Biochemical Markers Associated With Acute Vocal Fold

Wound Healing: A Rabbit Model

*Ryan C. Branski, *||Clark A. Rosen,
*||Katherine Verdolini, and *||Patricia A. Hebda
Pittsburgh, Pennsylvania

Summary: This study seeks to determine the ability of enzyme-linked immunosorbent assays (ELISAs) of vocal fold secretions to detect and describe the
acute tissue response to injury in a rabbit vocal fold model. Vocal fold
secretions were collected before the induction of a unilateral surgical injury
to the vocal fold and at 6 timepoints after injury (1, 5, 7, 10, 14, and 21
days). Secretions were then subjected to ELISAs to assess concentrations of
interleukin-1 beta (IL-1) and prostaglandin-E2 (PGE-2). The results indicate
that ELISAs may be useful in documenting fluctuations in these markers
associated with the wound healing process in the rabbit model. The temporal
expression of both IL-1 and PGE-2 was consistent with their proposed roles
in the wound healing cascade in other systems, pointing to the potential that
surface secretions may be at least partial indicators of wound healing events
within the tissue.
Key Words: LarynxVocal foldsInjurySecretionsCytokinesWound
healing.

Accepted for publication April 26, 2004.

Presented at the Steven Gray Memorial Session of the Voice
Foundations 32nd Annual Symposium: Care of the Professional
Voice, Philadelphia, PA, June 2003.
Supported in part by NIH DC 005643 (NIDCD).
From the *Department of Communication Science and Disorders, University of Pittsburgh, Pittsburgh, PA; Department of
Otolaryngology, University of Pittsburgh, Pittsburgh, PA; University of Pittsburgh Voice Center, University of Pittsburgh,
Pittsburgh, PA; Otolaryngology Wound Healing Laboratory,
Department of Pediatric Otolaryngology, Childrens Hospital
of Pittsburgh, Pittsburgh, PA; ||McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA;
Department of Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, PA.
Address correspondence and reprint requests to Katherine
Verdolini, PhD, Communication Science and Disorders, School
of Health and Rehabilitation Sciences, 4033 Forbes Tower,
Pittsburgh, PA 15260. E-mail: kittie@csd.pitt.edu
Journal of Voice, Vol. 19, No. 2, pp. 283289
0892-1997/$30.00
2005 The Voice Foundation
doi:10.1016/j.jvoice.2004.04.003

INTRODUCTION
Wound healing in the skin requires a highly organized series of events including hemostasis, inflammation, re-epithelialization, cell proliferation,
matrix deposition, angiogenesis, and wound contraction. A general timeline for such events and key
mediators of each stage have been described in the
skin and result in the resolution of epithelial defects
as well as the reconstitution of functional tissue.
The long-term sequelae of wound healing in the vocal
folds, specifically, the formation of vocal fold scar
and the resultant alteration in biomechanical function, have been described.1,2 However, the acute
response to vocal fold injury has not yet been
investigated. Insight into the sequence of events associated with the acute response to injury in the
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RYAN C. BRANSKI ET AL

vocal folds might yield important information about

critical periods for intervention as well as prognostic
indicators for outcomes of treatment. The current
study is a component of a long-term research program in our laboratory addressing the acute wound
healing response in the vocal folds, as well as
wound healing itself.
A description of the sequence of wound healing
events in the skin serves as a foundation for the
current study. Initially, after acute injury, the inflammatory response orchestrates the cascade of
events associated with wound healing, and it ensures
immune competence after injury.3 The inflammatory
response is initiated by the extravasation of blood
constituents from damaged vessels. Chemoattractants, cytokines, and growth factors are released that
recruit neutrophils and monocytes to the injury site
and directly stimulate keratinocytes and fibroblasts
to initiate the repair process by assuming the repair
phenotype.4 Macrophages continue to clean and debride the wound, in addition to releasing numerous
cytokines key to fibroblast chemotaxis and proliferation. Those cytokines include platelet-derived
growth factor (PDGF), fibroblast growth factor (FGF),
and transforming growth factor-beta (TGF-).
In addition, macrophage-derived cytokines such
as interleukin-4 (IL-4) are responsible for tissue
formation (ie, collagen production in fibroblasts).5
The re-establishment of a functional epithelium
at the site of the wound is a key aspect of wound
healing. Clinically, a dermal wound is re-epithelialized when a water-impermeable seal is present over
the wound.4 Keratinocyte migration into the site of
injury from the periphery is stimulated by epidermal
growth factor and fibroblast growth factor released
by both inflammatory cells and tissue fibroblasts.
This process typically begins 24 to 48 hours after
injury.68 Wound contraction begins 4 to 5 days
after injury.9 Contraction is characterized by the centripetal movement of the wound edge toward the
center of the wound. Both myofibroblasts and fibroblasts are thought to be responsible for this
process.10,11 Scar remodeling is the final stage of
wound healing. The accumulation of collagen becomes stable at approximately 21 days after
injury.9,12 However, remodeling, involving the synthesis and degradation of collagen, may continue for
Journal of Voice, Vol. 19, No. 2, 2005

several months, and it contributes to the gradual

regain of tissue tensile strength.
As noted, the sequence of events is relatively well
documented in the skin, and little is known about
acute wound healing activities in the vocal folds.
Because the vocal fold has a unique subepithelial
structure and highly specialized biomechanical functions, the study of the processes associated with
acute wound healing in the vocal folds is warranted.
This issue is addressed in this present study.
Before the introduction of the experimental questions, background information regarding the methods used in the current investigation is relevant.
Previous investigation has suggested that analyses of
biochemical markers associated with wound healing
extracted from secretions collected from the vocal
fold surface may serve as a noninvasive means to
document the wound healing process in human vocal
fold injury.1315 In short, dramatic shifts in proinflammatory markers such as interleukin-1 (IL-1),
tumor necrosis factor-alpha, and matrix metalloproteinase-8 were noted in a single adult subject after
a 1-hour period of intense vocal loading that induced
prominent vocal fold edema and dysphonia.15 In
another study, differential profiles of both IL-1 and
PGE-2 were noted in surface secretions of patients
with various lesions of the vocal folds.14 The current
study seeks to build on the previous ones by investigating the time-dependent expression of key biochemical markers in vocal fold secretions acutely
after vocal fold injury, and to assess the extent to
which such expression might reflect processes within the tissue. To that end, two biochemical markers
were selected based on their known temporal regulation in the wound healing response in other tissues: interleukin-1 (IL-1) and prostaglandin-E2
(PGE-2).
Interleukin-1 is a prototypical pro-inflammatory
cytokine. It is the secreted isoform of the IL-1
family. It is produced by macrophages, monocytes,
dendritic cells, and tissue epithelial cells as an acute
response to injury and an initiator of the inflammatory process.16,17 PGE-2 is involved in several aspects of the wound healing cascade and is produced
by inflammatory cells, epithelial cells, and fibroblasts.1820 PGE-2 is a key inflammatory mediator,
but it is also involved in later processes of wound
healing, including inhibition of fibroblast migration,

BIOCHEMICAL MARKERS
stimulation of angiogenesis, and wound contraction.2124 Furthermore, IL-1 and PGE-2 expression
appears to be interrelated. IL-1 is responsible for
the induction of gene expression for several key
mediators associated with PGE-2 production. In fact,
many biological activities associated with IL-1 are
actually caused by increased levels of PGE-2 and
vice versa.16
The analysis of cytokines to document the severity
of mucosal injury is not novel. As early as 1976,
investigators found that concentrations of various
constituents in gingival crevicular fluid (GCF) could
be used to detect inflammation associated with active
periodontal destruction.25 The authors reported that
such assays should be useful for both clinicians and
patients with gingivitis. Patients could read their own
values on a GCF meter to self-evaluate their personal
periodontal condition and the effectiveness of their
home care progam.25 More recently, Faizuddin
et al26 demonstrated the ability to differentiate
among clinically healthy gingiva, gingivitis with
no attachment loss, and gingivitis with attachment
loss, based solely on IL-1 concentration in GCF.
The current study seeks to use similar methods
to document and quantify the inflammatory process
in the vocal folds. The experimental questions are
(1) can biochemical markers associated with wound
healing be detected in vocal fold secretions in a
rabbit vocal fold injury model? (2) If so, is there
a temporal pattern associated with marker expression? (3) If a temporal pattern of expression is noted,
is it consistent with the progression of events known
to be associated with wound healing in other systems? Affirmative answers to these questions might
provide insight into the acute wound healing process
in the vocal folds, and it would suggest utility in
biochemical markers collected from the vocal fold
surface as a noninvasive means to detect and quantify some aspects of the acute wound healing process. However, the current study does not attempt
to isolate the particular cell source of the markers.
At least initially, determining the specific source(s)
of the markers is not necessary to gauge the inflammatory process, which involves numerous cell types
capable of producing the markers of interest. The
current study seeks to describe the overall inflammatory milieu of the mucosal tissue, taking into
account multiple cellular sources of such markers.

285
METHODS

The study was approved by the University of

Pittsburgh Institutional Animal Care and Use Committee (IACUC) as a component of a broad investigation into the mechanics of acute wound healing
in the larynx. Eighteen female White New Zealand
rabbits were used. While the animals were under
general anesthesia, secretions were collected from
the superior and medial surface of the vocal folds
with small pieces of Gelfoam sheeting (Upjohn
Company, Kalamazoo, MI) held by forceps, using
endoscopic guidance during direct laryngoscopy.
The Gelfoam swabs containing the secretions were
then placed in microfuge tubes and stored at 80C
until analysis.
After collection of secretions, a unilateral vocal
fold injury was induced by surgical removal of the
mucosa down to the ligament upon gross examination. Three animals were then killed at each of
six timepoints after the induction of the injury: 1,
5, 7, 10, 14, and 21 days. The larynx was removed
for future histological analyses and secretions were
immediately collected from the surface of the injured
vocal fold using the technique described previously.
To extract the secretions from the Gelfoam swabs,
500 mL of sterile saline solution was added to the
microfuge tubes. The tubes were then mixed by
vortex for approximately 5 seconds and centrifuged
at 10,000 rpm for 3 minutes. The supernatant solution was collected in 1.5-mL microfuge tubes and
labeled accordingly. This procedure was repeated with
another 500 mL of saline solution, and the supernatant
solution was collected in the same 1.5-mL microfuge
tubes as in the previous wash. At that point, the
microfuge tubes contained approximately 1000 mL.
The volume of sample required for analysis was
aliquoted. The remaining sample was placed in a
microfuge tube and stored at 80C for later analysis. The entire procedure was performed cold to
minimize the risk of denaturation, as occurs at room
temperature and with repeated freezing and thawing.
IL-1 and PGE2 were assayed by using ELISA kits
according to the suppliers recommended protocols (R&D Systems, Minneapolis, MN).
Marker concentrations were standardized based
on secretion weight, because secretion volume could
not be accurately measured. Protein as a baseline
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RYAN C. BRANSKI ET AL

measure was also problematic. Previously, our laboratory reported on the potential utility of the highly
sensitive bicinchoninic acid (BCA) protein assay
(Sigma Chemical Co., St. Louis, MO) as a means
to standardize marker concentrations.27 However, it
appears that such analyses may be susceptible to
increased error when even a trace amount of blood is
present in the sample. Microfuge tubes were weighed
before secretion extraction. After extraction, the tube
with Gelfoam swab was placed in a vacuum desiccation chamber for 5 days. The tube with Gelfoam
swab was weighed again after desiccation, and the
weight of the difference was used as the weight of
the secretion. Concentrations for each biochemical
marker were then divided by the weight of the secretion. Therefore, all results are reported in terms of
picogram per gram of secretion.
STATISTICAL ANALYSIS
Two one-way between-subjects analyses of variance (ANOVA) were performed. For each analysis,
marker concentration (pg/g secretion of IL-1 or
PGE-2) was the dependent variable, and time
after injury (days) was the independent variable.
Posthoc comparisons were performed with the
Tukey method to determine the specific differences
in marker concentrations associated with the five
timepoints after injury. The investigation-wide alpha
level was set at 0.10 because of its exploratory
nature.28 The alpha level was then adjusted to account for multiple statistical tests ( 0.10/2).
Therefore, the alpha level was set at 0.05 for each
of the ANOVAs. An alpha of 0.05 was also used
for the posthoc comparisons.
RESULTS
Detectable levels of both IL-1 and PGE-2 were
obtained for all animals. Results are presented separately for each biochemical marker.
IL-1b
The main effect for IL-1 was significant
(P 0.001). Results are displayed in Figure 1.
That figure illustrates that IL-1 concentration increased markedly in the acute phase after injury.
Maximal expression occurred 1 day postinjury. Statistically, concentrations of IL-1 were significantly
Journal of Voice, Vol. 19, No. 2, 2005

different from preinjury levels for every timepoint

through day 5 (1 day, P 0.003; 5 days, P 0.011).
Beyond 5 days after injury, statistically significant
differences relative to preinjury levels were not obtained (7 days, P 0.552; 10 days, P 0.905; 14
days, P 0.235, and 21 days, P 1.00). Although
numerically the concentration appeared to increase
somewhat at day 14, that observation was not reliable because of a large standard error. It appears that
IL-1 concentration is elevated early and begins to
return to baseline level by 7 days after injury.
PGE-2
The main effect for PGE-2 was also significant
in the ANOVA (P 0.001). Results are displayed
in Figure 2. Similar to IL-1, PGE-2 expression
appeared to increase 1 day after injury; however, that
increase was not statistically significant (P 0.150).
Similar results were obtained 10 days after injury.
Posthoc comparisons revealed insignificant findings
for that timepoint (P 0.234). The data appear to
indicate increased PGE-2 concentrations at day 10.
The concentration of PGE-2 at all other timepoints
was significantly higher than at preinjury levels
(5 days, P 0.015; 7 days, P 0.001; 14 days,
P 0.041; and 21 days, P 0.005). Maximal expression occurred 7 days after injury. PGE-2 levels
did not return to preinjury concentrations by 21
days, the endpoint of the current study. Thus, elevated levels of PGE-2 extended into the later phases
of healing.
DISCUSSION
The primary experimental question was to determine whether biochemical markers associated with
wound healing could be detected from secretions in
a rabbit model of acute vocal fold injury. If detected,
the next experimental question was to determine
whether there was differential temporal expression
of such markers, and, if so, whether expression was
consistent with previous studies of the events associated with wound healing in other systems. Detectable levels of IL-1 and PGE-2 were obtained by
using the rabbit model. That result strengthens conclusions from previous studies indicating the utility
of the assay of vocal fold secretions to monitor and
describe wound healing in the vocal folds.14,15

BIOCHEMICAL MARKERS

287

FIGURE 1. Results for IL-1 across time. Error bars represent standard error of the mean (SEM).

Relative to the second experimental question, the

data indicated a distinct pattern of expression for
each of the two markers as a function of time
after injury. Both mediators displayed increased concentrations in vocal fold secretions in the early postinjury period. Levels of IL-1 returned to baseline
levels by 7 days after injury. In contrast, PGE-2
concentrations remained elevated through day 21
with maximum expression 7 days after injury. However, statistically significant differences were not
achieved between preinjury levels and levels taken
1 day and 10 days after injury. The data suggest a
trend of increased PGE-2 expression at those timepoints. PGE-2 elevation at day 21 (the study endpoint) may indicate a role for this mediator in the
later phases of the wound healing process.
Of relevance, a moderate degree of variability in
the data was observed. Such variability may be related to several factors. This variability may reflect
both within- and across-species disparities in response to injury. Animals may have responded differently to injury, altering the concentrations of
biochemical markers associated with wound healing.
Indeed, individual differences in the response to
injury have been identified in studies of other tissues.26 Our laboratory has shown that vocal loading

dramatically alters the profile of biochemical markers in mucosal secretions.15 The phonatory patterns
of the animals were not controlled after injury;
this may also contribute to variability in the data.
Relative to the third experimental question, the
present data displayed a distinct pattern of expression that corresponds to the established roles of the
two markers in the wound healing process. IL-1
is a key inflammatory mediator and a key initiator
of the wound healing cascade after acute injury. In
the current study, IL-1 expression was maximal
in the acute phase (1 day) after injury, with relatively
little expression at later time points (721 days). In
contrast, the data indicated that PGE-2 has a role
in both the inflammatory phase and later stages of
wound healing in the vocal folds. Increased levels
of PGE-2 were noted throughout the 21-day experiment with maximal concentrations 7 days after
injury. Increased PGE-2 concentrations at later time
points may be related to the inhibition of fibroblast
migration or the initiation of wound contraction
as previously suggested in both the skin and
airway.21,22 However, this hypothesis is purely conjecture at this point.
To date, no study has attempted to correlate concentrations of biochemical markers in secretions collected from the vocal folds with concentrations of
Journal of Voice, Vol. 19, No. 2, 2005

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RYAN C. BRANSKI ET AL

FIGURE 2. Results for PGE-2 across time. Error bars represent standard error of the mean (SEM).

the same biochemical markers within the tissue. This

is a potential weakness of the current study that
will be addressed in future reports. However, our
laboratory has found that concentrations of key inflammatory mediators in secretions are significantly
greater when collected from the site of injury versus
uninjured vocal folds, as well as other control
sites along the upper aerodigestive tract.14 These
findings enhance the validity of the current investigation. Clearly, concentrations of wound healing
markers in mucosal secretions capture some aspect
of the wound healing response. Furthermore, the
profile of biochemical markers in vocal fold secretions appears to correlate well with the expected
temporal expression within the tissue, thereby providing encouraging data to stimulate further study.
Although further investigation of the present issues is certainly warranted, the current study sought
to build on a series of studies suggesting the potential
investigational use of ELISAs to document the
wound healing process in the vocal folds. Results
from the present study suggest that biochemical
markers associated with wound healing can be detected in vocal fold secretions in a rabbit model
of acute vocal fold injury. Furthermore, a temporal
Journal of Voice, Vol. 19, No. 2, 2005

pattern of biochemical marker expression was found;

this was consistent with previous studies regarding
the specific role of both IL-1 and PGE-2 in the
wound healing cascade in other systems.
Acknowledgment: We thank Elaine Rubinstein, PhD,
for statistical consulting.

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Journal of Voice, Vol. 19, No. 2, 2005