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ORIGINAL ARTICLE

Prevention of Anterior Glottic Stenosis After


Bilateral Vocal Fold Stripping With Mitomycin C
Jong-Lyel Roh, MD, PhD; Yeo-Hoon Yoon, MD

Objective: To examine the effects of immediate appli- Results: The stripping procedure induced AGS, affect-
cation of topical mitomycin C (MMC) on the preven- ing 58% to 86% (mean, 72%) of the length from the an-
tion of anterior glottic stenosis (AGS) after microsurgi- terior commissure to the vocal process in the control
cal stripping of both vocal folds, including the anterior group. The application of MMC at the time of initial sur-
commissure, in a canine model. gery significantly lowered the incidence and extent of the
web formation (P = .004). The AGS lesions were re-
Design: Prospective randomized experimental study. solved by web lysis and treatment with MMC, without
significant local adverse effects. Histological staining for
Interventions: Twelve canine larynges were injured by collagen and elastin revealed that MMC treatment did not
a stripping procedure of both entire membranous vocal induce excessive fibrotic or atrophic changes in the lamina
folds. The dogs were randomly divided into 2 groups for propria of the vocal folds.
treatment with 1.0 mg/mL of MMC or with isotonic so-
dium chloride solution (control) for 5 minutes immedi-
ately after surgery. Three and 6 weeks after surgery, the Conclusion: Bilateral stripping of the membranous vo-
glottic webs were lysed and repeatedly treated with MMC cal folds induces significant AGS, which can be mini-
or isotonic sodium chloride solution. The glottic wound mized by use of MMC at initial surgery.
healing and AGS formation were examined every week.
Ten weeks after the initial surgery, all larynges were col-
lected and examined histologically. Arch Otolaryngol Head Neck Surg. 2005;131:690-695

A
NTERIOR GLOTTIC STENOSIS gery can be successfully performed for treat-
(AGS) or web is the forma- ment of benign or malignant lesions in-
tion of a bridge of scar tis- volving the anterior glottic commissure.2,3
sue covered by epithelium The conventional treatments of AGS
between the vocal folds and have included intralesional injection, en-
involving the anterior commissure. Ante- dolaryngeal procedures, and keel inser-
rior glottic stenosis commonly occurs sec- tion via laryngofissure.4-6 Recently, topi-
ondary to laryngeal injury resulting from cal mitomycin C (MMC) has been used to
laryngeal surgery. Unintended injury from prevent restenosis after the lysis of ante-
endoscopic or microsurgical manipula- rior glottic webs.7 Mitomycin C is an an-
tion involving both vocal folds simulta- tineoplastic agent that is known to in-
neously can induce anterior glottic scar- hibit proliferation of fibroblasts and tumor
ring and adhesions that disturb the delicate growth.8 Its proven antifibrotic effect has
workings of the vocal folds. Excessive AGS led to several experimental and clinical
causes significant impairment of phona- trials. Mitomycin C has been widely used
tion, ventilation, and airway protection. for prevention of stenosis or adhesion after
Therefore, aggressive endolaryngeal mi- otolaryngological surgical treatments.9,10
crosurgical management of lesions involv- In a previous experiment, MMC was used
Author Affiliations: ing the anterior glottic commissure has of- only to prevent recurrence after the lysis
Department of ten been avoided or discouraged. When of anterior glottic webs that were origi-
Otolaryngology–Head and Neck
Surgery, Cancer Research
required, it has been recommended that bi- nally induced by laser injury of the vocal
Institute, Chungnam National lateral surgical interventions be separated folds.7 These clinical and experimental
University College of Medicine, into 2 unilateral procedures staged at least studies of MMC suggest that MMC can be
Daejeon, Korea. 2 weeks apart.1 Nonetheless, reports have used for prevention of AGS following mi-
Financial Disclosure: None. suggested that endolaryngeal microsur- crosurgical removal of lesions involving

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Table 1. First Intervention for Vocal Fold Stripping*

MMC-Treated Group (Stripping ⴙ MMC) Control Group (Stripping ⴙ Isotonic Sodium Chloride Solution)

Week Dog 1 Dog 2 Dog 3 Dog 4 Dog 5 Dog 6 Dog 7 Dog 8 Dog 9 Dog 10 Dog 11 Dog 12
3 7% 8% 27% 42% 57% 46% 69% 82% 58% 72% 66% 86%

Abbreviation: MMC, topical mitomycin C.


*Percentages indicate the extent of the web formed, defined as the anterior to posterior length of the web divided by the entire length of the membranous vocal
folds and multiplied by 100.

Table 2. Second Intervention for Treatment of Anterior Glottic Stenosis*

Control Group

Web Lysis ⴙ Isotonic Sodium


MMC-Treated Group (Web Lysis ⴙ MMC) Web Lysis ⴙ MMC Chloride Solution

Week Dog 1 Dog 2 Dog 3 Dog 4 Dog 5 Dog 6 Dog 7 Dog 8 Dog 9 Dog 10 Dog 11 Dog 12
6 No web No web No web No web 24% 13% No web 43% 26% 42% 37% 47%

Abbreviation: MMC, topical mitomycin C.


*Percentages indicate the extent of the web formed, defined as the anterior to posterior length of the web divided by the entire length of the membranous vocal
folds and multiplied by 100.

both anterior vocal folds. However, to our knowledge, achieve significant effects, while avoiding necrosis at the site
there have been no reports of experiments in which MMC of local treatment, based on reports in the literature.7-10 The depth
was applied at the time of initial surgery to prevent the and extent of the vocal fold stripping were identical in all the
development of AGS. animals, and the treatment with MMC or isotonic sodium chlo-
ride solution was randomized at the time of the initial inter-
In this study, we first developed a canine model in
vention.
which AGS is induced by bilateral microsurgical strip- At weekly intervals from the original surgery until the end
ping of the epithelium from the entire length of both mem- of the experiment, each animal was anesthetized with propo-
branous vocal folds. We then used this model to exam- fol, and the vocal folds were examined and photographed. Three
ine the effects of MMC in preventing AGS after surgery. weeks after the initial injury, the animals that had developed
AGS underwent web lysis using microscissors and were treated
METHODS for 5 minutes with 1.0 mg/mL of MMC (dogs 1-9) or with iso-
tonic sodium chloride solution (dogs 10-12) (Table 2). Treat-
ment with MMC or isotonic sodium chloride solution after the
EXPERIMENTAL DESIGN first web lysis was also randomized in the 6 animals initially
treated with isotonic sodium chloride solution that had devel-
Twelve male beagles aged 1½ to 2 years and weighing 10 to 12 oped significant AGS. Six weeks after the initial surgery, all re-
kg each were used in this study. The animals were anesthe- sidual AGS lesions were relysed and treated for 5 minutes with
tized by intravenous administration of propofol, 6 mg/kg of body 1.0 mg/mL of MMC (dogs 5, 6, and 8-12) (Table 3).
weight. Maintenance anesthesia was achieved with a continu- All animals were housed in an approved animal care facil-
ous infusion of propofol at 0.4 mg/kg per minute, with addi- ity with water ad libitum and regular canine food until their
tional small boluses of 1 mg/kg if anesthesia appeared inad- humane killing 10 weeks after the original injury. All experi-
equate. Direct laryngoscopy was performed using a Kleinsasser ments were performed with the approval of the Animal Ex-
laryngoscope modified for exposure of the anterior commis- periment Committee of the Clinical Research Institute at Chung-
sure (8590 J; Karl Storz GmbH & Co, Tuttlingen, Germany) and nam National University Hospital.
an operating microscope (Carl Zeiss, Göttingen, Germany) to
visualize the canine glottis. Microforceps and microscissors (Karl
Storz GmbH & Co) were used to remove the entire epithelium GROSS AND HISTOLOGICAL EXAMINATIONS
and a layer of the lamina propria from both sides of the mem-
branous vocal folds, from the anterior commissure to the vocal All animals were examined under anesthesia each week. The
process, and from the upper lateral margin of the true vocal folds entire glottis was evaluated for the presence of mucosal scar-
to a point 2 mm below the edge of the vocal folds.7 ring, granulation tissues, and AGS using a 40-cm-long, 5-mm-
After the vocal folds were stripped, the anterior glottic area diameter, 0° telescope (Karl Storz GmbH & Co). The extent of
of the lesions was immediately treated for 5 minutes by appli- the web formation was determined by dividing the anterior to
cation of a cottonoid pledget soaked in a solution of 1.0 mg/mL posterior length of the web by the entire length of the mem-
of MMC in sterile water or soaked in isotonic sodium chloride branous vocal folds from the anterior commissure to the vocal
solution (control) (6 in each group) (Table 1). After treat- process. The glottis of each animal was documented with tele-
ment with MMC, the glottis was washed with a isotonic so- scopic photographs taken after careful examination.
dium chloride solution–soaked cottonoid pledget. The se- At the end of the 10-week follow-up, each animal was
lected MMC concentration was expected to be sufficient to humanely killed with a lethal dose of sodium pentobarbital

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Table 3. Third Intervention for Treatment of Residual Anterior Glottic Stenosis (Web Lysis ⴙ MMC)*

MMC-Treated Group Control Group

Week Dog 5 Dog 6 Dog 8 Dog 9 Dog 10 Dog 11 Dog 12


10 No web No web 12% No web 16% No web 12%

Abbreviation: MMC, topical mitomycin C.


*Percentages indicate the extent of the web formed, defined as the anterior to posterior length of the web divided by the entire length of the membranous vocal
folds and multiplied by 100.

administered by intravenous injection after induction of anes- 50% of the membranous vocal folds, and the other half
thesia. The larynges were excised and placed in 10% formal- developed only very small glottic webs (Figure 1B). On
dehyde for 24 hours. The samples were processed using stan- endoscopic examination, the anterior glottic movement
dard alcohol dehydration and paraffin embedding procedures. was decreased in animals with severe AGS. In addition,
The whole vocal folds, including the anterior commissure, were
the formation of granulation and scar tissue on the vo-
sectioned at 5-µm thickness at the level of the free margins and
stained with hematoxylin-eosin. Masson trichrome and van Gie- cal fold mucosa was found more frequently in the con-
son stains were used to assess the collagen and elastin con- trol group (4 of 6 animals) at 3 weeks after the initial sur-
tents, respectively, and the levels were graded from 0 to 3⫹ in gery compared with the MMC-treated group (2 of 6
the lamina propria. All preinjury vocal fold samples collected animals), but this difference was not statistically signifi-
at the time of initial surgery were processed and stained as con- cant (P=.56). However, wound healing in the injured vo-
trol specimens for comparison with the injured larynges col- cal folds seemed to be delayed in the MMC-treated group.
lected when the animals were killed. The anterior commissure
region of each glottis was compared between the treatment MMC TREATMENT AFTER LYSIS
groups and the preinjured larynges. The telescopic and histo-
OF GLOTTAL WEBS
logical examinations and the scoring of the larynges were per-
formed in a blinded fashion with regard to which animals re-
ceived MMC treatment after the initial surgery. Three weeks after the initial surgery, the dogs that had
developed AGS were treated by surgical lysis of the le-
STATISTICAL ANALYSIS sions and immediate application of MMC or isotonic so-
dium chloride solution. Among the dogs that had ini-
The extent of AGS formation after microsurgical stripping of tially been treated with only isotonic sodium chloride
the vocal folds and the extent of recurrence after web lysis were solution after the original surgery (dogs 7-12), anterior
compared between the MMC-treated group and the control glottic webs involving a mean of 42% of the vocal fold
group using the Mann-Whitney test (SPSS 11.0 for Windows; length had reformed 3 weeks after web lysis in all of the
SPSS Inc, Chicago, Ill). The degree of mucosal scarring or granu- animals treated with only isotonic sodium chloride so-
lation tissue formation and the histological grades of collagen lution at the time of lysis (dogs 10-12) (Figure 1C), and
deposition or elastin content were compared between the groups
webs involving a mean of 23% of the vocal fold length
using the Fisher exact test. P⬍.05 was considered significant.
had reformed in 2 of the 3 animals treated with MMC
(dogs 7-9), revealing no significant differences between
RESULTS the treatments (P=.28) (Table 2). However, the extent
of the web reformation after lysis was smaller than the
MMC TREATMENT AFTER extent of the webs before lysis. In the group treated with
VOCAL FOLD STRIPPING MMC after the initial surgery (dogs 1-6), the recurrence
of anterior glottic webs after lysis and reapplication of
All of the animals survived all procedures during the ex- MMC was less prominent; no webs reformed in 4 of the
perimental period of 10 weeks, and all of the surgical 6 animals, yielding a significantly lower rate of recur-
wounds healed without hemorrhage or infection. Micro- rence compared with the group initially treated with iso-
surgical stripping of the epithelium from the bilateral vo- tonic sodium chloride solution (dogs 7-12) (P =.02).
cal folds induced AGS in all animals within 2 weeks af- Ten weeks after initial surgery, all of the injured
ter wounding when only isotonic sodium chloride solution vocal folds had healed well, without severe scarring,
was applied immediately after stripping. Three weeks af- granulation tissues, or webs. Small webs were found in
ter surgery, the AGS lesions in the control group af- 3 of the 6 animals that were treated with isotonic so-
fected 58% to 86% (mean, 72%) of the length of the vo- dium chloride solution after the initial surgery. No glot-
cal folds from the anterior commissure to the vocal process tic webs were found in the group initially treated with
(Table 1 and Figure 1A). In contrast, the animals treated MMC (Figure 1D). On gross examination, no signifi-
with MMC at initial surgery had smaller webs affecting cant atrophic changes in the vocal folds were found in
7% to 57% (mean, 31%) of the length of the vocal folds, the animals treated repeatedly with MMC. Although the
which was significantly less than the extent of web for- MMC appeared to induce delayed wound healing in the
mation in the control group (P = .004). All of the control injured vocal folds, there were no significant local ad-
group had extensive anterior glottic webs, but one half verse effects, such as necrosis or infection. No limita-
of the MMC-treated group had webs involving less than tion of vocal fold movement was found in any of the ani-

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A B

C D

Figure 1. Photographs of the canine glottises that underwent bilateral stripping of the epithelium and a layer of lamina propria from the entire length of the
membranous vocal folds. The stripping procedure induced severe anterior glottic stenosis (A, dog 12), but treatment with topical mitomycin C reduced the extent
of web formation (B, dog 2) within 2 weeks after the initial wounding. The web in the anterior glottis had reformed 3 weeks after web lysis and isotonic sodium
chloride solution application (C, dog 12). Ten weeks after the initial wounding, the glottis showed no web or granulation tissue formation and only mild scarring
(D, dog 2).

mals on endoscopic examination 10 weeks after the initial significantly different from that in the MMC-treated group
surgery. and the preinjury vocal folds (P =.21 and P =.46, respec-
tively). Elastin fibers were observed in the lamina pro-
HISTOLOGICAL EXAMINATION pria of the vocal folds, with particularly dense elastin stain-
ing seen in the superficial layer (Figure 2C). However,
Histological examination 10 weeks after the initial sur- the degree of elastin staining in the injured vocal folds
gery revealed that the vocal folds had mild collagen depo- and anterior commissure was not significantly different
sition and fibroblast proliferation, although the colla- from that in the preinjury vocal folds (P=.59) (Figure 2D).
gen content was not prominently increased in the
wounded vocal folds of the animals initially treated with
COMMENT
MMC. When present, collagen fibers were found in the
lamina propria of the vocal folds, particularly around the
anterior glottis and the anterior commissure on Masson Bilateral stripping of the entire length of the membra-
trichrome staining (Figure 2A). However, in the ani- nous vocal folds induced significant AGS in this study.
mals treated with isotonic sodium chloride solution af- This finding supports previous observations that surgi-
ter the initial surgery, irregular, thickened epithelium was cal manipulation of both anterior vocal folds can lead to
found in some of the vocal folds and was accompanied postoperative AGS.1,7 Our animal model of AGS was cre-
by collagen deposition, especially in the superficial layer ated to simulate such clinical situations. The extent and
of the lamina propria (Figure 2B). Nonetheless, the over- depth of injury to the glottis that we used in the model
all grade of collagen content in the control group was not were suggested by a study11 of endolaryngeal microsur-

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A B

C D

Figure 2. Photomicrographs of the canine vocal folds that underwent stripping and web lysis. Ten weeks after the initial wounding, the vocal folds had healed
without significant web formation or fibrosis (A, Masson trichrome stain, original magnification ⫻15). However, thickening and irregularity of the vocal fold
epithelium were sometimes found, accompanied by collagen deposition in the lamina propria, in the animals not treated with mitomycin C at the initial surgery
(B, Masson trichrome stain, original magnification ⫻40). The degree of elastin staining in the lamina propria of the vocal folds was not significantly altered 10
weeks after injury (C, van Gieson stain, original magnification ⫻40) compared with the preinjury vocal fold tissue samples (D, van Gieson stain, original
magnification ⫻200).

gery for laryngeal cancer, in which subligamental cor- gical injury. The atrophy of the vibratory surface re-
dectomy (type II) was performed on both sides accord- sulted in diminished mucosal wave vibration in the vocal
ing to a proposed classification of endoscopic cordectomy folds after treatment with MMC. However, another ex-
of the European Laryngological Society. The results of periment showed that although MMC delayed the heal-
that study showed that a single microsurgical stripping ing of surgical wounds in rats up to the fourth week fol-
of the entire length of the membranous vocal folds in- lowing treatment, the degree of fibrosis was comparable
duced more severe AGS than had been induced in a pre- in the treated and untreated wounds after 12 weeks.13 This
vious experiment using repeated laser vaporization of the latter finding was supported by the gross and histologi-
mucosa in the same area.7 cal observations in the present study, in which the in-
The present study also focused on the prevention of jured vocal folds showed no significant atrophy and ac-
AGS following bilateral endolaryngeal surgery of the an- ceptable amounts of scarring. Instead, MMC appeared to
terior vocal folds. The conventional methods used to pre- prevent severe, broad scarring of the vocal fold lamina
vent AGS4-6 are listed in the introduction. In this study, propria, which is attributable to abundant and disorga-
we applied MMC to the injured anterior glottis imme- nized collagen deposition that is a characteristic feature
diately after bilateral vocal cord stripping. The applica- of surgically injured vocal folds.14 In addition, no sig-
tion of MMC significantly lowered the incidence and ex- nificant local adverse effects were induced by use of 1.0
tent of AGS. In addition, MMC had a preventive effect mg/mL of MMC, a dose that may be considered as being
on restenosis after lysis of anterior glottic webs. All of in the upper range of typical clinical doses of MMC.8-10
the animals initially treated with MMC were AGS-free The concentration used in this study was less than the
after web lysis and the reapplication of MMC. 10 mg/mL of MMC concentration reported by Spector
A previous study12 showed that injured canine vocal et al7 but is comparable to the concentrations used clini-
folds became atrophic 4 weeks after MMC treatment ow- cally. The safe use of MMC is also supported by a recent
ing to decreased connective tissue response to the sur- report showing that the use of MMC at concentrations

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in excess of 0.4 mg/mL did not alter the protein concen- missure: controversies and observations. Ann Otol Rhinol Laryngol. 2000;
109:385-392.
trations in the extracellular matrix or the apoptosis rate.15
3. Steiner W, Ambrosch P, Rodel RM, Kron M. Impact of anterior commissure in-
The present study thus confirms that MMC at a typical volvement on local control of early glottic carcinoma treated by laser microresection.
clinical dose can be effectively and safely used to treat Laryngoscope. 2004;114:1485-1491.
the anterior vocal folds at the time of initial surgery. 4. Dedo HH. Endoscopic Teflon keel for anterior glottic web. Ann Otol Rhinol Laryngol.
In conclusion, we suggest that MMC can be used to 1979;88:467-473.
minimize the development of AGS in the injured ante- 5. Parker DA, Das Gupta AR. An endoscopic Silastic keel for anterior glottic webs.
J Laryngol Otol. 1987;101:1055-1061.
rior glottis when applied at the end of endolaryngeal mi-
6. Kojima H, Omori K, Shoji K, Kanaji M. Reconstruction of the anterior commis-
crosurgery involving the bilateral anterior vocal folds. Pro- sure with a free mucosal flap. Am J Otolaryngol. 1993;14:339-342.
phylactic use of the drug may be advocated in clinical 7. Spector JE, Werkhaven JA, Spector NC, Huang S, Sanders D, Reinisch L. Pre-
settings for surgical management of lesions involving the vention of anterior glottic restenosis in a canine model with topical mitomycin-C.
anterior glottic commissure, although further random- Ann Otol Rhinol Laryngol. 2001;110:1007-1010.
ized clinical studies will be required. 8. Khaw PT, Doyle JW, Sherwood MB, Grierson I, Schultz G, McGorray S. Pro-
longed localized tissue effects from 5-minute exposures to fluorouracil and mi-
tomycin C. Arch Ophthalmol. 1993;111:263-267.
Submitted for Publication: January 24, 2005; final re- 9. Chung JH, Cosenza MJ, Rahbar R, Metson RB. Mitomycin C for the prevention
vision received March 9, 2005; accepted March 9, 2005. of adhesion formation after endoscopic sinus surgery: a randomized, controlled
Correspondence: Jong-Lyel Roh, MD, PhD, Depart- study. Otolaryngol Head Neck Surg. 2002;126:468-474.
ment of Otolaryngology–Head and Neck Surgery, Can- 10. Rahbar R, Shapshay SM, Healy GB. Mitomycin: effects on laryngeal and tra-
cheal stenosis, benefits, and complications. Ann Otol Rhinol Laryngol. 2001;
cer Research Institute, Chungnam National University
110:1-6.
College of Medicine, 640 Daesa-Dong, Chung-Gu, Dae- 11. Remacle M, Eckel HE, Antonelli A, et al. Endoscopic cordectomy: a proposal for
jeon 301-040, Korea (rohjl@cnu.ac.kr). a classification by the Working Committee, European Laryngological Society. Eur
Funding/Support: This work was supported by Chung- Arch Otorhinolaryngol. 2000;257:227-231.
nam National University Hospital Research Fund 2004. 12. Garrett CG, Soto J, Riddick J, Billante CR, Reinisch L. Effect of mitomycin-C on
vocal fold healing in a canine model. Ann Otol Rhinol Laryngol. 2001;110:25-
30.
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