Nerve-intact Grafts Research Articles

Motor unit properties of nerve-intact extensor digitorum longus muscle grafts in young and old rats.

Impaired reinnervation has been implicated as the cause of the threefold disparity in the recovery of maximum force (P0) of standard muscle grafts in old compared with young rats. The specific, null hypothesis of this study is that compared with age-matched control extensor digitorum longus (EDL) muscles, nerve-intact EDL muscle grafts in young and old rats show no evidence of an age-related impairment in reinnervation. Nerve-intact grafts were performed in 3-month-old and 23-month-old rats and were evaluated 60 days postoperatively. Compared with age-matched control EDL muscles, nerve-intact grafts in young and old rats showed no difference in muscle mass or motor unit numbers. The mean motor unit P0 for nerve-intact graft muscles in both age groups was significantly lower than that of age-matched control muscles. These data support our hypothesis that if axons are allowed to regenerate in an endoneurial environment, there is no evidence of an age-related impairment in muscle reinnervation.

Myosin Heavy Chain Expression in Skeletal Muscle Autografts under Neural or Aneural Conditions

Background.Our purpose was to investigate (1) the heterogeneity of satellite cells derived from adult fast-twitch and slow-twitch skeletal muscles, (2) the influence of innervation on muscle regeneration, and (3) the differences between developmental myoblasts and satellite cells with regard to myosin heavy chain (MHC) expression.Materials and methods.Autografts under neural (nerve-intact graft;brief denervation interval) or aneural (aneural graft;prolonged denervation interval) conditions of the fast-twitch extensor digitorum longus (EDL) muscle or the slow-twitch soleus muscle were performed in adult rat hindlimbs. MHC expression during skeletal muscle regeneration was determined sequentially using immunocytochemistry.Results.After grafting, most muscle fibers in the EDL and soleus underwent ischemic degeneration and regeneration; at the periphery of each muscle, a few adult fibers survived. All regenerating fibers initially expressed embryonic/fetal (developmental) MHC alone, and subsequently both developmental and fast MHC. During the first week, no expression of slow MHC was observed in regenerating fibers in either the EDL or the soleus. In nerve-intact grafts, regenerating fibers expressed slow MHC as early as the second week; under aneural conditions, no regenerating fibers expressed slow MHC even 4 weeks after grafting. On the other hand, some persisting fibers in aneural grafts could maintain expression of slow MHC 4 weeks after grafting; other fibers underwent MHC transformation induced by denervation. No significant difference in MHC expression during regeneration was observed for slow compared with fast muscles, under either neural or aneural condition.Conclusions.These data suggest that regenerating adult skeletal muscle fibers, derived only from satellite cells, cannot express slow MHC without motor innervation, and that persisting muscle fibers, derived from both myoblasts in fetal development and satellite cells, may be intrinsically distinct from regenerating fibers. Satellite cells derived from slow and from fast muscles may be a single, homogenous population and may be the same population as fetal (secondary) myoblasts with regard to MHC expression.

Non-neural and neural expression of myosin heavy chains by regenerated intrafusal fibers of rats

Expression of myosin heavy chain (MHC) isoforms was studied in rat soleus (SOL) and extensor digitorum longus (EDL) muscles which regenerated in the presence or absence of innervation. Frozen sections of two 5 day denervated SOL and EDL grafts, two 40 day denervated SOL and EDL grafts, and two reinnervated 40 day SOL and EDL grafts were processed for demonstration of motor endplates, sensory endings, myosin adenosine triphosphatase (mATPase) and for expression of 4 MHCs. No qualitative differences in MHC expression were noted between 5 day or 40 day denervated grafts of the SOL and EDL muscles. All regenerated intrafusal and extrafusal myotubes or myofibers reacted to antibodies against neonatal and fast-twitch MHCs, but not to antibodies against slow-twitch and slow-tonic MHCs in these grafts. These data indicate that MHCs expressed by regenerated intrafusal myotubes do not parallel those expressed by myotubes which give rise to the three types of intrafusal fibers during development and that MHC expression by regenerated intrafusal myotubes parallels that of regenerated extrafusal myotubes prior to innervation. However, some regenerated intrafusal fibers in 40 day nerve-intact grafts bound antibodies to slow-twitch and slow-tonic MHCs, thus expressions of these two MHCs are nerve-dependent in regenerated muscle spindles.

Prior running reduces hypertrophic growth of skeletal muscle grafts

Run training can increase the mass of soleus muscle grafts, yet values remain lower than nongrafted muscle even with continued training. Thus we tested the hypothesis that nerve-implant soleus grafts of rats previously run trained would be refractory to the hypertrophic stimulus of ablation of synergistic muscle. We also compared the magnitude of growth of the nerve-implant soleus graft after ablation with that reported by others for the nerve-intact soleus graft. We studied eight groups that differed relative to the combination and order of treatments (running and ablation of synergistic muscle) and the graft age at the time of the ablation operation and study. Graft mass, protein concentration, and histochemical fiber composition were measured. Compared with grafts from cage-sedentary rats, the mass and protein content of the nerve-implant soleus grafts were higher (16-63%) at all times after ablation. When the ablation operation was performed at 56 days postgrafting, there was a 33% increase in protein content of the soleus graft by 84 days for cage-sedentary animals. This increase was twofold greater (P less than or equal to 0.02) than the 15% increase that followed ablation for the grafts from the animals that had been run trained before the ablation operation. Four weeks of run training before the ablation operation impaired the adaptive response of muscle grafts to the ablation of synergistic muscles, which may reflect alterations in motor unit recruitment and/or satellite cell activity. Ablation of synergistic muscles resulted in an absolute growth of the nerve-implant soleus grafts that was comparable with that reported for nerve-intact soleus grafts.

Glucocorticoid-induced glycogen increases in extensor digitorum longus and soleus grafts in the rat.

The purpose of the present study was to compare dexamethasone-induced glycogen increases in normal EDL and SOL muscles with that in free muscle grafts. Glycogen in mature EDL and SOL grafts in the rat equalled control concentrations irrespective of whether the graft was a nerve-intact (NI), nerve-crushed (NC), reimplanted, or cross-transplanted graft. The grafts also possessed the glycogen-regulatory mechanisms to respond to the glucocorticoid dexamethasone (DEX), which increases muscle glycogen. The increase in glycogen induced by DEX in the EDL and SOL grafts resembled that of the EDL and SOL muscles, respectively, whether the grafted muscle was originally an EDL or SOL. DEX induced an approximate twofold increase in glycogen concentration in control muscles and nerve-intact SOL grafts, and a smaller but significant increase in all other free grafts. Nerve crushing prior to grafting resulted in no significant change in muscle weight, glycogen concentration, or DEX-induced glycogen increase in these grafts. The data suggest that skeletal muscle grafts are qualitatively similar to normal muscles in terms of metabolic responsiveness to hormones. Leaving the nerve intact during grafting quantitatively enhances the graft's hormonal sensitivity but the technique of nerve crushing prior to grafting has no such effect.

Contractile properties of standard and nerve‐intact muscle grafts in the rat

The contractile properties of standard and nerve-intact grafts of extensor digitorum longus muscles of rats were compared in vitro. Fourteen days after grafting, the time to peak tension and the half-relaxation times of nerve-intact grafts were shorter than those of standard grafts, but both were longer than control values. By 60 days, these variables attained normal values. At every sample period, the tetanic tensions of nerve-intact grafts were higher than those of standard grafts. Even at the early sampling periods, the twitch-tetanic tension ratios of nerve-intact grafts were close to normal values, whereas those of standard grafts were higher than normal. Stabilized nerve-intact grafts had a larger mass and greater maximum tetanic tension development than standard grafts, but were more fatigable. Compared with control EDL muscles, stable nerve-intact grafts show no differences except for lessened fatigability, whereas standard grafts demonstrate significant functional deficits.

Calcium uptake by sarcoplasmic reticulum from nerve-intact and standard skeletal muscle grafts.

A freely grafted rat soleus muscle exhibits a decrease in velocity and capacity of SR calcium uptake. This deficit is not prevented by maintaining neural connections (nerve-intact graft) during grafting. Thus the greater mechanical capability of nerve-intact grafts, relative to standard grafts, is not accompanied by any enhancement of the SR tubules.

Motor unit function in skeletal muscle autografts of rats

Standard and nerve-intact grafts of the extensor digitorum longus (EDL) muscles of rats do not fully recover control values for maximum tetanic tension (P o). We compared the physiologic properties of motor units from standard and nerve-intact EDL grafts of rats with those of control EDL muscles. Standard grafts were completely removed and replaced in their original site. For nerve-intact grafts all physical connections were severed except for the nerve. Isometric contractile properties of whole muscles and single motor units were measured in situ 75 and 50 days after surgery for standard and nerve-intact grafts, respectively. Motor units from both types of grafts showed a mean and distribution for time-to-peak twitch tension (TPT) not different from control motor units. The absolute P o of nerve-intact grafts showed a greater recovery than the standard grafts, but was still significantly lower than the control value. The average decrease in the P o of motor units from nerve-intact grafts of 16% was not different from the decrease in the P o of the total EDL graft which suggests no loss of motor units in nerve-intact grafts. In standard grafts, the 57% decrease in P o for the whole muscle was attributable to a 20% decrease in the average tension development per motor unit and a 45% decrease in the number of motor units. These differences suggest a more complete reinnervation of the nerve-intact grafts than standard grafts.

Characteristics of cat skeletal muscles grafted with intact nerves or with anastomosed nerves

Grafting of 3-g extensor digitorum longus (EDL) muscles of cats may be made with (i) severence of the nerve with spontaneous reinnervation, termed standard grafts (ii) severence of the nerve with reinnervation facilitated by anastomosis of the nerve, termed nerve-anastomosed grafts; and (iii) preservation of the nerve, termed nerveintact grafts. In previous studies, standard grafts developed a maximum isometric tetanic tension (P 0) that was 22% of the value for control EDL muscles. We hypothesized that the low values of P 0 resulted from incomplete reinnervation of muscle fibers. To test this hypothesis, EDL muscles were grafted in cats with nerves intact and with nerves anastomosed. In standard grafts differences were observed in both structure and function at 120 compared with 240 days after grafting. Characteristics of the nerve-intact and nerve-anastomosed grafts did not change significantly between 120 and 240 days and the data were pooled for comparisons with control EDL muscles. Nerve-anastomosed and nerve-intact grafts developed P 0 values that were 34 and 64% of the control values, respectively. Nerve-intact grafts had a mass and fiber cross-sectional area not different from control EDL muscles. Compared with control values, all grafts had fewer fibers, more connective tissue, lower absolute and normalized P 0, reduced capillary density, and increased fatigability. The greater P 0 of nerve-intact compared with standard and nerve-anastomosed grafts supported our hypothesis that the degree of reinnervation is a factor that limits graft development. The presence of a necrotic core and the low tension development of even the nerve-intact grafts suggested that revascularization is a significant limitation as well.

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules. While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.