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Jennifer Y Lee, MD1,2; Sunil Singhal, MD1,2; Kevin Leahy, MD, PhD1; Noam Cohen MD, PhD1,2; Genevieve Philiponis, BA2; Ankona Ghosh, MD1,2; Natasha Mirza, MD1,2

1University of Pennsylvania, 2Philadelphia Veterans Administration Hospital

INTRODUCTION

METHODS AND MATERIALS

CONCLUSIONS

DISCUSSION

REFERENCES

Chart 1. RQ level of expression of inflammatory markers. p > 0.05

ABSTRACT

CONTACT

Laryngotracheal stenosis is a consequence of imbalance in wound healing leading to increased fibrosis and narrowing of the airway lumen. Inflammatory markers have been noted to be elevated in stenosis including IL-1 and TGF-beta. After injury, macrophages infiltrate the local site and produce chemoattractants such as TGF-beta which increase IL-1 and prostaglandins, pro-inflammatory agents.4 Other studies have shown expression of cyclooxygenase mediated by increased prostaglandins lead to mucosal fibroblasts activity in the airway.1 We hypothesized that expression of COX would be increased in our murine model of laryngotracheal stenosis by wire and acid injury. The expression of cycloxygenase was elevated in acid injury and wire injury LTCs compared to uninjured LTCs. However, it was not significant. The inhibition of COX using COX-2 inhibitor was only significant in acid injury compared to control LTCs. This study had many limitations. First, the size of the study was limited. Second, the expression of cyclooxygenase was not significantly elevated. COX expression is reported to be transient and that may have been missed in a three week study. Third, inhibition was COX with the inhibitor was limited.

ProcedureLaryngotracheal complexes (LTC) from C57BL6 mice were harvested by exposing the airway from the thyroid cartilage to approximately the third tracheal ring. (Figure 1A). A total of thirty two mice were used. Chemical or acid injury was performed on eight LTCsby injecting HCl acid (pH 3-4) within the lumen of the airway for five minutes prior to irrigation with saline. (Figure 1B) Traumatic or wire injury was performed on eight separate LTCs by inserting and removing a wire brush (2 mm in diameter) through the lumen twenty times. (Figure 1C) Eight separate LTCs were harvested without injury. One set of LTCs consisted of one uninjured LTC, one acid injured LTC and one wire injured LTC. Each set of three LTCs were placed into a recepient mouse’s dorsum. (Figure 1D.) After three weeks, the LTCs were harvested. InterventionMurine chow with cyclooxygenase inhibitor (0.1% weight to weight) were given as the only chow for three weeks for four recipient mice. Regular chow was given to the untreated four recipient mice. Amount consumed were measured by measuring remaining chow. Each recipient mouse ingested about 25% of its body weight in COX inhibitor in the course of the three weeks.HistologyTwo sets of LTCs were fixed in formalin, parafilm, sliced and stained with H&E. RT-PCRSix LTCs were harvested and RNA was extracted from the tissue. The RNA was reverse transcribed to cDNA, then quantitatively amplified using reverse transcription polymerase chain reaction or RT-PCR using primers for IL-1, COX2 and TGF beta. Statistical analysisStatistical significance was determined using the Student t-test with significance set at p < 0.05.

In this study, no significant difference in granulation formation, measured by lamina propria thickness was found between control and treatment laryngotracheal complexes. In addition, no significant change was noted in inflammatory markers such as IL-1 and TGF-beta in control versus treatment laryngotracheal complexes. This may be due to the limited sample size or may be secondary to inadequate inhibition.Further studies may elucidate the mechanism

Laryngotracheal stenosis is a narrowing of the airway secondary to activation of the inflammatory cascade from different types of injury. It is closely linked with inflammatory markers such as IL-1 or TGF-beta.1We have previously described a murine model of laryngotracheal stenosis that mimics physiologic injury such as laryngotracheal reflux or intubation.2 In this experiment, we aim to test the effects of inhibition of cycloxygenase on granulation formation in this murine model of laryngotracheal stenosis and on inflammatory cytokines.

1. Sandulache VC, Singh T, Li-Korotky HS et al. Prostagandin E2 is Activated by Airway Injury and Regulates Fibroblast Cytoskeletal Dyanmics. Laryngoscope. 2009, 119(7): 1365-1373.

2. Ghosh, A, Malaisrie N, Leahy KP, et al. Cellular Adaptive Inflammation Mediates Airway Granylation in a Murine Model of Subglottic Stenosis. Otolaryngolo Head Neck Surg. 2011, 144(6): 927-933.

3. Marin C, Wohlsen A, Uhlig S. Changes in airway resistance by simultaneous exposure to TNF-alpha and IL-1 beta in perfused rat lungs. Am J Physiol Lung Cell Mol Physiol. 2001, 280(4): L595-601.

4. Hirshoren N, Eliashar R. Wound-healing Modulation in Upper Airway Stenosis – Myths and Facts. Head and Neck. 2009, 31:111-126.

Figure 2. H &E stains of LTC.

Figure 1. A. Photo of exposed laryngotracheal complex. B. Photo of acid injury by injecting HCl acid. C. Photo of trauma injury by repeated insertion and removal of a

wire brush. D. Schematic of implantation of LTC on dorsum of recipient mouse.

Objective: The study explores possible therapies to decrease or prevent subglottic stenosis. We aim to study the effects of cyclooxygenase (COX) inhibition on laryngotracheal granulation and stenosis in a previously described murine model.

Methods: Laryngotracheal complexes (LTC) were transplanted into a previously established recipient mouse model. Treatment group (n=4) received COX inhibitor chow for three weeks. Control group (n=4) did not. After three weeks, LTCs were harvested and examined microscopically for comparison. Relative levels of IL-1, COX2, and TGF beta were compared using RT-PCR.

Results:Comparison of hematoxylin and eosin staining of granulation tissue showed no difference in lamina propria thickness between LTCs that received COX inhibitor or LTCs that did not. Also, no difference in lamina propria thickness was noted between different types of injury such as acid or wire brush. Furthermore, no further differences were noted in the RT-PCR results in the samples.

Conclusions:Inhibition of cyclooxygenase (COX) does not lead to significant differences in granulation formation or in cytokine levels in the murine model of subglottic stenosis.

Jennifer Y Lee, MDDepartment of Otorhinolaryngology – Head and Neck SurgeryUniversity of Pennsylvania Email: jennifer.y.lee@uphs.upenn.eduPhone: 215-264-6067

Cyclooxygenase Inhibition of LTC Stenosis in the Mouse ModelCyclooxygenase Inhibition of LTC Stenosis in the Mouse Model

A. Control Uninjured 40x B. Control Acid 40x C. Control Wire 40x

D. COX I Uninjured 40x E. COX I Acid 40x F. COX I Wire 40x

B. Wire0.02 cm

Uninjured

AcidpH 3-4

A. LTC B. Acid injury C. Wire injury D.

RESULTS

Thickness of lamina propria were measured for control and treated mice. (Figure 2) Thickness of lamina propria was not significant p>0.05 between control and treatment mice.RT PCR levels were expressed in RQ levels. The IL-1 expression relative to GAPDH was 1.44 in control acid injury LTC, while IL-1 relative to GAPDH was 1.84 in treated acid injury LTCs (p =0.55). The expression of COX was significantly inhibited in the acid injury compared to the untreated acid injury (p = 0.009). The expression of inflammatory cytokines, IL-1 and TGF-beta, were not inhibited for either mechanisms of injury (p > 0.05).