| Objective: That long-term denervated posterior cricoarytenoid muscles(PCAMs)could regain their functions by reinnerations made us to evaluated morphological and related factors alteration in long-term denervated PCAMs and to find the appropriate time point of reinneration.Methods: 38 patient with vocal paralysis were recruited into four groups, the 6-12 months denervation group , the 1-2 yr denervation group, the 2-3yr denervation group and the over 3 yr denervation group. 12 adults served as a control group. The morphological alteration was evaluated using Trichrome Masson staining ,SDH and AchE histochemistry stain.The change in expression of related factors such as Myogenin, TGF-β1 , GDNF and GFR-α1 were observed using immunofluorescence stain and realtime PCR. Results:①The number of white muscles that showed a low intensity of SDH activities was decreased , but the number of red muscles that showed a high intensity of SDH activities was increased after denervation. The levels of AchE expression in myoceptors were deseased with the denervated time course extension and the AchE expression in the myoceptors was disappeared after 1 year.②Immunostaining with antibody against Myogenin and image analysis showed no staining in innervated, however, by 6-12 months of muscle denervation there was a significant accumulation of myogenin protein in myonuclei . There was a peak in expression of Myogenin in 1-2 years denervated muscles. After 2 years of denervation, expression of myogenin protein in myonuclei was decreased significantly(P< 0.01), but after 3 years of denervation, we can not found any expression of Myogenin protein in myonuclei. Myogenin mRNA was observed an 4-fold (P< 0.05) increase from 0.5 to 2 yrs, 64-fold (P< 0.01) increase from 0.5 to 3 years. After 3 years ,Myogenin mRNA decreased ,0.5-fold to 6-12 months denervated muscles.③The secars of myofibers were deseased,but the secars of collagen fibers were increased with the denervated time course extension. The ratio of secars of myofibers to secars of collagen fibers were progressively decrease with increased time course of denervation and more decrease within 2 years,but there was still 48%secar of myofibers remained after 3 years of denervation . TGF-β1 showed no staining in innervated, however, by 6-12 months and 1-2 years of muscle denervation there was a significant accumulation of TGF-β1 protein in endochylema of myofiber, After 2 year of denervation, expression of TGF-β1 protein in endochylema of myofiber, was decreased significantly, but after 3 year of denervation, we can not found any expression of TGF-β1 protein in endochylema.④GDNF and GFR-α1 showed no staining in innervated, however, by 6-12 months of muscle denervation there was a significant accumulation of GDNF and GFR-α1 protein in endochylema and cytolemma of myofiber, After 1 year of denervation, expression of GDNF and GFR-α1 protein in endochylema and cytolemma of myofiber, was decreased significantly, but after 2 years of denervation, we can not found any expression of GNNF and GFR-α1 protein in endochylema and cytolemma. Conclusion:①The alteration in the type of muscles indicated their alteration because of adapting to the denervated situation. The AchE expression in the myoceptors observed within 1 year indicated there were structures of myoceptors within 1 year.②The change in expression of Myogenin, a mark of muscles regeneration,indicated there was a muscles regeneration within 3 years.③The morphological alteration and change in expression of TGF-β1,a important factor of myofibrosis, indicated there was a high amplitude of myofibrosis within 2 years,but there is a morphological basis of muscles to regain their function within 3 years denervated muscles .④The change in expression of GDNF and his acceptor GFR-α1 ,a powerful neurotrophic factor, hinted a good nervous regenerated microenvironment in PCAMs within 2 years.In conclusion,our experiment indicated denervated posterior cricoarytenoid muscles is able to regain their functions by reinneration within 3 years.
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