An Animal Model for Laryngotracheal Injuries: An Experimental Study

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1 The Laryngoscope VC 2014 The American Laryngological, Rhinological and Otological Society, Inc. An Animal Model for Laryngotracheal Injuries: An Experimental Study Saravanam P. Kumar, DLO, DNB, MNAMS; Arunchalam Ravikumar, MS, DNB, DLORCS, MAMS; J. Thanka, MD Objectives/Hypothesis: To establish an animal model for post-intubation laryngotracheal injuries resulting in significant laryngotracheal insult without compromising the survival of the animal for a minimum period of 12 weeks post-extubation. To study the extent of injury based on morphometric and histopathological change seen at 12 weeks post-extubation. Study Design: Randomized block, single-blinded, experimental study. Methods: Twelve New Zealand white rabbits were used in this study. The rabbits were randomly divided into three groups depending on the duration of intubation (6 hours, 4 hours, and 2 hours, respectively). The technique of intubation was standardized. Post-extubation, these rabbits were humanely sacrificed after observation for 12 weeks or when they developed severe respiratory distress. The larynx and trachea were subjected to gross and histopathological examination. Results: Histopathological examination and statistical analysis showed significant subglottic injury in all group B rabbits (intubated for 4 hours), and all of them survived for 12 weeks. Conclusion: In this study, an animal model for inducing and studying laryngotracheal injuries due to endotracheal intubation has been established. This study has the potential applications in research on etiopathogenesis and management of post-intubation laryngotracheal injuries. Key Words: Laryngotracheal injuries, laryngotracheal stenosis, airway model. Level of Evidence: N/A. Laryngoscope, 125:E23 E27, 2015 INTRODUCTION In a multivariate analysis of patients who had undergone endotracheal intubation, Rangachari et al. observed that about 80% of all airway intubations resulted in laryngotracheal injuries, and 20% of these resulted in a permanent sequelae. 1 The endotracheal tube size and duration of intubation at the end of 3 weeks were the only predictors of laryngeal sequelae. 1 Post-intubation injury is the most common cause of laryngotracheal stenosis. 2 The other causes for laryngotracheal stenosis are tracheostomy, external trauma, autoimmune process, and tumors. 2 The incidence of postintubation subglottic stenosis is reported to be as high as 11.38% in children. 3 Because the subglottis is the narrowest part of the laryngotracheal lumen in infants and children, it is more prone to injury. This problem is compounded by the formation of a scab, granulation, or postintubation edema, which leads to a respiratory distress that requires emergent intervention. From the Department of ENT, Head and Neck Surgery (S.P.K., A.R.); and the Department of Pathology (J.T.), Sri Ramachandra Medical College and Research Institute, Porur, Chennai-116, India. Editor s Note: This Manuscript was accepted for publication July 14, The authors have no funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Dr. Major Saravanam Prasanna Kumar, Associate Professor, Department of ENT, Head and Neck Surgery, Sri Ramachandra Medical College and Research Institute, Porur, Chennai-116, India. sprasannakumar10@gmail.com DOI: /lary Prospective, experimental, and histopathological studies of the pathogenesis and therapeutic management of laryngotracheal injuries are lacking and are almost impossible to conduct in humans. Many experimental animal models of laryngotracheal injuries have been described in literature. 4 7 All studies have induced injury with a sharpened metal rod, 4 wire scrappings, 5 laser, 6 electrocautery, or cyrocautery 7 in the subglottis and trachea. However, this is not the usual mode of injury that occurs in patients who develop postintubation laryngotracheal stenosis. Hence, these experimental animal studies have failed to initiate a process similar to the post-intubation laryngotracheal injuries found in humans. 8 Most animal experimental models have sacrificed the animals after a short period post-injury. These studies only report the acute changes following laryngotracheal injuries. In an experimental rabbit model, Otteson et al. have described the chronic changes following CO2 laser induced injury in the subglottis. 9 These rabbits were then followed up for 84 days, at which point they were sacrificed to study histopathological changes. 9 Thus, it is necessary, to study the chronic changes and the temporal sequence of wound healing, including the remodelling phase of wound repair, which usually takes 12 weeks. Aim and Objective To establish an animal model for post-intubationlaryngotracheal injuries resulting in significant E23

2 TABLE I. Follow-Up Chart of the Three Groups Post-extubation. Group A (6 hr.) Group B (4 hr.) Group C (2 hr.) Total no. rabbits Airway obstruction/respiratory distress Early death/euthanasia 3 Nil 1* Number of weeks at sacrifice/death 3,12, 4,8, Average: , 12, 4*,12 Average: 10 *One rabbit in group C died at 4 weeks due to unknown cause (not due to respiratory distress). laryngotracheal insult without compromising the survival of the animal for a minimum period of 12 weeks post-extubation. To study the extent of injury based on morphometric and histopathological change seen at 12 weeks post-extubation. MATERIALS AND METHODS This study was done at the Central Animal Facility for Toxicology and Developmental Research at Sri Ramachandra University, Chennai, India. Institutional animal ethics committee approval was obtained. The initial studies were carried out in postmortem specimens of New Zealand white rabbits weighing 2.0 to 2.5 kg. The cross-sectional diameter of the trachea and subglottis were measured. It was observed that the narrowest part of the rabbit s laryngotracheal airway was the subglottis, measuring on an average 5.8 mm in anteroposterior diameter and 5.4 mm in transverse diameter. This was a randomized block, single-blind study. Twelve New Zealand white rabbits were used in this study. Three rabbits from the same colony formed one block. They were randomly divided into three groups depending on the duration of intubation (i.e., group A, group B, and group C rabbits were intubated for 6 hours, 4 hours, and 2 hours, respectively). Randomized block design was used to reduce the variability that may occur in different rabbit colonies and the environmental and nutritional variability that can occur over a period of time. The pathologist reporting the morphological and histopathological examination was blinded to the group to which the specimen belonged. A 4.0 cuffed endotracheal tube with an outer diameter of 5.4 mm was used for intubation. An experienced veterinary doctor performed the intubation in the operating room. Method of Intubation The technique of intubation was standardized. The direct laryngoscopic method was used for intubation. Prior to intubation, injection ketamine 30 mg/kg and injection midazolam 0.75mg/kg were administered intramuscularly. When the rabbits were partly sedated and docile, they were preoxygenated. An assistant held the rabbit in a supine position with head extension, and a silk thread was used to open the mouth by looping it around the incisors. The tongue was gently pulled out to the left side, and a paediatric laryngoscope was used to visualize the larynx. A 4.0 mm cuffed Portex endotracheal tube (Smiths Medicals, Dublin, OH 43017, U.S.A) was introduced via the glottis under direct vision. A little resistance was felt when the cuff was passing just below the glottis in all the rabbits; this was gently negated. The rabbits were maintained on spontaneous respiration with oxygen at 4 litres and isoflurane at 0.75% to 1.25%. They were kept warm in an incubator for the intended period of time (6 hours, 4 hours, and 2 hours, respectively). The endotracheal tube was rotated, moved up and down after deflating the cuff at hourly intervals. The rabbits were then extubated and monitored in a temperature-controlled chamber and later transferred to their respective cages, where they were monitored for 12 weeks. These rabbits were humanely sacrificed using injection ketamine 100 mg/kg which was given intravenously as a bolus at the end of 12 weeks or in cases of severe respiratory distress occurring within 12 weeks. The larynx and trachea were harvested from the hyoid above to 3 cm of the trachea below. These specimens were coded, preserved in 10% formalin, and submitted for morphological and histopathological examination. They were grossed and sectioned at three levels:the glottis; subglottis; and the trachea 1.5 cm below the glottis, which corresponded to the level of the inflated cuff. Hematoxylin and Eosin stain and Masson s Trichrome stain were used. The following parameters were recorded: The internal transverse and anteroposterior diameter of the lumen were measured in micrometres (mm). The mucosal ulceration, submucosal capillary thickness, granulation, submucosal thickness, and submucosal fibrosis were all graded on a scale of 0 to 3 using a continuous high-power field per section by the pathologist (grade 0: no significant changes; grade 1: 0% 25 %; grade 2: 25% 50%; grade 3: > 50%). The measurements and scores obtained from the histopathological examination were statistically analysed using nonparametric testing, initially using the K independent test and applying the Kruskal-Wallis test, which were significant for the internal dimension at the level of subglottis. In addition, the two independent sample tests and the Mann-Whitney test were used to compare the two groups (IBM SSPS Statistics for Windows, Version 20, Armonk, NY). RESULTS All rabbits in group A (6-hour intubation) developed respiratory distress within 12 weeks. Of those, three could not survive the requisite 12 week period and had to be sacrificed at the third week, fourth week, and eighth week post-intubation, respectively. Two of the 4 rabbits from group B (4-hour intubation) developed respiratory distress, but all survived the 12-week period. One rabbit in group C (2-hour intubation) had a premature death due to an undetermined cause at fourth-week post-extubation. The rest of the rabbits survived the 12 week period (Table I). There was no statistically significant difference in the histopathological score between group A and group B specimens. A statistically significant narrowing in the subglottic lumen was seen in group A when compared with group C (P value ). Similarly, there was a statistically significant narrowing in the subglottic E24

3 Fig. 1. (a) Boxplot of the intraluminal dimension anteroposterior and lateral in mm at the level of subglottis for the three groups (mean measurements). (b) The score across groups at the subglottic level for mucosal ulceration, submucosal thickness, submucosal fibrosis, submucosal capillaries, and granulations. [Color figure can be viewed in the online issue, which is available at com.] lumen in group B compared with group C (P value ). Significant narrowing of the lumen, increase in submucosal thickness, and submucosal fibrosis was noted in group A and group B as compared to group C. The graphical representation of the comparison between the three groups at the level of the subglottic are shown in Figures 1a and 1b. The histopathological section of group A, group B, and group C specimens at the level of the subglottis are shown in Figures 2a and 2b. DISCUSSION Ten-Hallers et al. have defined an animal model as a living organism with one or more anatomical or physiological aspects that may be compared with a healthy or diseased human based on the reaction to an external stimulus. 10 Many factors can influence the choice of animal model for experimental research. These include ease of handling, accessibility, cost of procuring and maintaining, and knowledge and expertise of the person handling them. 10 The experimental animal model for laryngotracheal injuries has been widely described in literature. The most common animal models used in laryngotracheal research are piglets, dogs, and rabbits. 11 Supance et al. have described a canine model in which the puppies were intubated for 7 and 14 days. Those intubated for 14 days showed a 40% to 50% reduction in subglottic lumen. 8 In order to mimic the endotracheal cuff pressure induced tracheal wall damage, a porcine model in which piglets were intubated for 48 hours was described by Nseir et al. 12 They could not find any statistically significant result between the continuous controlled cuff-pressure group and the manually controlled cuff-pressure group. Belson et al. observed that prolonged intubation with a cuffed endotracheal tube resulted in pressure-induced injury to the trachea, which on histopathological examination showed squamous metaplasia. 13 All these studies, although congruent with the pathogenesis of laryngotracheal injury, are laborious and at times handling large animals can be unpredictably dangerous. In the past, an airway model using rabbits have been successfully described to yield reproducible results. 14,15 For this study, the New Zealand white rabbit was preferred because it is docile and easier to handle compared to larger animals. Moreover, the rabbit larynx and trachea are almost similar to those of an infant and children younger than 2 years of age. Delayed onset tracheal stenosis was reported by Grint et al. in three rabbits that were intubated for neutering. 16 Subglottic and tracheal mucosal epithelial erosions and submucosal inflammation were seen in rabbits intubated for 1 hour. 17 Subglottic injury and inflammation were observed in rabbits intubated with the inflated cuff kept just below the vocal cords for 2 hours. 12 A rabbit model in which the subglottis and upper trachea were stented using an endotracheal tube that was cut and positioned below the vocal cord, fixed to the trachea with sutures, and left in situ for a week was described by Kelly et al. 18 Three of the eight rabbits that underwent the procedure were excluded due to morbidity and mortality, the others were sacrificed 1 week post-extubation to study the morphometric and histopathological sections at the level of subglottis that showed submucosal thickening, fibrosis, and perichondritis. The direct laryngoscopic visualization technique for intubation was preferred. Whatever the technique of intubation, it must be noted that when using a snugly fitting endotracheal tube, there will be resistance as the glottis is entered. This resistance must be gently negated with a force not more than 30 g. 19 E25

4 Fig. 2. (a) Hematoxylin and Eosin stain (1.53) at the level of subglottis across all three groups, showing significant narrowing of lumen, submucosal thickness, submucosal fibrosis, and submucosal vascularity in group A and group B compared to group C. (b) Masson Trichrome staining (1.53) at the level of subglottis, showing significant narrowing of lumen, submucosal thickness, and submucosal fibrosis in group A and group B compared to group C. Note evidence of perichondritis and cartilage deformity (yellow arrow). [Color figure can be viewed in the online issue, which is available at In our study, it was noted that the maximum injury and histopathological changes were seen in the subglottis with intraluminal narrowing, submucosal thickening, collagen deposit, cartilage deformity, and perichondritis in all the rabbits of group A and B, as seen in Figure 2b. None of the rabbits in the three groups had any histological evidence of injury in the tracheal wall. The subglottis of the rabbit being the narrowest, and also surrounded by the only complete ring in the airway, is predisposed to injury similar to that seen in infants and children. The trachea of the rabbit is expandable to a certain limit because of the V-shaped cartilage and the connecting trachealis muscle; thus, the tracheal mucosa is able to sustain the cuff pressure without mucosal injury. The limitation of this study is the small number of rabbits used. Thus, the results of this study should be interpreted with caution while extrapolating them to human patients The objective of this study was to produce intubation-induced injury in the laryngotracheal airway. Endotracheal tubes that snuggly fit the lumen were used, and the tubes were moved every hour to induce injury. This experimental model establishes the fact that a snuggly fitting endotracheal tube in the subglottis can produce subglottic injury. The severity of subglottic injury depends on the duration of intubation. Many studies have pointed out that the size of the endotracheal tube is a major risk factor for the formation of post-intubation subglottic stenosis in neonates. 20,21 The group B (4-hour intubation) model is the most suitable experimental model for study of the post-intubation sequela in a temporal sequence over a period of 12 weeks. This model can be used to study the various risk factors that are implicated in post-intubation laryngotracheal stenosis, such as prolonged intubation, the size of the endotracheal tube, movement of the endotracheal tube, and gastroesophageal reflux. 22,23 This model can also be used to study the efficacy of drugs that would modify the wound-healing process in a temporal sequence over a 12-week period and prevent the formation of stenosis. E26

5 CONCLUSION In this study, an animal model for inducing and studying laryngotracheal injuries due to endotracheal intubation has been established. The animal model described in this study is similar to laryngotracheal injuries that occurs in infants and children following intubation. Potential applications are to study the ethiopathogenesis and management of post-intubation laryngotracheal injuries in a temporal sequence of events. BIBLIOGRAPHY 1. Rangachari V, Sundararajan I, Sumathi V, Krishna Kumar K. Laryngeal sequelae following prolonged intubation: A prospective study. Indian J Crit Care Med 2006;10: George M, Lang F, Pasche P, Monnier P. Surgical management of laryngotracheal stenosis in adults. Eur Arch Otorhinolaryngol 2005;262: Schweiger C, Marostica PJ, Smith MM, Manica D, Carvalho PR, Kuhl G. Incidence of post-intubation subglottic stenosis in children: prospective study. J Laryngol Otol 2013;127: doi: /S X. 4. Mitskavich MT, Rimell FL, Shapiro AM. Porcine model of airway mucosal injury. Am J Otolaryngol 1997;18: Nakagishi Y, Morimoto Y, Masanori F, Ozeki Y, Maehara T, Makoto K. Rabbit model of airway stenosis induced by scraping the tracheal mucosa. Laryngoscope 2005;115: Eliashar R, Eliaschar I, Esclamado R, Gramlich T, Strome M. Can topical mitomycin prevent laryngotracheal stenosis? Laryngoscope 1999;109: Mayer T, Matlak ME, Dixon J, Johnson DG, McCloskey D. Experimental subglottic stenosis: histopathologic and bronchoscopic comparison of electrosurgical, cryosurgical, and laser resection. J Pediatr Surg 1980; 15: Supance JS, Reilly JS, Doyle WJ, Bluestone CD, Hubbard J. Acquired subglottic stenosis following endotracheal intubation: a canine model. Arch Otolaryngol 1982;108: Otteson TD, Sandulache VC, Barsic M, DiSilvio GM, Hebda PA, Dohar JE. Acute and chronic changes in the subglottis induced by graded CO2 laser injury in the rabbit airway. Arch Otolaryngol Head Neck Surg 2008;134: ten Hallers EJ, Rakhorst G, Marres HA, et al., Animal models for tracheal research. Biomaterials 2004;25: Review. 11. Roh JL, Lee Y-W, Park CI. Can mitomycin C really prevent airway stenosis? Laryngoscope 2006;116: doi: /01.mlg Nseir S, Duguet A, Copin MC, et al. Continuous control of endotracheal cuff pressure and tracheal wall damage: a randomized controlled animal study. Crit Care 2007;11:R109. doi: /cc Belson TP. Cuff induced tracheal injury in dogs following prolonged intubation. Laryngoscope 1983;93: Kil HK, Alberts MK, Liggitt HD, Bishop MJ. Dexamethasone treatment does not ameliorate subglottic ischemic injury in rabbits. Chest 1997; 111: Roh JL, Kim DH, Rha KS, Sung MW, Kim KH, Park CI. Benefits and risks of mitomycin use in the traumatized tracheal mucosa. Otolaryngol Head Neck Surg 2007;136: Grint NJ, Sayers IR, Cecchi R, Harley R, Day MJ. Postanaesthetic tracheal strictures in three rabbits. Lab Anim 2006;40: Squire R, Siddiqui SY, CiNunzio G, et al. Quantitative study of the early effects of tracheostomy and endotracheal intubation on the rabbit tracheobronchial tree. Ann Otol Rhinol Laryngol 1990;99: Kelly NA, Murphy M, Giles S, Russell JD. Subglottic injury: a clinically relevant animal model. Laryngoscope 2012;122: Kruger J, Zeller W, Schottmann E. A simplified procedure for endotracheal intubation in rabbits. Lab Anim 1994;28: Contencin P, Narcy P. Size of endotracheal tube and neonatal acquired subglottic stenosis. Study Group for Neonatology and Pediatric Emergencies in the Parisian Area. Arch Otolaryngol Head Neck Surg 1993; 119: Sherman JM, Lowitt S, Stephenson C, Ironson G. Factors influencing acquired subglottic stenosis in infants. J Pediatr 1986;109: Michael JR, Robin TC. Airway management in infant and child. In: Ballenger s Otorhinolaryngology: Head and Neck Surgery. Centennial Edition. Snow JB Jr, Wackym PA, eds. Shelton, CT: People s Medical Publishing House; 2009: Loi HD, Parhr AS, Subramaniam SK, Choo KE, Ng HP Neonatal postintubation subglottic stenosis. Med J Malaysia 2004;59: E27