Acta Oto-Laryngologica. 2015; 135: 840–845
ORIGINAL ARTICLE
Novel experimental rabbit model of anterior glottic web formation
SEONG KEUN KWON1*, DONG WOOK KIM1*, YOON-JONG RYU1, SOO YEON KIM1, HYUN CHANG2, MYUNG WHUN SUNG1 & J. HUN HAH1 1
Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital & Seoul National University College of Medicine, Seoul and 2Department of Otorhinolaryngology-Head and Neck Surgery, Dongnam Institute of Radiological & Medical Sciences, Busan, Korea
Abstract Conclusion: The rabbit model of anterior glottic web (AGW) formation using the laryngofissure technique resulted in reproducible and stable AGW formation that may facilitate research into this area. Objective: To introduce and validate a novel experimental animal model of AGW formation using the rabbit. Methods: The inner larynges of eight New Zealand white rabbits were exposed through the laryngofissure technique. The mucosa of the bilateral true vocal fold was stripped off using the bevel of a needle tip. On the basis of the laryngoscopic findings at 8 weeks postoperatively, the extent of AGW was measured, and the success of this procedure was validated. Laryngeal specimens were sampled at 8 weeks for high-speed recording and histological analysis. Results: In seven (87.5%) rabbits, laryngoscopic examination revealed the formation of a scar band involving the anterior commissure. The mean extent of AGW ratio on the left and right sides was 0.58 ± 0.073 and 0.55 ± 0.075, respectively. The symmetric formation of AGW (p = 0.655, p = 0.128) and stability of the AGW procedure (p = 0.491, left; p = 0.501, right) were statistically validated. On high-speed recording, the vocal mucosal wave was hindered by AGW formation. Histologically, fibro-connective tissue, especially collagen fiber, was observed in the anterior commissure.
Keywords: Stenosis, larynx, laryngofissure technique, anterior commissure
Introduction Acquired anterior glottic webs (AGWs) are the most common type of glottic web, and can occur from sequelae of endolaryngeal lasers or instrumental procedures [1,2]. AGW refers to the formation of a fibrotic scar band covered by epithelium, between the vocal folds, that involves the anterior commissure. According to the severity and size of the web, AGW may result in abnormally elevated phonatory pitch, dysphonia, and airway restriction. Symptomatic AGW can be managed in various ways including by keel insertion via laryngofissure, which is recognized as the definitive treatment [3,4]. However, due to the invasiveness of this procedure, other more conservative and non-invasive treatments have been attempted. These treatments are diverse and range
from conventional cold instrumental dissection and intralesional injection to endoscopic CO2 laser with adjuvant mitomycin C application [2,5–8]. Despite a great deal of attention being focused on preventing restenosis, or preventing recurrence after management for AGW, this poses a therapeutic problem for otolaryngologists not only because of technical difficulties inherent in successful repair, but also because of the choice of several treatment methods. Since studies related to AGW are limited to a few case series reports and other studies [2,3,5,9–13], management of AGW is usually performed depending on the surgeon’s preference and clinical experience. To compare the different treatment options for AGW and to establish suitable management strategies, welldesigned studies involving suitable animal models are needed.
Correspondence: J. Hun Hah, MD PhD, Associate Professor, Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea. Tel: +82 2 2072 3649. Fax: +82 2 745 2387. E-mail: [email protected] *These authors contributed equally to this work.
(Received 9 February 2015; accepted 3 March 2015) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1028594
Novel rabbit model of anterior glottic web In the literature, there are several animal studies on AGW with large animals [2,6,12,14,15]. Although experiments on the larynx with large animals including dog, cow or pig have advantages, such as easy accessibility to the larynx and resemblance to humans in size, these could be more cumbersome than with small to mid-sized animals in terms of technique, feasibility, and expenses, including laboratory facility fees. Even though we thoroughly tried to find a small animal model for laryngeal stenosis or web, we could find only a few rabbit models of posterior glottic stenosis [5,16,17]. This lack of small animal models for AGW might be because of the difficulty of access to the larynx, especially to the anterior portion. Herein, we introduce and validate a novel experimental model of AGW formation using the laryngofissure in a rabbit. Material and methods The experiments performed in the present study were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (approval no. 14-0005-S1A0) and performed in
841
accordance with the ethical guidelines of the committee. Anterior glottic web formation in rabbit Eight male New Zealand white rabbits (Koatech Laboratory Animal Company, Gyeonggi, Korea) weighing 3.5 kg were anesthetized with intramuscular Zoletil 50 (50 mg/kg; Virbac Korea, Seoul, Korea). Through a 3 cm vertical midline skin incision at the level of the thyroid notch, the subcutaneous fat and the strap muscles were separated in the midline. By retracting the strap muscles using a self-curved mastoid retractor (Karl Storz, Tuttlingen, Germany), the hyoid, thyroid cartilage, and upper tracheal rings were further exposed by dissection (Figure 1A). To create a thyrohyoid window, a small vertical incision was made on the thyrohyoid membrane along the upper thyroid notch using a scalpel (no. 15). In this procedure, meticulous hemostasis of vessels in the pre-epiglottic space was performed with epinephrine (1:1000) soaked cotton or micro electro-cauterization (HIT1, Change-A-Tip, Bovie Medical Co., Purchase, NY, USA). Next, the whole length of perichondrium of the
A
B
C
D
E
Figure 1. Procedures for anterior glottic web (AGW) formation in rabbit, via a laryngofissure approach. (A) Exposed large pre-epiglottic vessel (arrowhead) after midline skin incision and strap muscle dissection. (B) Direct inspection of the bilateral vocal fold in rabbit (arrowheads) through laryngofissure. (C, D) Right and left vocal fold stripping from the vocal process (arrowheads) to the anterior commissure with an 18 gauge needle tip. (E) Bilaterally stripped vocal mucosa (arrowheads).
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thyroid cartilage was incised with a scalpel (no. 15), and the thyroid cartilage was split with monopolar electrocauterization at the midline, by a procedure similar to the laryngofissure approach [18]. Because the inner mucosa of the larynx should be incised at exactly the mid-point of the anterior commissure at the vocal fold, the laryngofissure procedure was performed following inspection through the window of thyrohyoid membrane. After splitting the thyroid cartilage, both sides of the inner mucosa on the true and false vocal fold were sufficiently exposed (Figure 1B). The true vocal mucosa just anterior to the vocal process tip was penetrated by a bevel of a needle tip (18 gauge) and stripped off along the whole length of the vocal fold from the vocal process to the anterior commissure (Figure 1C, D). For determination of exact depth of injury to vocal ligament, this procedure was performed under magnification using surgical microscopes. Bilateral mucosae of the true vocal folds were removed, and the bleeding was controlled with epinephrine-soaked cotton (Figure 1E). The split thyroid cartilages were closed tightly with Vicryl 4-0, and the suture site was covered by a strap muscle and adipose tissue layer by layer. The subcutaneous tissue and the skin were closed using surgical staples. After the procedure, animals were closely examined for signs of immediate postoperative complications such as bleeding and subcutaneous emphysema. Antibiotics (gentamicin sulfate, 80 mg/2 ml) were administered subcutaneously at 0.2 ml/kg/day for 7 days. All animals were monitored daily, with specific attention to their weight, cough, sputum production, wheezing, and dyspnea by professional laboratory animal keepers at the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) accredited institutions: Clinical Research Institute of Seoul National University Hospital (accreditation no. 001169).
Image J software, it was adjusted by dividing this number by the length of each vocal fold, from the anterior commissure to the vocal process, and this adjusted value was redefined as an AGW ratio (Figure 2). By evaluation of the AGW ratio in each rabbit and on each side of the vocal folds, the reliability and stability of the AGW formation procedure were determined. Recording of induced vocal fold vibration with high-speed camera and histologic evaluation At 8 weeks after AGW formation, the animals were humanely euthanized with intravenous injection of KCl under anesthesia and the larynges were removed by laryngectomy. Functional analysis with high-speed camera recording was followed by histological study. For better visualization of the vocal fold, the superior portion of the thyroid cartilages and the supraglottic structures were removed. The arytenoid cartilages were sutured together using a Prolene 6-0 suture to close both vocal folds. The trimmed larynx was mounted on a holder and the vocal fold vibration was generated by humidified air injection using a medical oxygen generator. Imaging of vocal fold vibration during induced phonation was accomplished by a MotionXtra NR4S2 high-speed video camera (DEL Imaging Systems, Cheshire, CT, USA). High-speed video data were recorded at 8000 images per second and a spatial
A
Laryngoscopic evaluation At 2, 4, and 8 weeks after AGW formation, rabbits were anesthetized as described previously and a laryngeal endoscopic examination was performed using a 4.0 mm 30 rigid endoscope (Richards, Knittlingen, Germany). Images of each animal’s vocal folds were taken with a digital camera (E4500, Nikon, Tokyo, Japan) attached to the rigid endoscope. By laryngoscopic imaging at 8 weeks, the extent of AGW was evaluated by measuring the length from the anterior commissure to the posterior end of the scar band formation at the right and left sides of the vocal folds with Image J imaging software (National Institute of Mental Health, Bethesda, MD, USA) [19]. Because this measured value was defined by pixels on the
C
B
AC , using Image J AB software: A, anterior commissure; B, vocal process; C, the end of the web in each vocal fold. Figure 2. Anterior glottic web (AGW) ratio =
Novel rabbit model of anterior glottic web resolution of 256 horizontal 512 vertical pixels. Illumination was provided by a 300 W xenon light source. Each high-speed video data segment consisted of 8000 images per second [20]. After high-speed camera recording, laryngeal specimens were fixed for 24 h in 10% formalin, embedded in paraffin, and sliced at a thickness of 2 mm using a microtome. The sections were deparaffinized and dehydrated in graded ethanol. Hematoxylin and eosin (H&E), Masson’s trichrome (MT), Alcian blue (AB), and Verhoeff-Van Gieson (VVG) stains were used to evaluate histologic changes associated with AGW formation. Statistical analysis Experimental data are expressed as means ± standard deviation (SD). For evaluation of symmetric AGW formation, paired comparison of right and left side AGW ratio from each individual experimental animal was conducted by the Wilcoxon signed rank test. Also, to validate the consistent formation of AGW among all the experimental rabbits, the distributions of AGW ratio from the eight experimental rabbits were statistically evaluated by the Kolmogorov– Smirnov test. The statistical tests in the current study were used for comparison using SPSS 20.0 statistical software and statistical significance was accepted at p < 0.05. Results All animals survived without complications following surgery. Successful AGW formation was observed in seven of eight rabbits by endoscopic examination (87.5%). Some granulated tissue and irregular margins of the scar band at the anterior commissure were observed during week 2 by endoscopic examination. However, the granulation was resolved and AGW covered with normal mucosa developed over time. Consequently, compared with normal larynx (reference laryngoscopic image of normal rabbit), which Normal
2 week
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showed an anterior commissure in an inverted ‘V’ shape and at an acute angle, AGW had a broad angle margin and an inverted ‘U’-shaped scar band by 8 weeks (Figure 3). The extent of AGW was evaluated by AGW ratio based on the images at 8 weeks. The mean AGW ratio of the right side from eight rabbits was 0.55 ± 0.075 and that of the left side was 0.58 ± 0.073 (p = 0.655 by Mann–Whitney U test). In paired comparison of right and left side AGW ratio from each individual experimental rabbit, the symmetric AGW formation was statistically revealed (p = 0.128 by Wilcoxon signed rank test). To validate the reliably consistent AGW formation, the right and left AGW from all eight animals was evaluated and showed statistically consistent AGW formation (right side, p = 0.501; left side, p = 0.491 by Kolmogorov– Smirnov test). Normal vocal mucosal waves were not detected by evaluation of high-speed video data. In particular, amplitude of vibration was decreased by AGW that extended to the mid portion of the vocal fold (Figure 4). On histologic examination, a fibrotic band and collagen fiber deposits were detected by H&E and MT stains at the anterior commissure of the glottis. Although these deposited fibers shared some continuity of normal vocalis muscular fiber and partially elongated transversely along the thyroid cartilage, almost all AGW had an amorphous pattern of structural fiber aggregation. Elastin fibers were not actively stained in AGW formation by VVG study and hyaluronic acid which is normally sited in the lamina propria around the anterior commissure was diminished on AB staining (Figure 5). Discussion Here, we describe a novel rabbit model of AGW formation with a laryngofissure approach. To the best of our knowledge, this is the first animal model of AGW formation using a small animal. The success 4 week
8 week
Figure 3. Representative laryngoscopic images of normal rabbits (reference laryngoscopic image), and laryngoscopic images in experimental rabbits at 2, 4, and 8 weeks after anterior glottic web (AGW) formation.
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Normal
AGW Figure 4. Representative high-speed recording images of normal control and anterior glottic web (AGW).
x 12.5, HE
x 40, HE
x 40, AB
x 40, MT
Normal x 12.5, HE
x 40, HE
x 40, AB
x 40, MT
AGW Figure 5. Histologic evaluation of normal rabbit larynx (reference image) and anterior glottic web (AGW) formation at 8 weeks. Stains: AB, Alcian blue; H&E, hematoxylin eosin; MT, Masson’s trichrome.
rate of AGW formation using the presented method was high (87.5%) and the AGWs were consistent and symmetric. In particular, AGW created by this method had sufficient extent at both vocal folds to hinder normal mucosal waves on high speed recordings. The laryngofissure approach was easily performed by a laryngologist who had expertise in laryngeal anatomy. Researchers could quickly become acquainted with the following factors. (1) The large pre-epiglottic vasculature of rabbits (Figure 1A). When the large vessels are exposed at the midline of the pre-epiglottic space, meticulous hemostasis is required before midline thyroid cartilaginous incision. Inadequate control of bleeding would delay the procedure. (2) Precise midline thyroid cartilaginous
incision and inner laryngeal mucosa incision for laryngofissure approach. Based on our preliminary animal study (unpublished data), if the cartilaginous incision was not performed at the exact midline during the laryngofissure approach, wound dehiscence or inward dislocation of cutting edges could occur after approximation and suturing of the thyroid cartilage. This model of AGW formation has several advantages over previously published models using large animals [5,6]. First, stripping of the bilateral vocal mucosa under direct inspection of laryngeal inner side through a laryngofissure approach could make the procedures more accurate and thus make the resulting webs consistent. Second, the present procedure for AGW formation can be easily performed without special preparation such as a laryngeal microscope,
Novel rabbit model of anterior glottic web microlaryngeal instruments, and tracheal intubation. It can be performed with basic surgical instruments such as a scalpel, forceps, and scissors. Additionally, the experiments with small animals are cost-effective. The purchase cost of experimental canines is 10 to 20 times higher than that of rabbits and the experimental care fees for large animals are 5–6 times as much as that for rabbits in our institution. Although all of the animals used in this study survived without any complications related to the procedure, the laryngofissure approach is more invasive than conventional methods using laryngeal micro instruments. Due to the invasiveness of experimental procedures, close postoperative care with specific attention to the airway problems should be provided. Also, prolonged postoperative antibiotics (for 7 days in the present study) might be needed to prevent postoperative wound complications. Conclusion This is the first experimental report of AGW formation using rabbits. This model generated reproducible and reliable formation of an anterior web on laryngoscopic examination. It showed sufficient extent of webs to hinder normal mucosal waves during functional analysis. This novel rabbit model of AGW formation could be helpful to carry out the related research with lower costs. Acknowledgments This research was supported by the grants of the Seoul National University Hospital (No. 0620121480 and 0420133110) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI14C1541). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Benjamin B. Chevalier Jackson Lecture. Congenital laryngeal webs. Ann Otol Rhinol Laryngol 1983;92:317–26. [2] Fang R, Sun J, Wan G, Sun D. Comparison between mitomycin C and chitosan for prevention of anterior glottic steno after CO2 laser cordectomy in dogs. Laryngoscope 2007;117: 2057–62.
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[3] Liyanage SH, Khemani S, Lloyd S, Farrell R. Simple keel fixation technique for endoscopic repair of anterior glottic stenosis. J Laryngol Otol 2006;120:322–4. [4] Montgomery WW, Gamble JE. Anterior glottic stenosis. Experimental and clinical management. Arch Otolaryngol 1970;92:560–7. [5] Roh JL, Yoon YH. Prevention of anterior glottic stenosis after transoral microresection of glottic lesions involving the anterior commissure with mitomycin C. Laryngoscope 2005;115: 1055–9. [6] Roh JL, Yoon YH. Prevention of anterior glottic stenosis after bilateral vocal fold stripping with mitomycin C. Arch Otolaryngol Head Neck Surg 2005;131:690–5. [7] Strong MS, Jako GJ. Laser surgery in the larynx. Early clinical experience with continuous CO2 laser. Ann Otol Rhinol Laryngol 1972;81:791–8. [8] Devgan BK, Lampros WP, Leach W. Endolaryngeal surgery for anterior glottic stenosis. Ear Nose Throat J 1976;55: 377–81. [9] Cheng AT, Beckenham EJ. Congenital anterior glottic webs with subglottic stenosis: surgery using perichondrial keels. Int J Pediatr Otorhinolaryngol 2009;73:945–9. [10] Rice DH. Simple keel fixation technique for endoscopic repair of anterior glottic stenosis. J Laryngol Otol 2008; 122:764; author reply 764. [11] Roh JL, Kim DH, Park CI. The utility of second-look operation after laser microresection of glottic carcinoma involving the anterior commissure. Laryngoscope 2008; 118:1400–4. [12] Spector JE, Werkhaven JA, Spector NC, Huang S, Sanders D, Reinisch L. Prevention of anterior glottic restenosis in a canine model with topical mitomycin-C. Ann Otol Rhinol Laryngol 2001;110:1007–10. [13] Tunkel DE. A novel stent for treatment of combined anterior glottic web-subglottic stenosis. Int J Pediatr Otorhinolaryngol 2005;69:893–6. [14] Mehl ML, Kyles AE, Pypendop BH, Filipowicz DE, Gregory CR. Outcome of laryngeal web resection with mucosal apposition for treatment of airway obstruction in dogs: 15 cases (1992–2006). J Am Vet Med Assoc 2008;233: 738–42. [15] Sun G, Tang H, Sprecher AJ, MacCallum JK, Sun N, Fang Q. Transoral laser surgery for anterior commissure involvement: a study in canines. Auris Nasus Larynx 2010;37:601–8. [16] Yoon YH, Rha KS, Koo BS, Park JY, Kim YM, Park YH. The preventive effect of halofuginone on posterior glottic stenosis in a rabbit model. Otolaryngol Head Neck Surg 2008;139:94–9. [17] Roh JL, Lee YW, Park CI. Can mitomycin C really prevent airway stenosis? Laryngoscope 2006;116:440–5. [18] Jackson CL. Laryngofissure. Trans Am Acad Ophthalmol Otolaryngol 1954;58:16–21. [19] Ohno S, Hirano S, Kanemaru S, Kitani Y, Kojima T, Ishikawa S. Transforming growth factor beta3 for the prevention of vocal fold scarring. Laryngoscope 2012;122: 583–9. [20] Kwon SK, Kim HB, Song JJ, Cho CG, Park SW, Choi JS. Vocal fold augmentation with injectable polycaprolactone microspheres/pluronic F127 hydrogel: long-term in vivo study for the treatment of glottal insufficiency. PLoS One 2014;9:e85512.
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ORIGINAL ARTICLE
Novel experimental rabbit model of anterior glottic web formation
SEONG KEUN KWON1*, DONG WOOK KIM1*, YOON-JONG RYU1, SOO YEON KIM1, HYUN CHANG2, MYUNG WHUN SUNG1 & J. HUN HAH1 1
Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital & Seoul National University College of Medicine, Seoul and 2Department of Otorhinolaryngology-Head and Neck Surgery, Dongnam Institute of Radiological & Medical Sciences, Busan, Korea
Abstract Conclusion: The rabbit model of anterior glottic web (AGW) formation using the laryngofissure technique resulted in reproducible and stable AGW formation that may facilitate research into this area. Objective: To introduce and validate a novel experimental animal model of AGW formation using the rabbit. Methods: The inner larynges of eight New Zealand white rabbits were exposed through the laryngofissure technique. The mucosa of the bilateral true vocal fold was stripped off using the bevel of a needle tip. On the basis of the laryngoscopic findings at 8 weeks postoperatively, the extent of AGW was measured, and the success of this procedure was validated. Laryngeal specimens were sampled at 8 weeks for high-speed recording and histological analysis. Results: In seven (87.5%) rabbits, laryngoscopic examination revealed the formation of a scar band involving the anterior commissure. The mean extent of AGW ratio on the left and right sides was 0.58 ± 0.073 and 0.55 ± 0.075, respectively. The symmetric formation of AGW (p = 0.655, p = 0.128) and stability of the AGW procedure (p = 0.491, left; p = 0.501, right) were statistically validated. On high-speed recording, the vocal mucosal wave was hindered by AGW formation. Histologically, fibro-connective tissue, especially collagen fiber, was observed in the anterior commissure.
Keywords: Stenosis, larynx, laryngofissure technique, anterior commissure
Introduction Acquired anterior glottic webs (AGWs) are the most common type of glottic web, and can occur from sequelae of endolaryngeal lasers or instrumental procedures [1,2]. AGW refers to the formation of a fibrotic scar band covered by epithelium, between the vocal folds, that involves the anterior commissure. According to the severity and size of the web, AGW may result in abnormally elevated phonatory pitch, dysphonia, and airway restriction. Symptomatic AGW can be managed in various ways including by keel insertion via laryngofissure, which is recognized as the definitive treatment [3,4]. However, due to the invasiveness of this procedure, other more conservative and non-invasive treatments have been attempted. These treatments are diverse and range
from conventional cold instrumental dissection and intralesional injection to endoscopic CO2 laser with adjuvant mitomycin C application [2,5–8]. Despite a great deal of attention being focused on preventing restenosis, or preventing recurrence after management for AGW, this poses a therapeutic problem for otolaryngologists not only because of technical difficulties inherent in successful repair, but also because of the choice of several treatment methods. Since studies related to AGW are limited to a few case series reports and other studies [2,3,5,9–13], management of AGW is usually performed depending on the surgeon’s preference and clinical experience. To compare the different treatment options for AGW and to establish suitable management strategies, welldesigned studies involving suitable animal models are needed.
Correspondence: J. Hun Hah, MD PhD, Associate Professor, Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea. Tel: +82 2 2072 3649. Fax: +82 2 745 2387. E-mail: [email protected] *These authors contributed equally to this work.
(Received 9 February 2015; accepted 3 March 2015) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1028594
Novel rabbit model of anterior glottic web In the literature, there are several animal studies on AGW with large animals [2,6,12,14,15]. Although experiments on the larynx with large animals including dog, cow or pig have advantages, such as easy accessibility to the larynx and resemblance to humans in size, these could be more cumbersome than with small to mid-sized animals in terms of technique, feasibility, and expenses, including laboratory facility fees. Even though we thoroughly tried to find a small animal model for laryngeal stenosis or web, we could find only a few rabbit models of posterior glottic stenosis [5,16,17]. This lack of small animal models for AGW might be because of the difficulty of access to the larynx, especially to the anterior portion. Herein, we introduce and validate a novel experimental model of AGW formation using the laryngofissure in a rabbit. Material and methods The experiments performed in the present study were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (approval no. 14-0005-S1A0) and performed in
841
accordance with the ethical guidelines of the committee. Anterior glottic web formation in rabbit Eight male New Zealand white rabbits (Koatech Laboratory Animal Company, Gyeonggi, Korea) weighing 3.5 kg were anesthetized with intramuscular Zoletil 50 (50 mg/kg; Virbac Korea, Seoul, Korea). Through a 3 cm vertical midline skin incision at the level of the thyroid notch, the subcutaneous fat and the strap muscles were separated in the midline. By retracting the strap muscles using a self-curved mastoid retractor (Karl Storz, Tuttlingen, Germany), the hyoid, thyroid cartilage, and upper tracheal rings were further exposed by dissection (Figure 1A). To create a thyrohyoid window, a small vertical incision was made on the thyrohyoid membrane along the upper thyroid notch using a scalpel (no. 15). In this procedure, meticulous hemostasis of vessels in the pre-epiglottic space was performed with epinephrine (1:1000) soaked cotton or micro electro-cauterization (HIT1, Change-A-Tip, Bovie Medical Co., Purchase, NY, USA). Next, the whole length of perichondrium of the
A
B
C
D
E
Figure 1. Procedures for anterior glottic web (AGW) formation in rabbit, via a laryngofissure approach. (A) Exposed large pre-epiglottic vessel (arrowhead) after midline skin incision and strap muscle dissection. (B) Direct inspection of the bilateral vocal fold in rabbit (arrowheads) through laryngofissure. (C, D) Right and left vocal fold stripping from the vocal process (arrowheads) to the anterior commissure with an 18 gauge needle tip. (E) Bilaterally stripped vocal mucosa (arrowheads).
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thyroid cartilage was incised with a scalpel (no. 15), and the thyroid cartilage was split with monopolar electrocauterization at the midline, by a procedure similar to the laryngofissure approach [18]. Because the inner mucosa of the larynx should be incised at exactly the mid-point of the anterior commissure at the vocal fold, the laryngofissure procedure was performed following inspection through the window of thyrohyoid membrane. After splitting the thyroid cartilage, both sides of the inner mucosa on the true and false vocal fold were sufficiently exposed (Figure 1B). The true vocal mucosa just anterior to the vocal process tip was penetrated by a bevel of a needle tip (18 gauge) and stripped off along the whole length of the vocal fold from the vocal process to the anterior commissure (Figure 1C, D). For determination of exact depth of injury to vocal ligament, this procedure was performed under magnification using surgical microscopes. Bilateral mucosae of the true vocal folds were removed, and the bleeding was controlled with epinephrine-soaked cotton (Figure 1E). The split thyroid cartilages were closed tightly with Vicryl 4-0, and the suture site was covered by a strap muscle and adipose tissue layer by layer. The subcutaneous tissue and the skin were closed using surgical staples. After the procedure, animals were closely examined for signs of immediate postoperative complications such as bleeding and subcutaneous emphysema. Antibiotics (gentamicin sulfate, 80 mg/2 ml) were administered subcutaneously at 0.2 ml/kg/day for 7 days. All animals were monitored daily, with specific attention to their weight, cough, sputum production, wheezing, and dyspnea by professional laboratory animal keepers at the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) accredited institutions: Clinical Research Institute of Seoul National University Hospital (accreditation no. 001169).
Image J software, it was adjusted by dividing this number by the length of each vocal fold, from the anterior commissure to the vocal process, and this adjusted value was redefined as an AGW ratio (Figure 2). By evaluation of the AGW ratio in each rabbit and on each side of the vocal folds, the reliability and stability of the AGW formation procedure were determined. Recording of induced vocal fold vibration with high-speed camera and histologic evaluation At 8 weeks after AGW formation, the animals were humanely euthanized with intravenous injection of KCl under anesthesia and the larynges were removed by laryngectomy. Functional analysis with high-speed camera recording was followed by histological study. For better visualization of the vocal fold, the superior portion of the thyroid cartilages and the supraglottic structures were removed. The arytenoid cartilages were sutured together using a Prolene 6-0 suture to close both vocal folds. The trimmed larynx was mounted on a holder and the vocal fold vibration was generated by humidified air injection using a medical oxygen generator. Imaging of vocal fold vibration during induced phonation was accomplished by a MotionXtra NR4S2 high-speed video camera (DEL Imaging Systems, Cheshire, CT, USA). High-speed video data were recorded at 8000 images per second and a spatial
A
Laryngoscopic evaluation At 2, 4, and 8 weeks after AGW formation, rabbits were anesthetized as described previously and a laryngeal endoscopic examination was performed using a 4.0 mm 30 rigid endoscope (Richards, Knittlingen, Germany). Images of each animal’s vocal folds were taken with a digital camera (E4500, Nikon, Tokyo, Japan) attached to the rigid endoscope. By laryngoscopic imaging at 8 weeks, the extent of AGW was evaluated by measuring the length from the anterior commissure to the posterior end of the scar band formation at the right and left sides of the vocal folds with Image J imaging software (National Institute of Mental Health, Bethesda, MD, USA) [19]. Because this measured value was defined by pixels on the
C
B
AC , using Image J AB software: A, anterior commissure; B, vocal process; C, the end of the web in each vocal fold. Figure 2. Anterior glottic web (AGW) ratio =
Novel rabbit model of anterior glottic web resolution of 256 horizontal 512 vertical pixels. Illumination was provided by a 300 W xenon light source. Each high-speed video data segment consisted of 8000 images per second [20]. After high-speed camera recording, laryngeal specimens were fixed for 24 h in 10% formalin, embedded in paraffin, and sliced at a thickness of 2 mm using a microtome. The sections were deparaffinized and dehydrated in graded ethanol. Hematoxylin and eosin (H&E), Masson’s trichrome (MT), Alcian blue (AB), and Verhoeff-Van Gieson (VVG) stains were used to evaluate histologic changes associated with AGW formation. Statistical analysis Experimental data are expressed as means ± standard deviation (SD). For evaluation of symmetric AGW formation, paired comparison of right and left side AGW ratio from each individual experimental animal was conducted by the Wilcoxon signed rank test. Also, to validate the consistent formation of AGW among all the experimental rabbits, the distributions of AGW ratio from the eight experimental rabbits were statistically evaluated by the Kolmogorov– Smirnov test. The statistical tests in the current study were used for comparison using SPSS 20.0 statistical software and statistical significance was accepted at p < 0.05. Results All animals survived without complications following surgery. Successful AGW formation was observed in seven of eight rabbits by endoscopic examination (87.5%). Some granulated tissue and irregular margins of the scar band at the anterior commissure were observed during week 2 by endoscopic examination. However, the granulation was resolved and AGW covered with normal mucosa developed over time. Consequently, compared with normal larynx (reference laryngoscopic image of normal rabbit), which Normal
2 week
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showed an anterior commissure in an inverted ‘V’ shape and at an acute angle, AGW had a broad angle margin and an inverted ‘U’-shaped scar band by 8 weeks (Figure 3). The extent of AGW was evaluated by AGW ratio based on the images at 8 weeks. The mean AGW ratio of the right side from eight rabbits was 0.55 ± 0.075 and that of the left side was 0.58 ± 0.073 (p = 0.655 by Mann–Whitney U test). In paired comparison of right and left side AGW ratio from each individual experimental rabbit, the symmetric AGW formation was statistically revealed (p = 0.128 by Wilcoxon signed rank test). To validate the reliably consistent AGW formation, the right and left AGW from all eight animals was evaluated and showed statistically consistent AGW formation (right side, p = 0.501; left side, p = 0.491 by Kolmogorov– Smirnov test). Normal vocal mucosal waves were not detected by evaluation of high-speed video data. In particular, amplitude of vibration was decreased by AGW that extended to the mid portion of the vocal fold (Figure 4). On histologic examination, a fibrotic band and collagen fiber deposits were detected by H&E and MT stains at the anterior commissure of the glottis. Although these deposited fibers shared some continuity of normal vocalis muscular fiber and partially elongated transversely along the thyroid cartilage, almost all AGW had an amorphous pattern of structural fiber aggregation. Elastin fibers were not actively stained in AGW formation by VVG study and hyaluronic acid which is normally sited in the lamina propria around the anterior commissure was diminished on AB staining (Figure 5). Discussion Here, we describe a novel rabbit model of AGW formation with a laryngofissure approach. To the best of our knowledge, this is the first animal model of AGW formation using a small animal. The success 4 week
8 week
Figure 3. Representative laryngoscopic images of normal rabbits (reference laryngoscopic image), and laryngoscopic images in experimental rabbits at 2, 4, and 8 weeks after anterior glottic web (AGW) formation.
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Normal
AGW Figure 4. Representative high-speed recording images of normal control and anterior glottic web (AGW).
x 12.5, HE
x 40, HE
x 40, AB
x 40, MT
Normal x 12.5, HE
x 40, HE
x 40, AB
x 40, MT
AGW Figure 5. Histologic evaluation of normal rabbit larynx (reference image) and anterior glottic web (AGW) formation at 8 weeks. Stains: AB, Alcian blue; H&E, hematoxylin eosin; MT, Masson’s trichrome.
rate of AGW formation using the presented method was high (87.5%) and the AGWs were consistent and symmetric. In particular, AGW created by this method had sufficient extent at both vocal folds to hinder normal mucosal waves on high speed recordings. The laryngofissure approach was easily performed by a laryngologist who had expertise in laryngeal anatomy. Researchers could quickly become acquainted with the following factors. (1) The large pre-epiglottic vasculature of rabbits (Figure 1A). When the large vessels are exposed at the midline of the pre-epiglottic space, meticulous hemostasis is required before midline thyroid cartilaginous incision. Inadequate control of bleeding would delay the procedure. (2) Precise midline thyroid cartilaginous
incision and inner laryngeal mucosa incision for laryngofissure approach. Based on our preliminary animal study (unpublished data), if the cartilaginous incision was not performed at the exact midline during the laryngofissure approach, wound dehiscence or inward dislocation of cutting edges could occur after approximation and suturing of the thyroid cartilage. This model of AGW formation has several advantages over previously published models using large animals [5,6]. First, stripping of the bilateral vocal mucosa under direct inspection of laryngeal inner side through a laryngofissure approach could make the procedures more accurate and thus make the resulting webs consistent. Second, the present procedure for AGW formation can be easily performed without special preparation such as a laryngeal microscope,
Novel rabbit model of anterior glottic web microlaryngeal instruments, and tracheal intubation. It can be performed with basic surgical instruments such as a scalpel, forceps, and scissors. Additionally, the experiments with small animals are cost-effective. The purchase cost of experimental canines is 10 to 20 times higher than that of rabbits and the experimental care fees for large animals are 5–6 times as much as that for rabbits in our institution. Although all of the animals used in this study survived without any complications related to the procedure, the laryngofissure approach is more invasive than conventional methods using laryngeal micro instruments. Due to the invasiveness of experimental procedures, close postoperative care with specific attention to the airway problems should be provided. Also, prolonged postoperative antibiotics (for 7 days in the present study) might be needed to prevent postoperative wound complications. Conclusion This is the first experimental report of AGW formation using rabbits. This model generated reproducible and reliable formation of an anterior web on laryngoscopic examination. It showed sufficient extent of webs to hinder normal mucosal waves during functional analysis. This novel rabbit model of AGW formation could be helpful to carry out the related research with lower costs. Acknowledgments This research was supported by the grants of the Seoul National University Hospital (No. 0620121480 and 0420133110) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI14C1541). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Benjamin B. Chevalier Jackson Lecture. Congenital laryngeal webs. Ann Otol Rhinol Laryngol 1983;92:317–26. [2] Fang R, Sun J, Wan G, Sun D. Comparison between mitomycin C and chitosan for prevention of anterior glottic steno after CO2 laser cordectomy in dogs. Laryngoscope 2007;117: 2057–62.
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[3] Liyanage SH, Khemani S, Lloyd S, Farrell R. Simple keel fixation technique for endoscopic repair of anterior glottic stenosis. J Laryngol Otol 2006;120:322–4. [4] Montgomery WW, Gamble JE. Anterior glottic stenosis. Experimental and clinical management. Arch Otolaryngol 1970;92:560–7. [5] Roh JL, Yoon YH. Prevention of anterior glottic stenosis after transoral microresection of glottic lesions involving the anterior commissure with mitomycin C. Laryngoscope 2005;115: 1055–9. [6] Roh JL, Yoon YH. Prevention of anterior glottic stenosis after bilateral vocal fold stripping with mitomycin C. Arch Otolaryngol Head Neck Surg 2005;131:690–5. [7] Strong MS, Jako GJ. Laser surgery in the larynx. Early clinical experience with continuous CO2 laser. Ann Otol Rhinol Laryngol 1972;81:791–8. [8] Devgan BK, Lampros WP, Leach W. Endolaryngeal surgery for anterior glottic stenosis. Ear Nose Throat J 1976;55: 377–81. [9] Cheng AT, Beckenham EJ. Congenital anterior glottic webs with subglottic stenosis: surgery using perichondrial keels. Int J Pediatr Otorhinolaryngol 2009;73:945–9. [10] Rice DH. Simple keel fixation technique for endoscopic repair of anterior glottic stenosis. J Laryngol Otol 2008; 122:764; author reply 764. [11] Roh JL, Kim DH, Park CI. The utility of second-look operation after laser microresection of glottic carcinoma involving the anterior commissure. Laryngoscope 2008; 118:1400–4. [12] Spector JE, Werkhaven JA, Spector NC, Huang S, Sanders D, Reinisch L. Prevention of anterior glottic restenosis in a canine model with topical mitomycin-C. Ann Otol Rhinol Laryngol 2001;110:1007–10. [13] Tunkel DE. A novel stent for treatment of combined anterior glottic web-subglottic stenosis. Int J Pediatr Otorhinolaryngol 2005;69:893–6. [14] Mehl ML, Kyles AE, Pypendop BH, Filipowicz DE, Gregory CR. Outcome of laryngeal web resection with mucosal apposition for treatment of airway obstruction in dogs: 15 cases (1992–2006). J Am Vet Med Assoc 2008;233: 738–42. [15] Sun G, Tang H, Sprecher AJ, MacCallum JK, Sun N, Fang Q. Transoral laser surgery for anterior commissure involvement: a study in canines. Auris Nasus Larynx 2010;37:601–8. [16] Yoon YH, Rha KS, Koo BS, Park JY, Kim YM, Park YH. The preventive effect of halofuginone on posterior glottic stenosis in a rabbit model. Otolaryngol Head Neck Surg 2008;139:94–9. [17] Roh JL, Lee YW, Park CI. Can mitomycin C really prevent airway stenosis? Laryngoscope 2006;116:440–5. [18] Jackson CL. Laryngofissure. Trans Am Acad Ophthalmol Otolaryngol 1954;58:16–21. [19] Ohno S, Hirano S, Kanemaru S, Kitani Y, Kojima T, Ishikawa S. Transforming growth factor beta3 for the prevention of vocal fold scarring. Laryngoscope 2012;122: 583–9. [20] Kwon SK, Kim HB, Song JJ, Cho CG, Park SW, Choi JS. Vocal fold augmentation with injectable polycaprolactone microspheres/pluronic F127 hydrogel: long-term in vivo study for the treatment of glottal insufficiency. PLoS One 2014;9:e85512.
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