agents in normal rats (de Groat
revealed that NMDA and non-NMDA glut- and Yoshimura, 2001). On the other hand, a re-
amatergic (AMPA) receptors are involved in the duction in detrusor hyperreflexia in spinal cord-
supraspinal and spinal reflex pathways controlling injured rats has been noted after administration of
voiding, and that AMPA receptor mechanisms are NK-1 and NK-2 tachykinin receptor antagonists
most important. Intrathecal or intravenous ad- (Abdel-Gawad et al., 2001). The role of NK1 re-
ministration of NMDA or AMPA antagonists in ceptor-expressing spinal neurons in C-fiber-affer-
urethane-anesthetized rats depressed bladder con- ent mediated bladder activity was also evaluated
tractions and electromyographic activity of the by destroying these neurons in the L6-S1 spinal
external urethral sphincter (Yoshiyama et al., cord by intrathecal administration of a ribosome-
1993, 1995). In spinal cord injured-rats, external inactivating toxin, saporin conjugated with a
urethral sphincter electromyographic activity was specific NK1 receptor ligand that promotes the
more sensitive than bladder reflexes to glut- binding and internalization of the toxin in NK1
amatergic antagonists (Yoshiyama et al., 1993, receptor-expressing neurons. In treated animals,
1997), raising the possibility that the two reflex NK1 receptor immunoreactivity was reduced in
pathways have different types of receptors. This lamina I of the spinal cord and the bladder
was confirmed with in situ hybridization tech- hyperactivity induced by intravesical instillation
niques which revealed that sacral parasympathetic of capsaicin was reduced, but cystometric param-
preganglionic neurons in the rat express high eters in awake rats were not changed (Seki et al.,
78

2005). Preliminary experiments in spinal cord-in- functions are regulated by a complex neural con-
jured rats indicate that detrusor hyperreflexia is trol system located in the brain and spinal cord.
also reduced by saporin treatment (Seki et al., This control system performs like a simple switch-
2004a). These studies raise the possibility that NK1 ing circuit to maintain a reciprocal relationship
receptor antagonists might be useful clinically in between the reservoir (bladder) and outlet compo-
treating bladder dysfunction in people with spinal nents (urethra and urethral sphincter) of the uri-
cord injury. nary tract. Spinal cord injury disrupts voluntary
control and the normal reflex pathways that coor-
Peripheral muscarinic mechanisms dinate bladder and sphincter function. Studies in
animals indicate that recovery of bladder function
Muscarinic receptor antagonists are the first line following spinal cord injury is dependent upon the
therapy for detrusor hyperreflexia induced by spi- reorganization of reflex pathways in both the pe-
nal cord injury because bladder contractions are ripheral and central nervous system. Part of this
induced by activation of postjunctional muscarinic reorganization may be influenced by neural-target
receptors in the detrusor muscle by acetylcholine organ interactions that are mediated by neurotro-
released from parasympathetic postganglionic phic factors released by the peripheral organs.
nerves (Kim et al., 1997; Stohrer et al., 1999;
Chapple, 2000). Although the M2 receptor is the
predominant subtype in the bladder (approximate- Acknowledgments
ly 80%), the contractions of the bladder are me-
diated by the M3 receptor subtype, which is The authors’ research is supported by NIH
therefore considered the primary target of drug research grants (DK49430, DK 57267, P01 HD
therapy (Hegde and Eglen, 1999; Chapple, 2000). 39768).
However, other receptors may play a role in trig-
gering bladder activity after spinal cord injury. References
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 6

Spinal mechanisms contributing to urethral striated


sphincter control during continence and micturition:
‘‘How good things might go bad’’

Susan J. Shefchyk

Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB R3E 3J7, Canada

Abstract: The external urethral sphincter motoneurons in the sacral ventral horn control the striated
external urethral sphincter muscles that circle the urethra. Activity in these motoneurons and muscle
normally contribute to continence but during micturition, when urine must pass through the urethra, the
motoneurons and striated muscle must be silenced. Following injury to descending pathways in the spinal
cord, the ability to inhibit sphincter activity is disrupted or lost, resulting in bladder–sphincter dyssynergia
and functional obstruction of the urethra during voiding. This chapter will first review the various reflex
pathways and neuronal properties that contribute to continence, and which must be modulated during
micturition in the spinal intact animal. A discussion about how the dyssynergia seen with spinal cord injury
may be produced will then be presented.

Discussions about the neural control of the lower the urethra is open to allow the passage of
urinary tract usually concentrate on the autonomic urine from the contracting bladder. The sacral
control of the bladder and urethra smooth muscle ventral horn motoneurons innervating the striated
with less focus on the role of the striated urethral sphincter muscle fibres are thus subject to differ-
sphincter muscle. However, the coordination ential control during bladder filling and micturit-
between autonomic and somatic motor systems ion. The manifestation of abnormal micturition
is fundamental to both continence and micturition. patterns involving both autonomic and sphincter
While the striated sphincter is not under auto- somatic systems creates major challenges in
nomic control per se, the convergence and coor- clinical management, particularly in individuals
dination of various autonomic and somatic with spinal cord and supra-spinal disease or
sphincter reflex pathways unite the two systems damage.
closely. For the purpose of this review, the discus- We will see that the neural control of sphincter
sion will be limited to the spinal neural control motoneuron output is not unlike that of limb
of the striated external urethral sphincter muscle. motoneuron, including the interplay of descending
Contraction of the sphincter muscle contributes and segmental reflex pathways as well as major
to continence by closing the urethra. In contrast, contributions from the anatomical and electrical
sphincter muscle activity is suppressed during properties of the motoneurons themselves. The
micturition when the sacral parasympathetic abnormal striated sphincter activity, commonly
bladder preganglionic neurons are activated and referred to as sphincter dyssynergia (Andersen and
Bradley, 1976; Blaivas et al., 1981; Koyanagi et al.,
Corresponding author. Tel.: +204-789-3736; 1982; Kruse et al., 1993; Pikov and Wrathall,
Fax: +204-789-3930; E-mail: sjs@scrc.umanitoba.ca 2001), occurs as a consequence of various spinal

DOI: 10.1016/S0079-6123(05)52006-5 85
86

cord and supra-spinal lesions. This review will interneurons such as Renshaw cells (Hultborn
address the mechanism(s) contributing to the et al., 1988; Alvarez et al., 1999). This lack of an
recruitment of the sphincter motoneurons and anatomical substrate for recurrent inhibition is
muscles during continence, the de-recruitment of consistent with the absence of any evidence for
these motoneurons and muscle during micturition, functional recurrent inhibition of the sphincter
and the possible underlying factors responsible for motoneurons (Mackel, 1979). Although a variety
the abnormal sphincter motor patterns observed of anatomical and electrophysiological evidence
following spinal cord injury. By first reviewing shows descending inputs from various supra-
what is known about the control of the urethral spinal regions (Mackel, 1979; Nakagawa, 1980;
sphincter reflex, a groundwork for a discussion Holstege et al., 1987; Hermann et al., 1998), the
about how good things can go bad can begin. functional contributions of these descending path-
ways to the dynamic control of the sphincter
motoneurons is not clear. Figure 1 summarizes the
Continence major inputs to the sphincter motoneurons.
The location and morphology of the ventral
In humans, as well as cats and rats, continence is horn motoneurons, sometimes referred to as
facilitated by closure of the urethra achieved via external urethral sphincter pudendal motoneu-
contraction of the smooth muscle of the bladder rons, innervating the external striated sphincter
neck and urethra and activity of the striated muscle in humans, cats, rats and monkeys have
sphincter muscles surrounding the urethra been described in some detail (Onuf, 1899;
(reviewed in de Groat et al., 2001). In addition, Schroder, 1981; Roppolo et al., 1985; McKenna
the muscles of the pelvic floor may also contribute and Nadelhaft, 1986; Beattie et al., 1990; Sasaki,
to continence. The source of excitation to the 1994; Pullen et al., 1997). As mentioned earlier,
sphincter motoneurons has been the subject of these motoneurons have axon collaterals that
investigation for over half a century. Much of the project to the region of the cell somas (Sasaki,
focus has been on the segmental sensory inputs to 1994). Studies have also shown that the dendrites
the sphincter motoneurons, a sensory input that of these motoneurons form bundles that travel
is largely excitatory and associated with perineal rostral-caudally between clusters of sphincter
and pudendal cutaneous, urethral (Bradley and motoneuron somas (Beattie et al., 1990). There is
Teague, 1972, 1977; Mackel, 1979; Fedirchuk no evidence to date of gap junctions or coupling
et al., 1992; Shefchyk and Buss, 1998; Buss and between dendrites or somas.
Shefchyk, 1999) and pelvic visceral (Garry et al., The passive electrical membrane properties of
1959; McMahon et al., 1982) afferents. In contrast cat sphincter motoneurons have been described in
to hindlimb motoneurons, there appears to be no detail by several laboratories (Hochman et al.,
significant monosynaptic afferent input to sphinc- 1991; Sasaki, 1991; Shimoda et al., 1992). Sphincter
ter motoneurons (Jankowska et al., 1978; Mackel, motoneurons have a very high membrane input
1979; Fedirchuk et al., 1992; Buss and Shefchyk, resistance and low rheobase making them easily
1999), and direct feedback from the muscle itself recruited. They also display a depolarizing ‘‘sag’’
is almost nonexistent (Chennells et al., 1960; in the membrane response to hyperpolarization,
Lassmann, 1984). In addition, there is no electro- which can contribute to rebound excitation fol-
physiological evidence in sphincter motoneurons lowing hyperpolarization. Their action potentials
for any crossed or reciprocal inhibition as de- are followed by relatively short duration after
scribed for other sacral motoneuron populations hyperpolarizations, suggesting that they are well
(Jankowska et al., 1978). Sphincter motoneurons suited to fire tonically. These properties are con-
have axon collaterals (Sasaki, 1994), but these sistent with the need for a continuous motoneuron
appear to terminate directly in the region of the output to drive the sphincter muscles tonically
sphincter motor nucleus and do not project during the long periods of bladder filling (Garry
medially to the region of recurrent inhibitory et al., 1959; McMahon et al., 1982).
87

Fig. 1. A summary of the descending and segmental sensory systems described anatomically or physiologically as having inputs to the
external urethral sphincter motoneurons. The size of the arrow reflects the hypothesized functional strength of the input to the
motoneurons.

For over 20 years now non-linear membrane dorsal horn neurons (Russo et al., 1997; Morisset
responses to depolarizing current injection, and Nagy, 1999; Derjean et al., 2003) and sacral
referred to as plateau potentials or bistable mem- preganglionic neurons (Derjean et al., 2005). Other
brane properties, have been described and studied putative transmitter systems, including substance
in spinal hindlimb motoneurons and interneurons P and acetylcholine, have also been implicated in
in a variety of vertebrates (Hultborn and Kiehn, the facilitation of plateau properties (Delgado-
1992; Morisset and Nagy, 1999). Similar motor Lezama et al., 1997; Russo et al., 1997). On the
responses have been documented in humans other hand, metabotropic GABAergic receptors
(Collins et al., 2001). When expressed, these prop- are thought to suppress plateaux and bistable
erties function to amplify or prolong a neuron’s properties (Derjean et al., 2003).
output in response to a brief excitatory input. More recently, for the first time in cat sphincter
Furthermore, the expression of these properties motoneurons, the presence of similar active mem-
may be controlled by a balance of metabotropic brane properties that are sensitive to neuromodu-
excitatory and inhibitory neuromodulatory sub- latory systems have been described (Paroschy and
stances. For instance, serotonin and noradrenalin Shefchyk, 2000). Paroschy and Shefchyk (2000)
have been shown to facilitate plateau property reported that brief trains of perineal or pudendal
expression in ventral horn motoneurons afferent stimulation produced sustained firing in
(Hounsgaard et al., 1988; Russo et al., 1998), cat sphincter motoneurons. This type of sustained
and activation of metabotropic glutamate type 1 response had been noted years ago by McMahon
receptors can facilitate their expression in deep et al. (1982). Using intracellular recordings,
88

Paroschy and Shefchyk (2000) showed that a brief which for this purpose more closely mimics human
intracellular depolarizing current injection could micturition.
produce a train of action potentials characterized It has been recognized that when the bladder
by an accelerating firing rate, or a membrane de- contracts during micturition and urine flow begins,
polarization, that persisted well beyond the period the urethra is unobstructed to facilitate the flow of
of current injection. As with cat hindlimb moto- fluid out of the body. For many years, a variety of
neurons, the expression of these non-linear re- reflex feedback loops recruited during the bladder
sponses to depolarizing current injection was contraction and initial flow of urine into the ure-
facilitated by the intravenous administration of thra (Barrington, 1914; Garry et al., 1959) were
serotonin precursors or noradrenalin. Paroschy conceived as responsible for the decrease in sphinc-
and Shefchyk (2000) concluded that at least some ter activity. However, it was not until examination
proportion of sphincter motoneurons could alter of the pattern of sphincter activity changes during
their excitability and firing characteristics (i.e., filling and micturition in the de-afferented animal
express non-linear responses manifested as persist- was done (Shefchyk, 1989) did it become evident
ent firing or sustained membrane depolarization), that a central micturition circuitry contributed to
and that such properties could contribute to the the sphincter silencing in the absence of sensory
tonic activity of the sphincter motoneurons during feedback. That is not to say that segmental reflexes
continence. This non-linear property may reduce originating from the bladder and urethra do not
the need for a continuous synaptic excitation of contribute to the sphincter silencing, but that a
the sphincter motoneuron to maintain a given central circuitry is also available.
muscle force because it could enhance and extend
any periodic excitatory actions of segmental or
descending synaptic inputs during bladder filling. Direct inhibition of sphincter motoneurons
during micturition

Micturition The initial question raised was whether the


absence of tonic or evoked sphincter activity in
During micturition, the external urethral sphincter the cat model during the void was due to a periodic
muscle relaxes and the urethra is opened allowing absence of excitation or an active inhibition of the
urine to flow freely. When the sacral parasympa- motoneurons by the micturition circuitry. Shimoda
thetic bladder preganglionic neurons are recruited, et al. (1992) and Fedirchuk et al. (1993), using
a parallel system is engaged to ensure that sphincter intracellular recordings from sphincter motoneurons
motoneurons do not fire and that the sphincter during distension- and brainstem-evoked micturition
muscle activity ceases for a period of time during showed that the membrane of the urethra sphincter
the void. In humans (Andersen and Bradley, 1976; motoneurons hyperpolarized during micturition
Blaivas et al., 1981; Dyro and Yalla, 1986) and when the firing of sphincter motor axons, recorded
cats (Barrington, 1914; Rampal and Mignard, in the pudendal nerve, and the sphincter muscle
1979b; Sackman and Sims, 1990), there is normally electromyogram were silenced. Furthermore,
a complete silencing of sphincter muscle activity during this hyperpolarization an increase in
during micturition while in the rat, the sphincter membrane conductance could be detected and
displays a pattern of rhythmic bursting during the intracellular injection of chloride ions could
bladder contraction (Kakizaki et al., 1997; Streng reverse the hyperpolarization (Fedirchuk et al.,
et al., 2004). The mechanism responsible for this 1993). Together, these data lead to the suggestion
rhythmic activity in the rat has not been identified, that an active inhibitory chloride conductance at
but while it does not appear to be a detriment to the motoneuron membrane was contributing to
bladder emptying in the rat, such patterns in the inhibition of the motoneurons (Fedirchuk
humans or cats are considered pathological. This et al., 1993; Shefchyk, 1998). Shefchyk et al. (1998)
review will focus on results from the cat model, went on to show that in the cat, the sphincter
89

motoneuron inhibition was sensitive to the neurons in the dorsal commissure of the cat sacral
glycinergic antagonist, strychnine, and that the spinal cord. More recently, Sie et al. (2001)
motoneuron somas and proximal dendrites were extended this observation to show that some of
immunopositive for gephyrin, a protein that has these spinal interneurons were glycinergic. Based
been shown to be associated with glycinergic on the facts that these interneurons were positive
receptors in the spinal cord (Fyffe et al., 1995). for GABA and glycine and that micro-stimulation
Immunohistochemical evidence for GABAergic in the area of these neurons decreased intra-ure-
terminals on cat sphincter motoneurons has also thral pressure (Blok et al., 1998), it was hypoth-
been obtained (Ramirez-Leon et al., 1994), but a esized that these neurons mediated the inhibition
functional examination of the GABAergic actions of the sphincter motoneurons during micturition.
directly on the motoneurons or sphincter activity Buss and Shefchyk (2003), using extracellular
has not been reported. Unfortunately, in the rat recordings from single units in the dorsal commis-
the sphincter system has not been subject to a sure and deep dorsal horn, identified a group of
similar examination, and it is not known whether neurons that were excited during reflex distension-
the rhythmic sphincter muscle activity during evoked or pontine micturition center-evoked void
micturition is due to: (1) a periodic inhibition of responses, but not by bladder distension alone.
sphincter motoneurons during voiding, (2) the These neurons may be those described by Blok and
expression of intrinsic oscillatory properties in the co-workers. In addition, the anatomical identifi-
motoneurons during voiding, or (3) a combination cation within this region of a variety of neurons
of both tonic inhibition of some motoneurons as in linked to the sphincter motoneurons has been
the cat and human, with only a subpopulation described using viral tracing methods (Nadelhaft
phasically active during voiding. and Vera, 1996; for review of sacral interneurons,
The membrane hyperpolarization observed in see Shefchyk, 2001).
the cat sphincter motoneurons during micturition
functions to decrease the excitability of the cells by
moving the membrane potential away from the Evidence for premotoneuronal inhibition
firing threshold. As the expression of the non- in excitatory reflex pathways to
linear membrane properties described by Paroschy sphincter motoneurons
and Shefchyk (2000) is voltage-dependent (thresh-
old around 43 mV), such membrane hyperpolar- Fedirchuk et al. (1994) observed that polysynaptic
ization could function to turn off any expressed excitatory postsynaptic potentials in hindlimb
non-linear responses (see Fig. 6 in Paroschy and motoneurons, within the first sacral segment, were
Shefchyk, 2000). The fact that perineal, pudendal diminished in amplitude, or completely sup-
(Fedirchuk et al., 1994) and urethral afferent- pressed, during voiding in the absence of a postsy-
evoked (Buss and Shefchyk, 1999) excitatory naptic motoneuron membrane hyperpolarization.
postsynaptic potentials in sphincter motoneurons This led to the hypothesis that the excitatory path-
are decreased in amplitude during voiding, when ways from the perineal and pudendal afferents
the membrane conductance of the sphincter moto- may be gated out during micturition at sites pre-
neurons increases is consistent with a conductance motoneuronal, that is, at the segmental excitatory
shunting effect to diminish the excitatory poten- interneurons mediating the excitation or at the
tials. Such postsynaptic inhibition in the spinal primary afferent terminals themselves.
cord is usually mediated by local interneurons. The possibility that perineal and pudendal pri-
Evidence for the location and identity of spinal mary afferents were subjected to presynaptic inhi-
inhibitory interneurons that may be part of the bition, or primary afferent depolarization, was
circuitry activated during micturition was provid- examined by monitoring the excitability of single
ed by Blok et al. (1997), who reported a descend- identified afferents in the dorsal horn of the sacral
ing projection from the brainstem pontine spinal segments during micturition (Angel et al.,
micturition center to a population of GABAergic 1994; Buss and Shefchyk, 1999). Angel et al. (1994)
90

observed that while bladder distension in the ab- differed in their activity during micturition. One
sence of micturition did not produce excitability group, already mentioned earlier, were recruited
changes in pudendal or perineal afferents, during during micturition, and may be the inhibitory
distension-evoked micturition reflexes, over one- neurons we are discussing. A second population,
third of the afferents tested displayed an increase which were excited by perineal and pudendal af-
in excitability during the void. It was concluded ferents were inhibited during micturition and
that primary afferent depolarization could be de- could be the excitatory interneurons receiving in-
creasing transmitter release from these afferents hibition during micturition (Buss and Shefchyk,
and diminishing the efficacy of the afferent exci- 2003).
tatory actions during voiding. Buss and Shefchyk As summarized in Fig. 2, the central micturition
(1999) examined the perineal and pudendal affer- circuitry has access to a circuitry of spinal neurons
ents separately from the urethral pudendal affer- that coordinate various inhibitory pathways dur-
ents during micturition evoked by both distension ing micturition. The need to ensure the suppres-
of the bladder and by electrical stimulation of the sion of sphincter motoneuron output must be
pontine micturition center. The results obtained a high priority for the system as reflected in the
during both types of evoked micturition were sim- redundancy in the mechanisms responsible for this
ilar: 50% of the dorsal penile/clitoral afferents un- inhibition. The relative contributions of each of
derwent primary afferent depolarization during these inhibitory systems is not known at this time,
the void, while almost 60% of the urethral affer- but it can by hypothesized that disruption of any
ents examined showed excitability increases during of these components may contribute to undesired
voiding. Biphasic changes in excitability were not- motoneuron hyperactivity and dyssynergia during
ed in 4/11 afferents, that is, they underwent pri- bladder emptying.
mary afferent depolarization early during the void
then showed a decrease in excitability as the
sphincter activity returned following the void. It So, how might good things go bad?
was hypothesized that the spinal circuitry and in-
terneurons mediating presynaptic inhibition of the Depending upon the extent of damage to the white
perineal, pudendal and urethral sacral afferents matter of the spinal cord, a variety of changes may
was organized in a way to recognize the different occur in lower urinary tract function. With supra-
functions of the perineal/pudendal versus urethral sacral injuries, bladder and sphincter dyssynergia
afferents during the voiding cycle. Specifically, is commonly encountered and has been character-
Buss and Shefchyk (2003) proposed the existence ized in humans (Andersen and Bradley, 1976), as
of at least two subgroups of interneurons recruited well as cat (Rampal and Mignard, 1976b) and ro-
during micturition to accomplish this selective dent (Pikov and Wrathall, 2001) models. Although
sensory modulation. The GABA-containing spinal the details may vary somewhat, the common fea-
neurons that Blok and co-workers (Blok et al., ture of the dyssynergia is the presence of activity in
1997,1998; Sie et al., 2001) described would be ex- the sphincter muscle during bladder contractions,
cellent candidates for mediating the presynaptic an activity that effectively limits the amount of
inhibition of the primary afferents. urine that can be expelled. Various attempts to
In addition to presynaptic inhibition of primary decrease the neural drive to the sphincter muscle,
afferents, it is also possible that the excitatory in- including denervating the muscle physically, or
terneurons interposed between the primary affer- chemically with botulinum toxin (Smith et al.,
ents and the sphincter motoneurons could be the 2002), are directed to the problem of too much
targets of direct inhibitory inputs during micturit- sphincter tone and reflex activity. But, such ap-
ion. Buss and Shefchyk (2003) found two popula- proaches are obviously limited and do not address
tions of neurons in the sacral dorsal commissure of the basic issue, that is, the identity of the mech-
the cat that responded to electrical stimulation anisms contributing to the inappropriate sphincter
of perineal, pudendal and urethral afferents, but motoneuron activity.
91

Fig. 2. Summary of the inhibitory mechanisms thought to contribute to the suppression of sphincter motoneuron activity during
micturition in the spinal intact animal.

In humans and some of the animal models stud- disrupted. If we consider the factors discussed so
ied, the loss of descending pathways controlling far in this chapter, we can identify some potential
motor neurons, below the spinal cord lesion, results mechanisms for the abnormal sphincter activity
in loss of voluntary striated muscle control. How- during micturition following spinal cord injury.
ever, this is often accompanied by hyper-reflexia and The loss of descending pathways that facilitate
spasms in the ‘‘paralyzed’’ muscles. For limb motor spinal inhibitory circuits may be part of the prob-
systems, the spasms and hyperactivity appear to in- lem. The loss of descending pathways that have
volve changes in the control of transmission through tonic inhibitory control over all excitatory seg-
excitatory segmental reflex pathways and adapta- mental reflex pathways, including those to the
tions in the intrinsic properties of the motoneurons sphincter motoneurons, might contribute to in-
themselves (for discussion, see Gorassini et al., creased reflex activation of the sphincter during
2004). Even with incomplete lesions of the spinal both continence and micturition. This increased
white matter, in particular the dorsolateral fun- activity would demand even more powerful inhi-
iculus, the release of a variety of segmental excita- bition to prevent the recruitment of sphincter
tory reflexes in motor systems below the level of the motoneurons during micturition. Furthermore, a
lesion (Cavallari and Petersson, 1989) may contrib- decrease in excitability of a subset of interneurons
ute to increased reflex motor output. The striated specifically mediating the micturition-related
sphincter muscle appears to be no different from postsynaptic inhibition of the sphincter motoneu-
limb muscles in terms of the increased tone and rons or of the excitatory interneurons interposed
reflex activity following cord injury. Not only is between segmental afferents and the motoneurons,
there an apparent increase in excitability and output could lead to the sphincter dyssynergia during mi-
of the sphincter motoneurons in general, but in cturition. We are addressing excitability changes in
addition the mechanisms normally used by the the neurons responsible for postsynaptic inhibi-
central micturition circuitry to inhibit sphincter tion, and these same concerns may be raised for
activity appear to be either absent or greatly the spinal interneurons mediating presynaptic
92

inhibition of the primary afferents. It has been contributing to excitability changes (Derjean
documented that spinal cord presynaptic inhibito- et al., 2003, 2005). These exciting possibilities
ry pathways are disrupted with loss of descending remain to be tested. Whether such systems can
systems (Carpenter et al., 1963; Hongo and enhance the expression of non-linear membrane
Jankowska, 1967), and this could contribute to properties and/or membrane excitability in the
increased reflex transmission through many affer- neurons remains to be tested in both spinal cord
ent systems. This possibility has been addressed in intact and lesioned animal models. Furthermore,
studies using baclofen, an agonist of spinal GABA we must look beyond the motoneurons themselves.
ergic systems, to treat spinal cord-injured subjects It is known that unidentified interneurons can ex-
with sphincter hyperactivity and dyssynergia press plasticity in their firing characteristics, in-
(Hachen and Krucker, 1977). Although the target cluding membrane oscillations and plateau
may be afferent excitatory pathways to the sphinc- potentials/enhanced firing (Derjean et al., 2003,
ter system, the clinical results revealed that this 2005). It would be exciting to determine if only
approach was not selective and can also decrease excitatory interneurons have this capability or if
bladder afferent transmission to the point of com- inhibitory spinal neurons also express such plas-
promising the bladder contractions (Steers, 1989). ticity and by what mechanisms. If inhibitory neu-
Turning now to the possible effects of loss of rons can, then we might develop strategies to
neuromodulators released from descending sys- ensure their expression should it be diminished or
tems, we will consider the modulation of intrinsic lost following spinal cord injury. Regardless of
electrical properties and excitability changes in whether we are considering motoneurons or inter-
spinal interneuron and sphincter motoneuron sys- neurons, the reality of the presence of plasticity of
tems involved in continence and micturition. It has firing pattern and excitability should now be care-
been established in rat tail motoneurons that fully addressed in both intact and chronic lesion
changes in various membrane currents occur fol- models for the spinal neurons involved in lower
lowing lower lumbar spinal cord injury and that urinary tract function. We need to determine: (1) if
these changes may contribute to an increased re- such properties are expressed in all spinal cell
cruitment of motoneurons and spastic motor ac- types; (2) what ion channels and second messenger
tivity in the tail musculature (Li et al., 2004). systems mediate the plasticity; and (3) which
Gorassini and co-workers have described results neuromodulators facilitate and inhibit the chan-
suggestive of similar mechanisms in limb moto- nels and systems involved in the expression of the
neurons of spinal cord injured people (Gorassini property. This knowledge may provide some ex-
et al., 2004). With the loss of descending serotonergic citing new insights into selectivity and organiza-
and adrenergic systems to the sphincter motoneu- tion of the systems and cellular mechanism.
rons known to promote non-linear properties and In summary, the control of the external urethral
increased motoneuron output (Paroschy and sphincter is not simple. The integration of various
Shefchyk, 2000), one might predict decreased, segmental inputs, interneuron activity and moto-
not increased sphincter reflex activity. However, neuron properties must be considered when ad-
it is known that substances intrinsic to the spinal dressing the changes in sphincter motor function
cord may also facilitate the expression of similar following spinal cord injury. Although the sphinc-
membrane currents and excitability changes. For ter system differs in many ways from limb muscle
instance, acetylcholine can produce plateau poten- control, there are similarities that are significant
tials (Svirskis and Hounsgaard, 1998) and a strong and we may do well to consider the well-developed
intrinsic cholinergic source to the sphincter moto- literature on limb reflex control and spinal cord
neurons may be directly from their own axon injury. The circuitry controlling the sphincter is
collaterals (Sasaki, 1994). Furthermore, changes in largely intrinsic to the spinal cord, and thus may
glutamatergic inputs and receptors following cord still be available for manipulation and participa-
injury (see Llewellyn-Smith et al., 1997), may tion in the normalization of lower urinary tract
result in glutamatergic metabotropic receptors function following spinal cord injury.
93

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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 7

Neurochemical plasticity and the role of


neurotrophic factors in bladder reflex pathways after
spinal cord injury

Margaret A. Vizzard

Departments of Neurology and Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington,
VT 05405, USA

Abstract: Transection of the spinal cord that interrupts the spinobulbospinal micturition reflex pathway,
abolishes voluntary voiding and initially produces an areflexic bladder with complete urinary retention.
However, depending upon the species, reflex bladder activity slowly recovers over the course of weeks or
months. In chronic spinal animals, reflex mechanisms in the lumbosacral spinal cord are capable of du-
plicating many of the functions performed by reflex pathways in animals with an intact spinal cord and can
induce bladder hyperreflexia. However, the bladder does not empty efficiently due to a loss of blad-
der–sphincter coordination (bladder–sphincter dyssynergia). In contrast to normal animals in which the
sphincter relaxes during voiding, animals with a spinal cord injury exhibit sphincter contractions during
voiding, an increase in urethral outlet resistance, urinary retention, bladder hyperreflexia, bladder over-
distension, and an increase in bladder afferent cell size. Changes in electrophysiological or neurochemical
properties of bladder afferent cells in the dorsal root ganglia and of spinal pathways could contribute to the
emergence of the spinal micturition reflex, bladder hyperreflexia and changes in the pharmacologic re-
sponses of reflex pathways in the lumbosacral spinal cord after spinal cord injury. Urinary bladder hype-
rreflexia after spinal cord injury may reflect a change in the balance of neuroactive compounds in bladder
reflex pathways. This review will detail: (1) changes in the neurochemical phenotype of bladder afferent
neurons and of spinal neurons mediating micturition reflexes after spinal cord injury, with an emphasis on
three neuroactive compounds, neuronal nitric oxide synthase (nNOS), galanin, and pituitary adenylate
cyclase activating polypeptide (PACAP); (2) possible functional consequences on bladder reflexes of
changes in spinal cord neurochemistry after spinal cord injury, and (3) the potential role of neurotrophic
factors expressed in the urinary bladder or spinal cord after spinal cord injury in mediating these neuro-
chemical changes.

Neural control of micturition and the smooth and striated muscle of the urethral
sphincters (Kuru, 1965; Klück, 1980; de Groat and
The storage and periodic elimination of urine re- Steers, 1990) (Fig. 1). Three neural pathways reg-
quires a complex neural control system that coor- ulate the lower urinary tract (Fig. 1): (1) sacral
dinates the activities of a variety of effector organs parasympathetic (pelvic) nerves provide excitatory
including the smooth muscle of the urinary bladder input to the bladder; (2) thoracolumbar sympathetic
(hypogastric) nerves provide an inhibitory input to
Corresponding author. Tel.: +802-656-3209; the bladder and an excitatory input to the bladder
Fax: +802-656-8704; E-mail: margaret.vizzard@uvm.edu neck and urethra; and (3) sacral somatic (pudendal)

DOI: 10.1016/S0079-6123(05)52007-7 97
98

Fig. 1. Neuroanatomy of micturition reflex circuitry. Postganglionic neurons that innervate the urinary bladder may be located in
intramural ganglia within the detrusor wall or in some species (e.g., rattus norvegicus), postganglionic neurons are located in pelvic
ganglia located in close proximity to the urinary bladder. From Fernandez (2002) copyright 2002 with permission.

nerves which innervate the striated muscles of the pathways are inactive (Kuru, 1965; de Groat and
sphincters and pelvic floor (Kuru, 1965; Klück, Kruse, 1993; de Groat et al., 1993; de Groat
1980; de Groat and Steers, 1990; Middleton and et al., 1997). During reflex or voluntary micturition,
Keast, 2004). Each of these sets of nerves contains the activity patterns are reversed such that para-
afferent (sensory) as well as efferent (motor) axons sympathetic pathways are excited and somatosym-
(Morrison, 1987; Lincoln and Burnstock, 1993). pathetic pathways are inhibited thereby promoting
The central neural pathways controlling the urine flow (Middleton and Keast, 2004) (Fig. 2).
lower urinary tract exhibit ‘‘all-or-none’’ or The lower urinary tract reflex mechanisms, or-
‘‘switch-like’’ characteristics reflecting the storage ganized at the level of the lumbosacral spinal cord,
and elimination functions of the lower urinary tract are modulated predominantly by supraspinal con-
(de Groat and Kruse, 1993; de Groat et al., 1993; trols (Kuru, 1965; de Groat, 1975; de Groat and
de Groat et al., 1997) (Fig. 2). During urine storage, Kruse, 1993; de Groat et al., 1993; de Groat et al.,
somatic and sympathetic pathways to the sphinc- 1997; Middleton and Keast, 2004). These mecha-
ters and sympathetic inhibitory inputs to the blad- nisms can be summarized as follows: (1) storage re-
der are tonically active, whereas parasympathetic flexes (parasympathetic and somatic) are organized
99

Fig. 2. Diagram illustrating the switch-like properties of the micturition reflexes. During urine storage, a low level of bladder afferent
activity activates efferent input to the external urethral sphincter (sympathetic and somatic nerves). A high level of afferent activity
induced by urinary bladder distention activates the switching circuit in the central nervous system resulting in the activation of efferent
pathways to the urinary bladder detrusor muscle (parasympathetic nerves), inhibition of efferent outflow to the sphincter and urine
outflow. From de Groat (1993) copyright 1993 with permission.

at the spinal level; (2) elimination reflexes (para- Emergence of automatic micturition after spinal
sympathetic) are organized at a supraspinal site in cord injury may be dependent on multiple factors,
the pons; and (3) spinal storage reflexes are mod- including: (1) elimination of bulbospinal inhibitory
ulated by inputs from the rostral pons. pathways; (2) strengthening of existing synapses, or
formation of new synaptic connections due to ax-
Spinal voiding reflexes after spinal cord injury onal sprouting in the spinal bladder reflex path-
ways; (3) changes in synthesis, release, or action of
Spinal cord injury above the lumbosacral spinal cord neurotransmitters in bladder reflex pathways;
(upper motoneuron injury) alters the coordination (4) alteration in afferent input from peripheral or-
between urinary bladder and sphincter and gans; or (5) central (spinal cord) and peripheral
chronically impairs micturition in humans, and in (urinary bladder) changes in the expression of
experimental animals (Kuru, 1965; de Groat et al., neurotrophic factors, which, in turn, would act by
1993). Spinal cord injury produces an initial period their influence on factors 2–5. A number of labo-
of urinary bladder areflexia that persists for several ratories have demonstrated electrical (Yoshimura,
weeks to months depending on the species. This 1999) and organization changes (Vizzard, 2000b)
period is followed by the emergence of a micturition in the central and peripheral components of the
reflex at the spinal level and the appearance of spon- micturition reflex pathways after spinal cord injury
taneous, involuntary bladder contractions (de Groat that may underlie the emergence of the spinal mi-
et al., 1993). However, the micturition reflex in cturition reflex with associated bladder dysfunction.
chronically spinalized animals is characterized by si- The changes that occur in spinal voiding reflexes
multaneous bladder and external urethral sphincter after spinal cord injury appear to be similar in
contractions (bladder–sphincter dyssynergia) leading humans and experimental animals and are begin-
to inefficient bladder emptying, large residual urine ning to provide important insights into a variety of
volumes and bladder hypertrophy (de Groat et al., neurogenic disorders of the lower urinary tract
1993). It has been suggested that bladder–sphincter (de Groat and Kruse, 1993; de Groat et al., 1993).
dyssynergia results from the loss of brainstem A major breakthrough has been the recognition
coordination mechanisms (de Groat et al., 1993). that C-fiber bladder afferents can reflexly trigger
100

bladder hyperactivity (de Groat et al., 1981, 1990, 1993; Vizzard et al., 1995b; de Groat et al., 1997)
1993). In cats with transected spinal cords, the have been used to examine the properties of uro-
properties of C-fiber bladder afferents are altered, genital afferent pathways in the lumbosacral spinal
so that they become mechanosensitive and re- cord (L6–S1). Anterograde transganglionic tracing
spond to bladder distension (de Groat et al., 1981, using horseradish peroxidase or wheat germ ag-
1990, 1993). In chronic spinal cord injury, C-fiber glutinin-conjugated horseradish peroxidase re-
afferent evoked bladder reflexes emerge. However, vealed that bladder afferents in rats pass through
in cats with an intact spinal cord, myelinated (A-q) the dorsal roots into Lissauer’s tract at the apex of
afferents activate the micturition reflex (de Groat the dorsal horn and then give off collaterals which
and Ryall, 1969; de Groat, 1975; de Groat et al., extend ventromedially and ventrolaterally along
1993). de Groat and colleagues (de Groat et al., the superficial layers of the dorsal horn to the
1990, 1993; de Groat and Kruse, 1993) have dem- dorsal commissure and to the area of the sacral
onstrated that systemically administered capsaicin, parasympathetic nucleus (laminae V–VII), which
a C-fiber neurotoxin, blocked bladder hype- contains preganglionic parasympathetic neurons.
rreflexia in the chronic paraplegic cat but was These afferents do not extend into the center of the
without effect in cats with intact spinal cords. In dorsal horn or into the ventral horn. The most
the rat, both the spinal and supraspinal micturit- prominent pathway is located in lamina I on the
ion reflexes are activated by capsaicin-resistant Aq lateral edge of the dorsal horn in a region termed
afferents (Mallory et al., 1989); by contrast, caps- the lateral collateral pathway of Lissauer’s tract.
aicin-sensitive afferents do appear to modulate Afferents from the uterine cervix, and from the
micturition under certain conditions (Maggi, 1991, urethra and external urethral sphincter, also
1993). In rats, C-fiber bladder afferents are not project heavily into the lateral collateral pathway.
necessary for eliciting bladder reflexes after spinal In contrast, afferents from the clitoris or penis
cord injury but do contribute to the appearance of project almost exclusively to the dorsal commis-
non-voiding bladder contractions (i.e., increases in sure. Thus, it has been concluded that excretory
bladder pressure not associated with release of reflexes depend on spinal processing in the regions
urine) after spinal cord injury (Cheng et al., 1995). of the lateral collateral pathway and sacral
The mechanisms underlying the emergence of the parasympathetic nucleus, whereas sexual reflexes
C-fiber evoked reflex are unknown. However, recent are processed in the dorsal commissure.
experiments have begun to examine changes in the The neuroactive compounds in the afferent
electrical properties of afferent neurons innervating pathways from the urogenital tract have been
the urinary bladder of the adult rat before and after examined using histochemical techniques (Donovan
spinal cord injury (Yoshimura, 1999). These studies et al., 1983; de Groat et al., 1986; Keast and de
suggest an ionic mechanism underlying the relative Groat, 1992; Vizzard et al., 1993b, c, 1994b;
inexcitability of C-fiber bladder afferents in normal Vizzard and de Groat, 1996). Bladder afferents
animals and the increased excitability of these af- contain a variety of neuropeptides, including
ferents after spinal cord injury (Yoshimura, 1999). calcitonin gene-related peptide, substance P,
Changes in the electrophysiological properties of vasoactive intestinal polypeptide, cholecystokinin,
bladder afferent neurons after spinal cord injury and enkephalins (Donovan et al., 1983; de Groat
may occur concomitantly with the changes in ne- et al., 1986; Vizzard, 2000d, 2001). Multiple
urochemical properties reviewed here. neuropeptides are present in the same cells sug-
gesting that transmission at afferent terminals in
Neurochemistry and morphology of afferent and the spinal cord and in the target organs is likely to
spinal pathways to the urogenital tract be complex and involve multiple neurotransmitters.
Our recent studies (Zvarova et al., 2004, 2005)
Axonal tracing and immunocytochemical tech- have also demonstrated that bladder afferent cells
niques (de Groat et al., 1981, 1986; Donovan et al., express pituitary adenylate cyclase-activating
1983; Steers et al., 1991a; de Groat and Kruse, polypeptide (PACAP) and galanin and that this
101

expression is increased after chronic cyclophosph- 1991; Vizzard et al., 1994a). In both the rat and
amide-induced cystitis or spinal cord injury (see cat, NADPH-d is present in a prominent afferent
below). With the exception of calcitonin gene- bundle projecting from Lissauer’s tract to the
related peptide, all of these substances are predom- region of the parasympathetic nucleus (Vizzard
inantly expressed in small (presumably C-fiber) et al., 1993a, c, 1994a, 1995a). This afferent path-
afferents (Donovan et al., 1983; de Groat et al., way closely resembles the central projections of the
1986; Keast and de Groat, 1992; Vizzard et al., afferent neurons innervating the pelvic viscera
1993b, c, 1994b; Vizzard and de Groat, 1996). (Steers et al., 1991a). In the cat, the NADPH-d
afferent pathways closely resemble the vasoactive
Neurochemical plasticity in bladder afferent cells in intestinal polypeptide-containing afferent projec-
dorsal root ganglia after spinal cord injury tions to the sacral spinal cord (Basbaum and
Glazer, 1983; Honda et al., 1983; Kawatani et al.,
The neuroactive compounds in the afferent path- 1985).
ways from the lower urinary tract and those in the Although NADPH-d was present in the primary
central pathways (interneurons, preganglionic neu- afferent neurons (Aimi et al., 1991; Vizzard et al.,
rons) exhibit either excitatory or inhibitory ac- 1993b, 1994b) and in their central projections in
tions. Pathological conditions can alter the known the rat (Vizzard et al., 1993a, c) and the cat
balance of these neuroactive compounds either in (Vizzard et al., 1994a, c), nNOS-IR was not iden-
the periphery or in the central pathways, conceiv- tified (Vizzard et al., 1994c, 1995a). These data
ably shifting the balance to a hyper- or hypo-active indicate that in pelvic afferent neurons in normal
state. Urinary bladder hyperreflexia, after spinal rats, NADPH-d is not a marker for nNOS and
cord injury (upper motoneuron injury), may reflect that NO is not a transmitter, or that immunore-
this change in the balance of neuroactive com- activity for nNOS is not as sensitive as NADPH-d
pounds in bladder reflex pathways. histochemistry. A similar situation exists for so-
matic afferent neurons in the adjacent lumbar
Nitric oxide (L4–L5) dorsal root ganglia that contain relatively
large numbers of NADPH-d positive neurons
Neuronal nitric oxide synthase (Ruda et al., 1994) but few nNOS-IR neurons in
Previous histochemical and pharmacological stud- rats (Verge et al., 1992; Zhang et al., 1993). However,
ies have raised the possibility that nitric oxide (NO) following sciatic nerve injury (Verge et al., 1992;
is a transmitter in autonomic reflex pathways. Zhang et al., 1993), pelvic nerve injury (Vizzard
In the rat, nicotinamide adenine dinucleotide et al., 1995a), or chronic bladder irritation
phosphate diaphorase (NADPH-d) activity (a pre- (Vizzard and de Groat, 1996) the levels of nNOS
sumed indicator of the presence of neuronal nitric protein or nNOS mRNA are markedly increased
oxide synthase) (Dawson et al., 1991; Hope et al., in the lumbosacral dorsal root ganglia. Increased
1991) and neuronal nitric oxide synthase (nNOS) NADPH-d staining or nNOS-IR has also
immunoreactivity (IR) have been identified in been noted following axotomy in motoneurons
sympathetic (Valtshanoff et al., 1992) and para- (Wu et al., 1994) and in some parasympathetic
sympathetic preganglionic neurons (Vizzard et al., preganglionic neurons (Vizzard et al., 1993a, 1995a).
1993c, 1995a; Saito et al., 1994) in the spinal cord Thus, expression of nNOS is plastic and neurons
and in some parasympathetic postganglionic neu- that do not normally synthesize nNOS can synthe-
rons in the peripheral ganglia. In cats, NADPH-d size the protein after injury or chemical stimulation.
activity is present in sympathetic preganglionic The possibility that nNOS participates in the
neurons but not in parasympathetic preganglionic pathophysiology of spinal cord injury has recently
neurons (Vizzard et al., 1994c). NADPH-d activity been investigated (Guizar-Sahagun et al., 1996;
is also present in a large percentage of visceral Sharma et al., 1996). Changes in the expression of
afferent neurons in dorsal root ganglia at various nNOS or NADPH-d activity in the myenteric
levels of the spinal cord of the rat (Aimi et al., plexus or thoracic spinal cord after spinal cord
102

injury have been demonstrated (Guizar-Sahagun parasympathetic nucleus in the L6–S1 spinal seg-
et al., 1996). Acute (1 day) spinal cord injury re- ments were significantly increased. In the L6–S1
sulted in increased numbers of NADPH-d positive sacral parasympathetic nucleus of animals with
cell bodies in the myenteric plexus. However, no intact spinal cords, an average of 5.270.4 cell
increase in NADPH-d positive cell bodies in the profiles/section (L6) and 4.870.6 cell profiles/sec-
myenteric plexus was observed after chronic (10 tion (S1) were nNOS-IR, whereas in animals with
weeks) injury (Guizar-Sahagun et al., 1996). Focal spinal cord injury, the L6–S1 sacral parasympa-
trauma to the dorsal horn of the thoracic thetic nucleus had an average of 9.370.9 cell pro-
(T10–T11) spinal cord of the rat significantly in- files/section (L6) and 10.370.4 cell profiles/section
creased the numbers of nNOS-IR spinal neurons (S1) that were nNOS-IR (Vizzard, 1997). In con-
in the perifocal T9 and T12 segments of the spinal trast, no difference in the numbers of nNOS-IR
cord (Sharma et al., 1996). Topical application of cells in the region of the intermediolateral cell nu-
nNOS antiserum, 2 min after injury, prevented the cleus in the L1–L2 segments was detected after
upregulation of nNOS-IR (Sharma et al., 1996). spinal cord injury (8 cell profiles/section in L1
These studies have raised the possibility that: (1) and 6 cell profiles/section in L2) (Vizzard, 1997).
nNOS participates in the pathogenesis of second- Following complete spinal cord transection, the
ary spinal damage after spinal cord injury and (2) number of nNOS-IR neurons increased approxi-
changes in nNOS expression in the gastrointestinal mately 30–70-fold in L6 and S1 dorsal root ganglia
tract may be relevant to its reduced motility after and 2–4-fold in L1 and L2 dorsal root ganglia.
spinal cord injury. However, there was not a significant change in the
numbers of nNOS-IR cells in the L5 dorsal root
ganglia (Vizzard, 1997). After spinal cord injury,
nNOS expression in lower urinary tract pathways the increase in nNOS-IR in the L6 and S1 dorsal
after spinal cord transection at the 8th thoracic root ganglia was highly significant (pp0.001;
segment 12–19 nNOS-IR cell profiles/section) as was the
Following spinal cord injury, nNOS-IR fibers were increase in nNOS-IR in the L1 and L2 dorsal root
detected along the lateral edge of the dorsal horn ganglia (p p 0.001; 15–40 cell profiles/section)
extending from Lissauer’s tract to the region of the (Vizzard, 1997). Following spinal cord injury,
sacral parasympathetic nucleus in the lumbar (L6) nNOS-IR was not restricted to the small and the
and sacral (S1) spinal segments (Vizzard, 1997). medium sized dorsal root ganglia neurons as noted
These fibers were not present in the adjacent spinal in control animals. After spinal cord injury,
segments (L4, L5, or S2) nor were they present at nNOS-IR was present in the medium (25–30 mm)
the rostral lumbar (L1–L2) spinal levels before or and in the large (430 mm) sized dorsal root
after spinal cord injury. The nNOS-IR fiber stain- ganglion neurons although occasional small
ing in the lateral collateral pathway was not dorsal root ganglion cells still exhibited nNOS-
present in every transverse section suggesting that IR (Vizzard, 1997).
the nNOS-IR fibers may occur intermittently To determine if the increase in nNOS-IR in
along the rostral–caudal axis as noted for viscer- dorsal root ganglion neurons was occurring after
al afferent projections labeled with wheat germ spinal cord injury in the urinary bladder afferent
agglutinin horseradish peroxidase or horseradish neurons, fluorogold was injected into the urinary
peroxidase (Steers et al., 1991a). The general lo- bladder of spinal cord injured animals 5–7 days
cation of the nNOS-IR fibers in lamina I and their prior to euthanasia. In the L6 and S1 dorsal root
selective segmental distribution are very similar to ganglia of spinal cord injured animals, an average
the central projections of the visceral afferents in of 31.373.0 cell profiles/section and 17.571.4 cell
the pelvic nerve, designated the lateral collateral profiles/section, respectively, were fluorogold-labe-
pathway of Lissauer’s tract (de Groat et al., 1986). led after injection of dye into the bladder (Vizzard,
Following spinal cord injury, the numbers 1997). In these ganglia, an average of 41.277.8%
of nNOS-IR cells in the region of the sacral (L6) and 36.370.9% (S1), respectively, of
103

fluorogold-labeled bladder afferent neurons were this hyperreflexia at the spinal cord level. The
nNOS-IR (Vizzard, 1997). In contrast, no fluoro- present results indicate that NO may have addi-
gold-labeled bladder afferent neurons were nNOS- tional functions in bladder primary afferent path-
IR in spinal cord intact animals. At rostral lumbar ways following spinal cord injury. It is possible
levels, a higher percentage of bladder afferents that NO may play a role in the sensitization of the
normally express nNOS-IR in comparison to bladder afferents or in changes in the central
lumbosacral dorsal root ganglia (5%). Follow- processing of afferent input that could contribute
ing spinal cord injury, a significantly greater per- to the pathologically induced alterations in lower
centage of dye-labeled bladder afferents in the L1 urinary tract function (de Groat et al., 1993;
and L2 dorsal root ganglia exhibited nNOS–IR de Groat and Kruse, 1993).
compared to spinal cord intact animals and the
percentage of fluorogold-labeled cells that were
Pituitary adenylate cyclase activating
nNOS-IR (20–55%) was similar to the percent-
polypeptide (PACAP)
age of fluorogold nNOS-IR cells in the L6–S1
dorsal root ganglia (Vizzard, 1997).
PACAP belongs to the vasoactive intestinal
polypeptide/secretin/ glucagon family of bioactive
peptides, and was isolated from the hypothalamus
Role of nitric oxide in lower urinary tract pathways
based on its stimulation of anterior pituitary
after spinal cord injury
adenylyl cyclase activity (Arimura, 1998). Two
The function of the NO formed by the enhanced
p-amidated forms of PACAP arise from alterna-
expression of nNOS in bladder afferent cells and
tive post-translational processing; PACAP38 has
lumbosacral preganglionic neurons following
38 amino acid residues [proPACAP(131–168)],
spinal cord injury is uncertain. However, the role
while PACAP27 corresponds to the N-terminus
of NO in the lower urinary tract after spinal cord
of PACAP38 [proPACAP(131–157)]. PACAP27
injury may be similar to its suggested role follow-
exhibits 68% homology with vasoactive intestinal
ing chronic chemical irritation/inflammation of the
polypeptide (Kimura et al., 1990). The relative
urinary bladder (Kakizaki and de Groat, 1996;
levels of the two forms are tissue-specific, although
Vizzard and de Groat, 1996). Although NO does
PACAP38 predominates in most tissues (Arimura,
not appear to be involved in the normal micturit-
1998). The rat PACAP precursor protein consists
ion reflex in the rat (Rice, 1995; Kakizaki and
of 175 amino acid residues (Kimura et al., 1990;
de Groat, 1996; Vizzard and de Groat, 1996) NO
Arimura, 1998; Braas et al., 1998); PACAP38 is
does appear to play a role in the facilitation of the
identical among mammalian species, suggesting
micturition reflex by noxious chemical irritation of
similar physiologically important roles for this
the bladder (Rice, 1995; Kakizaki and de Groat,
peptide. These peptides are abundantly expressed
1996). Bladder hyperreflexia induced by either
and have diverse functions as regulators, signaling
acetic acid (0.1%) (Kakizaki and de Groat, 1996)
modulators, and trophic factors in the nervous and
or turpentine (Rice, 1995) was partially antago-
endocrine systems (Arimura, 1998).
nized by intrathecal spinal cord administration of
NOS inhibitors. This suggests that NO is involved
at the spinal level in the facilitation of the mi- PACAP expression in lower urinary tract pathways
cturition reflex by nociceptive bladder afferents. after spinal cord transection at the 8th thoracic
The spinal micturition reflex pathway can also segment
produce bladder hyperreflexia in paraplegic and In rats with an intact spinal cord, PACAP is
urethral obstructed animals. Thus, NO may also expressed in nerve fibers in the superficial laminae
be involved in the facilitation of the spinal of the dorsal horn and dorsal commissure in all
micturition reflex following spinal cord injury. thoracic, lumbar, and sacral segments examined.
Increased expression of nNOS in lumbosacral Some PACAP staining in the intact spinal cord
preganglionic neurons could also contribute to was unique to specific levels with PACAP staining
104

being present in the lateral horn in L1–L2, and (Zvarova et al., 2005). At various times after
L6–S1 spinal segments. spinal cord injury (48 h to 6 weeks), PACAP–IR
At 6 weeks after spinal cord injury, PACAP–IR was significantly (pp0.001) increased in the rostral
increased in several regions in the rostral lumbar lumbar (L1–L2) and lumbosacral (L6–S1) dorsal
L1–L2 spinal cord compared to that in rats with root ganglia (Zvarova et al., 2005). Both small
intact spinal cords. The density of PACAP–IR (16.873.5 mm) and medium (24.072.0 mm) sized
increased in the superficial laminae (I–II) of the dorsal root ganglion cells expressed PACAP–IR in
dorsal horn having a denser distribution through- animals with intact or injured spinal cords.
out the entire medial (3.0-fold increase) to the lat- PACAP–IR was occasionally observed in larger
eral (7.0-fold increase) extent of the laminae (X30 mm) sized dorsal root ganglion cells. No
(Zvarova et al., 2005). Increased (17.0-fold in- change in numbers of cells expressing PACAP–IR
crease) PACAP–IR fiber staining was also present, was observed in the L4–L5 dorsal root ganglia at
after spinal cord injury, in a small fiber bundle any time after spinal cord injury.
extending laterally from Lissauer’s tract (LT) in To determine if the increase in PACAP–IR in
lamina I into the dorsolateral funiculus (Zvarova the lumbosacral dorsal root ganglia neurons after
et al., 2005). No dramatic changes in PACAP–IR cord injury was occurring in bladder afferent cells,
were observed in the region of the intermediolat- Fast Blue (FB) was injected into the urinary blad-
eral nucleus following spinal cord injury. der to label bladder afferent cells retrogradely in
PACAP–IR was unchanged in the L4–L5 the L1, L2, L6, S1 dorsal root ganglia (Fig.
segments after spinal cord injury in every region 4A–4C) (Zvarova et al., 2005). In animals with
examined: dorsal horn, dorsal commissure, or intact spinal cords, approximately 45% of bladder
lateral horn regions. In contrast, significant afferent cells in the L6–S1 dorsal root ganglia ex-
changes in PACAP–IR were detected in the hibited PACAP–IR (Fig. 4D). A similar percent-
L6–S1 spinal cord after spinal cord injury (Fig. age (40%) of bladder afferent cells in rostral
3B,D). In the L6 spinal segment, PACAP–IR was lumbar dorsal root ganglia (L1–L2) of control an-
dramatically increased in the dorsal horn (1.4–7.4- imals also exhibited PACAP–IR. After spinal
fold increase), dorsal commissure (11.7-fold in- cord injury (6 weeks), the percentage of bladder
crease), sacral parasympathetic nucleus (15.0-fold afferent cells exhibiting PACAP–IR significantly
increase), and lateral collateral pathway (17.0-fold (p p 0.001) increased in the L6 (88.872.2%) and
increase) (Fig. 3E) (Zvarova et al., 2005). Changes S1 dorsal root ganglia (80.272.5%) and in the
in PACAP–IR in the S1 segment were comparable L1–L2 dorsal root ganglia (L1, 74.873.5%; L2,
to those in the L6 segment after spinal cord injury 69.573.2%) (Fig. 4D) (Zvarova et al., 2005).
(Fig. 3D) (Zvarova et al., 2005). In some trans- Increases in the percentage of bladder afferent cells
verse sections of the L6–S1 spinal cord, PACA- expressing PACAP–IR after spinal cord injury were
P–IR axons in the lateral collateral pathway observed at the earliest time point after spinal cord
terminated at the base of the dorsal horn (Fig. 3B) injury (48 h) and were maintained up to 6 weeks
whereas in others, they extended medially toward after spinal cord injury with little variation with
the central canal in distinct bundles through time after injury (Fig. 4D) (Zvarova et al., 2005).
laminae V–VII (Fig. 3D) (Zvarova et al., 2005).
In contrast to PACAP–IR in the spinal cord, PACAP neuronal functions in the lower urinary
PACAP–IR in the dorsal root ganglia (L1–S1) was tract
expressed by neuronal cell bodies and fibers PACAP have diverse functions in the endocrine,
throughout each dorsal root ganglion examined. nervous, gastrointestinal, and cardiovascular sys-
In control animals, PACAP–IR was present in tems (Braas and May, 1996; Arimura, 1998). High
small numbers of cells in the L1–S1 dorsal root levels of PACAP and vasoactive intestinal poly-
ganglia. The number of PACAP–IR cells among peptide expression have been identified in many
the dorsal root ganglia examined was comparable CNS neurons and in sensory and autonomic gan-
(range 20–24 PACAP–IR cell profiles/section) glia (Sundler et al., 1996; Brandenburg et al., 1997;
Fig. 3. Fluorescence photomicrographs showing PACAP–IR in the dorsolateral quadrant of L6 (A,B) and S1 (C,D) spinal segments in control animals (A, C) and after
spinal cord injury (SCI, 6 weeks, B, D). Increased density of PACAP–IR was observed in the medial to lateral extent of the superficial laminae (I–II) of the dorsal horn
(DH) following SCI (A vs. B). Increased PACAP–IR was present in a fiber bundle (B) extending from Lissauer’s tract in lamina I along the lateral edge of the DH to the
region of the sacral parasympathetic nucleus (SPN) (lateral collateral pathway of Lissauer, LCP). Although this fiber bundle was present in control tissue sections, the
staining was less intense (C) and was less frequently observed in transverse sections compared to that after SCI. Faint PACAP–IR was present in the region of the SPN in
control sections (A,C) and was increased after SCI (B,D). Some PACAP–IR fibers in the LCP appeared to terminate in the region of the SPN, whereas others projected
medially toward the central canal (D, arrows). CC, central canal; DCM, dorsal commissure. Calibration bar represents 125 mm. (E). Histogram summarizing changes in
PACAP staining density in specific regions of the L6 spinal cord segment after spinal cord injury (SCI, 6 weeks). The spinal cord inset depicts the areas analyzed: medial
dorsal horn (MDH), lateral dorsal horn (LDH), lateral collateral pathway of Lissauer (LCP), sacral parasympathetic nucleus (SPN), dorsal commissure (DCM), and
ventral horn (VH). The density of PACAP–IR was significantly increased in the LDH, MDH, SPN, DCM, and LCP of the L6 spinal cord segment. Similar changes were
observed in the S1 spinal cord segment. *pp0.001. Reprinted from Zvarova et al. (2005) copyright 2005 with permission from Elsevier.

105
106
Fig. 4. Effect of spinal cord injury on PACAP expression by dorsal root ganglion cells. PACAP–IR in the L6 dorsal root ganglion (DRG) after spinal cord injury (SCI)
(A, B, C). (A) Fast Blue (FB) labeled bladder afferent cells in a L6 DRG section after SCI. (B) Same L6 DRG section shown in (A) immunostained for PACAP–IR.
PACAP–IR was primarily located in small and medium sized DRG cells. Bladder afferent cells expressing PACAP–IR are indicated by white arrows (A, B). (C) Merged
image of panels (A, B) with FB cells pseudocolored blue and PACAP–IR cells pseudocolored red. FB cells (presumptive bladder afferents) expressing PACAP–IR appear
pinkish-purple (white arrows). Some PACAP–IR cells do not show FB (red cells, yellow arrows). (D) After SCI, a significantly greater percentage (85%) of FB-labeled
bladder afferent cells expressed PACAP–IR at all time points examined; however, not all bladder afferent cells expressed PACAP after SCI. In addition, not all
PACAP–IR in the L6 DRG is accounted for by bladder afferent cells (B,C, yellow arrows). Calibration bar represents 40 mm in (A, B, C). *pp0.001. Reprinted from
Zvarova et al. (2005) copyright 2005 with permission from Elsevier.
107

Arimura, 1998; Braas et al., 1998). In neurons, 2000d) and spinal cord injury (Zvarova et al.,
PACAP facilitates calcium ion flux, induces mem- 2005) may represent a principal component of
brane depolarization, increases spike frequency, bladder hyperreflexia, by increasing excitability of
activates potently adenylyl cyclase and phospholi- sensory neurons in the bladder reflex arc.
pase C signaling, and stimulates neurotransmitter
secretion (May and Braas, 1995; Braas and May,
Galanin
1996, 1999; May et al., 1998; Beaudet et al., 2000).
Several immunocytochemical studies using a spe-
Previous studies have demonstrated that galanin
cific monoclonal antibody against PACAP
has a potent neuromodulatory action on the isolat-
demonstrate widespread PACAP–IR in nerve
ed human detrusor muscle where galanin suppresses
fibers along the rat urinary tract including the
the cholinergic component of the response to elec-
bladder smooth muscle, suburothelial plexuses,
tric field stimulation (Maggi et al., 1987). Thus, an
and blood vessels (Fahrenkrug and Hannibal,
inhibitory action for galanin on neurotransmitter
1998). Neonatal capsaicin (C-fiber neurotoxin)
release has been suggested in smooth muscle tissues
treatment significantly reduced this distribution
and may also pertain to the urinary bladder (Maggi
in adults (Fahrenkrug and Hannibal, 1998) sug-
et al., 1987). Galanin-IR was expressed in identical
gesting that the majority of the fibers are derived
spinal cord regions in animals with intact or injured
from small sensory neurons. Dorsal root ganglia
spinal cords. However, the intensity and the overall
with high PACAP expression demonstrate dra-
distribution of the staining were increased in specific
matic neurochemical plasticity during altered
spinal cord segments and regions after cord injury
physiological states (Vizzard, 2000d; Zvarova
(Zvarova et al., 2004). Increases in galanin expres-
et al., 2005). Many studies have demonstrated
sion in bladder afferent cells in the dorsal root gan-
changes in PACAP expression in sensory neurons
glia may therefore act to oppose the actions of
following nerve injury (i.e., axotomy) (Zhang
PACAP and nNOS, whereas a decrease would re-
et al., 1996; Larsen et al., 1997). A limited number
inforce these actions in micturition reflex pathways
of studies have examined PACAP expression fol-
after spinal cord injury (Vizzard, 1997; Vizzard
lowing the induction of inflammatory states. Pre-
et al., 2003). Significant changes in galanin expres-
vious studies have suggested an association of
sion were found after spinal cord injury in specific
PACAP expression with inflammation in sensory
regions of the L1, L2, L4, and S1 spinal segments,
neurons following either somatic (hindpaw) or oc-
suggesting a modulatory role for galanin in spinal
ular inflammation (Wang et al., 1996; Zhang et al.,
micturition reflex pathways.
1998). Our laboratory was the first to demonstrate
an up-regulation of PACAP levels in bladder
afferent cells and spinal cord projections follow- Galanin expression in lower urinary tract pathways
ing cyclophosphamide-induced cystitis (Vizzard, after spinal cord transection at the 8th thoracic
2000d) or spinal cord injury (Zvarova et al., 2005). segment
Cyclophosphamide-induced cystitis is character- Galanin-IR was expressed in identical spinal cord
ized by an increased frequency of voiding in awake regions in animals with intact or injured spinal
rats and by urinary bladder overactivity in anest- cords. However, the intensity and the overall dis-
hetized rats (Lecci et al., 1994). Spinal cord injury tribution of the staining were increased in specific
rostral to the lumbosacral spinal cord results in spinal cord segments and regions after cord injury
bladder hyperreflexia and bladder–sphincter dys- (Zvarova et al., 2004). In the intact spinal cord,
synergia (Kruse et al., 1993; Vizzard, 1997, 2000a, galanin-IR was present in nerve fibers but not in
b). As studies have shown that PACAP facilitates neuronal cell bodies as in the injured spinal cord.
spontaneous bladder contractions in control ani- In the L1 spinal cord segments, the density of
mals (Ishizuka et al., 1995), the observed increase galanin-IR was significantly decreased (pp0.001)
in PACAP expression in bladder afferent cells and in the superficial laminae (I–II) (2-fold) of the
spinal cord projections during cystitis (Vizzard, dorsal horn and in the lateral collateral pathway
108

following spinal cord injury. In contrast, in the L2 Role of neurotrophic factors in neuronal
spinal segments the only changes in galanin-IR were plasticity and lower urinary tract dysfunction after
increases in the intermediolateral nucleus (Zvarova spinal cord injury
et al., 2004). In the L4–L6 segments galanin-IR was
increased in the dorsal commissure region of the L4 Neurotrophic factors
segment after cord injury. In the S1 spinal segment,
galanin-IR was increased in the dorsal commissure A potential mechanism underlying the neurochem-
(1.2-fold), lateral collateral pathway (1.4-fold), and ical changes (Vizzard and de Groat, 1996; Vizzard,
sacral parasympathetic nucleus (1.4-fold). 2000b, c, d) in bladder afferent neurons after spinal
In contrast to galanin-IR in the spinal cord, cord injury (described above) may involve ne-
galanin-IR in the dorsal root ganglia (L1–S1) was urotrophic factors expressed in the urinary bladder
expressed consistently by both neuronal cell bodies or spinal cord or changes in neural activity
and fibers (Fig. 5A1, B1). In control animals, (Vizzard, 2000a). The concept of trophic interac-
galanin-IR was present in modest numbers of cells tions between nerve cells and their targets is clearly
in the L1–S1 dorsal root ganglia (Fig. 5A1, B1, C) demonstrated during embryonic or postnatal
(Zvarova et al., 2004). The number of galanin-IR development (Oppenheim et al., 1991). Recent
cells among the dorsal root ganglia examined was experiments from several laboratories have
comparable (range 12–29 galanin-IR cell profiles/ demonstrated the influence of target organ–neuron
section). At 6 weeks after spinal cord injury, interactions in the adult animal (Steers and
galanin-IR was significantly (pp0.001) increased de Groat, 1988; Steers et al., 1991a, b; Tuttle and
in the rostral lumbar (L1) and sacral (S1) dorsal Steers, 1992; Tuttle et al., 1994; Vizzard, 2000a).
root ganglia (Fig. 5A). Both small (17.574.2 mm) A large number of studies have demonstrated
and medium (23.573.5 mm) sized dorsal root that pathological changes in a target organ after
ganglion cells expressed galanin-IR in control spinal cord injury can alter the neurochemical
animals following spinal cord injury. No change in (Vizzard, 1997; Yoshimura, 1999; Vizzard et al.,
numbers of cells expressing galanin-IR was 2003), electrical (Yoshimura, 1999), and organiza-
observed in the L2, L4–L6 dorsal root ganglia tional (Vizzard, 2000b) properties of micturition
following spinal cord injury (Fig. 5C) (Zvarova reflex pathways. A possible mechanism underlying
et al., 2004). We examined galanin-IR after acute these changes may involve neurotrophic factors or
spinal cord injury (o1 week) to determine if we neural activity arising in the bladder. Previous
had missed an earlier increase in galanin expres- experiments have demonstrated target organ to
sion that might have returned to control levels by 6 neuron interactions in the adult animal (Steers and
weeks after spinal cord injury. The number of de Groat, 1988; Steers et al., 1991a, b,1996; Tuttle
galanin-IR cells in the dorsal root ganglia after et al., 1994; Zvara et al., 2002). Furthermore,
acute spinal cord injury (o1 week) was not a recent study from this laboratory has demon-
different from control (Fig. 5C). strated changes in mRNA or protein expression
To determine if galanin-IR was expressed in of neurotrophic factors in the urinary bladder after
bladder afferent cells, F B dye was injected into the complete spinal cord injury, including nerve
urinary bladder to retrogradely label bladder growth factor, brain-derived neurotrophic factor,
afferent cells in the L1, L2, L6, S1 dorsal root glial-derived neurotrophic factor, neurotrophin-3
ganglia (Fig. 5A2, B2). In control animals, and -4 (Vizzard, 2000a). Both acute and chronic
approximately 1.5% of bladder afferent cells in spinal cord injury (4–6 weeks) resulted in signifi-
the L1, L2, L6, or S1 dorsal root ganglia exhibited cant increases in nerve growth factor, brain-derived
galanin-IR (Fig. 5B2, 5D) (Zvarova et al., 2004). neurotrophic factor, glial-derived neurotrophic
Following spinal cord injury (6 weeks), the factor, neurotrophin-3 and -4 transcript expres-
percentage of bladder afferent cells exhibiting sion as well as in increased nerve growth factor
galanin-IR significantly increased in the L1–L2, protein expression in urinary bladder (Vizzard,
L6, and S1 dorsal root ganglia (Fig. 5D). 2000a).
109

Fig. 5. (A, B) Bladder afferent cells in lumbosacral dorsal root ganglia (DRG) express galanin (Gal)-immunoreactivity (IR). (A1, B1).
Fluorescence photographs of Gal-IR cells (arrows and arrowheads) in the L1 DRG from control (A1) and spinal cord injured (SCI)
(B1) rats. Bladder afferent cells in the DRG were labeled by retrograde transport of Fast Blue (FB; A2, B2). Some bladder afferent cells
express Gal-IR before (A1, A2, arrowheads) and after SCI (B1, B2, arrowheads). Note that not all bladder afferent cells express Gal-
IR (B2, arrow) and not all Gal-IR cells are bladder afferent cells (A1, B1; arrows). Calibration bar represents 100 mm. (C) Histogram
depicting the number of Gal-IR DRG cells per section in DRG examined in control (spinal intact) and SCI rats 6 weeks or less than 1
week (o week) after SCI. No changes in the numbers of Gal-IR cells in DRG examined were observed less than 1 week after SCI. In
contrast, significant increases in the numbers of Gal-IR cells in the L1 and S1 DRG were observed 6 weeks after SCI. (D) Histogram
depicting the percentage of bladder afferent cells in the DRG expressing Gal-IR in control or SCI rats. Six weeks after SCI, the
percentage of bladder afferent cells expressing Gal-IR significantly increased in all DRG examined. *pp0.001. Reprinted from
Zvarova et al. (2004) copyright 2004 with permission.

It has also been reported that nerve growth fac- growth factor content in spinal segments immedi-
tor levels increase in the transected spinal cord ately rostral to the T8 transection site (Fig. 6A).
(Krenz and Weaver, 2000; Brown et al., 2004) after However, some of the spinal segments caudal to
spinal cord injury. Our work (Zvarova et al., 2004) the transection site (T9–T10; T13–L1; L6–S1)
also demonstrated a significant increase in nerve exhibit decreased nerve growth factor protein
110

Fig. 6. (A). Changes in total spinal cord nerve growth factor (NGF) as detected with a NGF ELISA (Enzyme Linked Immunosorbant
Assay) after spinal cord injury (SCI) (o 1 week or 6 weeks). The line drawing at the top represents the spinal cord and the vertical line
indicates the position of the spinal cord transection at (T8). A significant increase in total NGF in the T7–T8 spinal segments was
present 6 weeks after SCI. Significant decreases in total NGF in the T9–T10, T13–L1, and L6–S1 spinal segments were present after
acute or chronic SCI compared to control values. *pp0.001. (B) Changes in total spinal cord brain-derived neurotrophic factor
(BDNF) as detected with a BDNF ELISA after SCI o1 week or 6 weeks. The line drawing at the top represents the spinal cord and the
vertical line indicates the position of the spinal cord transection at (T8). A significant increase in total BDNF from the T7–T8, T9–T10,
T13–L1, and L6–S1 spinal segments were detected after acute (o1 week) or chronic (6 weeks) SCI. Significant increases in spinal cord
BDNF were also seen acutely after SCI in the T11–T12 and L4–L5 spinal segments but not after chronic SCI. *pp0.001. Reprinted
from Zvarova et al. (2004) copyright 2004 with permission.
111

content with acute or chronic spinal cord injury At sites of tissue injury, inflammation or target
(Fig. 6A). In contrast, brain-derived neurotrophic organ hypertrophy, cytokines and growth factors
factor protein content significantly increased in the are up-regulated and this can result in the up-
majority of spinal segments examined (Fig. 6B). regulation of nerve growth factor (Lewin and
No spinal segments exhibited a decrease in brain- Mendell, 1993; Woolf et al., 1997) (Fig. 7). Nerve
derived neurotrophic factor protein content after growth factor activates TrkA receptors on axon
spinal cord injury (Fig. 6B). Thus, bladder afferent terminals in the urinary bladder or spinal cord re-
neurons may have at least two potential sources of sulting in internalization and retrograde transport
increased brain-derived neurotrophic factor fol- of activated TrkA (Kuruvilla et al., 2004) to affer-
lowing spinal cord injury: (1) central terminals in ent cells in the dorsal root ganglia. Excess nerve
the spinal cord and (2) peripheral terminals in the growth factor within the dorsal root ganglia may
urinary bladder (Vizzard, 2000a). The results of induce increased production of neuropeptides
these studies may focus new attention on the po- (i.e., substance P, calcitonin gene-related peptide
tential role of brain-derived neurotrophic factor in and PACAP) in sensory neurons (Gary and
micturition reflex plasticity after spinal cord injury. Hargreaves, 1992; Woolf et al., 1997) (Fig. 7). An
A model of nerve growth factor-dependent increase in the levels of neuroactive compounds
sensory consequences of tissue damage and in- (e.g., enkephalin (Lewin and Mendell, 1993),
flammation in the somatic system has been pro- dynorphin (Ruda et al., 1988), calcitonin gene-
posed (Lewin and Mendell, 1993). This proposed related peptide (Gary and Hargreaves, 1992; Woolf
scheme has been modified for this review to dem- et al., 1997; Vizzard, 2001), substance P (Ruda
onstrate how an excess of nerve growth factor or et al., 1988; Gary and Hargreaves, 1992; Lewin and
another neurotrophic factor in the urinary bladder Mendell, 1993; Vizzard, 2001), neuropeptide Y
or spinal cord could alter lower urinary tract (Lewin and Mendell, 1993), nNOS (Vizzard et al.,
pathways after spinal cord injury, and is shown in 1995a; Vizzard and de Groat, 1996; Vizzard, 1997),
Fig. 7. In addition to peripheral afferent changes and PACAP (Jongsma et al., 2000; Vizzard, 2000d)
after spinal cord injury, it is clear that central reflex following noxious peripheral stimulation, cyclophos-
mechanisms are also changed. phamide-induced cystitis (Vizzard, 2000c, d, 2001;

Fig. 7. Proposed involvement of urinary bladder or spinal cord neurotrophic factors in plasticity of micturition reflexes after spinal
cord injury (SCI).
112

Vizzard and de Groat, 1996), or spinal cord injury mentorship and support. Gratitude is also ex-
(Vizzard and de Groat, 1996; Vizzard, 2000b) has pressed to Dr. Victor May and Dr. Karen Braas,
also been demonstrated in dorsal root ganglion cells University of Vermont, for encouraging me to
as well as in spinal cord neurons. Furthermore, in- explore the role of PACAP in lower urinary tract
travesical administration of exogenous nerve growth reflexes. Many former and current members of
factor in animals may facilitate afferent firing and my laboratory have contributed to the studies
induce bladder hyperreflexia that is blocked by anti- discussed in this review; including Dr. Li-ya Qiao,
nerve growth factor treatment (Dmitrieva Dr. Katarina Zvarova, Mr. Dana J. Dunleavy,
et al., 1997). Over-expression of nerve growth fac- Ms. Elaine Murray, and Ms. Susan Malley.
tor in bladder smooth muscle in spontaneously hy-
pertensive rats leads to bladder hyperinnervation References
and bladder overactivity (Clemow et al., 1998).
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lumbosacral spinal cord suppresses detrusor–sphinc- Localization of NADPH-diaphorase containing neurons in
ter dyssynergia in spinal cord-injured rats (Seki et sensory ganglia of the rat. J. Comp. Neurol., 306: 382–392.
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 8

Effect of injury severity on lower urinary tract


function after experimental spinal cord injury

Jean R. Wrathall and Gregory S. Emch

Department of Neuroscience, Georgetown University Medical Center, TRB EP04, Washington, DC 20057, USA

Abstract: Lower urinary tract dysfunction is a serious burden for patients following spinal cord injury.
Patients are usually limited to treatment with urinary drainage catheters, which can lead to repeated urinary
tract infections and lower quality of life. Most of the information previously obtained regarding lower
urinary tract function after spinal cord injury has been in completely transected animals. After thoracic
transection in the rat, plasticity of local lumbosacral spinal circuitry establishes a ‘‘reflex bladder,’’ which
results in partial recovery of micturition, albeit with reduced voiding efficiency. Since at least half of cord-
injured patients exhibit neurologically incomplete injury, rat models of clinically relevant incomplete con-
tusion injury have been developed. With respect to lower urinary tract function, recent anatomical and
physiological studies have been performed after incomplete thoracic contusion injury. The results show
greater recovery of lower urinary tract function that varies inversely with the severity of the initial trauma
and is positively correlated with time after injury. Recovery, as measured by coordination of the bladder
with the external urethral sphincter, occurs between 1 and 4 weeks after spinal cord injury. It is associated
with normalization of: serotonin immunoreactivity and glutamate receptor subunit mRNA expression in
the dorsolateral nucleus that innervates the external urethral sphincter muscle, the response to glut-
amatergic pharmacological probes administered at the lumbosacral spinal cord level, and c-Fos activation
patterns in the lumbar spinal cord. Understanding the mechanisms involved in this recovery will provide a
basis for enhancing lower urinary tract function in patients after incomplete spinal cord injury.

Introduction often with resistant organisms (Trautner and


Darouiche, 2002; Garcia Leoni and Esclarin De
Spinal cord injury results in lower urinary tract Ruz, 2003). Recently, there have been advances in
dysfunction that contributes to patients’ morbidity the treatment of other types of lower urinary tract
and mortality and profoundly limits their quality disorders, such as stress incontinence (Thor, 2003).
of life. Currently, there are few options to treat These have been based on understanding the ne-
lower urinary tract dysfunction after spinal cord uroanatomical pathways and transmitter systems
injury. Most patients are limited to the use of uri- involved in lower urinary tract function. With re-
nary drainage catheters transiently or permanent- spect to spinal cord injury, detailed studies of the
ly. The altered voiding dynamics, repeated use of changes that occur after complete spinal cord
catheters, and frequent exposure to antibiotic transection have been performed and are reviewed
agents predispose individuals with spinal cord in- elsewhere in this book. However, at least half of all
jury to recurrent episodes of urinary tract infection spinal cord injury patients have incomplete injuries
(Bracken et al., 1990), and much less is known
Corresponding author. Tel.: +202-687-1196; about the alterations in lower urinary tract func-
Fax: +202-687-0617; E-mail: wrathalj@georgetown.edu tion after incomplete spinal cord injury. Recent

DOI: 10.1016/S0079-6123(05)52008-9 117


118

studies from our laboratory, reviewed below, dem-


onstrate that the effect of incomplete spinal cord Pontine micturition
injury on lower urinary tract function depends centers
upon the severity of the injury. Under certain con-
ditions considerable recovery of lower urinary
tract function can occur (Pikov and Wrathall,
2001). Understanding the mechanisms involved in
this recovery may provide a basis for new thera-
peutic approaches to enhance lower urinary tract
function after clinical spinal cord injury. CRF GLU

Bladder
Normal Control of lower urinary tract function and detrusor SPN
effect of spinal cord transection 5-HT GLU

EUS
The bladder and external urethral sphincter com- DL
prise a coordinated visceral system that has been
well studied both in normal and spinal transected Fig. 1. Spinal cord and brainstem control of micturition in the
animals (Tiseo and Yaksh, 1990; de Groat, 1995; rat. Neurons innervating the bladder detrusor reside in the
sacral parasympathetic nucleus (SPN) at spinal segments
Morrison, 1997; de Groat et al., 1998). Urine stor-
L6–S1, while those innervating the external urethral sphincter
age and voiding are the two main functions of the (EUS) muscle are part of the dorsolateral nucleus (DL) in the
lower urinary tract. Urine storage and release from spinal cord at L6/S1. Afferents from the bladder convey pres-
the bladder depend on sympathetic and parasym- sure information to the pontine micturition centers, which
pathetic innervation, respectively. The pre-gang- stimulate the sacral parasympathetic nucleus to cause bladder
contraction and the dorsolateral nucleus to activate, intermit-
lionic sympathetic neurons are located in the rostral
tently, the external urethral sphincter in a coordinated fashion.
segments of the lumbar spinal cord and the pre- Both afferent and efferent pathways are glutamatergic (GLU).
ganglionic parasympathetic neurons in the rostral Serotonin (5-HT) and corticotrophin releasing hormone (CRF)
portion of the sacral spinal cord (de Groat, 1995). are also involved in descending motor control pathways.
The urethral sphincter is responsible for outlet re-
sistance during storage. Micturition in rats is pro- tegmentum of the pons and the periaqueductal
duced by coordinated contraction of the smooth gray are considered to be important in triggering
muscle of the urinary bladder and contraction of the the initiation of voiding (Kruse et al., 1990, 1991).
striated muscle of the external urethral sphincter The external urethral sphincter-controlling motor
intermittently, in 4–6 Hz frequency bursts, which neurons, located in the dorsolateral nucleus of
appears to facilitate voiding (Mersdorf et al., 1993). L6-S1 (Schroder, 1980), receive direct and indirect
If external urethral sphincter activity is prevented by (via spinal interneurons) supraspinal projections
neuromuscular blockade, efficient voiding is abol- mostly from the pontine micturition center
ished (Maggi et al., 1986a; Mersdorf et al., 1993; (Vizzard et al., 1995; Nadelhaft and Vera, 1996;
Kakizaki et al., 1997). As shown in Fig. 1 effective Marson, 1997), the D-region just ventral to
voiding, therefore, requires interaction of spinal au- Barrington’s nucleus (Ding et al., 1995), and the
tonomic reflexes with supraspinal micturition-con- ventrolateral pontine periaqueductal gray (Marson,
trolling centers to coordinate urine expulsion from 1997; Ding et al., 1998; Matsuura et al., 1998).
the bladder with low-frequency intermittent con- Other brainstem nuclei with identified connec-
tractions of the somatically innervated external ure- tions to bladder and external urethral sphincter
thral sphincter muscle (Holstege et al., 1986; pathways are the raphe magnus, raphe pallidus,
Holstege and Tan, 1987; Kakizaki et al., 1997). parapyramidal medullary reticular formation,
Among the brainstem centers involved in the subcoeruleus pars alpha, locus coeruleus, and the
control of micturition, the pontine micturition A5 and A7 nuclei (Vizzard et al., 1995; Marson,
center (Barrington’s nucleus) in the dorsolateral 1997). Of these, cells in the raphe nuclei and
119

nucleus paragigantocellularis in the medullary re- ‘‘complete’’ may have some residual connections
ticular formation produce serotonin (5-hydroxy- with supraspinal control centers. For example,
tryptamine, 5-HT) (Marson, 1997). Thus, 5-HT is with respect to lower urinary tract function, 15%
a marker of direct supraspinal projections to the of people classified as complete can consciously
dorsolateral nucleus (Ramirez-Leon et al., 1994; detect bladder filling and/or electrical stimulation
Tang et al., 1998). Because of the importance of indicating the persistence of afferent connections
these descending control pathways, a lesion of the with the cerebral cortex (Wyndaele, 1991). Because
spinal cord above the lumbar level would be ex- of the importance of incomplete spinal cord injury,
pected to leave spinal micturition reflexes intact rat models of clinically relevant incomplete con-
and compromise lower urinary tract function by tusion injury have been developed and character-
affecting bladder–external urethral sphincter sy- ized (e.g., Wrathall et al., 1985; Gruner, 1992). In
nergy of activation (Chancellor et al., 1990). these models, spinal cord injury is produced by the
Previous studies of complete spinal cord transec- impact of a weight onto the exposed dura after a
tion in the cat (de Groat, 1990), and in the rat laminectomy, usually at a mid-thoracic location.
(Kruse et al., 1993; Mimata et al., 1993), have The impact produces mechanical destruction of
shown that lower urinary tract function varies with tissue and hemorrhage that is maximal in the cen-
time after injury. Initially, during the phase of spinal tral gray matter and the white matter just above it
shock, the bladder is areflexic and urinary retention in the dorsal funiculus (Noble and Wrathall,
occurs (Hassouna et al., 1984). During this stage, 1989). However, a peripheral rim of white matter
the bladder becomes tonically overdistended and is spared, the thickness of which depends on the
noncompliant. Then the spinal reflex activity reap- severity of the impact. A 10 g weight impacting
pears (Tiseo and Yaksh, 1990), in an exaggerated onto the exposed dura at thoracic level T8 will
(spastic) mode with hyperreflexic bladder detrusor spare nearly a complete peripheral rim if dropped
muscle contractions (Osborn et al., 1990). This is from 12.5 or 25 mm, but only residual white matter
believed to be due to a lack of supraspinal inhibition in the most ventrolateral region remains after a
and/or an increase of afferent signaling (Cheng 50 mm impact, as shown in Fig. 2. However, the
et al., 1995) as well as plasticity of afferents (Kruse spared white matter is far from normal, as there is
et al., 1995) resulting from the enlarged bladder. a preferential loss of the larger axons from even
The external urethral sphincter, which in uninjured the most peripheral regions and the myelination
animals is under supraspinal control and works in and glial microenvironment remain abnormal
synergy with the detrusor muscle, becomes contin- chronically for at least 2 months after spinal cord
uously active and therefore dyssynergic with the injury (Wrathall et al., 1998; Rosenberg et al.,
emerging automatic bladder contraction reflex 2005). Thus, there is complete loss of long de-
(Schalow et al., 1995). Thus, although spinal cir- scending and ascending axons in some regions of
cuits alone are capable of establishing automatic the spinal cord at the injury epicenter and partial
bladder control after transection (de Groat et al., loss in other regions of white matter.
1998), detrusor–external urethral sphincter coordi- There is now considerable data documenting re-
nation that is mediated via a spino-bulbo-spinal re- covery of hind limb sensory and motor function
flex (Holstege et al., 1986; de Groat, 1990) does not after experimental contusion of the thoracic spinal
recover after complete spinal cord transection. cord (Wrathall, 1994). The extent of recovery is
inversely related to the severity of the initial trauma
Incomplete spinal cord injury and lower urinary and, for the most part, positively correlated with
tract function time after injury. The recovery phase following in-
itial hind limb areflexia and complete paralysis is
Large-scale clinical trials demonstrate that at least characterized by the return of segmental reflexes in
half of cord-injured people have neurologically a modified state and increasingly effective use of the
incomplete injury (Bracken et al., 1990). Further, hind limbs in coordinated movements that are
a significant proportion of those classified as known to mediate postural control, swimming
120

Fig. 2. White matter sparing at the spinal cord injury epicenter. Top, bottom left: Photomicrographs of representative sections through
the lesion epicenter from one rat of each of the three T8 spinal cord injury (SCI) groups and from an uninjured control stained with
eriochrome cyanine to label myelin. The dorsal, lateral, and ventral funicular white matter of the normal spinal cord is heavily stained,
whereas little myelin staining is seen in the gray matter. The cross-sectional profiles of the injured spinal cords are reduced in diameter.
The center of the injured cords contains cavities and an abnormal loose network of cells, but no myelin staining is apparent.
A peripheral rim of residual white matter is seen. Myelin staining is present but reduced compared with normal white matter, consistent
with the chronic hypo-myelination of residual axons. Bottom right: The average areas of myelinated white matter from the ventral and
lateral funicular zones at the lesion epicenter in the injury groups. SCI height indicates height from which weight is dropped, i.e.,
severity of injury. * Indicates a significant difference from the 12.5 mm weight drop group, based on p.o.0.05 in Tukey’s post hoc test
after ANOVA. Scale bar, 250 mm. Taken from Pikov and Wrathall (2001) with permission.

movements and elements of locomotion. This re- The difference in function between these spinal
covery phase plateaus at 3–4 weeks in the adult rat cord-injured animals at a few days after injury and
with the standard tests revealing no significant ad- 4 weeks later is remarkable — from almost com-
ditional recovery between 4 and 8 weeks (Noble plete paralysis to quite effective, albeit still abnor-
and Wrathall, 1989) or even by 6 months (J.R. mal, use of their hind limbs. Thus, considerable
Wrathall, unpublished). With respect to open field natural recovery of hind limb sensory-motor func-
locomotion after an incomplete contusion, rats tion occurs in rats that retain only 10–20% of spi-
show a stereotypical pattern of recovery of loco- nal cord tissue at the injury epicenter.
motion consisting of early, intermediate, and late These spinal cord-injured rats also demonstrate
stages (Basso et al., 1995). During the first stage abnormalities of lower urinary tract function and
there is increasing joint movement in the hind are initially unable to urinate. A ‘‘reflex bladder’’
limbs. In the second, rats become capable of plan- develops with time as seen after spinal cord tran-
tar stepping and bearing weight on their hind limbs. section. However, depending on the severity of the
In the last phase of recovery, exhibited by the least lesion, coordinated function of the bladder and the
severely injured rats, there is consistent coordinated external urethral sphincter may also recover after
weight-bearing locomotion with increasingly nor- incomplete spinal cord injury (Pikov et al., 1998;
mal positioning of the paws, the trunk, and the tail. Pikov, 2000; Pikov and Wrathall, 2001, 2002).
121

Evaluating recovery of lower urinary tract function


after experimental contusion spinal cord injury

Our initial interest in recovery of lower urinary


tract function stemmed from the observation that
the length of time required for manual expression
of the bladder after contusion injury appeared to
be reduced with acute treatments that enhanced
white matter sparing and recovery of hind limb
function. Groups of rats treated with the gluta-
mate receptor antagonist NBQX demonstrated a
dose-related sparing of white matter at the injury
epicenter and a dose-related decrease in the
number of days required for them to acquire a
reflex bladder (Wrathall et al., 1994). Similarly,
when a sodium channel blocker was used to reduce
axonal loss, the number of days required to attain Fig. 3. Time course of recovery of spontaneous voiding. The
a reflex bladder after spinal cord injury was re- bars on the curve show the urine volumes (71 standard error of
duced (Teng and Wrathall, 1997). the mean) manually expressed every 12 h from the urinary
bladder, plotted against time after spinal cord injury (SCI). The
Evaluating the volume of urine expressed over
volume collected increased between days 0 and 4, presumably
time after three different severities of thoracic con- due to an increase in urine production during the immediate
tusion injury revealed an initial increase in expressed post-operative period. The expressed volume then decreased
volume followed by a decrease, as the spinal bladder after day 4, presumably due to recovery of spontaneous void-
reflex was established (Pikov and Wrathall, 2001). ing. After day 4, progressively less urine was expressed from the
bladders of the rats with the (milder) 12.5 mm weight drop cord
However, more severe spinal cord injury, associated
injury, indicating a faster recovery of spontaneous voiding in
with greater loss of white matter at the injury site, as this group than in the other two more severely injured groups.
shown in Fig. 2, was reflected in a slower establish- Taken from Pikov and Wrathall (2001) with permission.
ment of spontaneous reflex voiding (Fig. 3).
To study recovery of lower urinary tract func- In initial studies average values of external ure-
tion further, we used a urodynamic procedure that thral sphincter spiking activity during bladder fill-
allows a rapid collection of data over a large ing and voiding were calculated, and threshold (at
number of voiding cycles (Maggi et al., 1986b). As the initiation of contraction) and maximal in-
shown in Fig. 4, bladder intravesical pressure was travesical pressures during voiding were measured
recorded with a transurethral bladder catheter for each voiding cycle over a 20-min period in each
(polyethylene-50) during continuous perfusion of the animals (Pikov et al., 1998). The most useful
with warm saline (0.22 ml/min). During the blad- measure was found to be the change in external
der detrusor contractions, fluid was released by urethral sphincter spiking activity calculated from
flowing around the catheter in the urethra. The the raw electromyography data. This was obtained
signal from the pressure transducer was amplified, by counting the number of peaks above the base-
sampled at 1 kHz and acquired on-line using Bio- line at 100 ms intervals with a custom-written peak
Bench 1.0 software (National Instruments, Austin, detection macro in Microsoft Excel (Pikov and
TX). For electromyography, two fine (50 mm) Wrathall, 2001). The change in external urethral
epoxy-coated platinum–iridium wire electrodes sphincter spiking activity was measured during
were placed percutaneously in the sphincter area bladder filling and emptying as illustrated in
of the urethra to record external urethral sphincter Fig. 5. Although the catheter and electrodes were
electrical activity. The electromyographic activity inserted under anesthesia, the rats were then al-
was pre-amplified, sampled at 1 kHz, and acquired lowed to recover so that the urodynamic record-
on-line simultaneously with intravesical pressure. ings were done on awake restrained animals
122

Fig. 4. Experimental design for urodynamic recordings. A transurethral bladder catheter (polyethylene-50) is implanted in an anest-
hetized rat. After recovery from anesthesia, urodynamic recordings are performed under light anesthesia. The catheter is connected to
an infusion pump and a pressure transducer. Bladder intravesical pressure is recorded during continuous perfusion with room
temperature saline (CMG, cystometrogram). During bladder contractions, fluid is released by flowing around the catheter in the
urethra. For electromyography (EMG), two fine platinum wire electrodes are placed percutaneously in the sphincter area of the urethra
to record external urethral sphincter (EUS) electrical activity. The EMG activity is amplified, sampled at 1 kHz, and acquired on-line
simultaneously with intravesical pressure. Taken from Pikov et al. (1998) with permission.

because anesthesia markedly reduces the efficiency In order to investigate anatomical evidence of
of voiding (Yoshiyama et al., 1994, 1999). supraspinal involvement in lower urinary tract func-
Comparing urodynamic measures of normal rats tion, pseudorabies virus was injected into the blad-
to those after thoracic contusion or complete tran- der wall in normal and spinal cord-injured animals.
section injury, we found that rats after both tran- The rats were allowed to survive long enough for
section and contusion spinal cord injury showed transneuronal tracing to the brainstem. As shown in
evidence of reflexive bladder contractions in week 2 Fig. 6, labeling was present in the pontine micturit-
after injury, but only the contused groups demon- ion center and in the periaqueductal gray. As might
strated some recovery of coincidental activation of be expected, there was much lower labeling in the
the external urethral sphincter (Pikov et al., 1998). contused cord-injured animal as compared to unin-
Bladder weight was measured, showing a six-fold jured control, illustrating an anatomical basis for
increase at week 1, an eight-fold increase at week 2 the reduced supraspinal control of lower urinary
after incomplete contusion, and an 11.6-fold in- tract function in rats after injury.
crease in cord-transected animals. Voided volume,
or the amount that is released from the bladder in
each contraction, was found to decrease in both Effect of injury severity on chronic lower urinary
contused and transected animals. Voiding efficiency tract function after incomplete spinal cord injury
(volume voided/capacity  100), due to increase in
bladder capacity and decrease in voided volume was More information was obtained from an injury
very low (2.8–3.5%) in both groups of cord-injured dose–response study in which groups of rats were
animals as compared to uninjured controls (58%). subjected to spinal cord injury with the widely
The inter-contraction interval (time between de- used Multicenter Animal Spinal Cord Injury Study
trusor contractions) was also lower in the spinal cord (MASCIS) injury device (Gruner, 1992) and pro-
injury groups (40–70 vs. 115 s in control animals). duced by the impact of a 10 g weight dropped from
Fig. 5. Urodynamic analysis of detrusor–external urethral sphincter coordination in representative uninjured animals (A, D, G and J), and animals with 12.5 mm weight
drop (B, E, H and K) and 50 mm weight drop (C, F, I and L) spinal cord injury (SCI) at 8 weeks. (A–C) Bladder intravesical pressure (IVP) recordings during one voiding
cycle. Solid horizontal line in A and B indicates the duration of stream-like voiding, and the dashed line in C indicates the drop-by-drop voiding. (D–F) external urethral
sphincter EMG recordings, showing activation of external urethral sphincter EMG in relation to the voiding cycle in uninjured and 12.5 mm SCI animals but not in the
50 mm SCI animal. (G–I) Power spectrum analysis of external urethral sphincter EMG activity as a function of time. A broad band of frequencies (5–40 Hz) shows an
increased power during the voiding phase in uninjured and 12.5 mm cord-injured animals but not in the 50 mm cord-injured animal. (J–L) Peak detection analysis of the
external urethral sphincter spiking activity (ESA). Peaks were detected in 1 ds (ds ¼ 101 s) intervals. An increase in spiking activity occurred at the time corresponding to
the voiding phase in uninjured and 12.5 mm cord-injured animals, but there was no change in the level of spiking activity in the 50 mm cord-injured animal. Taken from
Pikov and Wrathall (2001) with permission.

123
124

Fig. 6. Pseudorabies virus tracing from the bladder. (A and B) Micrographs of Pseudorabies virus-labeled neurons in the pontine
micturition center of a normal rat (A) and a rat on day 12 after a 10 g  25 mm weight drop contusion (B). The large neurons of the
mesencephalic nucleus of the fifth cranial nerve on the left of each field demonstrate some nonspecific staining. Bar ¼ 100 mm. (C–F)
Schematized images of Pseudorabies virus retrograde transneuronal labeling from the bladder. Dorsolateral tegmentum (C and D) and
periaqueductal gray (E and F) in uninjured (C and E) and SCI animals (D and F). Abbreviations: Mes5-mesencephalic trigeminal
nucleus, PAG-periaqueductal gray, SCP-superior cerebellar peduncle. Taken from Pikov et al. (1998) with permission.

a height of 12.5, 25 or 50 mm onto the dura after a bladder pressure during urodynamic evaluation. In
laminectomy at T8 (Pikov and Wrathall, 2001). contrast the 12.5 mm group, although showing
Table 1 shows a comparison of bladder weights, similar tendencies, was not significantly different
volumes and pressure (intravesical pressure) at 8 from controls in these parameters of lower urinary
weeks after injury. Compared to uninjured con- tract function.
trols, the 25 and 50 mm groups demonstrated sig- As shown in Fig. 7A, the 12.5 mm spinal cord-
nificantly greater bladder weight and volume injured group recovered bladder–external urethral
chronically. The most severely injured (50 mm) sphincter coordination (as measured by the change
group also had significantly decreased maximal in external urethral sphincter spiking activity) by 8
125

Table 1. Changes in lower urinary tract parameters 8 weeks after spinal cord injury

Parameter Spinal cord injury severity (weight drop in mm)

0 (n ¼ 6) 12.5 (n ¼ 7) 25 (n ¼ 4) 50 (n ¼ 7)

Bladder weight (g) 0.1170.01 0.2370.09 0.37*70.13 0.41*70.18


Bladder volume (ml) 3.871.5 9.176.0 14.9*76.2 26.1**78.1
Intravesical pressure amplitude (mmHg) 25.477.7 19.477.6 14.173.0 13.0*75.1

The number of animals in each group is shown within parentheses. Urodynamic evaluation was performed at 8 weeks after SCI or laminectomy. The
bladder was then weighed after blot-drying, and its length and width were measured to calculate volume. The amplitude of the bladder pressure was
calculated as the difference between the maximal intravesicular pressure during voiding and the pressure just before voiding was initiated. Mean
values7standard error are presented for each measurement. *po0.05, indicates significantly different from values in control rats (0 weight drop). **,
significantly different from both the control and 12.5 group. Data from Pikov and Wrathall (2001).

Fig. 7. Recovery of detrusor–external urethral sphincter coordination at 8 weeks after spinal cord injury (SCI) depends upon injury
severity (A), and is correlated to white matter sparing at the epicenter (B), to the amount of serotonin immunoreactivity (5HT-IR) in
the dorsolateral nucleus (C), and to CRF immunoreactivity (CRF-IR) in the sacral parasympathetic nucleus (SPN; D). In panel A, SCI
height indicates distance of weight drop, i.e., severity of spinal cord injury. Bars represent means and standard errors. Vertical axis is
change in external urethral sphincter spiking activity (sESA) during voiding. N ¼ 6, 7, 5 and 7 for the 0, 12.5, 25, and 50 mm groups,
respectively (ds ¼ 101 s). Correlation coefficients are shown at the top left corner of B–D. In A, symbols indicate a significant
difference from the uninjured group (*) or from both the uninjured and 12.5 mm groups (**). (po0.001). Taken from Pikov and
Wrathall (2001) with permission. Data from Pikov and Wrathall (2001).

weeks to an extent statistically indistinguishable well as chronic white matter sparing at the injury
from uninjured controls, whereas the 25 and 50 mm epicenter (Fig. 7B). Correlation with spared de-
spinal cord-injured groups did not. Furthermore, scending control pathways from the brainstem was
at 8 weeks there was a significant correlation be- indicated by quantification of the relative immuno-
tween the degree of recovery of lower urinary tract reactivity for 5-HT associated with the dorsolateral
function in terms of bladder–external urethral nucleus motor neurons that innervate the external
sphincter coordination and the initial impact as urethral sphincter (Fig. 7C), and corticotrophin
126

Table 2. Expression of NMDA (NR1, NR2A, NR2B) and AMPA (GluR1, GluR2, GluR3, GluR4) subunit mRNA in dorsolateral
nucleus motoneurons at 8 weeks after spinal cord injury

Subunit Spinal cord injury severity (weight drop in mm)

0 ðn ¼ 6Þ 12.5 ðn ¼ 7Þ 25 ðn ¼ 3Þ 50 ðn ¼ 5Þ

NR1 39.478.7 43.776.0 43.176.4 45.175.3


NR2A 6.871.5 9.972.7 12.4*74.1 12.3*72.9
NR2B 1.670.4 2.370.5 2.270.5 2.270.2
GluR1 1.370.2 1.670.5 1.670.4 2.070.6
GluR2 4.671.1 6.372.2 10.5*71.6 8.2*72.6
GluR3 3.272.1 5.871.3 4.971.6 5.972.6
GluR4 2.470.6 2.970.6 2.070.3 2.370.2

The data are presented as the number of grains per square micrometer of cell area (mean 7 standard error). Significant difference from the uninjured
group is indicated by asterisk (*) and bold font and is based on po0.05 in Tukey’s post hoc test after ANOVA.

releasing factor immunoreactivity associated with c-Fos, which is expressed in neurons after intense
the sacral parasympathetic nucleus that innervates or prolonged activation (Rinaman et al., 1993), in
the bladder detrusor muscle (Fig. 7D). response to bladder filling in normal and spinal
Glutamate receptors are utilized in spinal circuits cord-injured rats at 8 weeks after injury (Emch
controlling the detrusor and external urethral et al., 2003). Rats that had not recovered normal
sphincter (Matsumoto et al., 1995a, b; Iwabuchi, levels of external urethral sphincter spiking activity
1997), and thus changes in properties of these re- (25 mm injury group) demonstrated extensive
ceptors may be involved in altered lower urinary c-Fos activation (Fig. 8), as previously reported
tract function after spinal cord injury. Comparing for rats after complete transection (Vizzard, 2000).
groups of rats that did or did not recover blad- In contrast, the mild (12.5 mm) group that recov-
der–external urethral sphincter coordination at 8 ered bladder–external urethral sphincter coordina-
weeks after spinal cord injury showed significant tion had a c-Fos activation pattern at 8 weeks that
differences in the expression of mRNAs for gluta- was similar to uninjured controls. Thus, aspects of
mate receptors in the dorsolateral nucleus motoneu- recovered lower urinary tract function after this
rons that innervate the external urethral sphincter severity of spinal cord injury include normal blad-
(Table 2). The 25 and 50 mm groups that did not der–external urethral sphincter coordination, nor-
recover normal external urethral sphincter spiking mal expression of glutamate receptor mRNAs in
activity also exhibited abnormally high expression of dorsolateral nucleus neurons and a normal local
NR2A and GluR2, as determined by in situ hybrid- segmental response to bladder stimulation as in-
ization autoradiography. Assuming these changes in dicated by c-Fos expression.
chronic mRNA levels produce altered functional
glutamate receptors, these alterations may be related
to the aberrant hyperactivity of these motoneurons What occurs during recovery of bladder–external
as in the spontaneous spastic activity seen chroni- urethral sphincter coordination after mild
cally in lumbosacral somatic motoneurons after contusion injury?
spinal cord injury (Hiersemenzel et al., 2000; Little
et al., 2000). In contrast the 12.5 mm group that did In order to study the mechanisms that may be in-
recover normal external urethral sphincter spiking volved in this recovery of lower urinary tract func-
activity expressed normal levels of the receptor sub- tion we compared animals after mild spinal cord
unit mRNAs in the dorsolateral nucleus motoneu- injury at 5 days after injury (non-recovered; sub-
rons that innervate the external urethral sphincter. acute), when the bladder reflex can be detected
Recently, we investigated, by immunohistoche- without any coordinated activation of the external
mical identification, the proto-oncogene product urethral sphincter, to animals at 8 weeks (recovered;
127

A B

L6 spinal cord
55 +
50
Fos count/10 um section

45 +
40
35
30
25 +
*
20
15
10
5
0
C SPN DCM MDH LDH 12.5mm 8wk 25mm 8wk

Fig. 8. After spinal cord injury c-Fos immunoreactivity in the L6 spinal cord segment is altered in animals with abnormal lower
urinary tract function and returns to normal when animals recover detrusor–external urethral sphincter coordination. The bladders of
lightly anesthetized rats were catheterized and room temperature saline was perfused continuously for 2 h to stimulate the voiding
reflex. Spinal cords were sectioned and c-Fos immunohistochemistry was performed with methods adapted from Emch et al. (2001). In
animals with abnormal detrusor–external urethral sphincter coordination as measured in our EMG preparation, the pattern of c-Fos
expression was altered in 4 areas of the L6 spinal cord: the sacral parasympathetic nucleus (SPN), the dorsal gray commissure (DCM),
the medial dorsal horn (MDH), and the lateral dorsal horn (LDH). Increases in c-Fos expression normalized by 8 weeks after injury in
animals that recovered lower urinary tract function, i.e. the mild injury group (10 g  12.5 mm weight drop). The pattern of c-Fos
expression in animals that did not recover lower urinary tract function, i.e. the moderate injury group (10 g  25 mm weight drop), did
not normalize. (A and B) Sections of the L6 DCM tissue stained for c-Fos-IR in a control rat (A) and in a rat 8 weeks after the injury
produced by a 10 g weight dropped 25 mm (B). Note the large number of immunoreactive cells in B. (C) Histograms of c-Fos counts in
the four L6 areas listed above. Open bars indicate controls. Gray and black bars show counts in the same regions in rats injured with
the 12.5 and 50 mm weight drop, respectively. +, significantly different from both control and the 12.5 mm injury group. *Significantly
different from control only.

chronic), when the external urethral sphincter about 30% at the earlier time point (subacute) with
spiking activity is indistinguishable from uninjured some recovery by 8 weeks (chronic), when the
controls (Fig. 9). Bladder weight at both subacute change in intravesical pressure was not significantly
and chronic time points was higher than in unin- different from that in uninjured animals. The in-
jured animals, and there was no difference in blad- crease and decrease in intravesical pressure during
der weight between these two time points. The detrusor contraction and relaxation, respectively,
change in bladder pressure during contraction occurred more slowly in injured than in uninjured
(change in intravesical pressure) was decreased by animals. The external urethral sphincter spiking
128

To test the hypothesis that altered glutamate


receptor function is involved with altered lower
urinary tract function after spinal cord injury, the
intravesical pressure and external urethral sphinc-
ter spiking activity were measured during urody-
namic evaluation in the presence of the NMDA
receptor antagonist 3(2-carboxypiperazin-4-yl)-
propyl-1-phosphonic acid (CPP) and the (R,S)-2-
amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic
acid (AMPA) receptor antagonist 2,3-Dihydro-
xy-6-nitro-7-sulphamoylbenzo(f)-quinoxaline 6-
Nitro-7-sulphamoylbenzo(f)-quinoxaline-2,3-dione
(NBQX) (Pikov and Wrathall, 2002). The drugs
were given intrathecally to the lumbosacral spinal
cord with increasing dosages administered during
the urodynamic procedures. The range of doses
was chosen so as not to affect the detrusor itself.
We found that external urethral sphincter spik-
ing activity was mildly affected by low doses of
either drug and was dramatically inhibited by high
doses. The mean values of intravesical pressure
change were unaffected by either drug except for
Fig. 9. Time course of recovery of detrusor and external ure-
high doses of CPP in the uninjured and chronic
thral sphincter muscle function after mild spinal cord injury.
The increase in intravesical pressure (dIVP, top panel) and in groups. In contrast, the external urethral sphincter
external urethral sphincter spiking activity (dESA, bottom pan- spiking activity was decreased with each drug in a
el) during voiding was measured in uninjured rats and in rats at dose-dependent manner, with maximal inhibition
5 days (subacute) and 8 weeks (chronic) after spinal cord injury of external urethral sphincter spiking activity
produced by dropping at 10 g weight 12.5 mm unto the dura at
(60–70%) seen at the highest doses used. The in-
T8 (ds ¼ 101 s). Symbols indicate a significant difference from
the uninjured group (*) or from both the uninjured and chronic hibitory dose (ID)50 values for individual animals
groups (**). Adapted from Pikov and Wrathall (2002) with were used to calculate the average ID50 values
permission. for NBQX and CPP. There was no effect of
injury on the ID50 of NBQX between experimental
activity was significantly inhibited at the subacute groups. In contrast, the subacute group exhibited
time point (5 days) and recovered by 8 weeks after a 50% lower ID50 for CPP than uninjured
this mild spinal cord injury (Fig. 9). controls. By 8 weeks, this effect was no longer
In examining the dorsolateral nucleus motoneu- evident.
rons we found that 5-HT immunoreactivity at 5 Our finding of changes only in NMDA receptor
days is significantly below that of uninjured con- function is consistent with evidence suggesting that
trols, and, by 8 weeks, it has recovered to normal NMDA and non-NMDA receptors are part of
levels. This suggests that sprouting of raphespinal parallel, but functionally separate, synaptic circuits
fibers spared by the injury may be involved in the that are important in micturition (Yoshiyama
recovery of external urethral sphincter spiking ac- et al., 1995). An intriguing hypothesis to explain
tivity shown in Fig. 9. In contrast, corticotrophin our results is an alteration in NMDA receptor sub-
releasing factor associated with the sacral para- unit composition during recovery of lower urinary
sympathetic nucleus was about 60% of the normal tract function. CPP, a potent NMDA receptor an-
at both 5 days and 8 weeks, consistent with the tagonist, has different affinities to NMDA recep-
ability of a reflex bladder to be established in the tors depending upon the NR2 subunit present as
absence of supraspinal connections. part of the receptor complex, ranking in the order
129

NR2A4NR2B4NR2D while the affinity to may serve as the basis for novel pharmacological
glutamate is in the opposite order (Monaghan strategies to enhance functional recovery in the
et al., 1998). Thus, increased sensitivity to CPP subacute period after spinal cord injury.
could be due to a shift toward a higher proportion Subsequent studies have confirmed that at 5 days
of NR2A at 5 days after spinal cord injury. If such after a mild spinal cord injury, NMDA receptors
receptor composition shifts are confirmed and are on dorsolateral nucleus motoneurons may contain
typical after spinal cord injury, this information a higher proportion of NR2A than after recovery

Fig. 10. (Upper panel) Photomicrograph depicting Neutral Red-stained dorsolateral (DL) nucleus motoneuronal cell bodies with
overlying NR2A mRNA grains. Uninjured animals exhibit normal levels of mRNA, whereas 5 days after a MASCIS mild contusion
injury (12.5 mm weight drop), the number of grains are increased. Scale bar ¼ 5 mm. (Lower panel) Quantitative comparison of in situ
hybridization net grain counts for the NMDA subunits NR1, NR2A, NR2B, and the AMPA subunit GluR2 in the DL nucleus. Data
are expressed as net grain counts/mm2 cell body. NR2A and GluR2 net grain counts from injured animals are significantly higher than
uninjured controls, po0.01, whereas those for NR1 and NR2B do not differ significantly between injured and uninjured animals.
Symbols represent data from individual rats; the bar represents the mean value for the group.
130

at 8 weeks. Slides containing the dorsolateral nu- 22.5


cleus from animals at 5 days after mild MASCIS 20.0
contusion injury and from uninjured control ani- 17.5
mals were hybridized with 35S-ATP labeled anti- 15.0
sense oligonucleotides to the NMDA subunit

dESA
12.5
mRNA for NR1, NR2A, and NR2B and the
AMPA subunit mRNA for GluR2. The density of
10.0 ∗
7.5
the grains over the dorsolateral nucleus neurons 5.0 ∗ ∗
was then calculated as a ratio of grains/mm2 and 2.5 ∗
was corrected for background by subtracting the
0.0
density of grains from adjacent areas devoid of tis-

k
s

k
8w
ol

1w

1w

4w

4w

8w
sue. The results (Fig. 10) indicate that the mRNAs

tr
on

m
m

m
m

m
m
for GluR2 and NR2A are abnormally expressed

.5

25

.5

25

.5

25
12

12

12
(upregulated) at 5 days as found chronically in
more severely injured groups as shown in Table 2. Fig. 11. Recovery of detrusor–external urethral sphincter co-
GluR2 is generally found as part of functional ordination occurs by 4 weeks after mild spinal cord injury. At 1
AMPA receptors but the presence of NR2A in week after cord injury both mild (12.5 mm) and moderate
NMDA receptors is associated with altered recep- (25 mm) injury groups exhibit changes in external urethral
sphincter spiking activity (dESA) during bladder contraction
tor sensitivity as mentioned above. Our current
that are significantly decreased from uninjured controls. By 4
data suggest that altered sensitivity of dorsolateral weeks, animals in the mild group exhibit dESA that have re-
nucleus motoneurons may be a general occurrence covered to control levels. *po0.001, ANOVA with Tukey’s
in the subacute period after spinal cord injury but is post hoc testing.
normalized if and when recovery of bladder–exter-
nal urethral sphincter coordination occurs.
Recently, we have undertaken studies to further to the lumbar spinal cord (Lecci et al., 1992).
define the time of recovery of lower urinary tract Administration of 5-HT antagonists, such as
function after mild spinal cord injury as measured WAY-100635 increases bladder capacity and in-
by the normalization of external urethral sphincter hibits the voiding reflex (Kakizaki et al., 1997;
spiking activity. We have found that complete re- Testa et al., 1999). In fact, modulation of the se-
covery is consistently found by 4 weeks after spinal rotonergic influence on micturition is under clin-
cord injury, as shown in Fig. 11. Further, this func- ical investigation for application in stress-induced
tional recovery is mirrored by recovery of 5-HT urinary incontinence. The drug duloxetine, an in-
immunoreactivity of the dorsolateral nucleus moto- hibitor of both serotonin and norepinephrine re-
neurons (Fig. 12). Are these correlations indications uptake appears especially promising for reducing
of a functional connection? Future studies will fo- stress-induced urinary incontinence by facilitating
cus on this question, further testing the temporal external urethral sphincter activity (Thor, 2003).
relationship between recovery of external urethral We speculate that pharmacological support of
sphincter spiking activity and 5-HT immunoreac- serotonergic neurotransmission after incomplete
tivity and extending the studies to pharmacological spinal cord injury may modulate the extent of ex-
evaluation of the functional role of the serotonin ternal urethral sphincter activation and thus nor-
system in recovery of lower urinary tract function. malize the bladder–sphincter dyssynergia for
The serotonin system has been shown to alter efficient voiding.
the micturition reflex in both normal rats and
those after a complete spinal cord transection.
Administration of 8-OH-DPAT (a selective Conclusion
5-HT1A agonist) facilitates the voiding reflex re-
gardless of whether drug administration is intra- On the basis of our studies, we postulate that spinal
peritoneal, intracerebroventricular or intrathecal cord injury initially affects the lower urinary tract
131

Fig. 12. 5-HT immunoreactivity in the dorsolateral (DL) nucleus recovers by 4 weeks after mild SCI. 5-HT pixel density was quantified
in the DL nucleus using METAMORPHs software in controls (A), at 1 week (B), and at 4 weeks (C) after mild and moderate spinal
cord injury. # indicates DL motor neurons in A–C. (D) 5-HT pixel density is significantly decreased in both injury groups at 1 week
post-injury (*po0.001), but normalizes by 4 weeks in the mild group. Bars represent mean+SEM of 5-HT pixel density with N ¼ 6 for
controls and 5 for each injury group. ANOVA with Tukey’s post hoc testing.

function similarly after a wide range of severities of interneuronal alterations that results in the devel-
thoracic spinal cord injury ranging from a mild opment of a reflex bladder (Kruse et al., 1993;
contusion through a complete surgical transection. de Groat, 1995), as described elsewhere in this
The loss of normal descending control inhibits volume. We postulate that the same mechanisms
normal micturition requiring manual bladder occur after incomplete contusion injury in the first
expression in the rat or catheterization for a week(s) after spinal cord injury. However, the
cord-injured person. The bladder responds by sparing of key long tract axons then allows a sec-
enlarging, and in transection models this is known ond form of recovery to occur, which is only seen
to initiate the afferent plasticity and subsequent with incomplete spinal cord injury. Changes occur
132

in some spared descending control pathways for de Groat, W.C., Araki, I., Vizzard, M.A., Yoshiyama, M.,
lower urinary tract function, such as the raphespi- Yoshimura, N., Sugaya, K., Tai, C. and Roppolo, J.R. (1998)
nal system, that allow simultaneous activation of Developmental and injury induced plasticity in the micturit-
ion reflex pathway. Behav. Brain Res., 92: 127–140.
the external urethral sphincter with bladder con- Ding, Y.Q., Takada, M., Tokuno, H. and Mizuno, N. (1995)
traction. With time this coordination improves Direct projections from the dorsolateral pontine tegmentum
and some of the initial changes associated with loss to pudendal motoneurons innervating the external urethral
of lower urinary tract function normalize, such as sphincter muscle in the rat. J. Comp. Neurol., 357: 318–330.
altered glutamate receptor subunit expression on Ding, Y.Q., Wang, D., Nie, H., Guan, Z.L., Lu, B.Z. and Li,
J.S. (1998) Direct projections from the periaqueductal gray to
dorsolateral nucleus motoneurons, 5-HT immuno- pontine micturition center neurons projecting to the lumbo-
reactivity in the dorsolateral nucleus, and perhaps sacral cord segments: an electron microscopic study in the
increased c-Fos activation patterns. rat. Neurosci. Lett., 242: 97–100.
A challenge for future studies will be identifying Emch, G., Lund, IV., Gandy, V., Lytle, J.M. and Wrathall, J.R.
the key causal factors in this secondary recovery (2003) Glutamate receptor plasticity during recovery of lower
urinary tract function after spinal cord injury. Soc. Neurosci.
phase that produces bladder–external urethral
Abs., 954: 914.
sphincter coordination. With this information, Emch, G.S., Hermann, G.E. and Rogers, R.C. (2001) TNF-
we may learn how to increase the efficiency of alpha-induced c-Fos generation in the nucleus of the solitary
voiding and thus reduce the long-term deleterious tract is blocked by NBQX and MK-801. Am. J. Physiol.
effects of abnormal lower urinary tract function Regul. Integr. Comp. Physiol., 281: R1394–R1400.
Garcia, Leoni, M.E. and Esclarin De Ruz, A. (2003) Manage-
after incomplete spinal cord injury.
ment of urinary tract infection in patients with spinal cord
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Gruner, J.A. (1992) A monitored contusion model of spinal
Acknowledgments cord injury in the rat. J. Neurotrauma, 9: 123–126 discussion
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Hassouna, M., Galeano, C., Abdel-Rahman, M. and Elhilali,
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Holstege, G., Griffiths, D., de Wall, H. and Dalm, E. (1986)
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 9

Role of the urothelium in urinary bladder


dysfunction following spinal cord injury

Lori A. Birder

Departments of Medicine and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

Abstract: A consequence of spinal cord injury is a change in bladder reflex pathways resulting in the
emergence of detrusor hyperreflexia and increased activity of the urethral sphincter. A basis for some of
these alterations could be changes in the environment of bladder sensory nerve endings at the target organ.
Recent evidence suggests that the urothelium (the lining of the urinary bladder) plays a prominent role in
modulating bladder sensory nerve ending excitability. It is conceivable that factors and processes affecting
the plasticity of bladder neurons after spinal cord injury may be partly due to changes occurring in the
urothelium. Although the urothelium has classically been thought of as a passive barrier to ions/solutes, a
number of novel properties have been recently attributed to these cells. Our work and that of others clearly
demonstrates that the urothelium exhibits both ‘‘sensor’’ (expression of sensor molecules or response to
thermal, mechanical and chemical stimuli) as well as ‘‘transducer’’ (release of factors/transmitters) prop-
erties. Taken together, these and other findings discussed in this chapter suggest a sensory function for the
urothelium and that alterations in urothelial properties may contribute to afferent abnormalities following
spinal cord injury.

The urothelium: an effective barrier against solutes accelerated proliferation can occur in pathology.
and pathogens For example, using a model which creates a selec-
tive injury of apical urothelial cells (protamine
The bladder urothelium is a specialized lining of sulfate), it has been shown that, in response to
the urinary tract, extending from the renal pelvis injury, the urothelium undergoes both functional
to the urethra. The urothelium is composed of at and structural changes in order to restore the bar-
least three layers: a basal cell layer attached to a rier (Lavelle et al., 2002).
basement membrane, an intermediate layer and a The umbrella cells function as a barrier against
superficial apical layer with large (diameters of most substances found in urine thus protecting the
25–250 mm) hexagonal ‘‘umbrella’’ cells (Lewis, underlying tissues (Negrete et al., 1996; Zeidel,
2000; Acharya et al., 2004). It has been reported 1996; Lewis, 2000; Apodaca, 2004). When this
for some species that both the umbrella and per- function is compromised during injury or inflam-
haps intermediate cells may have projections to the mation, it can result in the passage of toxic sub-
basement membrane (Martin, 1972; Hicks, 1975; stances into the underlying tissue (neural/muscle
Apodaca, 2004). The basal cells, which are thought layers) resulting in urgency, frequency and dysuria.
to be precursors for other cell types, normally ex- The superficial or umbrella cells play a prominent
hibit a low (3–6 months) turnover rate; however, role in maintaining this barrier, and exhibit a
number of unique properties including specialized
Corresponding author. Tel.: +412-383-7368; membrane lipids, asymmetric unit membrane par-
Fax: +412-648-7197; E-mail: lbirder@pitt.edu ticles and a plasmalemma with stiff plaques (Lewis,

DOI: 10.1016/S0079-6123(05)52009-0 135


136

2000; Hu et al., 2002; Apodaca, 2004). These cells properties that could allow reciprocal communi-
are also interconnected with extensive junctional cation with neighboring urothelial cells as well
complexes which include cytoskeletal elements, as nerve fibers or other cell types (i.e., immune,
and cytoplasmic and transmembrane proteins, myofibroblasts, inflammatory) in the bladder wall.
some of which play a role in cell–cell adhesion Recent studies have shown that afferent as well as
(Lewis, 2000; Acharya et al., 2004; Apodaca, autonomic axons are located in close proximity to
2004). This ‘‘water-tight’’ function of the apical the urothelium (Birder et al., 2001; Beckel et al.,
cell membrane is partly due to the above-mentioned 2004). Peptide- and TRPV1-immunoreactive nerve
specialized lipid molecules and uroplakin proteins, fibers have been found localized throughout the
a major protein component of the apical cell urinary bladder musculature and in a plexus
membrane, which reduce the permeability of beneath, and extending into, the urothelium. This
the urothelium to small molecules (water, urea, suggests that the release of a number of mediators
protons). Tight junction complexes are thought to from the urothelium could alter bladder nerve
reduce the movement of ions and solutes between excitability and, in turn, release of mediators from
cells (Tammela et al., 1993; Lewis, 2000; Wang nearby bladder nerves may also impact urothelial
et al., 2003; Apodaca, 2004). function. In support of this idea is evidence that
ATP (released from urothelial cells during stretch)
can activate a population of suburothelial bladder
Sensor and transducer functions of urothelium afferents expressing P2X3 purine receptors (Ferguson
et al., 1997; Burnstock, 2001), signaling changes in
Urothelial cells exhibit a number of properties bladder fullness and pain (Vlaskaovaka et al.,
similar to neurons (nociceptors/mechanorecep- 2001). Accordingly, P2X3 null mice exhibit urinary
tors): both types of cells use diverse signal-trans- bladder hyporeflexia, suggesting that this receptor
duction mechanisms to detect physiological and neural–epithelial interactions are necessary for
stimuli. Examples of ‘‘sensor molecules’’ (i.e., re- normal bladder function (Cockayne et al., 2000).
ceptors/ion channels) associated with neurons that Thus, the activation of bladder nerves and
have been thus far identified in urothelium include urothelial cells could modulate urinary bladder
receptors for: bradykinin (Chopra et al., 2005), function directly or indirectly via the release
neurotrophins (trkA and p75) (Wolf-Johnston of chemical factors in the urothelial layer. This
et al., 2004), purines (P2X and P2Y) (Lee et al., type of regulation may be similar to the epithelial-
2000; Birder et al., 2004a; Sun and Chai, 2004; dependent secretion of chemical factors in airway
Tempest et al., 2004), norepinephrine (a- and b-) epithelium thought to modulate submucosal
(Birder et al., 1998, 2002b), acetylcholine (mu- nerves and bronchial smooth muscle tone
scarinic and nicotinic) (Beckel et al., 2002, 2004; (Homolya et al., 2000; Jallat-Daloz et al., 2001).
Chess-Williams, 2002). Other sensor molecules The cellular mechanism(s) by which stretch
identified in urothelial cells are protease-activated evokes the release of ATP from epithelial, end-
receptors (D’Andrea et al., 2003), mechanosensi- othelial or other cell types is unclear. One hypoth-
tive Na+ channels (Lewis and Hanrahan, 1985; esis is that cellular distension causes intracellular
Wellner and Isenberg, 1993; Smith et al., 1998; vesicles rich in ATP to fuse with the urothelial cell
Carattino et al., 2005) and a number of transient membrane promoting ATP release and an increase
receptor potential (TRP) channels (TRPV1, in umbrella cell surface area during bladder filling.
TRPV2, TRPV4, TRPM8) (Birder et al., 2001, This mechanism would allow the bladder to ex-
2002a; Barrick et al., 2003; Stein et al., 2004). pand its epithelial surface area as urine accumu-
The ability of urothelial cells to express ‘‘sensor lates. Consistent with this possibility, mechanical
molecules’’ and release chemical mediators (nitric distension of the excised bladder has been shown
oxide, adenosine triphosphate (ATP), acetylcho- to trigger an increase in the membrane capacitance
line, substance P, prostaglandins) suggests that of the apical urothelial surface (Truschel et al.,
these cells exhibit specialized sensory and signaling 2002).
137

Impact of spinal cord injury on urothelial cell barrier prevents the ‘‘acute’’ cord injury-induced disrup-
function and morphology tions in both epithelial morphology and barrier
function (Figs. 2A–D). These and other findings
Spinal cord injury (transection of the spinal cord suggest the involvement of the autonomic nervous
in rats at level T8-9) resulted in changes in both system in the acute effects of cord injury on
urothelial morphology and barrier function the bladder urothelium. Studies have also shown
(Apodaca et al., 2003). At 24 h post spinal cord that the release of stress hormones (i.e., nor-
injury, there is a decrease in transepithelial resist- epinephrine) can disrupt the urothelial tight junc-
ance and an increase in permeability to water and tions with loss of urothelial cells (Veranic and
urea. The urothelium also exhibits a number of Jezernik, 2000). Thus, it is possible that transec-
regions which lack umbrella cells (Figs. 1E and F). tion of the spinal cord could induce the release of
The alterations in ultrastructure and function ‘‘stress-hormones’’ which could contribute to the
worsen within a few days following spinal cord changes observed in the urothelium at early time
injury, when significant disruptions in the urothe- points following spinal cord injury. We have found
lium are observed (Figs. 1G and H) correlating that intravesical administration of norepinephrine
with decreased transepithelial resistance and in- significantly altered epithelial permeability
creased permeabilities. Although some of these (decrease in transepithelial resistance) compared
changes could be the downstream consequence of to controls (Birder et al., unpublished results).
barrier disruption, some of the alterations could be Although the mechanism for these changes is
due to cord injury-induced urinary retention and under investigation, one possibility is adrenergic-
bladder overdistension. The bladders were not induced release of a soluble factor such as nitric
cannulated to avoid catheter-induced injury/in- oxide from urothelial cells (Birder et al., 2002b),
flammation, but were manually expressed several which in excess levels can alter the barrier function
times a day. Following recovery of the spinal reflex of the urothelium (Truschel et al., 2002). Alterna-
pathways and emergence of automatic micturition tively, cord injury-dependent neurotransmitter re-
14–28 days after spinal cord injury, barrier func- lease from efferent nerves could stimulate mast cell
tion was re-established, although the morphology release of histamine, bradykinin, prostaglandins,
of the urothelium was altered and the superficial leukotrienes and proteases, all of which could
urothelial cells were smaller (Figs. 1I–L). stimulate urothelial cells and contribute to tissue
Examination of urothelial morphology and damage and inflammation. In addition to the re-
function at earlier time points (1–2 h after tran- lease of catecholamines from bladder efferent
section) revealed significant changes in urothelial nerves, other modulators released from immune
morphology including areas of urothelium which cells might also play a role in the loss of barrier
lacked apical cells (Figs. 1C and D). These findings function after spinal cord injury.
correlated with decreased transepithelial resist- Reports suggest that capsaicin-sensitive nerves
ance, which suggests disruption of the tight junc- may contribute to mucosal protection following
tions and cell–cell contact. In contrast, only minor injury or inflammation (Abdel-Salam et al., 1999).
alterations in urea and water permeabilities oc- The neurotoxin capsaicin was therefore used to
curred at this time. The reason the permeabilities evaluate the involvement of capsaicin-sensitive
did not change is unknown. One possibility is the bladder afferents in changes in mucosa ultrastruc-
underlying cells still provide an adequate barrier ture and permeability after acute spinal cord injury
during the acute phase of spinal cord injury. (Figs. 2E–H). In these studies, capsaicin-pretreat-
A number of mechanisms may contribute to ment did not prevent functional changes but en-
these acute urothelial changes including increased hanced the susceptibility of the mucosa to injury
autonomic activity (i.e., release of catecholamines by decreasing transepithelial resistance compared
from stimulated efferent nerves) following spinal to untreated cord-injured animals or capsaicin-
cord injury. We have shown that pretreatment with treated controls. It has been suggested that the
the ganglionic blocking agent, hexamethonium, effect of capsaicin may be due to alterations in
138

Fig. 1. Scanning electron micrograph (SEM) of urothelial images taken from control and at various time points following spinal cord
injury (SCI). (A and B), Images taken from sham-treated animals; or 2 h (C and D), 24 h (E and F), 3 days (G and H), 14 days (I and J)
or 28 days (K and L) following SCI. Panels on the right depict a higher magnification view of inset regions shown on the left. (With
permission from Apodaca et al., Am. J. Physiol., 2003.) Arrows in F indicate small shrunken cells associated with epithelium.
139

Fig. 2. Scanning electron micrograph (SEM) of images depicting effects of either hexamethonium (50 mM) or capsaicin (100 mg/kg s.c.,
4 days prior) on acute changes in urothelial ultrastructure 2 h following spinal cord injury (SCI). (A and B) pretreatment with
hexamethonium before SCI; (C and D) hexamethonium pretreatment prior to sham surgery; (E and F) capsaicin pretreatment (4 days
prior) then SCI; (G and H) capsaicin pretreatment then sham surgery. Panels on the right depict a higher magnification view of inset
regions shown on the left. (With permission from Apodaca et al., Am. J. Physiol., 2003.)

substance P/calcitonin gene-related peptide con- leading to increased permeability may not be
tent in capsaicin-sensitive nerves (Szolcsanyi and evident using the employed morphological
Bartho, 2001). Although we did not detect a approaches.
significant difference in the surface architecture Thus, in addition to affecting the bladder de-
between treated animals and controls, dilation of trusor muscle and its innervation (de Groat, 1995),
extracellular spaces or alterations in tight junctions spinal cord injury also leads to a rapid disruption
140

of urothelial barrier function. This is evident by a been shown in sensory nerves that ATP can po-
loss of cell–cell interactions, decreased transepi- tentiate the response of vanilloid receptors (caps-
thelial resistance and increased water and urea aicin, protons and moderate heat act on vanilloid
permeabilities. Our results indicate that release of receptors) by lowering the threshold for responses
neurotransmitters by bladder efferent nerves is at to these stimuli (Tominaga et al., 2001). This rep-
least partially responsible for the disruption of the resents a novel mechanism through which the large
urothelium. Although the neurotransmitters and/ amounts of ATP released from damaged or sen-
or inflammatory mediators that disrupt barrier sitized cells in response to injury or inflammation
function are not well characterized, our observa- may trigger increased excitability of afferent
tions indicate that bladder nerves play an important nerves. These findings have clinical significance
role in regulating and maintaining barrier function. and suggest that alterations in afferents or epithe-
lial cells in pelvic viscera may contribute to the
sensory abnormalities in a number of pelvic dis-
Impact of spinal cord injury on urothelial cell sensor orders, including interstitial cystitis, a chronic
and transducer properties painful condition of the urinary bladder (Nickel,
2003). In a comparable disease in cats, termed fe-
Sensitization of urothelial cells and afferents can line interstitial cystitis (Buffington et al., 1999) we
be triggered by various mediators (nerve growth reported alterations in stretch-evoked release of
factor, ATP, nitric oxide, prostaglandins) released urothelium-derived ATP (Birder et al., 2003), con-
by both neuronal and non-neuronal cells (urothe- sistent with the augmented release of ATP from
lial cells, fibroblasts, mast cells) located near the urothelial cells from some patients with interstitial
luminal surface of the bladder. Increased endog- cystitis (Sun et al., 2001). We have recently found
enous levels of nerve growth factor and/or similar results in chronic spinal cord-injured cats
urothelial receptors for nerve growth factor (p75; (Birder et al., unpublished results), in which the
trkA) have been detected in the target organ augmented stretch-evoked ATP release from urothe-
(smooth muscle and urothelium) in a number of lial cells may contribute to bladder hyperreflexia.
bladder pathologies (Steers et al., 1991; Vizzard, The urothelium maintains a tight barrier to ion/
2000; Jallat-Daloz et al., 2001; Wolf-Johnston solute flux and augmented release of mediators
et al., 2003; Wolf-Johnston et al., 2004). More- such as nitric oxide from urothelial cells and/or
over, altered nerve growth factor levels (even in the nearby bladder nerves may play a role in the
absence of inflammation) have been linked to maintenance and regulation of this urothelial bar-
changes in the properties of afferent pathways rier function. Our previous studies have demon-
(Kornblum and Johnson, 1982; Dmitrieva and strated an upregulation in inducible nitric oxide
McMahon, 1996; Lamb et al., 2004). Thus, nerve synthase and elevated basal levels of nitric oxide
growth factor may play a significant role in en- measured in the bladder mucosa in cats with in-
hancing the sensitivity of a number of ‘‘sensor terstitial cystitis (Birder et al., 2005). Similar find-
molecules’’ within both the urothelium and senso- ings have been obtained clinically, as some patients
ry neurons. These findings suggest that targeting with classic interstitial cystitis also demonstrate
nerve growth factor and/or nerve growth factor elevated release of nitric oxide (Hosseini et al.,
signaling mechanisms may provide important in- 2004). As increased nitric oxide has been linked to
sight into new therapies for urinary bladder dys- cellular damage and alterations in epithelial bar-
function caused by inflammation or injury. rier function (Salzman et al., 1995; Arkovitz et al.,
Another important component of the injury/in- 1997), and it has been demonstrated that feline
flammatory response is ATP release from various interstitial cystitis is accompanied by changes in
cell types including the urothelium, which can in- bladder permeability and urothelial ultrastructure
itiate painful sensations by exciting purinergic (Lavelle et al., 2000), we examined whether nitric
(P2X) receptors on sensory fibers (Cockayne oxide levels might also be altered in rats following
et al., 2000; Burnstock, 2001). Recently, it has spinal cord injury, which results in changes in both
141

urothelial morphology and function (Apodaca of events that are thought to be part of symptoms
et al., 2003). In urothelium from normal rats, associated with urinary tract infections.
basal nitric oxide release (measured from the urinary Taken together, modification of the urothelium
bladder mucosal surface) remained undetectable and/or loss of epithelial integrity in a number of
(o10 nM nitric oxide release). However, after bladder pathologies could result in passage of tox-
chronic spinal cord injury, we detected elevated ic/irritating urinary constituents through the epi-
levels of basal nitric oxide release (200–500 nM) thelium leading to changes in the properties of
recorded from the mucosal surface of the urinary sensory pathways.
bladder (Truschel et al., 2001).
To evaluate the impact of elevated mucosal ni-
Therapeutic options for spinal cord injury that could
tric oxide levels on epithelial function, we exam-
target the urothelium
ined the effects of prolonged exposure to high
concentrations (2.5–5 mM) nitric oxide donors
An emerging body of evidence indicates that
(S-nitroso-N-acetyl penicillamine or sodium nit-
urothelial cells exhibit ‘‘polymodal’’ properties,
roprusside) on cultured urothelial cells. It has been
i.e., can be activated by chemical, thermal or me-
previously shown that cultured urothelial cells ex-
chanical stimuli, and that their activation can po-
hibit properties similar to native urothelium
tentially evoke the release of a myriad of
(Truschel et al., 1999). Normally, these urothelial
transmitters which can impact afferent activity
cultures exhibit a high transepithelial resistance
and ultimately bladder function. While the urot-
and low urea and water permeabilities. However,
helium has been historically viewed as primarily a
the administration of high concentrations of nitric
‘‘barrier,’’ it is becoming increasingly clear that it
oxide resulted in a significant decrease in transep-
is a responsive structure capable of detecting phys-
ithelial resistance (90% decrease as well as 3–5-
iological and chemical stimuli, and of releasing a
fold increase in permeability to water and urea), as
number of signaling molecules. The following is a
compared to controls (Truschel et al., 2001). This
summary of various therapies, most given intrave-
response was reversible upon washout of the nitric
sically, which are traditionally thought to target
oxide donor. Similar findings were obtained using
bladder nerves. It is conceivable that a number of
excised urinary bladder from rodents, in which
these treatments could also target urothelial re-
application of high concentrations of nitric oxide
ceptors and/or release mechanisms.
also decreased transepithelial resistance by 60%
compared to untreated control (Truschel et al.,
2001). Although their mechanism is unknown, Intravesical vanilloid compounds
these effects are reminiscent of similar observa-
tions in epithelia of other organs (lung, gut) in One example of a urothelial ‘‘neuronal-like’’ sen-
which excess production of nitric oxide has been sor molecule is the TRP channel TRPV1, known
linked to changes in epithelial integrity (Ding to play an important role in nociception and in
et al., 2004; Han et al., 2004). urinary bladder function (Szallasi, 2001). It is well
Disruption of epithelial integrity in some blad- established that the painful sensations induced by
der pathologies may also be due to substances such capsaicin, the pungent substance in hot peppers,
as antiproliferative factor, which has been shown are caused by stimulation of vanilloid receptor-1
to be secreted by bladder epithelial cells from in- (TRPV1), an ion channel protein which is activat-
terstitial cystitis patients and can inhibit epithelial ed by vanilloid compounds such as capsaicin,
proliferation thereby adversely affecting barrier moderate heat and protons (Caterina et al., 1997;
function (Keay et al., 1999, 2004). Uropathogenic Caterina, 2001). TRPV1 is highly expressed in
Escherichia coli can also bind to uroplakin pro- urinary bladder unmyelinated axons (C-fiber) that
teins present on the apical surface of superficial detect bladder distension or the presence of irritant
umbrella cells (Schilling and Hultgren, 2002). This chemicals (Chancellor and de Groat, 1999).
is thought to be an initial step leading to a cascade Intravesical instillation of vanilloid compounds
142

Fig. 3. (A) Confocal image of basal cells depicting TRPV1-immunoreactivity (cy-3, red) and cytokeratin-17, a marker for these cells
(Fluorscein isothiocyanate or FITC, green) immunoreactivity. (B) Confocal image of urinary bladder urothelium reveals TRPV1-
positive (cy-3, red) nerve fibers located in close proximity to basal urothelial cells (FITC, green). Punctate TRPV1 staining in urothelial
cells was electronically subtracted to facilitate imaging of the TRPV1-IR nerve fiber. (With permission from Birder et al., Proc. Natl.
Acad. Sci. USA, 2001.)

such as capsaicin or resiniferatoxin, which leads to ATP release and membrane capacitance as well as
desensitization of bladder nerves, has been shown a decrease in hypotonic or stretch-evoked ATP
to improve voiding efficiency significantly in cord- release from cultured TRPV1 null urothelial cells.
injured animals as well as in patients with detrusor Thus, the functional significance of these receptors
hyperactivity (Szallasi and Fowler, 2002; Kim in the bladder extends beyond pain sensation to
et al., 2003). include participation in normal bladder function.
One of the more remarkable findings in our own These receptors are also essential for normal me-
studies is that TRPV1 is not only expressed by chanically evoked, purinergic signaling by the
afferent nerves that form close contact with urothelium. In addition to the known effects on
urothelial cells but also by the urothelial cells bladder nerves, intravesical use of vanilloids could
themselves (Fig. 3) (Birder et al., 2001). Further, also target TRPV1 on urothelial cells, where
TRPV1 receptor expression correlates with sensi- persistent activation of urothelial TRPV1 might
tivity to vanilloid compounds, as exogenous ap- lead to receptor desensitization or depletion of
plication of capsaicin or resiniferatoxin increases urothelial-derived transmitters.
intracellular calcium and evokes the release of
transmitters (nitric oxide, ATP) in cultured ur- Antimuscarinic drugs
othelial cells. These responses are dependent upon
TRPV1 expression (Birder et al., 2001, 2002a). In Antimuscarinic drugs are widely regarded as a
neurons, TRPV1 is thought to integrate/amplify standard treatment in patients with neurogenic
the response to various stimuli and thus plays an lower urinary tract dysfunction (Andersson and
essential role in the development of inflammation- Yoshida, 2003). By targeting muscarinic receptors
induced hyperalgesia. Thus, it seems likely that on bladder smooth muscle, these agents prevent
urothelial-TRPV1 might participate in a similar receptor stimulation by acetylcholine released
manner, in the detection of irritant stimuli follow- from bladder efferent nerves and promote in-
ing bladder inflammation or infection. creased bladder capacity. However, these drugs are
While anatomically normal, TRPV1 null mice ex- thought to be effective during bladder storage when
hibited a number of alterations in bladder function parasympathetic nerves are silent. Since various
including a reduction of in vitro, stretch-evoked stimuli have been shown to release acetylcholine
143

from urothelial cells (Andersson and Yoshida, Diagnostic test for spinal cord injury: the ice
2003; Beckel et al., 2004), it is postulated that this water test
release from non-neural stores (i.e., urothelium)
could also contribute to detrusor overactivity It has been reported that intravesical instillation of
(Andersson and Yoshida, 2003). Thus, the effec- cold solutions can unmask the presence of de-
tiveness of some of these agents may be partly due trusor reflex activity in people with spinal cord
to targeting urothelial receptors and/or release injury (Balmaseda et al., 1988). This ‘‘bladder
mechanisms. Although muscarinic receptor sub- cooling reflex’’ is thought to be due to activation of
types have been detected on urothelial cells a subset of cold-sensitive C-fiber-type bladder
(Hawthorn et al., 2000; Beckel et al., 2004), a role afferents, which are sensitive to both cold temper-
for these receptors in bladder function has not yet atures and menthol (Jiang et al., 2002).
been established. Taken together, these data sug- Some TRP ion channels can be activated by a
gest that the bladder urothelium may be an addi- wide range of temperatures as well as by natural
tional source of acetylcholine that influences products (capsaicin, menthol), which can elicit
bladder contractility by modulating smooth mus- sensations of hot or cold (Patapoutian et al.,
cle tone and afferent activity. 2003). In contrast to TRPV1, which is a detector
of warm temperatures, TRPM8 has been shown to
be activated by cold temperatures as well as by
Botulinum toxin cooling agents (menthol) and is expressed in a
subset of sensory neurons (Clapham, 2003). Both
Recent studies have demonstrated that in- of these TRP channels are also expressed in blad-
tradetrusor injection of botulinum neurotoxin type der urothelium, suggesting that the urothelium can
A (Botox) is an effective therapy in a number of express a range of thermoreceptors underlying
lower urinary tract disturbances including the both ‘‘cold’’ and ‘‘heat’’ stimuli. While the func-
severe incontinence due to neurogenic detrusor tional role of these thermosensitive channels in
overactivity of spinal cord injury patients (Harper urothelium remains to be clarified, it seems likely
et al., 2004; Reitz and Schurch, 2004). Following that a primary role for these proteins may be
injection, the toxin binds to bladder cholinergic to recognize noxious stimuli in the bladder. For
nerve terminals and cleaves the protein, SNAP25, example, noxious cold solutions instilled into the
necessary for exocytosis and release of acetylcho- urinary bladder could, via stimulation of urothelial
line (Harper et al., 2004). In patients with spinal TRP channels, augment the release of urothelial-
cord injury, this treatment can lead to a significant derived mediators, thereby altering bladder affer-
reduction in episodes of incontinence and an in- ent excitability. Further studies are needed to
crease in maximum bladder capacity. elucidate fully the role of these TRP channels in
There is evidence that Botox can suppress the urothelium and influence on bladder function.
release of a number of mediators (acetylcholine,
ATP and neuropeptides) from both neural and
non-neural cells (Morris et al., 2001). Recent stud- Conclusion
ies have demonstrated that this agent can effec-
tively reduce both chemically and mechanically There is considerable interest in the putative role
evoked ATP release from cultured urothelial cells of urothelial receptors/ion channels and release
(Barrick et al., 2004). Alterations in the release of mechanisms in bladder function. By targeting
ATP or other transmitters from the urothelium various urothelial sensor molecules and/or modu-
could have a profound impact on neural excita- lating the release of transmitters/inflammatory me-
bility. Taken together, these findings suggest that diators, it may be possible to modulate afferent
targeting neural and non-neural release mechanisms activity and prevent the disruption of the urothe-
may be effective for the treatment of bladder lium that accompanies spinal cord injury and oth-
hyperreactivity in spinal cord injury. er bladder conditions. These results highlight the
144

need for additional studies in order to establish the Birder, L., Apodaca, G., de Groat, W.C. and Kanai, A.J. (1998)
physiological relevance of these urothelial targets. Adrenergic- and capsaicin-evoked nitric oxide release from
urothelium and afferent nerves in urinary bladder. Am. J.
Physiol., 275: F226–F229.
Acknowledgments Birder, L., Barrick, S.R., Roppolo, J.R., Kanai, A.J., de Groat,
W.C., Kiss, S. and Buffington, C.A. (2003) Feline interstitial
I thank Drs. A. Kanai and W. C. de Groat for cystitis results in mechanical hypersensitivity and altered
ATP release from bladder urothelium. Am. J. Physiol., 285:
critical comments and suggestions during prepa-
F423–F429.
ration of this chapter. The electron micrographs Birder, L., Kanai, A.J., de Groat, W.C., Kiss, S., Nealen, M.L.,
were prepared by W. Giovani Ruiz. This work was Burke, N.E., Dineley, K.E., Watkins, S., Reynolds, I.J. and
supported by grants to Lori A. Birder from the Caterina, M.J. (2001) Vanilloid receptor expression suggests
NIH (RO1-DK-54824 and RO1-DK-57284). a sensory role for urinary bladder epithelial cells. Proc. Natl.
Acad. Sci. USA, 98: 13396–13401.
Birder, L., Nakamura, Y., Kiss, S., Nealen, M.L., Barrick,
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 10

Plasticity in the injured spinal cord: can we use it


to advantage to reestablish effective bladder voiding
and continence?

Natasha D.T. Zinck1, and John W. Downie1,2

1
Department of Pharmacology, Faculty of Medicine, Dalhousie University, 5850 College St., Halifax, NS B3H 1X5,
Canada
2
Department of Urology, Faculty of Medicine, Dalhousie University, 5850 College St., Halifax, NS B3H 1X5, Canada

Abstract: Micturition is coordinated at the level of the spinal cord and the brainstem. Spinal cord injury
therefore directly interrupts spinal neuronal pathways to the brainstem and results in bladder areflexia.
Some time after injury, however, dyssynergic bladder and sphincter function emerges. The changes me-
diating the appearance of bladder function after spinal cord injury are currently unknown. Primary afferent
neurons have been shown to sprout in response to spinal cord injury. Sprouting primary afferents have been
linked to the pathophysiology of centrally manifested disorders, such as autonomic dysreflexia and ne-
uropathic pain. It is proposed that sprouting of bladder primary afferents contributes to disordered bladder
functioning after spinal cord injury. During development of the central nervous system, the levels of specific
neuronal growth-promoting and guidance molecules are high. After spinal cord injury, some of these
molecules are upregulated in the bladder and spinal cord, suggesting that axonal outgrowth is occurring.
Sprouting in lumbosacral spinal cord is likely not restricted to neurons involved in the micturition reflex.
Furthermore, sprouting of some afferents may be contributing to bladder function after injury, whereas
sprouting of others might be hindering emergence of function. Thus selective manipulation of sprouting
targeting afferents that are contributing to emergence of bladder function after injury is critical. Further
research regarding the role that neuronal sprouting plays in the emergence of bladder function may con-
tribute to improved treatment of bladder dyssynergia after spinal cord injury.

Introduction neurons exist within an environment rich in mole-


cules that are important for axonal guidance,
Plasticity within the spinal cord outgrowth and targeting (Drescher et al., 1997;
Dickson and Senti, 2002). These molecules act by
Axonal outgrowth and pathfinding are limited either attracting or repelling the leading edge of
within the adult central nervous system (CNS). growing axons (the growth cone), thus directing
During development, however, axonal outgrowth developing axons to their appropriate targets
and pathfinding are critical for a properly wired, (Gallo and Letourneau, 2004; Gordon-Weeks,
and thus, functioning nervous system (Crowley 2004). It has been proposed that there is a de-
et al., 1994; Maier et al., 1999). Developing crease or absence in growth-promoting molecules
once adulthood is reached, and that this is why the
Corresponding author. Tel.: +(902) 494-3459; adult CNS is unable to promote neuronal plastic-
Fax: +(902) 494-1388; E-mail: nzinck@dal.ca ity. For this reason, much research has focused on

DOI: 10.1016/S0079-6123(05)52010-7 147


148

reproducing the molecular environment that is Axonal remodeling occurs normally within the
present during development with the hopes of aid- developing micturition reflex
ing neuronal repair and regrowth after CNS injury.
On the other hand, CNS plasticity may also have The micturition reflex is subject to extensive re-
negative consequences. There is increasing evidence modeling during development. For approximately
that upregulation of key elements involved in aid- the first 3 weeks of life, micturition in rats and cats
ing axonal outgrowth contributes to the pathogen- is evoked by stimulation of somatic perineal af-
esis of centrally manifested disorders. These include ferents, via licking of the perineum by the mother
neuropathic pain (Theodosiou et al., 1999) as well (Maggi et al., 1986; Thor et al., 1989). The effe-
as autonomic dysreflexia (Krenz et al., 1999). Spi- rent limb, through the pelvic nerve, stimulates
nal cord injury, depending on its severity, affects detrusor contraction, thereby facilitating bladder
many centrally mediated visceral functions, includ- emptying. This somatic-bladder reflex is a spinal
ing bladder function. Normal micturition requires reflex that becomes progressively weaker through-
supraspinal integration at the level of the pontine out postnatal life to a point where there appears to
micturition center (Barrington, 1915). Suprasacral be a switch from spinally mediated to supraspin-
spinal cord injury disconnects the parasympathetic ally mediated micturition (de Groat et al., 1998).
spinal outflow from the pons rendering the bladder Micturition in these animals, as in adults, is in
areflexic. Bladder function is novel in that micturit- response to bladder stretch, having both afferent
ion emerges some time after spinal cord injury and efferent limbs in the pelvic nerve. This switch
without neuronal integration at the level of the is thought to be accompanied by major changes in
brainstem (Yoshiyama et al., 1999). However, post neuronal circuits used to elicit micturition.
spinal cord injury, voiding is dysfunctional due to Developing nervous systems undergo a great
lack of coordination between the detrusor muscle deal of synaptic strengthening and neuronal re-
and the external urethral sphincter, clinically finement. One process thought to play a major role
termed detrusor–sphincter dyssynergia (Kruse during development of the micturition reflex is
et al., 1993). For spinal cord-injured patients, this synaptic competition. The numbers of synapses on
results in hyperactive inefficient bladder function. spinal neurons derived from spinal and supraspinal
Treatments for these patients focus on alleviating sources change throughout development (Fig. 1).
hyperactive bladder dysfunction pharmacologically It is thought that synaptic input to the para-
via non-selective anti-muscarinic agents (Pannek sympathetic preganglionic nucleus from supraspi-
et al., 2000), reducing dyssynergy surgically nal centers increases during postnatal development
through external sphincterotomy (Reynard et al., and out competes sacral interneurons for the same
2003) and generating voiding on demand by sacral target (de Groat, 2002). This may explain the loss
anterior root stimulation (Schumacher et al., 1999). of spinally mediated micturition and the emer-
Spinal cord injury has been shown to induce gence of supraspinally mediated micturition dur-
neuronal sprouting within the spinal cord. Plastic- ing development.
ity of neurons within the micturition reflex circuit Coincidently, spinal cord injury in adult animals
is a possible mechanism by which bladder function triggers a switch back to micturition mediated via
emerges after spinal cord injury. Continued re- a spinal reflex (Kakizaki and de Groat, 1997;
search regarding the role that neuronal sprouting Shefchyk and Buss, 1998) thereby making synaptic
plays in bladder function after spinal cord injury competition an hypothesis not only for the emer-
will aid in the development of treatment methods gence of brain stem-mediated micturition during
which target the cause of bladder dysfunction after development, but also for the emergence of spinal
injury rather than its symptoms. In this chapter, reflex micturition after spinal cord injury.
we will discuss the evidence for neuronal plasticity If the alternative neuronal pathways for eliciting
occurring in the micturition reflex path and how bladder contraction already exist in the spinal
this may mediate the emergence of bladder dys- cord, why does it take so long for a spinal reflex to
function after spinal cord injury. emerge after spinal cord injury? Assuming that
149

transection (Yoshiyama et al., 1999) and in humans,


depending on the severity of the injury, it may not
emerge for months (Weld and Dmochowski, 2000).
This delay in emergence of the bladder function
implies that other changes associated with neurons
in the micturition reflex, both phenotypic and
anatomical, may be involved in emergence of
bladder function after spinal cord injury.

Bladder primary afferents and spinal cord injury

General characteristics

Retrograde tracers such as cholera toxin B subunit


and horseradish peroxidase have proven useful for
illustrating the pattern of bladder primary afferent
termination in the spinal cord (Morgan et al.,
1981; Nadelhaft and Booth, 1984; Wang et al.,
1998). Bladder primary afferents enter Lissauer’s
tract from which two major tracts form the path-
ways of entry into the gray matter of the dorsal
horn. The lateral collateral pathway extends from
superficial dorsal horn (lamina I and II) through
the dorsolateral funiculus and terminates densely
in the area of the parasympathetic preganglionic
nucleus. A few terminations are also seen to enter
the dorsal gray commissure from this pathway.
A second and less dense pathway, the medial col-
lateral pathway, sends fibers from the dorsomedial
border of the dorsal horn in laminae I and II
Fig. 1. Synaptic competition may explain the switch from spi- to lamina X. Terminating fibers from this pathway
nal to brainstem control of micturition during development. It can also be seen in medial laminae V and VI.
is proposed that sacral preganglionic neurons (PGN) in neon-
ates receive input from sacral interneurons (INT) as well as
Bladder primary afferents exhibit a periodical
from descending fibers from the brain stem. In young neonates rostrocaudal termination pattern within the
a spinal reflex elicits micturition because sacral interneurons spinal cord.
outcompete any descending modulation by the brainstem. As Bladder primary afferents consist of small un-
the neonate ages the number of synapses on preganglionic neu- myelinated C-fibers and thinly myelinated A-delta
rons from sacral interneurons decreases, and the number from
the brain stem increases. The strong synaptic input from
fibers. These subtypes can be divided further into
the brainstem is thought to underlie supraspinally controlled peptidergic and non-peptidergic groups. Pep-
micturition in the older neonate and throughout adulthood. tidergic fibers are characterized by the presence
From de Groat (2002). of calcitonin gene-related peptide and substance P.
Non-peptidergic primary afferents contain fluoride
spinal shock accounts for bladder areflexia during resistant acid phosphatase activity and are able to
the first few days after injury, reflex mediated bind the plant isolectin B4 (IB4) (Stucky and
micturition should occur soon after resolution of Lewin, 1999). Regionally, peptidergic afferents
areflexia. However, micturition does not emerge terminate in lamina I as well as lamina II outer,
in rats until around 2 weeks after spinal cord whereas non-peptidergic afferents terminate in
150

lamina II inner. 86% of all bladder primary affer- the density and distribution of trk receptors
ents innervating the bladder body are peptidergic thought to play a role in growth-promoting signa-
fibers (Yoshimura et al., 2003). The greatest pro- ling (Qiao and Vizzard, 2002) among L6/S1 dorsal
portion of non-peptidergic afferent innervation is root ganglion cells.
to the distal urethra where close to 30% of sensory
innervation is from IB4 positive fibers.
Sprouting and the factors implicated in this response
Further characterization of these two groups
reveals that peptidergic and non-peptidergic fibers
As well as phenotypic changes, anatomical reor-
possess different trophic factor receptors and
ganization of bladder primary afferents may con-
therefore respond to distinct neurotrophic factors.
tribute to emergence of bladder function after
Peptidergic primary afferents contain the trkA nerve
spinal cord injury.
growth factor receptor while non-peptidergics
do not contain trk receptors but contain glial
derived neurotrophic factor receptors Ret and Neurotrophic factors
GRFalpha1. Neuronal plasticity that follows spinal cord injury
is thought to be regulated largely by neurotrophic
factors. If neuronal plasticity is involved in the
Phenotypic changes emergence of bladder function after spinal cord
injury, then changes in trophic factor levels at both
After spinal cord injury, bladder primary afferents organ and spinal levels are likely to be involved.
may undergo changes in phenotype as well as a Nerve growth factor provides trophic support to
sprouting response. In the cat, under normal the majority of bladder primary afferents and
conditions, bladder sensory information travels to chemically increases the sensitivity of primary affe-
the spinal cord via mechano-sensitive A-delta rents (Lamb et al., 2004). Several studies have in-
fibers (Janig and Morrison, 1986). After spinal vestigated the role that nerve growth factor may
cord injury, sensory information is conveyed by play in bladder function after spinal cord injury.
what were once mechano-insensitive C-fibers Increases in spinal nerve growth factor have
(de Groat et al., 1990). This increase in C-fiber been linked to the development of disorders such
afferent excitability may be mediated by a decrease as neuropathic pain and autonomic dysreflexia. In
in tetrodotoxin-resistant and an increase in both disorders, nerve growth factor is increased
tetrodotoxin-sensitive Na+ channel expression predominantly within small diameter primary affe-
(Yoshimura and de Groat, 1997; Waxman et al., rent neurons. Neuronal nerve growth factor is in-
1999). A decrease in Nav 1.8 channels, a subtype of creased in lumbar and sacral (L6/S1) dorsal root
tetrodotoxin-resistant Na+ channels, is associated ganglia and L6 spinal cord after thoracic spinal
with bursting behavior of cerebellar Purkinje cord injury (Seki et al., 2002). Schwann cells,
neurons (Renganathan et al., 2003) as well as astrocytes and other microglia also upregulate
spontaneous activity of dorsal root ganglion their expression of nerve growth factor after spinal
cells (Renganathan et al., 2001). Decreased ion cord injury (Krenz and Weaver, 2000). In models
conductance across A-type potassium channels is of neuropathic pain, expression of nerve growth
also thought to contribute to increased afferent factor by glial cells sensitizes primary afferent
excitability (Sculptoreanu et al., 2004). nociceptors leading to hyperalgesia and allodynia.
Although overall afferent number remains the The spinal cord is not the only source of nerve
same, there is an increase in proportion of growth factor. In fact, the bladder has increased
myelinatated fibers (Yoshimura et al., 1998) as levels of nerve growth factor mRNA acutely after
well as dorsal root ganglion cell body size (Yu spinal cord injury. Upregulation of nerve growth
et al., 2003) among bladder primary afferents after factor protein and increased expression of its re-
spinal cord injury. Other injury-induced changes ceptor, trkA, also occur in the bladder and dorsal
to bladder afferent phenotype include changes in root ganglia 6 weeks post injury (Vizzard, 2000;
151

Qiao and Vizzard, 2002), well after the emergence unpublished data). Furthermore, administration
of voiding function in the rat. Thus bladder- of antibodies against nerve growth factor after
derived nerve growth factor may facilitate changes spinal cord injury leads to a decrease in density
that occur in bladder function at chronic time- and distribution of these peptidergic fibers in the
points after spinal cord injury but not the initial dorsal horn (Christensen and Hulsebosch, 1997).
emergence of bladder function. Nerve growth fac- Therefore increases in L6/S1 spinal calcitonin
tor acts on peptidergic C-fibers and has been gene-related peptide may be the consequence of
shown to induce the expression and secretion of peptidergic fiber sprouting elicited by increased
calcitonin gene-related peptide (Bowles et al., nerve growth factor. At both acute (3 days)
2004). Increased calcitonin gene-related peptide (N. Zinck, V. Rafuse, J. Downie, unpublished
distribution within the L6/S1 spinal cord segments data) and chronic (6 weeks) (Vizzard and Boyle,
has been demonstrated in spinal cord injury rats at 1999) time-points post spinal cord injury growth
timepoints before the emergence of bladder func- associated protein-43 is increased within L6/S1 spi-
tion (Fig. 2) (N. Zinck, V. Rafuse and J. Downie, nal cord segments, suggesting synaptic remodeling

Fig. 2. Calcitonin gene-related peptide-immunoreactive primary afferents sprout in rat lumbosacral spinal cord after spinal cord
injury. Inset shows the approximate level of the longitudinal sections. Increases in density and distribution of fiber terminations is seen
in rats 8 days post spinal cord transection at T10 when compared to non-injured (control) rats. Arrowhead indicates filling in of gaps
between primary afferent termination bundles. SPN, sacral parasympathetic nucleus; DCG, dorsal commissural nucleus. (N.D.T.
Zinck, V.F. Rafuse, J.W. Downie, unpublished.)
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is occurring both before and after emergence of the presence of nerve growth factor. Because nerve
bladder function. growth factor-stimulated brain-derived neurotro-
Although previous literature suggested that phic factor upregulation is associated with inflam-
non-peptidergic afferents do not sprout in re- matory processes in the peripheral nervous system,
sponse to injury (Belyantseva and Lewin, 1999), central inflammatory processes after spinal cord
recent studies report increased fiber branching of injury may not be contributing to increases in brain-
IB4 positive neurons within the dorsal root ganglia derived neurotrophic factor at the spinal level.
after spinal nerve transection (Li and Zhou, 2001).
Bladder mRNA levels of glial-derived neurotro- Cellular adhesion molecules
phic factor are also increased after spinal cord in- A critical aspect of neuronal outgrowth is appro-
jury in the rat (Vizzard, 2000) although direct priate synapse formation, facilitated largely by
studies of IB4 positive fiber density or distribution changes in adhesivity of growth cones. Several
in L6/S1 spinal cord segments after spinal cord factors have been implicated in mediating adhe-
injury have not been reported. Furthermore, un- sion of individual axon terminals to neighboring
like nerve growth factor, no studies have been cells or to the extracellular matrix. Three major
conducted that selectively target non-peptidergic players in the cell adhesion molecule family are
afferents via application of glial-derived neurotro- N-cadherin, the Ig cell adhesion molecules, L1 and
phic factor or antibodies to glial-derived neurotro- neural cell adhesion molecule (NCAM) (Kiryushko
phic factor in lumbosacral spinal cord making it et al., 2004). Homophilic binding of these members
difficult to determine what role this neurotrophin is necessary for proper adhesion and synapse
plays in the micturition reflex. formation throughout the development of the
Brain-derived neurotrophic factor is a member central nervous system.
of the nerve growth factor family and is also NCAM levels are greatly upregulated at critical
associated with small to medium diameter timepoints in neuronal development (Bruses et al.,
peptidergic primary afferents that terminate in 2002). Alternative splicing of the same gene results
lamina I and II of the dorsal horn (Luo et al., in three different isoforms of NCAM that have
2001). It is upregulated and synthesized in response been implicated in the creation of neuronal
to peripheral inflammation (Apfel et al., 1996). In networks during development and after injury in
fact, increases in nerve growth factor have been the adult nervous system (Kiss and Muller, 2001).
linked to increased expression of brain-derived Named by their molecular weights, NCAM-180
neurotrophic factor in trkA-containing sensory and -140 span the cell membrane whereas NCAM-
afferents and to heightened pain sensitivity associ- 120 has only an extracellular domain. NCAM can
ated with neuropathic pain (Obata et al., 2003). be separated from other members of the cell
Increased expression of brain-derived neurotrophic adhesion molecule family by their ability to bind
factor occurs throughout the spinal cord, including homopolymers of the carbohydrate polysialic acid.
L6/S1, after complete thoracic spinal cord injury. Polysialic acid-bound NCAM facilitates a decrease
Upregulation of trk B within bladder-specific in homophilic NCAM binding and thus a decrease
dorsal root ganglion neurons also occurs after in cellular adhesion and an increase in axonal
spinal cord injury (Qiao and Vizzard, 2002). defasciculation (Tang and Landmesser, 1993;
Unlike other neurotrophic factors studied, Monnier et al., 2001). Polysialic acid has been
increased spinal brain-derived neurotrophic factor shown to increase after central (Bonfanti et al.,
expression occurs both acutely and chronically 1996) and peripheral nerve (Franz et al., 2005)
after spinal cord injury. In fact, in the lumbosacral injury. Increases in polysialic acid have been asso-
cord, brain-derived neurotrophic factor upregula- ciated with regeneration of injured neurons of the
tion precedes that of nerve growth factor (Zvarova hippocampus (Aubert et al., 1998). During the first
et al., 2004), implying that in spinal cord injury, few weeks after birth neonatal rats use a spinal
the increased brain-derived neurotrophic factor pathway to elicit micturition. Approximately three
expression in primary afferents does not require weeks after birth bladder emptying is controlled by
153

supraspinal neurons. Polysialic acid-NCAM levels activation. NCAM is not only unique in its ability
are elevated in L6/S1 spinal cord segments of to bind polysialic acid, but has been shown to act
postnatal day 6 (P6) rats (Fig. 3) (N. Zinck, V. as a co-receptor with GFRa1, a glycosylpho-
Rafuse, J. Downie, unpublished data) when com- sphatidylinositol-linked receptor for glial-derived
pared to adult control, as suggested by the level of neurotrophic factor (Zhou et al., 2003). Glial-de-
smearing in lane one (P6) compared to lane two rived neurotrophic factor binding to this receptor
(control). This is a time when rat pups still use a complex has been associated with stimulating ax-
spinal reflex to elicit micturition implying that po- onal growth in vitro (Paratcha et al., 2003). Inter-
lysialic acid may have a role in the plasticity that action of brain-derived neurotrophic factor with
accompanies the transition from spinal to supra- its receptor trkB may be, in part, mediated by the
spinal micturition in rats. presence of polysialic acid-NCAM, as selective
NCAM has recently been shown to interact with enzymatic removal of polysialic acid decreases
brain-derived neurotrophic factor and glial-de- trkB phosphorylation (Vutskits et al., 2001). Be-
rived neurotrophic factor. NCAM, depending on cause both polysialic acid and NCAM have been
the isoform, signals through two main pathways, shown to interact with neurotrophic factors that
the Fyn/FAK pathway or through the fibroblast are upregulated in lumbosacral spinal cord after
growth factor receptor, both of which converge spinal cord injury (Zvarova et al., 2004), polysialic
upstream of the mitogen-activated protein kinase acid-NCAM may play a significant role in the
kinase MEK (Kiryushko et al., 2004). Signaling emergence of the spinal micturition reflex.
via the fibroblast growth factor receptor also in-
duces release of intracellular calcium stores, which
Inhibitors of neuronal outgrowth after injury
has been shown to mediate axonal outgrowth by a
The central nervous system contains several
variety of mechanisms, including by growth asso-
known inhibitors of neuronal sprouting. These
ciated protein-43 dependent phosphorylation and
molecules are expressed to stop neuronal out-
growth and aberrant synapse formation at the end
of development and are often upregulated after
nerve injury. Injury to the central nervous system
causes the formation of a glial scar that over ex-
presses both myelin-dependent and -independent
inhibitors of neuronal extension thereby impeding
neuronal sprouting and regeneration. Myelin-de-
pendent inhibitors of sprouting include Nogo-A,
myelin associated glycoprotein and oligodendro-
cyte myelin glycoprotein (Grados-Munro and
Fournier, 2003) whereas myelin-independent inhi-
bitors of sprouting are members of the proteo-
glycan family including chondroitin sulfate
Fig. 3. Neural cell adhesion molecule (NCAM) in neonatal proteoglycans (Bovolenta and Fernaud-Espinosa,
spinal cord is highly polysialated. Illustration shows a western 2000). Important signaling pathways of key mol-
blot of NCAM isoforms in rat L6/S1 spinal cord. Arrowheads ecules that inhibit sprouting will be discussed in
point out the level of three NCAM isoforms (NCAM-120, the section ‘‘Manipulation of neuronal sprouting’’.
NCAM-140, NCAM-180). The level of polysialation can be
inferred by the smearing of the NCAM bands due to variable
polysialic acid (PSA) binding to NCAM. At post-natal day 6 Do interneurons play a role in bladder function after
(P6) rats contain all three isoforms as well as showing smearing
spinal cord injury?
between these bands. Adult rats (control) have weak to no ex-
pression of NCAM-180 and little PSA expression as indicated
by a lack of smearing between bands. (N.D.T. Zinck, V.F. There are many neurons in the lumbosacral spinal
Rafuse, J.W. Downie, unpublished.) cord that respond to bladder afferent stimulation
154

(McMahon and Morrison, 1982; Honda, 1985; It is likely that spinal interneurons undergo
Coonan and Downie, 1999). Some may participate physiological and structural changes after spinal
in ascending transmission of bladder-related ac- cord injury and that these changes are contributing
tivity, either as part of the micturition reflex path- to emergence of micturition after injury. However,
way or in pain-related pathways (McMahon and until precise methods are developed to study these
Morrison, 1982; Milne et al., 1982; Ding et al., interneurons specifically, the contribution of spi-
1994). Also, activation of spinal interneurons is an nal bladder interneurons to bladder function after
important component of efficient micturition both spinal cord injury will remain elusive.
for bladder contraction and sphincter inhibition
(Shefchyk, 2001). These interneuron pools have Repairing the injured spinal cord to improve
not been well localized. However, it appears from bladder function
virus tracing and immediate early gene expression
studies that the dorsal gray commissure and the Cellular implants
region of the parasympathetic preganglionic nu-
cleus are important locations of bladder and Stem cells have been touted as a major aid in the
sphincter-related interneurons (Nadelhaft et al., treatment of many diseases, including Parkinson’s
1992; Nadelhaft and Vera, 1996; Marson, 1997; disease, diabetes and amyotrophic lateral sclerosis.
Grill et al., 1998; Vera and Nadelhaft, 2000). Because stem cells have the ability to differentiate
There is some suggestion of synaptic reorgani- into various tissue types, they also have been tested
zation in the lumbosacral spinal cord after spinal for a role in repairing the damaged spinal cord af-
cord injury in rats (Yu et al., 2003). On the other ter injury. Stem cells injected into severely injured
hand, evidence for change in the pelvic afferent rat spinal cord have shown cellular differentiation,
terminal arbor in the spinal cord is lacking (Kruse resulting in increased axonal regeneration and sig-
et al., 1995). There is evidence for reorganization nificant improvement in motor function below the
of motor neuronal pathways after spinal cord in- site of injury (McDonald et al., 1999). Few studies,
jury to facilitate hind limb locomotion (Grasso however, have addressed the impact of stem cell
et al., 2004). Thus one possibility is that spinal implantation on visceral function after spinal cord
interneuron reorganization may underlie the emer- injury. Injections of neural stem cells in the injured
gence of bladder activity after spinal cord injury. rat spinal cord has improved lower urinary tract
A second possibility is that emergence of blad- function by increasing voiding efficiency, although
der function after spinal cord injury is a matter of has not improved bladder–sphincter dyssynergia
developing access to an existing spinal circuit sub- (Mitsui et al., 2003).
serving micturition. The existence of such a circuit, Implantation of nerve grafts with cells genetically
analogous to the spinal pattern generator for lo- engineered to secrete growth factors has become a
comotion, is implied by the finding that micturit- common animal model to treat spinal cord injury.
ion can be evoked by perineal or urethral nerve The ability of the peripheral nervous system to
stimulation in some circumstances (Shefchyk and regenerate after nerve injury is far greater than that
Buss, 1998; Boggs et al., 2004). One problem with of the CNS. Because of this ability of peripheral
this circuit is that it appears not to be activated by nerves to regenerate, nerve grafts implanted in the
bladder distension and thus coordinated micturit- injured spinal cord are often made from peripheral
ion never emerges in spinal cord-injured cats. It is nerve tissue or peripheral nervous system specific
possible that sprouting of urethral or perineal af- cell types. For example, implantation of a Schwann
ferents is hindering the use of this circuit at later cell graft, secreting trkB activating neurotrophins,
time points post spinal cord injury. Thus encour- brain-derived neurotrophic factor and neurotro-
aging the sprouting of bladder afferents to target phin-3, into a severely contused rat spinal cord has
the spinal micturition circuit while suppressing the provided restoration of bladder function (Sakamoto
perineal inputs to the circuit may be an appropri- et al., 2002). Not every growth factor will have
ate approach to restoration of function. positive effects on bladder function. For example,
155

implantation of peripheral nerve grafts secreting cord injury, no definitive studies have been con-
acidic fibroblast growth factor in a patient with a ducted. The last decade has provided a wealth of
complete hemisection of the thoracic spinal cord knowledge with respect to specific extracellular
improved motor recovery although bladder function signaling pathways involved in neurite outgrowth
was unchanged (Cheng et al., 2004). Because differ- and extension. With a molecular knowledge of key
ent motor and visceral systems are reliant on specific elements responsible for axon regeneration, new
growth factors, it is likely that if we are to signif- targets have been uncovered for experimental ma-
icantly improve micturition by the addition of nipulation. The function of the lower urinary tract
neuronal growth factors, then we need first to could benefit from such investigation, providing
establish which trophic factors are pertinent to the new avenues of research that have the potential to
micturition reflex. answer questions regarding the neuronal plasticity
Olfactory ensheathing cells, as the name implies, of the micturition reflex after spinal cord injury.
ensheath fibers as they travel from the olfactory Neurotrophic factors, especially nerve growth fac-
bulb to the peripheral nervous system. These cells tor, have been shown to play a major role in bladder
are thought to provide a permissive environment function after spinal cord injury, thereby making
from the CNS to the peripheral nervous system by possible methods for interfering with signaling path-
remaining in contact with the olfactory nerve and ways of these molecules invaluable. Intrathecal ad-
inhibiting astrocytes from blocking entry into the ministration of immuno-neutralizing nerve growth
CNS. Injection of olfactory ensheathing cells into factor antibody into the L6/S1 spinal cord segments
deep dorsal horn regions after transection of dorsal results in a marked decrease in the number of non-
roots promotes afferent reentry into the dorsal spi- voiding bladder contractions, as well as increasing
nal cord and also facilitates the emergence of blad- voiding efficiency in rats with complete spinal cord
der function (Pascual et al., 2002). Although injury (Fig. 4). Electromyographic recording of
deafferentation is not a model of spinal cord inju- external urethral sphincter muscles in spinal rats
ry, these findings provide justification for further treated with this antibody also shows a decrease in
investigation on the effects that olfactory ensheath- detrusor–sphincter dyssynergia (Seki et al., 2004).
ing cells may have on spinally mediated bladder These results are very similar to the effects of
function. Recently, many investigators studying subcutaneous administration of capsaicin to spinal
bladder incontinence are turning to gene therapy cord-injured rats. Because capsaicin is selectively
as a possible way to treat urological dysfunction. neurotoxic to C-fibers, upregulation of nerve growth
This technique involves the use of a viral vector that factor is likely to occur in small diameter primary
encodes a particular gene of interest and is injected afferents (Cheng and de Groat, 2004).
into the bladder wall. Transport of the vector to the Saporin-tagged IB4 injected intrathecally at the
bladder afferent neurons in dorsal root ganglia as level of L6/S1 in a recent experiment caused selec-
well as to the spinal cord is accomplished due to tive reduction of IB4-positive (non-peptidergic) af-
retrograde virion transmission. This technique has ferents in L6 and a decrease in bladder overactivity
previously been used to transport molecules like in response to inflammation (Vulchanova et al.,
nerve growth factor as well as pre-proenkephalin to 2001; Nishiguchi et al., 2004). It was not clear
bladder primary afferents to treat bladder afferent which visceral population of IB4-positive afferents
neuropathy associated with diabetes (Goins et al., were destroyed after application of the cytotoxin
2001; Yoshimura et al., 2001). This may also be an saporin in this experiment, so the role of bladder
interesting avenue of research for treating bladder specific non-peptidergic fibers in detrusor overacti-
dysfunction after spinal cord injury. vity after inflammation is unclear. Injections of
saporin-tagged IB4 in the bladder wall or pelvic
Manipulation of neuronal sprouting nerve would aid in clarifying these results. Recent-
ly, saporin-tagged IB4 that had been injected into
Although it has been implied that neurons con- the sciatic nerve was detected in the spinal cord
trolling the micturition reflex sprout after spinal indicating that these more selective methods are
156

Fig. 4. Antagonism of nerve growth factor (NGF) normalizes aberrant bladder function after spinal cord injury. Bladder function is
abnormal 10 days after a mid-thoracic spinal cord injury and the administration of vehicle i.t. (A). However, 14 days after i.t.
administration of 10 ug NGF-Ab the non-voiding contractions are fewer and smaller (B). Arrows indicate voiding episodes. The
micturition pattern in panel B closely resembles that seen in uninjured rats. Infusion of saline ¼ 0.04 ml/min. From Seki et al. (2002)
with permission.

possible (Vulchanova et al., 2001; Tarpley et al., are elevated to a point at which myelin-associated
2004). The role that IB4 positive bladder afferents glycoprotein signaling is overridden. When cAMP
play in bladder overactivity associated with spinal levels decline, myelin-associated glycoprotein is
cord injury, however, has not yet been investigated. then able to promote growth cone collapse, ending
As described earlier, the peripheral nervous axonal outgrowth during development. Exogenous
system has a far greater ability to regenerate after elevation of cAMP (Neumann et al., 2002) or
neuronal assault than the CNS. The most studied vaccination against myelin (Huang et al., 1999) in
of all myelin-associated inhibitors is Nogo. The models of spinal cord injury has improved neuronal
receptor for Nogo, NgR, forms a receptor complex regenerative ability as well as increased functional
with the low-affinity neurotrophin receptor p75. recovery. In addition, inhibiting protein kinase A, a
Myelin-associated glycoprotein and oligodendro- downstream regulator of cAMP, decreases the
cyte myelin glycoprotein, along with Nogo, ability of peripheral nerve grafts to extend axons
mediate their inhibitory actions on neuronal out- into host spinal cord (Cai et al., 2001).
growth through the NgR-p75 receptor complex Signaling through NgR-p75 activates other
(Wang et al., 2002). The antagonist to the Nogo downstream effectors important in signaling inhi-
receptor, NEP (1–40), and exogenous addition of bition of neuronal outgrowth. A small group of
neurotrophins (Cai et al., 1999) have both signi- GTPases, known as Rho GTPases, are activated in
ficantly improved axon re-growth after nerve injury response to Nogo- and myelin-associated glyco-
(GrandPre et al., 2002). p75 receptor knockouts protein binding to the p75 receptor. It is becoming
have shown similar results (Wang et al., 2002). An increasingly evident that the low-affinity receptor
overview of signaling pathways that are critical in for all neurotrophins, p75, has a major role in reg-
regulating neuronal extension is presented in Fig. 5. ulation of neuronal extension and survival. Con-
During development, myelin-associated glyco- version of RhoGDP to its active GTP-bound state is
protein appears to play a key role in the switch thought to be mediated by a number of GTPase-
from neurite extension to arrest. Cyclic adenosine activating proteins, guanine nucleotide exchange
monophosphate (cAMP) levels appear to be critical factors, and guanine nucleotide dissociation inhib-
in this switch. During development, cAMP levels itor (GDI). Rho-GDI interacts directly with
157

Fig. 5. Major signaling pathways of myelin associated inhibitors of neuronal outgrowth. Myelin associated glycoprotein (MAG),
Nogo and oligodendrocyte myelin glycoprotein signal through the Nogo receptor (NgR)-p75 receptor complex to activate Rho and
Rho-kinase. Neurotrophic factors (NT) stimulate neuronal regeneration through increases in cAMP levels. MAG signaling is able to
inhibit cAMP induced regeneration via signaling through an inhibitory G protein. Arrest of axonal remodeling during development is
likely mediated by MAG signaling. From Grados-Munro and Fournier (2003) with permission.

the intracellular domain of the p75 receptor as well as the myosin binding subunit of myosin
(Yamashita and Tohyama, 2003) and thus is light chain phosphatase induces actomyosin as-
thought to play a key role in Rho signaling. In its sembly and growth cone collapse. Inhibition of
active state, RhoGTP acts to stabilize actin polym- Rho-kinase signaling with the specific Rho-kinase
erization, thereby facilitating growth cone collapse inhibitor, Y–27632 results in increased axonal ex-
and inhibiting axonal sprouting through activation tension and growth cone formation within dorsal
of several effector molecules (Bishop and Hall, root ganglia neurons (Borisoff et al., 2003).
2000). One of the most studied Rho effector mol- Most research directed at restoring function
ecules involved in actin reorganization is a serine after spinal cord injury seeks to enhance survival
threonine kinase known as Rho-kinase. Rho- and extension of injured and uninjured neurons.
kinase regulates axonal outgrowth via two main Although neuronal sprouting and synapse creation
pathways. Phosphorylation of myosin light chain is essential for reestablishing damaged connections
158

between neurons, it is critical that new synapses undergo. Some of these changes may play a role in
are functionally relevant. Increasing synaptogene- the emergence of bladder function after spinal cord
sis has the potential to do harm as well as good as injury. However, uncontrolled plasticity does not
seen in neuropathic pain and autonomic dysre- appear to provide functional outcomes that are
flexia. Upregulation of nerve growth factor has favorable to a spinal cord-injured person. Great
been linked to bladder hyperactivity after spinal advances have been made in unraveling the mo-
cord injury (Seki et al., 2002). Thus, if plasticity is lecular mechanisms of neuronal plasticity. These
mediating emergence of micturition after spinal findings have already contributed to a better
cord injury, then perhaps the goal for intervention understanding of how bladder afferents respond
in this process should be to maximize functional to spinal cord injury and how bladder function
outcome by selectively targeting sprouting to pro- might be dictated by that response. Because
duce efficient micturiton and avoid unwanted side bladder activity after spinal cord injury is
effects. Several molecules in the CNS (e.g. netrin, dysfunctional, specifically targeting bladder affer-
semaphorins and slits) are implicated in mecha- ent outgrowth may serve as a potential therapy for
nisms in guidance of growth cones to appropriate restoring ‘‘normal’’ bladder voiding and conti-
targets and formation of appropriate synapses (for nence. Although this presents a difficult task,
review see Dickson and Senti, 2002) However, research will be facilitated by the plethora of new
specific targeting of sprouting neurons to produce molecular techniques and the creation of knockout
efficient, non-dyssynergic function after spinal and transgenic animals. In conclusion, plasticity of
cord injury is currently not feasible because the bladder primary afferents after spinal cord injury is
intricate molecular interactions involved are not associated with bladder dysfunction, and therefore
well understood. The most practical strategy, for this dysfunction may be considered to be an
the present, may be to reduce neuronal sprouting unwanted consequence of plasticity. We suggest
in lumbosacral spinal cord in an attempt to alle- that, with more research, uncontrolled plasticity
viate bladder hyperreflexia. may be directed toward the more positive outcome
Current technology has provided ways to ma- of normal bladder function after spinal cord injury.
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L.C. Weaver and C. Polosa (Eds.)
Progress in Brain Research, Vol. 152
ISSN 0079-6123
Copyright r 2006 Elsevier B.V. All rights reserved

CHAPTER 11

Control of urinary bladder function with devices:


successes and failures

Robert A. Gaunt and Arthur Prochazka

Department of Biomedical Engineering and Center for Neuroscience, University of Alberta, 507 HMRC, Edmonton,
AB T6G 2S2, Canada

Abstract: The management of urinary tract dysfunction is crucial for the health and well-being of people
with spinal cord injury. Devices, specifically catheters, play an important role in the daily regime of bladder
management for most people with spinal cord injury. However, the high incidence of complications as-
sociated with the use of catheters, and the fact that the spinal segments involved in lower urinary tract
control remain intact in most cord-injured people, continue to motivate research into devices that could
harness the nervous system to provide greater control over lower urinary tract function. Mechanical devices
discussed in this review include catheters, artificial urethral sphincters, urethral stents and intraurethral
pumps. Additionally, many attempts to restore control of the lower urinary tract with electrical stimulation
have been made. Stimulation sites have included: inside the bladder, bladder wall, thigh, pelvic floor, dorsal
penile nerve, pelvic nerve, tibial nerve, sacral roots, sacral nerves and spinal cord. Catheters and sacral root
stimulators are two techniques whose efficacy is well established. Some approaches have proven less
successful and others are still in the development stage. Modifications to sacral root stimulation including
posterior root stimulation, anodal blockade and high-frequency blockade as well as new techniques in-
cluding intraspinal microstimulation, urethral afferent stimulation and injectable microstimulators are also
discussed. No single device has yet restored the control and function of the lower urinary tract to the pre-
injury state, but new techniques are bringing this possibility closer to reality.

Introduction spinal cord injury and account for the second


highest number of bed-days for readmitted pa-
People with spinal cord injury face many chal- tients (Savic et al., 2000; Middleton et al., 2004).
lenging medical problems. Inadequate post-injury While management of lower urinary tract dys-
management of lower urinary tract dysfunction function with devices, primarily catheters, has re-
can lead to many complications including renal duced mortality after spinal cord injury, the high
failure. This used to be the leading cause of death incidence of complications is largely due to the
after spinal cord injury, but has dropped to fourth limited success that these devices or other treat-
position in recent decades (Frankel et al., 1998) ment modalities have had in restoring normal
with improved treatment methods (Jamil, 2001). function to the neurogenic bladder. In addition to
However, complications of the genitourinary sys- these clinical considerations, effective management
tem, primarily urinary tract infections, are the of lower urinary tract dysfunction is generally
most common cause of rehospitalization after outranked in its importance to patients only by the
desire for hand function in people with quadriple-
Corresponding author. Tel.: +780-492-3783; gia and sex function in people with paraplegia
Fax: +780-495-1617; E-mail: arthur.prochazka@ualberta.ca (Anderson, 2004). These factors provide an

DOI: 10.1016/S0079-6123(05)52011-9 163


164

impetus to develop improved methods of manag- co-contractions of the external urethral sphincter.
ing lower urinary tract dysfunction after spinal This combination, termed detrusor–sphincter dys-
cord injury. synergia (Andersen and Bradley, 1976; Blaivas et al.,
1981), leads to incontinence, inefficient voiding
with high residual volumes and high intravesical
Lower urinary tract control pressures which in turn leads to ureteric reflux and
upper urinary tract deterioration.
The lower urinary tract has two functions: storing
urine (continence) and voiding urine (micturition).
The lower urinary tract is innervated by the so-
matic nervous system and both the sympathetic Why devices?
and parasympathetic branches of the autonomic
nervous system. Efferent parasympathetic inner- Any treatment for lower urinary tract dysfunction
vation of the detrusor, the muscular layer of the after spinal cord injury should create a bladder
bladder, arises from preganglionic neurons in the capable of storing large volumes of urine at low
sacral (S) parasympathetic nucleus in spinal seg- pressure, prevent incontinent episodes and allow
ments S2–S4. The preganglionic neurons send ax- periodic evacuation of urine at low pressure. Sur-
ons via the pelvic nerve to the pelvic plexus where gical treatments, such as bladder augmentation
they synapse with ganglionic neurons. Afferent in- using a section of intestine, ameliorate the problem
nervation of the bladder is also primarily via the of hyperreflexia and low storage volume (Hollander
pelvic nerve. Efferent somatic innervation of the and Diokno, 1993), while sphincterotomies
external urethral sphincter arises from motoneu- (cutting into the external urethral sphincter) im-
rons in Onuf’s nucleus in spinal segments S1–S3. prove detrusor–sphincter dyssynergia (Reynard
These efferent axons as well as the afferents of the et al., 2003). Anticholinergic medications are fre-
external urethral sphincter and urethra travel via quently used to relax the hyperreflexive bladder
the pudendal nerve. As the bladder fills during the but have undesirable side effects including a dry
storage phase, stretch-sensitive mechanoreceptors mouth and blurred vision (Wein, 1998). These
in the bladder wall transmit a sense of fullness to treatments address the symptoms of the ne-
both spinal and supraspinal centers. Once the urogenic bladder so that storage and evacuation
decision to void is reached, the external urethral of urine is achieved without upper urinary tract
sphincter is voluntarily relaxed and parasympa- damage, but they do not address the fundamental
thetic activity causes detrusor contractions. This loss of control associated with spinal cord injury.
synergistic activity, coordinated by the pontine mi- Devices present attractive alternatives to the
cturition center, results in micturition (Barrington, management of lower urinary tract dysfunction
1921, 1925). More details on the anatomy and after spinal cord injury as they attempt, at least
physiology of the lower urinary tract can be found partly, to restore the control of the neurogenic
in de Groat (1993) and de Groat et al. (2001). bladder. Additionally, while devices are locally in-
After spinal cord injury, supraspinal coordina- vasive to varying degrees, they do not generally
tion from the pontine micturition center is lost cause systemic complications as do pharmacolog-
leading to lower urinary tract dysfunction. Sacral ical treatments. Surgical procedures such as the
spinal cord or cauda equina lesions generally lead ones described above are usually irreversible and
to an areflexive bladder and sphincter paralysis. subsequently limit patients to a specified course of
Suprasacral lesions, however, spare sacral spinal treatment while possibly excluding new tech-
reflexes, and after a period of shock reflexive blad- niques. The devices described in this review, and
der contractions often occur at low bladder vol- those under development do not generally cause
umes. This condition, called detrusor hyperreflexia irreversible changes and therefore do not prevent
or neurogenic detrusor overactivity (reviewed in patients from taking advantage of improved treat-
Yoshimura, 1999), is often accompanied by reflexive ments in the future.
165

Many review articles have been published that (Comarr, 1972; Lapides et al., 1972), and this
focus on devices for bladder control (Schmidt, technique, along with generally improved medical
1983; Talalla et al., 1987; Lee, 1997; Rijkhoff et al., care, has caused urinary tract dysfunction to fall
1997b; Grill et al., 2001; Groen and Bosch, 2001; from the primary cause of death (22%) for pa-
Jamil, 2001; Jezernik et al., 2002; Van Kerrebroeck, tients injured between 1943 and 1972 to the fourth
2002; Middleton and Keast, 2004; Rijkhoff, 2004b; most common cause of death (9%) for patients
van Balken et al., 2004), so no attempt will be injured between 1973 and 1990 (Frankel et al.,
made to provide detailed descriptions of each of 1998).
these methods here. Rather, we will summarize the Chronically indwelling urethral and suprapubic
methods that have been devised over the years for catheters, condom catheters and clean intermittent
device-based management of the neurogenic blad- catheterization are common forms of catheteriza-
der secondary to spinal cord injury and summarize tion currently used in the management of spinal
current research on those devices and methods cord injury patients. Each method has its own ad-
that are likely to affect the field in the future. Ad- vantages and disadvantages (reviewed in Selzman
ditionally, we will attempt to identify the reasons and Hampel, 1993), but clean intermittent cathe-
that many methods and devices have ultimately terization is the form of bladder management
been unsuccessful, sometimes in spite of good clin- least likely to lead to complications (Weld and
ical results. Finally, we will summarize the prob- Dmochowski, 2000). Clean intermittent catheter-
lems that we feel should be addressed to improve ization is generally the most prescribed form
the effectiveness and adoption of devices in the of bladder management at hospital discharge
management of the neurogenic bladder. Both me- (Cardenas et al., 1995), and although a number
chanical and electrical devices will be described as of reports suggest that there is a trend for some
they have met with different levels of success and people to switch to other methods (Cardenas et al.,
failure and have the potential to offer solutions to 1995; Weld and Dmochowski, 2000), a more re-
a variety of problems faced by people with spinal cent study suggests that this trend may be revers-
cord injury. ing (Hansen et al., 2004). However, only 30% of
cord-injured people using clean intermittent cath-
eterization remain free of urinary tract infections.
Clean intermittent catheterization requires good
Mechanical devices for control of the lower hand function, preventing people with tetraplegia
urinary tract and impaired hand function as well as some people
with paraplegia from performing this procedure
Catheters themselves (Selzman and Hampel, 1993; Dahlberg
et al., 2004).
The use of catheters to manage urinary retention The critical role of catheter technology and
dates back to ancient Egypt (reviewed in Nacey techniques in the management of lower urinary
and Delahunt, 1993). During World War I, up tract dysfunction after spinal cord injury cannot be
to 80% of patients with spinal cord injury died overstated. The simplicity and clinical efficacy of
shortly after injury due to complications arising catheters in increasing life expectancy in people
from the neurogenic bladder (Kennedy, 1946). with cord injury make them arguably the single
However, improved management of the lower most important device for these people. However,
urinary tract using catheters during World War II the high incidence of urinary tract infections and
(Kennedy, 1946) and especially Guttmann’s other complications associated with catheter
technique of sterile intermittent catheterization use presents a continuous burden on patients and
(Guttmann and Frankel, 1966) helped reduce this the medical system. This, and the desire of people
figure significantly. Sterile intermittent catheteriza- with spinal cord injury for improved methods
tion was eventually modified to non-sterile clean in- (Anderson, 2004), is a motivation for new device
termittent catheterization for reasons of practicality development.
166

Artificial sphincters for patients with detrusor–sphincter dyssynergia.


Sphincterotomies are generally irreversible and
The concept of an artificial urethral sphincter was can cause hemorrhage, erectile dysfunction, blad-
first proposed by Foley (1947) to treat urinary in- der neck stenosis or stricture (reviewed in Reynard
continence. The artificial urethral sphincter devel- et al., 2003). Urethral stents are inserted into the
oped by Scott, Bradley and Timm (Scott et al., urethra and mechanically hold the external ure-
1974; Timm et al., 1974) has developed into the thral sphincter open. After sphincterotomy, or im-
commercially available AMS 800 artificial sphinc- plantation of a urethral stent, most cord-injured
ter (American Medical Systems, Minnetonka, people must wear a collection device such as a
MN, USA) (reviewed in Hajivassiliou, 1998). The condom catheter as the continence mechanism of
AMS 800 uses a pump to deflate a cuff placed the urethra is defeated.
around the bladder neck or urethra by transferring Several different urethral stent designs have
fluid to a pressure-regulated reservoir. The cuff re- been tested in various trials including the
inflates automatically over a period of several UroLumes (American Medical Systems, Minne-
minutes. Of reported studies using the AMS 800 tonka, MN, USA) (Chancellor et al., 1999b),
including 2606 subjects, 73% achieved full conti- Memokaths (Doctors & Engineers A/S Ltd.,
nence, 14% experienced device failure, 4.5% ex- Kvistgaard, Denmark) (Low and McRae, 1998;
perienced infections and 11.7% experienced Hamid et al., 2003), Memotherms (Bard Corp.,
urethral erosion from excessive pressure placed Covington, GA, USA) (Juan Garcia et al., 1999)
on the urethra by the cuff (Hajivassiliou, 1998). and Ultraflexs (Boston Scientific Corp., Natick,
Artificial urethral sphincters are primarily used MA, USA) (Chartier-Kastler et al., 2000). The
to treat patients with post-prostatectomy inconti- UroLume, Memotherm and Ultraflex are flexible
nence, but have been successful in managing wire mesh tubes while the Memokath is a helically
incontinence with other etiologies as well (Petrou wound wire. The devices are inserted into the ure-
et al., 2000). It was originally suggested that thra and positioned in the region of the external
detrusor hyperreflexia was a contraindication for urethral sphincter where the wire becomes largely
artificial urethral sphincter implantation as high covered by urothelium over time. The UroLume is
intravesicular pressures may cause deflation of the best studied of these devices and has similar
the pressure-regulated cuff (Scott et al., 1974). results, in terms of urodynamic parameters and
However, artificial urethral sphincters have been incidence of urinary tract infection to sphincter-
implanted in spinal cord injury patients with an otomies, but requires less hospitalization and is
overall success rate of 70% (Light and Scott, 1983), potentially reversible (Chancellor et al., 1999a). A
though device removal due to infections was high 5-year multi-center trial of the UroLume in 160
(24%). Currently, artificial urethral sphincters are cord-injured subjects showed that the treatment
not commonly used to manage incontinence after was successful in 84%, while 15% required ex-
spinal cord injury, but can be useful in people with plantation. Complications such as device migra-
lesions leading to a flaccid bladder and sphincter. tion were most common in the first 3 months
(Chancellor et al., 1999b). Although explantation
of the stent was possible, it has presented a variety
Urethral stents of challenges (Chancellor et al., 1999b; Wilson et al.,
2002). The Memokath was found to be suitable
Urethral stents were first developed to treat ure- for short-term implantation only as most devices
thral strictures, but shortly after, their use in spinal fail within 2 years (Hamid et al., 2003) and com-
cord injury patients with detrusor–sphincter dys- plications including migration, autonomic dysre-
synergia leading to hydronephrosis and vesicoure- flexia and stone formation on the stent can occur
teric reflux was described (Shaw et al., 1990). (Low and McRae, 1998). However, explantation
Urethral stents were proposed as an alternative to of this device is much simpler than the UroLume
sphincterotomies, the primary surgical treatment due to its helical design and thermosensitive
167

material which, when cooled with saline, becomes


soft and uncoils, making this device useful for
acute management of detrusor–sphincter dyssy-
nergia (Hamid et al., 2003).
Urethral stents represent a clinically successful
device for management of detrusor–sphincter
dyssynergia in people with spinal cord injury. Al-
though stents do not restore normal control of the
sphincter, their efficacy, simplicity and potential
reversibility makes them an attractive option for
people who would otherwise receive an irreversible
sphincterotomy (Chancellor et al., 1999b).

Intraurethral pump

In 1997, Nativ et al. (1997) described a device in-


corporating a miniature valve and pump that
could be inserted into the urethra to control both
continence and voiding in women. The In-FlowTM
intraurethral pump (SRS Medical Systems, Inc.,
Billerica, MA, USA) is designed to manage chron-
ic urinary retention caused by an atonic bladder or
urethral dysfunction. The device secures itself in
the urethra by means of flexible fins that open in
the bladder and a flange at the external urethral
meatus (see Fig. 1). The device is controlled by a
remote activator that is placed over the pubic area
and is magnetically coupled to the pump. Once
activated, the turbine actively pumps urine out of Fig. 1. The In-FlowTM intraurethral pump. (A) Photograph
the bladder at a rate of 6–12 ml/s until the bladder showing the unfolded petals that secure the device in the ure-
is empty. The device is easily inserted by a phy- thra and prevent migration. (B) Diagram showing the place-
sician and can be removed by the patients if they ment of the device in the urethra. Adapted from Madjar et al.
(1999) and Schurch et al. (1999).
wish. The device is designed to be replaced every
month, but successful usage to an average of 90
days has been reported, at which time the device
can become fouled by salt deposits (Madjar et al., reported success rates of 50% with average follow-
1999). up times of 3 and 7.6 months respectively. Most of
In a study of 18 women with spinal cord injury those patients that adopted this device for long-
and hyporeflexive bladders, only six continued to term usage were previously dependent on clean
use the device at follow-up (mean 9.6 months) intermittent catheterization and preferred the con-
(Schurch et al., 1999). Discomfort, incontinence, venience of this device. Intraurethral pumps are
urinary tract infections, technical failures, urethral very interesting from a technical viewpoint and
dilation and the possibility of long-term urethral further investigation with clearer indications for
damage were cited as reasons why this device was use, such as complete spinal cord injury, atonic
unsuitable for chronic use. Studies in 60 (Mazouni bladder and previous dependence on clean inter-
et al., 2004) and 92 (Madjar et al., 1999) patients mittent catheterization may improve the success
with voiding dysfunction from various etiologies rate among people with spinal cord injury.
168

Electrical stimulation devices for control of the is below the threshold required to elicit bladder
lower urinary tract contractions directly via stimulation of the efferent
portion of the pelvic nerve or of the detrusor
While mechanical devices are necessarily limited to myocytes themselves. Acute studies in rats and
treating symptoms of the neurogenic bladder, elec- cats have confirmed the hypothesis that in-
trical stimulation techniques allow devices to be travesical electrical stimulation acts by stimulat-
created that can exert control over spared muscles ing stretch-sensitive mechanoreceptors in the wall
and their neural control systems. Electrical cur- of the bladder that reflexively cause contractions
rent, passed between two electrodes, can be used to of the bladder (Ebner et al., 1992).
generate action potentials in surviving neurons in Few reports of intravesical electrical stimulation
the spinal cord or peripheral nerve below the le- studies in people with spinal cord injury exist, but
sion in spinal cord injury patients. These artificial- one dealing specifically with subjects with incom-
ly generated action potentials can lead directly to plete spinal cord injury reported improvements in
muscular contraction or they can modulate the bladder sensation, detrusor contraction and resid-
activity of neuronal networks and reflex pathways ual volumes in almost all subjects (Madersbacher
(termed neuromodulation). et al., 1982). A retrospective study on the effec-
The discussion below of devices and techniques tiveness of intravesical electrical stimulation for
that have been developed to control the lower people with spinal cord injury by the same author
urinary tract is organized by the location of stim- indicated that one third of the subjects experienced
ulation electrodes rather than by the neurophys- improvements in sensation, detrusor contractility
iological mechanisms on which the devices operate and voluntary control. This occurred only in in-
or by their intended function. Five primary loca- dividuals with preserved pain sensation in the
tions can be identified where electrical stimulation S2–S4 dermatomes (Madersbacher, 1990). This
electrodes can be placed: on or in the bladder, on would seem to be the only predictor of the efficacy
the skin, peripheral nerve, sacral roots and in the of this therapy. Additionally, patients require
spinal cord itself. Figure 2 shows the various stim- many hours of treatment before the effectiveness
ulation locations for devices discussed throughout of intravesical electrical stimulation can begin to
this review. be evaluated and the positive results reported by
some investigators (Kaplan, 2000) have not been
repeatable by others (Decter, 2000). While in-
Electrical stimulation of the bladder travesical electrical stimulation has been used to
treat patients with spinal cord injury, recent stud-
Intravesical stimulation ies have focused on children with underactive
Intravesical electrical stimulation was the first bladders (Gladh et al., 2003). Intravesical electrical
attempt at treating bladder dysfunction using elec- stimulation seems ultimately unattractive as a clin-
trical stimulation. In 1878, M.H. Saxtorph de- ical technique to improve micturition in people
scribed a technique in which stimulation between a with spinal cord injury as it only seems to work in
catheter-mounted electrode, passed into the blad- some with incomplete spinal cord injury, requires
der to act as the cathode (see Fig. 2A), and a sup- long treatments before effectiveness can be evalu-
rapubically placed indifferent electrode, was used ated and the results have not been repeatable
to treat urinary retention caused by an underactive among investigators.
bladder (reviewed in Madersbacher, 1990). In-
travesical electrical stimulation is essentially a ne-
uromodulation therapy intended to reinforce the Bladder wall stimulation
weak functioning of existing neural micturition Electrical stimulation of the exterior surface of the
pathways by stimulating mechanoreceptors in the bladder (see Fig. 2B) was first studied in the early
bladder wall to facilitate reflex bladder contrac- 1950s (Boyce et al., 1964). This marked the begin-
tions and improve sensation. Electrical stimulation ning of the development of electrical stimulation
169

Fig. 2. Electrode locations for controlling the lower urinary tract. The locations are numbered primarily by the order in which they are
discussed in the text. The location for a posterior rhizotomy is also indicated. (A) Intravesical, (B) bladder wall, (C) Thigh, (D) pelvic
floor, (E) dorsal penile nerve, (F) tibial nerve, (G) pelvic nerve, (H) intradural sacral anterior root, (I) extradural mixed sacral root, (J)
intradural sacral posterior root, (K) sacral nerve, (L) spinal cord, (M) intraurethral, (N) pudendal nerve, (O) sacrum.

devices to elicit voiding directly, in response to the enough to generate useful bladder contractions
high morbidity and mortality associated with cath- spread to surrounding structures causing co-
eterization (Bradley et al., 1962). Several groups activation of the external urethral sphincter and
developed implanted stimulators inductively pelvic floor musculature. It was noted that the
coupled to external transmitters with variations canine bladder is primarily an abdominal organ,
in the design, placement and number of electrodes whereas the human bladder is a pelvic organ and is
(Bradley et al., 1962; Hald et al., 1967; Stenberg in close proximity to the pelvic floor musculature,
et al., 1967; Susset and Boctor, 1967; Merrill and increasing pelvic floor susceptibility to contraction
Conway, 1974; Magasi and Simon, 1986). Initial by current spread (Bradley et al., 1963).
animal experiments demonstrated that dogs with Because of this problem, experimental and clinical
spinal cord transections were able to void regularly work was then directed toward obtaining sufficient
using the implanted stimulators without requiring contraction of the bladder while limiting current
additional procedures (Bradley et al., 1962, 1963; spread. Tape electrodes and more powerful stimula-
Kantrowitz and Schamaun, 1963). However, re- tors successfully elicited micturition, but infection
sults in spinal cord injury patients implanted with and technical failures prevented evaluation of their
these stimulators were much less successful (Bradley long-term effect (Bradley et al., 1963). Experience
et al., 1963; Hald et al., 1967; Stenberg et al., 1967; with the Avco stimulator, in which individual wires
Susset and Boctor, 1967; Merrill and Conway, were embedded into the bladder wall, were also
1974). The primary reason that these people were hampered by activation of urethral and pelvic floor
unable to void was that stimulation currents high musculature (Hald et al., 1967; Stenberg et al.,
170

1967). Another stimulator design, the Mentor


bladder stimulator, used two helical wire electrodes
sewn into the bladder wall. This was successful in
two of five people with upper motoneuron lesions,
but required subarachnoid injections of phenol
to abolish electrically induced detrusor–sphincter
dyssynergia that otherwise prevented micturition
(Merrill and Conway, 1974). Susset and Boctor
(1967) reported a successful implant that incorpo-
rated eight disc electrodes around the dome of
the bladder in a person with a complete lower
motoneuron lesion. These investigators considered
upper motoneuron lesions to be a contraindication
for implantation of these systems due to the un-
wanted activation of sphincter and pelvic floor mus-
cles. The most successful report of bladder wall
stimulation was made by Magasi and Simon (1986)
in which 29 of 32 subjects with neurogenic bladder
paralysis attained complete voiding with eight disc
electrodes implanted around the bladder (see
Fig. 3). However, the concomitant sphincter acti-
vation reported by most investigators, lead and
electrode breakage, receiver malfunction, bladder
perforation and pain caused failure in most human
studies. With the success of sacral root stimulation
(see below) for restoring micturition in people with
upper motoneuron lesions, and the multiple diffi-
culties in achieving successful clinical results with
bladder wall stimulation, recent work in this area
has focused on people with lower motoneuron
lesions who cannot benefit from sacral root stim-
ulation (Walter et al., 1999).

Transcutaneous electrical stimulation


Fig. 3. The bladder wall stimulator used by Magasi and Simon
Thigh stimulation (1986). (A) The intended positioning of electrodes on the blad-
der. (B) Actual positions of electrodes around the bladder in
In 1986 it was reported that electrical stimulation
one female subject. Reprinted from Magasi and Simon (1986)
through surface electrodes over the thigh muscles with permission from S. Karger AG, Basel.
(see Fig. 2C) could cause changes in the urody-
namic parameters of spinal cord injury patients spasticity in cord-injured people, noted that 16 of
(Wheeler et al., 1986). Stimulation was applied 32 subjects became continent (Shindo and Jones,
through bilateral quadriceps surface electrodes on a 1987) perhaps indicating a suppression of detrusor
daily basis for 4–8 weeks. Some people exhibited hyperreflexia. A more recent study examining uro-
persistent increases in bladder capacity and/or re- dynamic changes in response to thigh muscle stim-
ductions in bladder pressure, while others experi- ulation showed that 8 of 14 subjects, including one
enced the opposite result. Another study, examining person with spinal cord injury and neurogenic de-
hamstring and quadriceps stimulation to reduce trusor overactivity, increased their bladder volumes
171

by 450% (Okada et al., 1998). However, no meth- results in people with spinal cord injury for sup-
ods of identifying those people likely to respond pressing hyperreflexive bladder contractions are
positively to treatment exist. None of these studies mixed (reviewed in Previnaire et al., 1998). Given
noted any adverse side effects from the treatment. the side effects of maximal functional electrical
The effects of electrical stimulation of