Ganglion cells in the posterior cricoarytenoid muscle of the rat

OtolaryngologyHead and Neck Surgery

Research F o r u m - - M o n d a y

Volume 121 Number 2

in the rabbit. Our results suggest that multiple factors contribute to subglottic stenosis, as not all rabbits developed stenosis, nor did they all develop it to the same degree. Clinical Significance: An animal model for subglottic stenosis may help determine the pathogenesis of this disease and ultimately its prevention among patients. Poster 75

Blood Type Matching in Canine Laryngeal Transplantation KURTISC BIRUSINGH MD (presenter); MICHAEL JAKOBSEN MD; JOEL A SERCARZ MD; KEITH E BLACKWELL MD; MING YE MD; GERALD S BERKEMD; Los Angeles CA

Problem: Recent experience in the field of human solid organ transplantation indicates that orthotopic transplantation is possible when major histocompatibility complex (MHC) mismatches are present. Due to a relative shortage of organ donors compared with potential organ recipients, reliance on ABO blood type matching alleviates some of the difficulties associated with organ procurement. We undertook this study to investigate the feasibility of canine orthotopic laryngeal transplantation performed solely on the basis of a blood type match between the donor and recipient animals. Methods: Canine laryngeal transplantation was performed between 2 pairs of blood type-matched adult male beagles. Selective motor reinnervation of laryngeal adductor and abductor muscles using distal branches of the recurrent laryngeal nerve and superior laryngeal nerve sensory reinnervation was performed in the transplant recipients. Subsequent histocompatibility testing revealed DLA-DRB sequences with nonidentity at 14 positions and DLA-DQB sequences with nonidentity at 13 positions, indicating an MHC mismatch. Immunosuppression was achieved using FK-506 and steroids. Results: Two canines survived for 76 and 180 days postoperatively. Recovery of laryngeal function was seen in one animal, with return of a strong bark, normal deglutition, airway protection, and good exercise tolerance. Electrophysiologic testing at 6 months demonstrated physiologic laryngeal motion of both abductors and adductors with laryngeal stimulation and tube occlusion. In the second animal, 2 episodes of acute rejection were successfully treated with steroid pulses, although this animal subsequently developed ankylosis of the cricoarytenoid joint secondary to inflammation. Conclusion: These results indicate that laryngeal transplantation in the setting of blood type match with MHC mismatch is possible using FK-506 and steroid immunosuppression. However, MHC matching is recommended, given that episodes of acute rejection appear to compromise laryngeal function. Clinical Significance: This research will help refine and improve the surgical techniques and immunosuppressive regimen that will be used in human orthotopic laryngeal transplantation. (Supported by a VA Merit Review Grant.)

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Poster 76

Myosin Heavy Chain Isoform Expression in Laryngeal Muscle in Response to Thyroid Hormone Level YA ZHEN WU MD (presenter); MICHAEL J BAKER; ROGER L CRUMLEY MD; VINCENT J CAIOZZO PHD; Irvine CA; Irvine CA; Orange CA; Irvine CA

Problem: Myosin heavy chains (MHCs) are contractile proteins within skeletal muscle that are responsible for many mechanical properties of skeletal muscle. Since past studies have indicated that thyroid hormone (T3) has a powerful influence on MHC expression in some hindlimb muscles, the goal of this study was to determine the malleability of MHC expression in laryngeal muscle by manipulating thyroid hormone state. Methods: Female Sprague-Dawley rats (250-275 g) were randomly assigned to 1 of 3 groups: (1) control (CON), n = 5; (2) hypothyroid (-T3), n = 7; or (3) hyperthyroid (T3), n = 5. After 6 weeks, each rat was sacrificed, and the posterior cricoarytenoid (PCA) muscles were removed. Results: The MHC isoforrn expression within PCA single muscle fibers was then determined by gel electrophoresis (SDS-PAGE). Previously, 5 adult MHC isoforms had been identified in rat laryngeal muscle: slow type I, fast type IIA, fast type IIX, fast type IIB, and fast type IIL, in order of increasing speed of contraction. With respect to whole muscle MHC isoform expression, no significant differences existed between the 3 experimental groups for type I and type IlL. However, when examining type IIA, type IIX, and type IIB MHC isoform expression, significant differences were found among CON, -1"3, and T3 (P < 0.05). With respect to singlefiber MHC isoform expression, a large percentage of the fibers from the CON group (-90%) were pure type IIX, pure type IIB, or type IIX/IIB hybrid fibers. Hypothyroidism (-T3) resulted in a significant decrease in pure type IIB fibers and the production of a unique type IIXfIIB/IIL hybrid fiber population (P < 0.001). Hyperthyroidism (T3) resulted in a decrease in type IIX/IIB hybrid fibers and a concomitant increase in the pure type IIB fiber population. Conclusion: These results indicate that laryngeal muscle MHC isoform expression is far from static, and it appears that thyroid hormone holds the greatest impact on the expression of the type IIX and type IIB MHC isoforms. Poster 77

Ganglion Cells in the Posterior Cricoarytenoid Muscle of the Rat SIEJI KAWAKITA MD (presenter); EIJI YUMOTO MD PHD; MASAMITSU HYODO; Onsengun Ehime Japan; Kumamoto Japan; Onsengun Ehime Japan

Problem Addressed: The existence of the intralaryngeal ganglion cells has been frequently demonstrated in the connective tissue outside the intrinsic laryngeal muscles. How-

OtolaryngologyHead and Neck Surgery August T999

P1116 Research F o r u m - - M o n d a y

ever, there is little information concerning intramuscular ganglion cells of the intrinsic laryngeal muscles. We determined the distribution of the ganglion cells in the rat posterior cricoarytenoid (PCA) muscle and investigated the positional relationship to motor nerve fibers. Methods/Measures: The PCA muscles of 8 rats were used in this study. The PCA muscle was obtained from each of 5 rats and fixed with 3% glutaraldehyde. Serial sections, 0.5 gm thick, were cut and stained with 0.5% buffered toluidine blue. The distribution of the ganglion cells in and around the PCA muscle was examined with a light microscope. For histochemical observation, the PCA muscles taken from each of the other 3 rats were treated with an acetylcholine esterase stain. Sections, 10 ~tm thick, were cut and observed under a light microscope. Results: The PCA nerve bundle of the recurrent laryngeal nerve branched into 2 bundles. One bundle was found along the ventral surface of the muscle and entered the muscle at its middle portion to form neuromuscular contacts. In contrast, the other bundle, with 20 to 30 ganglion cells aggregated, was found in the dorsal surface at the top of the muscle. Several ganglion cells were scattered in the dorsal side of the PCA muscle, and they were also seen among muscle fibers near the bifurcations of arterioles. These ganglion cells were acetylcholine esterase positive. Conclusions: The intramuscular ganglion cells of the PCA muscle, suggesting a parasympathetic nature, mainly receive projections from the dorsal side of the rat PCA muscle, the opposite side of the location of the motor fibers. The intramuscular ganglion cells originated from the parasympathetic nerve via the recurrent laryngeal nerve and seemed to innervate vascular smooth muscle cells to regulate local blood flow. Clinical Significance: These ganglion cells may be involved in the regulation of the inner-environment of the larynx. These resuks may help understanding of possible roles of the intramuscular ganglion cells in the larynx. Poster 78

Relations between Carbon Monoxide and Nitric Oxide in the Canine Esophagus and Trachea HITOSHI BAMBA MD (presenter); YASUO HISA MD; TOSHIYUKI UNO MD; SHINOBU KOIKE MD; NOBUHISA TADAKI MD; KAZUHIRO SHOGAKI MD; Kyoto Japan

Problem: Carbon monoxide (CO) and nitric oxide (NO) have been suggested as messenger molecules in the central and peripheral nervous systems. CO is produced by heme oxygenase (HO). There are 2 isoforms of HO: HO-1 and HO-2. HO-1 is inducible type and HO-2 is constitutive type. NO is produced by neuronal NO synthase (n-NOS) in the neuronal cells, and NADPH-diaphorase (NADPH-d) histochemistry can be used as a reliable marker of n-NOS activity in both central and peripheral nervous systems. We have already reported that HO-2-1ike immunoreactive cells were

present in the canine esophageal myenteric plexuses and tracheal local ganglions. Methods: In this study we have investigated the coexistence of CO and NO in the canine esophageal myenteric plexuses and tracheal local ganglions by means of a doublestaining technique of HO-2 immunohistochemistry and NADPH-d histochemistry. Results: In the esophagus, the percentage of expression of HO-2 in NADPH-d-reactive cells was 80%, and the percentage of NADPH-d reactivity in HO-2-immunoreactive cells was 40%. In the tracheal local ganglions, coexistence of HO2 immunoreactivity and NADPH-d reactivity was detected in only a few neuronal cells. HO-2 immunoreactive cells were present much more than NADPH-d-reactive cells. Conclusion: These results suggest that CO and NO interact with each other in the esophagus and the trachea. Because the coexistent ratio of them is higher in the esophagus than in the trachea, CO and NO may play more important roles in the esophagus. Poster 79

Modulation of Myosin Heavy Chains in Rat Laryngeal Muscle AKIHIRO SHIOTANI MD (presenter); HIDEKI NAKAGAWA MD; PAUL W FLINT MD; Ise Mie Japan; Baltimore MD; Baltimore MD

Problem: Although myosin heavy chain (MHC) composition is predetermined by cell lineage, modulation of expression appears to be dependent on neural input. Within the developing rat larynx, changes in MHC composition can be correlated with onset of laryngeal function and reflect the functional characterization of each muscle in the adult animal. Moreover, MHC composition in denervated rat laryngeal muscle is characterized by a decrease in type IIB and IIL and an increase in IIA/IIX. This study will test the hypothesis that MHC composition is a biologic marker indicative of appropriate and functional reinnervation. Methods: In adult rats, MHC composition was assessed in 3 conditions, including transient recurrent laryngeal nerve (RLN) crush injury, RLN transection and repair, and cricoarytenoid joint fixation with an intact RLN. Animals survived for 30, 90, and 180 days. At each interval, vocal fold mobility was assessed by rigid microlaryngoscopy. Laryngeal EMG was performed prior to euthanasia. The thyroarytenoid and posterior cricoarytenoid muscles were then excised, and each muscle was processed for SDS-PAGE. Optical densitometry was used to determine MHC composition. Results: Thirty days following nerve crush injury, 3 of 6 animals regained vocal fold mobility and normal MHC composition. Impaired vocal fold motion in 3 of 6 animals was associated with MHC composition characteristic of denervation. At 90 and 180 days, normal vocal fold motion and normal MHC composition were observed in all animals. Following nerve transection and repair, impaired vocal fold