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Endodontic Topics 2010, 17, 1241 2010 r John Wiley & Sons A/S

All rights reserved ENDODONTIC TOPICS


1601-1538

Inflammatory nerve responses in


the dental pulp
INGE FRISTAD, ATHANASIA BLETSA & MARGARET BYERS

Tooth pulp has a dense innervation and a rich vascular supply to maintain homeostasis and to preserve the integrity
of the tissue. Function, trauma, and antigenic challenges make teeth and supporting tissues susceptible to tissue
injury and inflammation, partially due to the lack of collateral blood and nerve supply and to their low compliance.
This review focuses on dental nerve functions and adaptive changes in the pulpal nerve supply following
inflammation and peripheral injury. Overviews of dental innervation and its development and of the peptidergic
innervation of oral tissues are presented, followed by a discussion of peripheral and central changes after local insults
to teeth and peripheral nerve injuries. The functional implications of these adaptive changes are considered.

Received 13 February 2009; accepted 3 September 2009.

Innervation of teeth and supporting ing molecules in a spatial and time-dependent manner
tissues (3). Teeth are well-defined peripheral target organs, and a
highly sophisticated neural system is required for both
Sensory nerves: The trigeminal nerve, with neurons their function and protection. Dental trigeminal axon
located in the trigeminal ganglion, provides the sensory growth and patterning are tightly linked to advancing
supply to the oral tissues, including teeth and supportive tooth morphogenesis and cell differentiation. The dental
structures. The trigeminal nerve emerges from the ventral nerve fibers reach the tooth germ during a period of
surface of the pons, near its upper border. This cranial programmed cell death within the trigeminal ganglion
nerve has three divisions: the ophthalmic, the maxillary, neurons. During this period, the survival of the neurons is
and the mandibular branch. The maxillary and mandib- dependent on growth factors supported by the target
ular branches innervate the upper and lower jaws, field (4). Several studies, both in human and murine
respectively. The neurons supporting the upper and lower teeth, have shown that axon growth and patterning take
teeth are organized in distinct locations in the ganglion place in a spatio-temporally controlled manner, tightly
(Fig. 1). Sensory innervation of periodontal tissues also linked with advancing tooth morphogenesis (510).
comes from the trigeminal mesencephalic nucleus (1, 2). Innervation of dentin takes place during early tooth
Autonomic nerves: The autonomic innervation of development (8, 1113). At the start of root formation in
the pulp and periodontium can be subdivided into rat teeth, the pioneer sensory fibers are already approach-
sympathetic and parasympathetic nerves, although the ing the odontoblast layer and dentin in the regions where
presence, origin, and distribution of the latter are the largest amount of dentin is formed (8, 1113).
controversial and still debated. Post-ganglionic sympa-
thetic nerve fibers innervating oral tissues originate
Neurotrophins in tooth development,
from the superior cervical ganglion (SCG) (Fig. 1).
maturation, and aging
Developmental aspects When innervation commences, the nerve growth
factor (NGF) concentration in the tooth pulp is high,
Tooth development and innervation
but it declines afterwards (14). NGF therefore seems to
Teeth, like many other organs, develop as a result of play an important role in the establishment of pulpal
epithelialmesenchymal interactions, governed by signal- innervation, since a reduced number of both sympa-

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Inflammatory nerve responses

Fig. 2. Nerve growth factor (NGF) synthesis is retained


in adult teeth, especially in subodontoblast coronal pulp,
Fig. 1. Sensory nerves to teeth and supporting tissues and is up-regulated after injury. NGF-mRNA is up-
originate from the trigeminal ganglion (TG). Post- regulated in the mesial pulp horn 6 h after cavity
ganglionic sympathetic nerve fibers innervating oral preparation (asterisk). Reprinted with permission from
tissues originate from the superior cervical ganglion Taylor & Francis Ltd. (30).
(CSG). Max, maxillary area; Mand, mandibulary area.

important for the maintenance of neuronal phenotype


thetic and sensory axons has been found after admin- characteristics. During development, all dental fibers
istration of NGF-antibodies to neonatal rats (15). The appear to require NGF and TrkA at some stage (46),
pulpal sensory nerves are found to express the nerve whereas in adult teeth, the large trigeminal neurons are
growth factor receptor p75 (p75NGFR) and tyrosine kinase potentially dependent only on dental pulp-derived glial cell
A (TrkA) (13, 1619), that bind all known neurotrophins line-derived neurotrophic factor (GDNF) while small
with low affinity (20, 21). In addition, TrkB receptor of dental trigeminal neurons seem to be dependent on NGF
the high-affinity group, binding brain-derived neuro- (27, 47). This suggests that GDNF may function as a
trophic factor and neurotrophin-3 (NT-3) (20, 22), is neurotrophic factor for subsets of nerves in the tooth,
shown to be expressed by sensory nerves during tooth which apparently mediate mechanosensitive stimuli (Fig.
development (23). Few, if any, pulpal afferents express the 4), whereas NGF is suggested to support neurons res-
high-affinity NT-3 receptor TrkC (18). From recent ponsible for nociception (48). This phenomenon is found
research, different neurotrophins have been connected throughout the peripheral nervous system (4951), and
to the development of different neuron types, and hence has gained substantial support since the discovery of NGF
to the development and survival of the different fiber types (52). NGF is the most extensively investigated among the
(24). The discovery of receptors sensitive to a number of trophic factors that mediate target support of neurons
growth factors in teeth and supporting tissues indicates (53), and has come to be widely used as a model in the
that these growth-promoting signals may be involved in study of neurotrophic factors (54). Binding of target-
the regulation of nerve in-growth, and like NGF (25, 26), derived NGF is dependent on specific receptors located on
these growth factors are expressed in a spatial and temporal the axonal surface, with subsequent internalization and
manner in developing and mature teeth (2730) (Fig. 2). transport to the cell body, where the effects are mediated.
There is a considerable anterograde axonal transport in The retrograde and anterograde axoplasmatic trans-
dental sensory fibers (12, 3138), including neuropep- port systems represent bi-directional communication
tides and cytoplasmatic components (Fig. 3). There is between the neuron and the peripheral target organ,
also a robust retrograde axonal transport (39), which and can be utilized in tracing studies to establish
includes an active uptake of neurotrophic factors (15, peripheral and central projections of axons and nerve
27, 4042) and transport from peripheral target tissues endings (11, 15, 29, 3137, 39, 5560) (Fig. 5). The
to the cell body (27, 4345) (Fig. 4). neurotoxic drug capsaicin (an extract from red pepper),
Neurotrophic and target-derived factors regulate acting on vanilloid receptors expressed on sensory C
neuronal structure, survival and function, and are fibers (6062), has been reported to destroy the micro-

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Fristad et al.

Fig. 3. Trigeminal pulpal nerve fibers labeled by anterograde axonal transport of protein labeled with 3H-proline. Nerve
fibers branch in crown and innervate peripheral pulp and inner dentinal tubules of dog (A) and rat (B) teeth. Reprinted
with permission from Alan R. Liss Inc. (37, 38).

Fig. 4. Glial cell line-derived neurotrophic factor (GDNF) is produced in the dental pulp (A). Radioactive labeled
GDNF injected into the dental pulp is transported to neurons in the trigeminal ganglion by retrograde transport. GDNF
is found in a subset of nerves which apparently mediate mechanosensitive stimuli (B and C). OMR, ophthalmic-maxillary
region; BT, brain tissue (control); D, dentin; O, odontoblast layer; P, pulp; G, gingiva. Courtesy of Dr. I. Hals
Kvinnsland. Reprinted with permission from Blackwell Munksgaard (27).

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Inflammatory nerve responses

Fig. 5. Tracing and characterization of pulpal neurons in the trigeminal ganglion. A trigeminal ganglion section labeled
with d-opioid receptor (DOR) antibody (A) and pulpal neurons retrogradely labeled with Fluorogold (FG) (B).
Reprinted with permission from Elsevier (60).

tubule system, thus resulting in the blockage of both the Origin, axonal transport, and distribution
afferent and efferent axoplasmatic transport (63, 64). Sensory nerves
Although neurotrophins and their receptors are There are at least six distinct types of dental sensory nerve
expressed in a spatial and time-dependent manner fibers based on physiology: A-b, A-d-fast, A-d-slow, and
during tooth development (13, 23, 25, 29), several three kinds of C-fibers (17, 70, 71, 7793). Neuropep-
studies have shown that the neurotrophic property of tides including CGRP (Fig. 6) and peptides of the
the pulp is retained and may also be up-regulated in tachykinin group, such as SP and NKA, are synthesized
mature teeth (13, 23, 27, 30, 65) (Fig. 2). on ribosomes in the cell body of sensory neurons (94,
95). They are transported both centrally and in the
The peptidergic nerves peripheral axons. The first evidence for axonal transport
The discovery of neuropeptides as transmitters and sig- of sensory neuropeptides came from immunohistochem-
naling molecules has given new insight into neurobiology ical data 30 years ago for sensory fibers in general (96)
and the function of the peripheral nerves, including the and for dental axons in particular (66). The overall
structure and function of the nerve fibers in the pulp average transport rate for SP and CGRP in rodents has
dentin complex. The neuropeptide first demonstrated in since been found to be 11.25 mm/h, with 2030%
the dental pulp was substance P (SP) (66), a member of transported at a rate of 610 mm/h (97100). The
the tachykinin family. Since then a large number of neuropeptides are transported in afferent nerve fibers
neuropeptides including calcitonin gene-related peptide (Fig. 6) and stored in vesicles in the axons and in the
(CGRP), neurokinin A (NKA), neuropeptide Y (NPY), sensory nerve terminals (Fig. 7a).
vasoactive intestinal polypeptide (VIP), and somatostatin At an ultrastructural level, co-localization of tachy-
has been demonstrated immunohistochemically, and their kinins and CGRP in large dense-cored vesicles has been
origin, occurrence, and spatial distribution in teeth documented for both the central and peripheral
and supporting tissues have been studied and mapped projections of sensory neurons (101104), including
(8, 6676). co-existence in nerve fibers in the dental pulp of rats

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Fristad et al.

and cats (70, 105). In the gingiva, periodontal Sympathetic nerves


ligament and pulp, sensory nerve fibers are located The sympathetic innervation of teeth derives from the
both along blood vessels and as free nerve endings in SCG (106, 107). Post-ganglionic sympathetic nerves
the tissue. In the pulp periphery, sensory nerve fibers travel with the internal carotid nerve, join the
branch extensively with a dense network of nerve fibers trigeminal nerve at the ganglion, and supply teeth
found in the subodontoblast layer, often penetrating and supporting structures via the maxillary and
0.10.2 mm into the inner part of dentin as free nerve mandibular division of the trigeminal nerve (107,
endings (Figs. 7a and 8). 108). Besides the classical neurotransmitter noradre-
nalin (NA), sympathetic nerve fibers contain the
neuropeptide NPY (109). NPY is synthesized in
sympathetic neurons and supplied to nerve terminals
by axonal transport (110, 111) (Figs. 7b and 9). In
contrast, NA is mainly produced locally in the nerve
terminals. Ultrastructural analysis of sympathetic nerve
terminals has demonstrated that NA and NPY are
stored in small and large vesicles, the small containing
only NA or NPY and the large containing both NA and
NPY (112). Tyrosine hydroxylase (TH) is the rate-
limiting enzyme in the production of NA and is widely
distributed in peripheral sympathetic pulpal fibers (74).
Therefore, TH has also been used for the characteriza-
Fig. 6. Sensory neuropeptides, including calcitonin tion of these nerves. The presence of adrenergic nerves
gene-related peptide (CGRP), are synthesized in has been demonstrated in the dental pulp of various
neurons in the trigeminal ganglion, and transported to species including man (106, 107). Sympathetic nerves
oral tissues by axonal transport. Bar 5 100 mm. in pulp and oral tissues are sparse in number compared

Fig. 7. (A) In the pulp periphery, calcitonin gene-related (CGRP)-immunoreactive nerve fibers branch extensively with a
dense network of nerve fibers found in the subodontoblast layer, odontoblast layer and dentin. (B) Neuropeptide Y
(NPY)-immunoreactive fibers are generally found around blood vessels in the root pulp and deeper parts of pulp proper.
Bars 5 100 mm.

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Inflammatory nerve responses

and terminal axons which are crowded by synaptic


vesicles are 1520 nm in the smaller arterioles (10
15 mm in diameter).
Chemical sympathectomy, either by guanethidine or
6-hydroxydopamine, removes all TH-immunoreactive
fibers in teeth and supporting tissues. Surgical removal
of the sympathetic cervical ganglion or chemical
sympathectomy likewise eliminates almost all NPY-
immunoreactive nerve fibers in the dental pulp (68,
115, 116), indicating their sympathetic origin.

Parasympathetic nerves
The presence of cholinergic nerves in dental tissue has
Fig. 8. Sensory nerves fibers branch in the pulp been and is still controversial (117), although it has
periphery, penetrating up to 0.10.2 mm into the inner been concluded that there is an absence of parasympa-
part of dentine as free nerve endings. C-fibers (green) are thetic vasodilation in the cat dental pulp (118). It has
located in the pulp proper, whereas A-fibers (blue) been reported that VIP is localized in the parasympa-
innervate the inner part of the dentine.
thetic neurons (119, 120). The origin of these fibers is
uncertain, since no form of surgical denervation has
resulted in complete loss of VIP-containing fibers from
the peripheral target (121). VIP-immunoreactive
nerve fibers have been immunohistochemically dem-
onstrated in human (67) and feline (122, 123) dental
pulp. In rat dental pulp, however, evidence for the
presence of VIP has not been demonstrated either by
the use of immunohistochemical or by functional
methods under normal conditions; but VIP is up-
regulated in sensory neurons (124127), including the
dental pulp (127), following nerve injury (see Periph-
eral injury and neuronal plasticity).

Functions of peptidergic nerves


Fig. 9. Section from the cervical sympathetic ganglion.
Neuropeptide Y (NPY) is synthesized in sympathetic Sensory nerves
neurons and supplied to nerve terminals by axonal The tachykinins SP, NKA and neurokinin B (NKB),
transport (arrows). Bar 5 25 mm.
and CGRP are peptide molecules released from sensory
nerve cells. Upon release, these signaling molecules
to nerve fibers conveying CGRP and SP (8, 76). The exert a broad variety of regulatory functions both in the
distribution of the sympathetic fibers also differs from central nervous system (CNS) and in peripheral target
the sensory nerves, as NPY- and TH-immunoreactive tissues (128130). The biological effects of the
fibers are normally not found in the odontoblast layer tachykinins SP, NKA, and NKB are mediated by three
and dentin, but are mainly associated with larger blood distinct receptors, the tachykinin receptors neurokinin-
vessels in the root pulp and deeper parts of the pulp 1 (NK1), neurokinin-2 (NK2), and neurokinin-3
proper (8, 68, 73, 74) (Fig. 7b). An intimate structural (NK3). Based on pharmacological features of selective
association between nerves and microvessels in human agonists and antagonists in functional studies, CGRP
dental pulp has been shown (113, 114). The vasomo- acts via two main receptor subtypes, named CGRP1-
tor nerves are mostly adrenergic in nature, and and CGRP2-receptor (131). CGRP1-receptor is
arterioles are the most densely innervated vessels. In highly sensitive to the antagonist properties of
addition, the distances between smooth muscle cells CGRP8 37 (79, 132, 133). Apart from sensory

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Fristad et al.

Fig. 10. Immunohistochemical micrographs from the first rat molar pulp and gingiva showing CGRP-immunoreactive
nerve fibers after intermittent long-lasting tooth stimulation (A and B) and the corresponding unstimulated control (C
and D). Simultaneous recordings of systemic arterial pressure (PA) and pulpal blood flow (LDF) before, during, and after
repeated electrical tooth stimulation (arrows) of an innervated first molar (E) and 3 days after unilateral inferior alveolar
nerve axotomy (F). D, dentine; P, pulp; n, gingival; OE, oral epithelium; JE, junctional epithelium; PU, perfusion units.
Bars: (AFD) 5 100 mm. Reprinted with permission from Elsevier (134).

functions, the pulpal nerves seem to take an active part species in vitro. According to these results, the
in the maintenance of both function and structure. A mechanisms underlying smooth muscle relaxation are
wide range of local effector functions is exerted by the either endothelium-independent or endothelium-
sensory nerves in peripheral target tissues. After dependent. The endothelium-independent action
sensory nerve stimulation (Fig. 10), neuropeptides shown for CGRP (149153) seems to be mediated
are released in the tissue (134137), and mediate through cyclic adenosine monophosphate accumulation
sensory (138, 139) and vasoactive functions (79, 134, in smooth muscle cells. The endothelium-dependent
140) (see also the review on circulation in this issue). In mechanism shown for CGRP, SP, and NKA (154, 155)
addition, the CGRP and tachykinins are reported to may act through the production of nitric oxide (NO; also
promote mitotic effects on fibroblasts (141, 142), known as endothelium-derived relaxing factor) in
smooth muscle cells (143), and endothelial cells (144), endothelial cells, which mediates second messenger
and exert influence on bone metabolism (145147) systems in smooth muscle cells. A mechanism causing
and the micro-hardness of dentin (148). Furthermore, vasodilation has been proposed, acting through adeno-
CGRP stimulates bone formation in vivo (147) and sine triphosphate (ATP) sensitive K1 channels in the
proliferation of pulpal fibroblasts in vitro (142). The vascular smooth muscle cell membrane, since K1-ATP-
direct effects of such effector functions are dependent activated ion channel inhibitors prevent CGRP-induced
on receptor-mediated signaling between nerves and vasodilation (156, 157). In the dental pulp, however, a
target tissues. An indirect function is also possible, blockade of the K1-ATP-activated ion channels resulted
mediated by signaling molecules secreted by pulpal in a reduced resting blood flow, whereas the vasodilation
cells either in a paracrine or an autocrine fashion. induced by tooth stimulation was unchanged (158).
Effect on the vascular system: The effects of sensory Another mechanism is the indirect action on endothelial
neuropeptides on the vascular system are obtained cells via inflammatory mediators such as histamine,
from tissue preparations of blood vessels from different bradykinin, leukotriene, and prostaglandin. The intimate

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Inflammatory nerve responses

Fig. 11. Pulpal survival is much better in innervated rat molars (A) at 6 days after occlusal pulp exposure, than in
denervated molars (B). That difference was not found for injuries on the side of the crown. Adapted from Bayer &
Taylor (168). Reprinted with permission from International and American Associations for Dental Research.

relationship between nerves and the vascular system is kocytes has been shown in the pulp (134) (Fig. 12),
further strengthened by the finding that blood vessels giving further support for a role of the sensory nerves as
express receptors to NGF (17). immunomodulators.
Effect on the immune system: Reports of functional Transendothelial migration of immunocompetent cells
receptors for neuropeptides on immunocompetent into inflamed tissues requires adhesion molecule
cells (159163) have given further evidence that nerves mediated events with activation of both selectins and
are capable of modulating the inflammatory process, integrins (169171). Although up-regulation of adhe-
and thus take an active part in the cellular inflammatory sion molecules of both integrins and members of the
defense response after local injury. Intra-articular selectine family has been reported after intradermal
injection of SP exacerbates arthritis in rats (164), injection of SP, CGRP, and VIP (172) in human skin,
whereas sensory denervation with capsaicin attenuates conflicting results concerning a direct or an indirect
inflammation and nociception in arthritic rats (165), effect of neuropeptides on adhesion molecule activation
and prevents inflammation and accumulation of have been presented. It has been suggested that mast cell
immunocompetent cells in ligature-induced perio- degranulation after administration of SP may cause
dontitis (166). In the dental pulp, a less pronounced activation of adhesion molecules in vascular endothelium
recruitment of immunocompetent cells was found in (173) and be responsible for the granulocyte infiltration
the tissue subjacent to deep dentinal cavities in found in mouse skin (174, 175). However, the
denervated compared to innervated teeth (167), and uninjured dental pulp is almost devoid of mast cells
pulpal damage was much greater after small pulp (176, 177), and the recruitment of granulocytes
exposure injury to denervated rat molars compared to observed during experimental neurogenic inflammation
innervated teeth (168) (Fig. 11). (134) could be mediated indirectly through the release
Following neurogenic inflammation induced by of components from other target cells rather than via
long-term intermittent tooth stimulation in young mast cell degranulation. Macrophages are stimulated to
rat molars, accumulation of polymorphonuclear leu- release cytokine by CGRP and SP (178180), and

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Fristad et al.

Fig. 12. Immunohistochemical micrographs from the pulp proper of rat first molars. Polymorphonuclear leukocyte
(W3/13) labeled sections after intermittent long-lasting electrical tooth stimulation of an innervated (A) and
denervated (B) molar. D, dentine; n, odontoblast layer. Bars 5 100 mm. Reprinted with permission from Elsevier (134).

accumulated CGRP-like immuno-reactivity is located in neuropeptides on pulpal connective tissue and en-
inflamed rat dental tissue (181). Neuropeptide stimula- dothelial cells, and their vasoregulatory functions, may
tion causes the release of factors such as tumor necrosis be of crucial importance for tissue repair after injury to
factor and interleukin-1 (IL-1), which are important the pulpdentin compartment.
mediators for the activation of adhesion molecules on Effect on hard tissue cells: The possibility that
vascular endothelium (182186). odontoblasts are under nervous control, and that
Earlier, the uninflamed dental pulp was believed to be nerves affect the function of odontoblasts either
devoid of immunocompetent cells. However, studies of directly or indirectly, has been debated. Denervation
uninflamed rat and human teeth (187) have shown that studies have shown conflicting results concerning the
a variety of immunocompetent cells are distributed in effect of innervation on dentin formation. Enhanced
both the coronal and apical part of the pulp. These secondary dentin formation was found after denerva-
results have been confirmed in subsequent studies (188, tion and cavity preparation in cats (190), whereas a
189). Macrophages are present in large numbers in reduced secondary dentin formation has been shown
uninflamed rat dental pulp (167, 188, 189). Therefore after inferior alveolar nerve (IAN) axotomy and
macrophages may be a source of inflammatory mediator capsaicin treatment in rats (191). At an ultrastructural
release that is important for the induction of adhesion level, neonatal capsaicin treatment in rats resulted in
molecules on vascular endothelium. irregularities in dentinal tubuli, and measurements
As the dental pulp is submitted to continuous showed a reduced micro-hardness in the predentin
stimulation from masticatory forces and frequently (148). Recently, odontoblasts, in addition to other
exposed to antigenic components in the oral environ- pulp cells, have been shown to express receptors to
ment, a bi-directional communication between the sensory neuropeptides, including SP and NKA (192,
nerves and the immune system may be postulated. In 193) (Fig. 13). Odontoblasts are derived from neural
this way the pulpal nerves may have an important crest cells, and both developing and mature odonto-
protective function for the maintenance of homeostasis blasts in rats, cats, and humans express the neuro-
in the pulp. In addition, the stimulating effect of chemical marker protein gene product (PGP) 9.5 (8,

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Inflammatory nerve responses

increased number of osteoclasts (205), indicating that


SP has a role in hard tissue remodeling. In addition, it
has been suggested that osteoblasts control osteoclast
recruitment (206). Recently, NK1 receptor has been
identified in various bone cells in the rat (192, 209).

Sympathetic nerves
Sympathetic nerves have been reported to represent
approximately 10% of the intradental nerve supply
(210). Because the majority of fibers innervating the
dental pulp are of sensory origin, the focus on nerves
Fig. 13. Confocal microscopic image showing an and pulpal inflammation has traditionally been on
odontoblast labeled for neurokinin 2 (NK2) receptor sensory nerves. However, the sympathetic nervous
(red), which binds neurokinin A (NKA) with high
affinity. Broken line represents the border between pulp system (SNS) has been shown to modulate immune
and dentine. Reprinted with permission from Springer responses (211), and lately there has been a growing
Science and Business Media (192). interest in the role of sympathetic nerves in dental
inflammation [for a review, see (212)]. Furthermore, as
194, 195). PGP 9.5 belongs to the ubiquitin carboxyl- NA, the classic post-ganglionic neurotransmitter in
terminal hydrolases (196), which are found to be perivascular sympathetic nerves (213), is well known
involved in a variety of cellular functions (197, 198). for its vasoconstrictor role, the focus on sympathetic
Hydrolases have a modifying effect on receptor nerves in pulpal hemostasis has been connected to
function, including T-lymphocyte homing receptor circulation (111, 214216).
(199), platelet-derived growth factor receptor (200), Effect on the vascular system: The vasoregulatory
and growth hormone receptor (201). The expression functions of sympathetic nerves in dental tissues are in
of PGP 9.5 immunoreactivity in dental epithelium accordance with findings in other tissues. Activation of
seems to be paralleled by expression of the neurotro- sympathetic nerves may give rise to co-release of NPY
phin receptor p75NGFR in rats (13, 202) and humans and NA from sympathetic nerve terminals (110, 217).
(203). Thus, a function for PGP 9.5 in regulation of This event is followed by vasoconstriction in the dental
cell receptors and signaling seems plausible. pulp and oral mucosa (218, 219), causing a reduction
The complicated neuro-immune signaling in the dental in blood flow. The effects exerted by the sympathetic
pulp is further illustrated by the recent finding that the nerves are receptor mediated, and evidence has been
odontoblasts are also possible targets for antigens, as provided that the dental pulp is equipped with both a-
microbial pattern recognition receptors are shown to be and b-receptors (219221) and receptors for NPY
expressed by odontoblasts. In addition, odontoblasts are (222). The different responses on pulpal blood flow
able to respond to bacterial challenge by the production of using a1-, a2-, and b-antagonists further indicate the
cytokines (204). The different response to Gram-negative presence of a1-, a2-, and b-receptors in the smooth
and Gram-positive agents raises the possibility of their muscles of the pulpal vessels (223, 224). In the human
participation in innate immunity. dental pulp, however, evidence for b-adrenergic
It is well documented that hard tissue-forming cells vasodilation has not been found (225). NPY exerts
in oral tissues are closely supported by sensory nerves vasoconstriction via a calcium-dependent mechanism
conveying CGRP, SP, and NKA. Functionally, there is which is independent of adrenergic receptors (111,
growing evidence for the presence of different neuro- 214, 226228) and intact endothelium (229). In
peptide receptors on bone cells in vitro and in vivo addition, NPY exerts an angiogenic activity similar to
(205207). The regulating mechanisms behind the basic fibroblast growth factor, showing that sympa-
reported effects of SP on bone formation and resorp- thetic nerves may be important in angiogenesis during
tion are still unclear. SP containing nerve fibers have tissue development and repair (230).
been localized inside resorption lacunae in cementum Effect on the immune system: As both primary and
and dentin following inflammatory root resorption secondary lymphoid organs are innervated by sympathetic
(208), and enhanced local SP release induces an nerves, these organs and cells of the immune system are

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Fristad et al.

natural targets for neutrally derived NA and NPY (231). NPY is the most abundant (248). Osteoblasts and
Moreover, immunocompetent cells express adrenergic osteoclasts are equipped with adrenergic and peptider-
receptors, indicating that they are controlled by the SNS gic receptors, which suggests neuronal regulation of
(232234). Stimulation of adrenergic receptors, either by bone homeostasis (249). Studies involving either
locally released NA or NPY, or by circulating catechola- chemical or surgical sympathectomy provide useful
mines, has an effect on lymphocyte traffic, circulation, and information about the significance of sympathetic
proliferation, and also modulates cytokine production and nerves on hard tissue remodeling. In the majority of
the functional activity of lymphoid cells [for a review, see studies, sympathectomy reduced osteoblastic activity
(211)]. Although contradictory results have been re- and bone apposition, whereas the number of osteo-
ported concerning sympathetic nerves and inflammation, clasts, osteoclast surface area, and bone resorption
the vast majority of the reports indicate an anti- increased (147, 250254). Accordingly, larger peria-
inflammatory and inhibitory effect on immune mechan- pical lesions and an increased number of osteoclasts
isms and inflammation (235237). lining the inflamed area were found in sympathecto-
Activation of the SNS results in the mobilization of mized rats when periapical lesions were induced by
blood leukocytes, a phenomenon also referred to as pulp exposures (255). In the same study, reparative
adrenergic leukocytosis (238), and bilateral surgical dentin formation was reduced by sympathectomy,
sympathectomy is followed by down-regulation of macro- showing that the effect of the SNS on odontoblasts is
phage and neutrophil function, and reduced phagocytic similar to its effect on osteoblasts. Furthermore,
and chemotactic responses (239241). In the same sympathectomy increases root dentin resorption dur-
context, stimulation of sympathetic nerves causes recruit- ing orthodontic tooth movement (243), signifying the
ment of CD431 polymorphonuclear cells in the dental inhibitory effect of sympathetic nerves on odontoclasts
pulp (242) and sympathectomy impairs the recruitment of in the same manner as previously shown for osteoclasts.
these cells during pulpal inflammation (243).
Knockout mice, which cannot produce catechola- Cholinergic nerves
mines, have normal blood leukocyte numbers and
Co-localization and co-release of VIP and acetylcholine
normal T- and B-cell development and function in the
have been reported in oro-facial tissues (41, 119, 120).
absence of infection, but exhibit reduced recruitment
VIP is a potent vasodilator and induces vasodilation in
of neutrophils and impaired T-cell function, including
cat pulp when systemically infused (256). Similar to that
T-helper (Th) 1 cytokine production during infection
of CGRP, the action of VIP has been found to partially
(244). Several infectious and autoimmune diseases
depend on endothelium (257). The vascular effect of
have been attributed to the imbalance between pro-
VIP is reduced after administration of NO synthase
inflammatory (Th1) and anti-inflammatory (Th2)
inhibitor, indicating that VIP acts indirectly via NO and
cytokines (245), and an increasing body of evidence
secondary messenger systems (258).
suggests that the SNS can alter the Th1/Th2 balance
by a shift from a pro-inflammatory (Th1) to an anti-
inflammatory (Th2) response (211). As a periapical
lesion recapitulates the pulpal inflammatory response Plasticity in dental primary afferent
to bacterial infection, it provides an excellent model for
and sympathetic nerves
studying local immune responses. When IL-1a was
measured in rat periapical lesions, it was found that Primary sensory neurons have been extensively in-
sympathectomy increased the levels of this traditional vestigated as a model for neuronal plasticity. These
pro-inflammatory cytokine compared to the contra- experiments have shown that the sensory neurons
lateral non-sympathectomized side, again illustrating undergo phenotypic changes in response to subacute
the anti-inflammatory role of the SNS. and chronic peripheral injury. Recently, adaptive
Effect on hard tissue cells: Histochemical approaches changes have also been shown for the sympathetic
have revealed that bone and adjacent tissues (bone nerve supply (255) (Fig. 14). This section reviews the
marrow, periosteum) have a dense supply of sensory, plasticity of sensory and sympathetic innervation of
sympathetic, and parasympathetic nerves (246, 247). teeth in relation to various durations and recoveries
Quantification of neuropeptides in bone showed that from pulpitis, compared to irreversible pulpitis.

22
Inflammatory nerve responses

Fig. 14. (A) Sections from maxillary first molar 20 days after pulp exposure, showing neuropeptide Y (NPY)-
immunoreactive fiber sprouting in the pulp adjacent to reparative dentine (RD) formation. (B) Distal root apex of
mandibular first molar. Sprouting of NPY-immunoreactive fibers is seen surrounding the larger blood vessels in apical
periodontal ligament. Bars 5 50 mm. Courtesy of Dr. S. R. Haug. Reprinted with permission from Elsevier (255).

Dental nerves and inflammation modal nociceptors, a type of sensory peptidergic fiber
that promotes tissue protection, pro- or anti-inflam-
Sensory nerves
matory action and healing after injury, as well as pain
Injuries to teeth trigger barrages of action potentials and (261, 262). There is also evidence for extensive
altered neurochemistry in all of the subregions of the vasoregulation by pulpal nerve fibers (214, 263),
afferent neurons: dentinal and pulpal terminals, dental mechanosensitivity of fast conducting fibers (264,
and trigeminal axons, ganglion cell body and satellite 265), and specific thermal sensitivity (266, 267), and
cells, central axons and a myriad of synaptic connections a possibility that some of the dental innervation is
in the brainstem, primarily in the nucleus caudalis. Along participating directly in the innate immune defense by
with these neural changes, there are also specific reactions responding to bacterial antigens (268), or by release of
of peripheral and central glia that can enhance or neuropeptides that can mimic antimicrobial defensins
modulate pain, especially if the peripheral inflammation (269). When teeth become inflamed or have pulpal
persists and continues to cause changes in the afferent damage, all of the dental neural subtypes can be
input to the CNS (259, 260). As indicated above in affected, and if the inflammation or trauma includes
relation to peptidergic nerve functions, there are the periodontal tissues and alveolar bone, then sensory
numerous interactions of dental sensory nerve endings innervation to those tissues will also be drawn into the
with dentin, odontoblasts, pulp cells, immune cells, and cascade of neuraltissueimmune-vascular communi-
the pulpal vascular system which induce changes within cations and reactions.
the afferents. In turn, these affect all of the components Neural reactions cause a perception of pain for most
of pulp and the progress of inflammation, and contribute tooth injuries, although if the damage is slow or
to the tooths ability to limit initial damage, fight off shallow, as during initial caries or gradual occlusal wear,
infection, and repair itself. there is usually not any perceived sensation. As is true
Tooth pulp and dentin have evolved for supporting for all tissues, the type of neural reaction, its severity,
enamel, and a rich sensory innervation by peptidergic and its duration vary depending on the extent of
and non-peptidergic nerve fibers terminates in periph- damage and duration of inflammation (262). Thus,
eral coronal pulp and the inner 0.1 mm of dentinal dental neural responses to a mild injury which causes
tubules. Sensations of sharp pain involve the faster no significant pulp damage may resolve within hours or
conducting dentinal fibers, while a diffuse ache days, perhaps with some enhanced dentinogenesis to
primarily involves specific small nociceptors and poly- form reactionary dentin (270, 271) (Fig. 15a). As

23
Fristad et al.

Fig. 15. (A) Mild injury. No pulp damage at 4 days after unetched cavity. Sprouting CGRP fibers orient under the lesion.
Reprinted with permission from Elsevier (270). (B) Reversible pulpitis. Fourth day after etched deep cavity. Abscess is
present. Calcitonin gene-related peptide (CGRP) fibers sprout outside the developing fibrous barrier. (C) Healed
reversible pulpitis with reparative dentine at 3 weeks after injury. CGRP intensity has been reduced in nearby pulp
nerves. (B and C) reprinted with permission from Elsevier (272).

dentinal cavities go deeper and approach the pulp, peripheral inflammation can alter conduction rates and
there is increasing damage to the odontoblast layer, transganglionic conduction (283285), within the
nearby pulp, innervation, and vasculature. Those ganglion where there can be long-term satellite cell
changes cause neural reactions that persist for weeks changes (286, 287), within the brainstem, and in the
or months (272, 273) (Fig. 15b), and the initial injury thalamus and higher centers (288).
is more severe and more rapidly necrotic when the
sensory innervation is missing (168). If the pulp can
Sympathetic nerves
rebuild the pulpdentin border and seal it with
reparative dentin, then the neural reactions subside As previously described, dental neural responses to
(272) (Fig. 15c), but if irreversible pulpitis occurs, then injury vary according to the degree of damage and
reacting neural endings retreat from the advancing duration of inflammation. The dense sensory innerva-
damage while continuing to sprout and enhance the tion in the dental pulp has led to extensive investigation
neuropeptide levels around the lesion (181, 274) (Figs. of the involvement of sensory nerves in local inflam-
11 and 16). When pulpitis becomes chronic, associated matory responses. Nevertheless, recently there is
neural changes can persist for years (275, 276). If the increasing evidence for the involvement of sympathetic
peripheral changes induce persistent changes in the nerves as well.
CNS, then problems with referred pain, atypical facial Not only sensory but also sympathetic nerve sprout-
pain, trigeminal neuralgia and other neuropathic pain ing takes place during pulpal and periapical inflamma-
may occur (260), as well as the long-term expression of tion (255) (Fig. 14). However, the timing of the nerve
central regulators of gene expression such as c-Fos sprouting varies for the different fiber types. While
(277281) (Fig. 17) and long-term glial reactions sensory nerve sprouting occurs very early during pulp
(282). Thus, the neural contributions to pulpal injury, as early as 1 day (289, 290), sympathetic nerve
inflammation and wound healing include events at sprouting occurs around 3 weeks later (255), at which
the tooth (Figs. 15 and 16), along the nerve where point the sensory sprouting usually diminishes (290).

24
Inflammatory nerve responses

Fig. 16. (A) Nerves and vital pulp are still present in roots during irreversible pulpitis when there is already a periapical
lesion with CGRP nerve fiber changes at 14 days after injury. Reprinted with permission from Alan R. Liss Inc. (274). (B
and C). Normal apex and periapical abscess at 5 weeks after a pulp exposure injury. Nerve fibers with increased CGRP
sprout around the lesion and their incoming fibers in alveolar canals are also much intensified for CGRP. Reprinted with
permission from Elsevier (181).

speculate on the involvement of NPY in throbbing


pain, increased sensitivity during drilling above the
pulp horns in teeth with pulpitis, and persistent
percussion sensitivity in teeth with chronic pulpal and
periapical pathosis.
Furthermore, the sympathetic nerves serve important
effector functions in relation to tissue healing. The
angiogenic effect of NPY (230) may be of particular
significance in revascularization during repair, and
reparative dentin formation is a wound healing process
in the injured pulpdentin complex. Complementary
sprouting of NPY-containing fibers was prominent
adjacent to reparative dentin after small pulp exposures
Fig. 17. Neurons in medullary dorsal horn still express c- (255). Lately, the quantification of NPY levels in carious
FOS 2 weeks after a rat molar pulp exposure injury. Adapted
compared to non-carious human teeth has been
from (280). Reprinted with permission from Elsevier.
performed by radioimmunoassay (293). The overall
finding that NPY levels were significantly elevated in
A recent study in human teeth showed caries-associated carious compared to non-carious teeth is in line with
changes in the expression of NPY-immunoreactive previous work (291). However, within the caries group,
fibers, but any increases were limited to the tip of the NPY levels were significantly elevated in mildly/
pulp horn (291). The significance of this type of moderately carious teeth (where reparative dentin
sprouting is still not clearly understood. As NPY was formation is likely to be occurring) rather than in grossly
shown to contribute to hyperalgesia after nerve carious teeth. The finding that NPY-containing fibers
damage (292), it seems reasonable to assume that it were observed in the subodontoblastic layer close to the
may also be involved in pain sensation during pulpal predentin supports the hypothesis that NPY could have
and periapical inflammation. More specifically, one can a modulatory role in reparative dentin formation.

25
Fristad et al.

In addition, a caries-associated increase has been inflammation and nerve injury (298, 299). Even a
observed in both the number of VIP-containing fibers pulpectomy procedure implies a peripheral nerve injury
(291) and the levels of VIP (294) in human pulp. As with of up to 1800 unmyelinated axons in a fully developed
NPY, VIP levels were found to be higher in the pulps of single-rooted human tooth (300). The number of
moderately carious teeth compared to grossly carious unmyelinated axons entering the tooth is three to four
ones (295). Moderately carious lesions may imply a times the number of myelinated axons (300). Thus,
reversible inflammatory process in the pulp characterized pulpectomy implies deafferation of nerve fibers, which
by vasodilation and increased pulpal blood flow. Thus, is reported to result in an incidence of chronic pain in
the dynamic increase in VIP levels seen in carious teeth 25% of the cases (301303).
may be related to the vasodilatory role of VIP. Moreover, Peripheral injury to sensory nerves, including branches
teeth with reversible pulpitis must have the ability to of the trigeminal nerve in rats, is shown to induce up-
down-regulate and eventually reverse the sequelae to regulated neuronal transcription of NPY (59, 297, 304
inflammation, and VIP has been identified as a potent 307), VIP (126, 127, 308310), galanin (297, 309),
endogenous anti-inflammatory agent in several ways and pituitary adenylate cyclase-activating polypeptide
(296). The presence of the VIP receptor, vasoactive (PACAP) (305) (Figs. 18 and 19), whereas classical
intestinal polypeptide/pituitary adenylate cyclise-activat- neuropeptides are down-regulated. The altered metabolic
ing polypeptide receptor (VPAC1-R), in the pulp tissue state is also reflected in an up-regulation of growth-
(294) provides further support concerning the func- associated protein (GAP) 43 and its mRNA in sensory
tional role of VIP in pulpal health and disease. neurons and in Schwann cells after peripheral nerve injury
(311314) (Fig. 20). Up-regulated GAP 43 expression is
transported peripherally and is found in axonal growth
Dental nerve injury and regeneration cones and in regenerating axons (315317). Following
IAN axotomy in rats, NPY expression was located in
Peripheral injury and neuronal plasticity
trigeminal ganglion cells that also exhibited an increased
Nerve injury presents a very different survival challenge expression of GAP 43 (311) (Fig. 20), indicating a role for
for the neuron compared to tissue inflammation, and this protein after neuronal injury.
the responses in terms of altered gene expression and The functional consequences of the neuronal plasti-
axonal transport show a marked contrast. When a nerve city and neurochemical changes shown in injured
is injured, the only way to regain normal function is to neurons during regeneration have so far been scarcely
regrow the axon, and so the neuron converts to a elucidated. However, it has been suggested that VIP
growth metabolism and abandons its sophisticated promotes axonal regeneration and survival of neurons
production of molecular machinery for the endings. after nerve injury, and is up-regulated in pulpal nerves
For example, neuropeptides such as CGRP are reduced during regeneration (Fig. 19). In vitro studies have
after nerve injury compared to up-regulation of that shown that VIP may prevent neuronal cell death after
peptide when the nerve endings encounter inflamma- NGF deprivation (318) and act as a growth factor by
tion of the target tissue (297). stimulating NPY gene expression and axonal out-
Following nerve injury, a dramatic shift in the growth in rat sympathetic ganglion cells (319). In
transcription of neuropeptides has been documented. addition, VIP is a potent vasodilator (119). Together
The bi-directional contact between the nerve cell and with the reported stimulating effect on glucogenolysis
the peripheral target tissue is lost, and the neurons and increase in glucose utilization (320), VIP may
change into a state of either regeneration or neuronal serve nutritional functions important for nerve regen-
cell death. The impact on neurons in the trigeminal eration. A functional role for VIP after nerve injury and
ganglion is dependent on the injury site. A peripheral during regeneration of pulpal nerves is indicated as this
injury has less impact than a more centrally located one. neuropeptide is up-regulated in pulpal nerve fibers
However, even a small pulp exposure is shown to under these conditions (127).
induce neuronal changes both in the trigeminal gang- NPY has been reported to control or even inhibit
lion and at the second order neuronal level in the brain transmitter release, both at interneural synapses (321
stem (277, 280) (Fig. 17). This may have implications 323) and at peripheral nerve endings (324). In the dental
for hyperalgesia and chronic pain connected to pulp, NPY may act in a similar manner, since activation of

26
Inflammatory nerve responses

Fig. 18. Micrographs from the trigeminal ganglion (A and B) and dental pulp (C) after nerve injury. Classical
neurotransmittors such as substance P (SP) are down-regulated, whereas neuropeptide Y (NPY) is up-regulated (A and
B). The dental pulp is devoid of sensory nerve fibers after axotomy (C). Reprinted with permission from Elsevier and
Blackwell Munksgaard (59, 134, 311).

Fig. 19. After nerve injury, a pronounced up-regulation of vasoactive intestinal polypeptide (VIP) is seen in smaller-
sized neurons (arrows) in the trigeminal ganglion (left). During regeneration of sensory nerves, a coarse VIP-
immunoreactive fiber (arrows) is seen in the cuspal area approaching the odontoblast layer (right). P, pulp; D, dentine.
Bars 5 50 mm. Reprinted with permission from Elsevier (127).

27
Fristad et al.

Fig. 20. Micrographs from trigeminal ganglion (A), pulp (B), and apical periodontium (C) 4 days after nerve injury
(axotomy). Growth-associated protein (GAP) 43 is up-regulated in trigeminal ganglion neurons (A). In pulp (B) and apical
periodontium (C), thick coarse nerve profiles are GAP 43 immunoreactive. D, dentine; PDL, periodontal ligament; AB,
alveolar bone. Courtesy of Dr. S. M. Khullar. Reprinted with permission from Blackwell Munksgaard (311).

sympathetic nerves in rat incisors has been shown to flexor reflex in rats (330), and galanin seems to inhibit
counteract vasodilation and plasma extravasation induced the release of classical transmitters at least in the CNS
by the sensory nerves (325). Neurons in the trigeminal (331). Functional recovery is rarely complete for
and dorsal root ganglion of rats, rabbits, and monkeys are trigeminal nerves, though many axons find their way
found to express receptors for NPY (326). Therefore, back to the tooth with guidance from growth factors
NPY may regulate transmitter release during nerve such as NGF (332).
regeneration, thus inhibiting nociceptive input centrally
and influencing vasoregulation in peripheral targets.
Dental nerve injury and regeneration
NPY has been found to accumulate in peripheral
neuroma after tight ligation or transection of the rat Dental nerves can be injured in two ways, either by
sciatic nerve (327) and thus transport of NPY in sensory direct injury to specific trigeminal nerve bundles that
nerves has been suggested. Further evidence for the are en route to the teeth (nerve avulsion during tooth
transport of NPY in primary afferent nerves during IAN extractions, jaw trauma, etc.), or by the regression of
regeneration in oro-facial tissues has been shown by the tooth nerve endings away from advancing pulpal
increased expression of NPY in the trigeminal ganglion necrosis until the main nerve becomes involved in the
(Fig. 18), the IAN, and in the teeth and supporting root, periapex, and alveolar canal (Fig. 16). When an
tissues of rats (59, 328, 329). Double immunolabeling alveolar nerve in rats is transected or crushed, the cut
has shown the co-existence of CGRP and NPY in both axons find their regeneration path quickly and can
trigeminal neurons and pulpal nerves during regenera- return to the denervated pulp and dentin of rat molars
tion after IAN axotomy (59) (Fig. 21). in only 23 weeks in order to rebuild their initial
The functional role of the neuronal transcription of dentinal terminal in synchrony with the return of the
galanin and PACAP after injury is still not completely jaw opening reflex response to electric tooth stimula-
understood. However, PACAP has been reported to tion (333, 334). In that situation, the original pulp
induce a long-lasting suppression of a C-fiber evoked dentin complex will not have been damaged and

28
Inflammatory nerve responses

appears to be directing some aspects of the regeneration


through the release of growth factors from the
odontoblasts and/or nearby cells, since those growth
factors have the same density of expression as the
sensory innervation terminal patterns (335). Detailed
analysis of the electrophysiology of reinnervated teeth in
cats and ferrets has shown that there is excellent return
of sensory fibers but that they mostly have slower
conduction rates than under normal conditions (336).
The responses in a physically denervated tooth have
been reported to include increased dentinogenesis as if
the normal sensory innervation holds odontoblasts and/
or their associated vasculature in a retarded dentinogen-
esis state (190). With chemical denervation subsequent
to post-natal capsaicin exposure, there is dysplasia of the
dentinal tubules and the dentin matrix is softer than it
should be (148). The tooth replantation model is an
interesting denervation/regeneration model in which
young rat first molars are extracted and then immediately
replaced in the sockets, thereby denervating and
devascularizing the tooth (337). In that situation, the
sensory fibers and blood vessels begin to re-enter the
Fig. 21. Double immunofluorescence labeling showing pulp within days, and can rebuild a normal innervation if
co-localization of NPY and CGRP in pulpal nerve fibers the original odontoblasts and pulpdentin complex
(arrows) during regeneration of sensory nerve fibers.
survived the trauma (335) (Fig. 22), but more
Sections show the subodontoblast and odontoblast layer
(OB) of a first mandibular rat molar. Reprinted with commonly encounter reparative dentin or bone depend-
permission from Elsevier (59). ing on the pulpal reaction to extraction/replantation.

Fig. 22. (A) At 90 days after replantation of rat first molars, some of the pulps retain columnar odontoblasts (Od) and
associated innervation beneath reactionary dentine (small arrows), while other regions have reparative dentine (Rd) (B).
Nerve fibers (arrowheads) are sparse in the repaired dentine. Bars 5 100 mm. Reprinted with permission from John Wiley
and Sons Inc. (335).

29
Fristad et al.

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