Reinnervation and recovery of mouse soleus muscle after long-term denervation

Abstract Reinnervation and recovery of the mouse soleus muscle were studied 2–10 months after denervation periods of about 7 months. To maintain denervation the right sciatic nerve was frozen 14 times at 2-week intervals. Though initially intermittent muscle reinnervation occurred, contractile force of denervated muscles was reduced to less than 10% of the contralateral muscles by the fifth nerve freezing and further declined thereafter. Following reinnervation, recovery of soleus muscle force proceeded slowly to reach plateau values after 5–6 months. Tetanic muscle force reached on average 72% (range 58–86%, n = 12 ) of contralateral muscles after 5–10 months, ( P , t-test for absolute values) and 87% of unoperated animals after 10 months ( P , n = 5 ). Muscle fibre diameters were significantly reduced in reinnervated muscles, but frequency distributions were normal and similarly shaped in reinnervated and control muscles, suggesting complete muscle reinnervation and the absence of denervated fibres even at 2 months of reinnervation. Total numbers of muscle fibres were similar in reinnervated (842 ± 73 S.D. , n = 15 ), contralateral (854 ± 104 S.D. , n = 15 ) and control soleus muscles (853 ± 77 S.D. , n = 5 ). The number of myelinated axons in regenerating soleus nerves reached control values by 3 months after the last freezing, continued to increase till 6 months (150% of control), and declined thereafter (125% at 9–10 months). In the contralateral soleus nerves the number of myelinated axons remained constant during this period. Nerve fibre diameters remained abnormally small; even after 10 months of reinnervation fibre diameters were unimodally distributed with a mean diameter of 3.3μm in contrast to the bimodal distribution in intact nerves (mean values 3.9 and 9.0μm, respectively). Total fibre cross-section area per nerve increased with time but reached only 54% ± 6 S.D. , ( n = 3 ) of contralateral nerves by 10 months. The relative thickness of the myelin sheath (g-ratio) returned to normal after 9–10 months. Anatomically, muscle reinnervation appeared to be complete by 7–8 weeks since unusually small muscle fibre profiles were absent. Functionally, however, innervation was rendered immature for the following reasons: acetylcholine-induced contractures (50mg/l acetylcholine perchlorate) were still larger than normal ( 17% ± 4S.D. , n = 6 , of maximum tetanic force as compared to 3% ± 1S.D. in normal muscles, n = 12 ), nerve-evoked tetani (50 Hz for 2s) showed fatigue and peak values reached only 86% ± 6S.D. ( n = 5 ) of the muscle force obtained from direct muscle stimulation. Even by 3 months acetylcholine contractures were slightly elevated ( 8% ± 2 S.D. , n = 6 , P ) and block resistance of the nerve-evoked responses in high magnesium/low calcium Tyrode (safety margin of transmission) was reduced. No deficits in nerve-related parameters were found after 5–10 months of reinnervation. At 7–8 weeks of reinnervation fibre type distribution was abnormal since most muscle fibres stained for acid-stable myosin ATPase (Type I fibres, pH 4.5), only a few for alkaline-stable myosin ATPase (Type IIB, pH 10.3), and some for both (Type IIC). Consequently, the frequency of Type I fibres declined and by 3 months fibre type distribution approached values in normal control animals. Surprisingly, in contralateral muscles a similar surplus followed by a decline in Type I fibres was observed. Running in wheels during the denervation and reinnervation periods had moderately positive effects on muscle force of both reinnervated (plus 17%) and contralateral control muscles (plus 14%), while training during the reinnervation period only (5–6 months) surprisingly was not effective at all. In conclusion it is suggested that muscle recovery after prolonged periods of denervation, though incomplete, is remarkably successful, when proper motoneurons reinnervate in sufficient numbers. The strikingly slow progress in recovery of muscle force despite early full reinnervation could be due to deficits in the regenerated nerves which suffered a heavy loss in axonal volume; alternatively, muscle recovery itself might be impaired, e.g. due to exhaustion of the satellite cell pool.