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The effect of denervation and reinnervation on PCA cell cycle gene expression
Otolaryngology– Head and Neck Surgery Volume 131 Number 2
Research Posters P203
R154 The Effect of Denervation and Reinnervation on PCA Cell Cycle Gene Expression Ya Zhen Wu, MD (presenter); Michael J Baker, Caiozzo, PhD; Roger L Crumley, MD MBA Irvine CA; Irvine CA; Irvine CA; Orange CA
MS;
Vincent J
Problem: While it is known that denervation of skeletal muscle can induce atrophy, the effect of denervation on muscle cell cycle genes is unclear. Further, while attempted reinnervation of laryngeal muscle has thus far been unsuccessful in restoring function, cell cycle gene expression might be utilized to predict reinnervation success. The objective of this study was to examine the effects of denervation and reinnervation of the posterior cricoarytenoid (PCA) muscle on key genes involved in the cell cycle.
Methods: Female Sprague-Dawley rats were assigned to 1 of 3 groups: control (CON, n ⫽ 24); denervation (DEN, n ⫽ 24); or reinnervation (REIN, n ⫽ 24). DEN and REIN animals had their left recurrent laryngeal nerves transected and ligated at the fourth tracheal ring while REIN animals subsequently had their nerves repaired using 10 – 0 suture. Animals were then euthanized after 7, 14, and 30 days, and mRNA expression examined using RT-PCR. If significant group differences were seen using one-way ANOVA (P ⬍ 0.05), Tukey post-hoc tests were employed. Results: Expression of p21, p53, GADD45 alpha, GADD45 beta, GADD45 gamma, and cyclin D1 genes were compared. At all time points, cyclin D1 and p53 gene expressions were unchanged with DEN or REIN. However, DEN muscles were found to express significantly higher levels of p21, GADD45 alpha, and GADD45 beta than CON at all time points. Conclusion: Interestingly, while REIN p21, GADD45 alpha, and GADD45 beta expressions were found to be elevated at 7 and 14 days, GADD45 alpha and GADD45 beta levels at 30 days were not significantly different from CON, threreby suggesting a recovery of REIN muscle gene expression at later time points. Significance: Paradoxically, while the restoration of some cell cycle genes to normal levels in REIN muscles might be seen as an indication of successful reinnervation of the PCA, no significant functional recovery was associated. Support: None reported. R155 T-588 Prevents Motoneuron Loss after Recurrent Laryngeal Nerve Injury Yuko Mori, MD; Akihiro Shiotani, MD; Kazuhisa Moro, MD; Koji Araki, MD; Kaoru Ogawa; Masaya Nakagawa, PhD; Ken Ikeda, MD; Kazuhiko Watabe, MD PhD Tokyo Japan; Tokyo Japan; Tokyo Japan; Tokyo Japan; Tokyo Japan; Toyama Japan; Fuchu Japan; Fuchu Japan
Problem: Unsuccessful reinnervation or neurofunctional recovery after recurrent laryngeal nerve injury may attribute to the loss of motoneurons in nucleus ambiguus. To assess the possibility of new drug therapy for injury of these motoneurons, we examined the neuroprotective effect of a novel neuroprotective compound, T-588 on the motoneuron loss in nucleus ambiguus after adult rat vagal nerve avulsion. Methods: The left vagal nerves of 12-week-old SpragueDawley rats were avulsed and removed at the level of jugular foramen. After the operation, the animals were freely administrated water containing 0.05% T-588 (Toyama Chemical Co Ltd, Toyama, Japan) solution. Four weeks after the operation, the number of surviving motoneurons in nucleus ambiguus was counted after Nissl staining to evaluate the neuroprotective effect of T-588. Results: In T-588-treated animals, a significantly larger
POSTERS
toms associated with laryngeal paralysis. Although changes in myosin-heavy chain components have been described in denervated laryngeal muscle, little is known about alterations occurring in the general population of muscle proteins. Alteration of proteins in rat laryngeal muscle following denervation and reinnervation was therefore studied using proteomic techniques. Methods: Adult Sprague-Dawley rats were divided into normal control, denervation (RLN divided and ligated), and reinnervation (RLN divided and suture repaired) groups. The thyroarytenoid (TA) muscle was excised 60 days after surgery. Protein separation and identification were performed using 2-dimensional electrophoresis, MALDI-TOF mass spectrometry, and MASS/MASS with database search. Results: Forty-five proteins and their modified isoforms were found to have significant alteration in expression level after denervation. The majority of these proteins (60%), associated with energy metabolism, cellular proliferation and differentiation, signal transduction, and stress reaction, were found to have decreased levels of expression in denervated TA muscle. The remaining 40% of proteins, involved with degradation of cellular proteins, immunoreactivity and injury repair, regulation of muscle contraction and microtubular formation, were found to have increased levels of expression. Multiple spot patterns due to site modification by phosphorylation were detected in 16% of identified proteins. In the reinnervated group, only 8 protein spots identified were significantly different from normal controls. Changes in expression levels were similar to denervated muscle although to a lesser degree. Conclusion: Significant changes in protein expression occur in denervated laryngeal muscle. Fewer changes are observed in reinnervated muscle. Significance: Modulation of these protein shifts by trophic factors may provide potential therapeutic strategies for the treatment of laryngeal paralysis. Support: None reported.
Research Posters P203
R154 The Effect of Denervation and Reinnervation on PCA Cell Cycle Gene Expression Ya Zhen Wu, MD (presenter); Michael J Baker, Caiozzo, PhD; Roger L Crumley, MD MBA Irvine CA; Irvine CA; Irvine CA; Orange CA
MS;
Vincent J
Problem: While it is known that denervation of skeletal muscle can induce atrophy, the effect of denervation on muscle cell cycle genes is unclear. Further, while attempted reinnervation of laryngeal muscle has thus far been unsuccessful in restoring function, cell cycle gene expression might be utilized to predict reinnervation success. The objective of this study was to examine the effects of denervation and reinnervation of the posterior cricoarytenoid (PCA) muscle on key genes involved in the cell cycle.
Methods: Female Sprague-Dawley rats were assigned to 1 of 3 groups: control (CON, n ⫽ 24); denervation (DEN, n ⫽ 24); or reinnervation (REIN, n ⫽ 24). DEN and REIN animals had their left recurrent laryngeal nerves transected and ligated at the fourth tracheal ring while REIN animals subsequently had their nerves repaired using 10 – 0 suture. Animals were then euthanized after 7, 14, and 30 days, and mRNA expression examined using RT-PCR. If significant group differences were seen using one-way ANOVA (P ⬍ 0.05), Tukey post-hoc tests were employed. Results: Expression of p21, p53, GADD45 alpha, GADD45 beta, GADD45 gamma, and cyclin D1 genes were compared. At all time points, cyclin D1 and p53 gene expressions were unchanged with DEN or REIN. However, DEN muscles were found to express significantly higher levels of p21, GADD45 alpha, and GADD45 beta than CON at all time points. Conclusion: Interestingly, while REIN p21, GADD45 alpha, and GADD45 beta expressions were found to be elevated at 7 and 14 days, GADD45 alpha and GADD45 beta levels at 30 days were not significantly different from CON, threreby suggesting a recovery of REIN muscle gene expression at later time points. Significance: Paradoxically, while the restoration of some cell cycle genes to normal levels in REIN muscles might be seen as an indication of successful reinnervation of the PCA, no significant functional recovery was associated. Support: None reported. R155 T-588 Prevents Motoneuron Loss after Recurrent Laryngeal Nerve Injury Yuko Mori, MD; Akihiro Shiotani, MD; Kazuhisa Moro, MD; Koji Araki, MD; Kaoru Ogawa; Masaya Nakagawa, PhD; Ken Ikeda, MD; Kazuhiko Watabe, MD PhD Tokyo Japan; Tokyo Japan; Tokyo Japan; Tokyo Japan; Tokyo Japan; Toyama Japan; Fuchu Japan; Fuchu Japan
Problem: Unsuccessful reinnervation or neurofunctional recovery after recurrent laryngeal nerve injury may attribute to the loss of motoneurons in nucleus ambiguus. To assess the possibility of new drug therapy for injury of these motoneurons, we examined the neuroprotective effect of a novel neuroprotective compound, T-588 on the motoneuron loss in nucleus ambiguus after adult rat vagal nerve avulsion. Methods: The left vagal nerves of 12-week-old SpragueDawley rats were avulsed and removed at the level of jugular foramen. After the operation, the animals were freely administrated water containing 0.05% T-588 (Toyama Chemical Co Ltd, Toyama, Japan) solution. Four weeks after the operation, the number of surviving motoneurons in nucleus ambiguus was counted after Nissl staining to evaluate the neuroprotective effect of T-588. Results: In T-588-treated animals, a significantly larger
POSTERS
toms associated with laryngeal paralysis. Although changes in myosin-heavy chain components have been described in denervated laryngeal muscle, little is known about alterations occurring in the general population of muscle proteins. Alteration of proteins in rat laryngeal muscle following denervation and reinnervation was therefore studied using proteomic techniques. Methods: Adult Sprague-Dawley rats were divided into normal control, denervation (RLN divided and ligated), and reinnervation (RLN divided and suture repaired) groups. The thyroarytenoid (TA) muscle was excised 60 days after surgery. Protein separation and identification were performed using 2-dimensional electrophoresis, MALDI-TOF mass spectrometry, and MASS/MASS with database search. Results: Forty-five proteins and their modified isoforms were found to have significant alteration in expression level after denervation. The majority of these proteins (60%), associated with energy metabolism, cellular proliferation and differentiation, signal transduction, and stress reaction, were found to have decreased levels of expression in denervated TA muscle. The remaining 40% of proteins, involved with degradation of cellular proteins, immunoreactivity and injury repair, regulation of muscle contraction and microtubular formation, were found to have increased levels of expression. Multiple spot patterns due to site modification by phosphorylation were detected in 16% of identified proteins. In the reinnervated group, only 8 protein spots identified were significantly different from normal controls. Changes in expression levels were similar to denervated muscle although to a lesser degree. Conclusion: Significant changes in protein expression occur in denervated laryngeal muscle. Fewer changes are observed in reinnervated muscle. Significance: Modulation of these protein shifts by trophic factors may provide potential therapeutic strategies for the treatment of laryngeal paralysis. Support: None reported.