Modulation of myosin heavy chains in rat laryngeal muscle

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

OtolaryngologyHead and Neck Surgery Volume 121 Number 2

motion and MHC composition characteristic of denervation were observed in all animals, despite evidence of reinnervation by EMG. Following joint fixation, alteration in MHC composition consistent with denervation was observed only at 30 days, as seen in the nerve crush model. Conclusion: Temporary injury and vocal fold immobilization result in transient shifts in MHC composition. Nerve transection and repairs and subsequent reinnervation result in persistent alteration of MHC composition and vocal fold dysfunction. Clinical Significance: These findings suggest that expression of normal MHC composition is dependent on the condition of appropriate neural contact and functional reinnervation. In subsequent studies, MHC composition will be used as a biological marker to evaluate future strategies for the management of laryngeal paralysis. (Supported by NIDCD grant K08DC0081.) Poster 80

A New Technique for Studying Innervation Status of a Muscle MARK JOSEPH BILLANTE BS (presenter); KURT GARREN MD: RICARDO RODRIGUEZ MD; ROGER BANNISTER BS; DAVID L ZEALEAR PHD; Nashville TN; Aurora CO; Nashville TN; Nashville TN; Nashville TN

Problem: A useful technique for characterizing connectivity between nerve and muscle fibers involves electrical stimulation of nerves at high enough rates to deplete glycogen in their target muscle fibers. Depleted muscle fibers can then be identified in tissue sections stained by the periodic acid-Schiff (PAS) technique. Unfortunately, the reproducibility of the technique has come into question, with reports of nonspecific glycogen depletion or retention as high as 50%. In a preliminary study in our laboratory, no stimulus could be found of a sufficiently high rate to deplete glycogen in all muscle fibers without encountering failure of neuromuscular signal transmission. The purpose of this investigation was to evaluate a new technique for glycogen depletion of muscle fibers using epinephrine to inhibit glycogen synthase, thereby enhancing simulated depletion. Methods: This approach was evaluated in the hindlimb of the anesthetized rat. A stimulating cuff electrode was placed around the sciatic nerve, and 3 recording electrodes were positioned in the gastrocnemius muscle, the tibial protuberance, and the achilles tendon. Electrical stimulation was applied at a frequency ranging from 0.5 to 100 Hz. Epinephrine was delivered by continuous infusion intramuscularly at a rate ranging from 0.12 to 1.2 mg/hour. Any failure of synaptic transmission could be detected by a decrease of the evoked electromyographic response. Deterioration of the mechanical response due to glycogen usage was monitored by an accelerometer. Following an hour of stimulation, the hindlimb muscles were harvested, frozen, sectioned, and stained for glycogen using PAS. Muscles from the contralateral limb served as controls.

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

PI07

Results: Results of the investigation revealed a narrow frequency range from 2 to 5 Hz at an epinephrine infusion rate of 1.2 mg/hour, which reproducibly depleted glycogen in 99% to 100% of stimulated muscle fibers. Nonstimulated control muscles demonstrated only 0% to 2% nonspecific depletion. Conclusion: This technique should provide a reliable means for determining the number and types of muscle fibers undergoing denervation or reinnervation in a variety of muscle systems. Recently this paradigm has been successfully employed in studies of laryngeal innervation in the canine. Poster 81

Superior Laryngeal Nerve Contribution to Vocal Fold Adduction MICHAEL JAKOBSEN MD (presenter); KURTISC BIRUSINGH MD; JOEL A SERCARZ MD; KEITH E BLACKWELL MD; MING YE MD; GERALD S BERKE MD; Santa Monica CA; Los Angeles CA; Los Angeles CA; Los Angeles CA; Los Angeles CA; Los Angeles CA

Problem: Although the superior laryngeal nerve (SLN) is primarily involved in increasing the pitch of the larynx by lengthening of the vocal ligament, it can act as a vocal fold adductor. Previous studies in our laboratory have quantified the force produced with recurrent laryngeal nerve (RLN) stimulation in the canine. In this study, the adductory properties of SLN stimulation and cricothyroid muscle contraction were studied. Methods: Four mongrel dogs weighing 20 to 25 kg underwent in vivo force measurements with stimulation of both the SLN and RLN. Force was measured on the vocal process utilizing Shimpo Digital Force gauge (DF-0.5R, Shimpo American Corp). Results: The results indicate that in the canine model, the SLN not only acts to lengthen the vocal ligament but also is a powerful adductor, especially in the absence of RLN activation. When adductor force was measured on the vocal process, a force near that of the individual laryngeal adductors could be measured. In the presence of RLN function the SLN did not add significant adductory force. Conclusion: This work supports the notion that in the setting of isolated RLN injury, the SLN may via the cricothyroid muscle act to compensate as a powerful adductor. Clinical Significance: In patients with an isolated RLN injury, the SLN may act via the cricothyroid muscle to compensate as a powerful adductor. (Supported by a VA Merit Grant.) Poster 82

Neurotrophic Factors on Regeneration of Denervated Recurrent Laryngeal Nerve MASAHIRO KOMORI MD (presenter); EIJI YUMOTO MD PHD; MASAMITSU HYODO MD; SIEJIKAWAKITA MD; TAKAHIKOYAMAGATA MD; TETSUJI SANUKI MD; Hiroshima Japan; Kumamoto Japan; Onsengun Ehime Japan; Onsengun Ehime Japan; Onsengun Ehime Japan; Onsengun Ehime Japan