Regeneration of skeletal muscle after grafting in monkeys.

Twenty-five palmaris longus muscles were transplanted into the forearm (orthotopic autografts) or into the face (heterotopic autografts) in 15 Rhesus monkeys. These muscles were transplanted with or without anastomosis to a motor nerve. No significant difference was observed long-term between the grafts done with or without prior denervation of the muscle. The forearm muscle grafts without neurorrhaphies formed a static, fibrous sling which did not contract. The forearm autografts with neurorrhaphy and the grafts to the face, with or without neurorrhaphy, all developed regenerating skeletal muscle fibers and showed contractile activity.     

Functional deficits in skeletal muscle grafts

Individual skeletal muscle fibers degenerate and regenerate with minimal functional deficits. When whole skeletal muscles are grafted in rats or cats by standard grafting techniques, revascularization and reinnervation must occur spontaneously. Under these circumstances, contraction times and maximum velocities of shortening eventually return to control values, but a significant deficit is observed in maximum tetanic tension. Grafts made with anastomosis of nerves or with nerves left intact have smaller deficits in tension development than do standard grafts made without nerve repair. The measurement of contractile properties of single motor units in extensor digitorum longus (EDL) muscles and in EDL grafts in rats indicates that the decreased maximum tetanic tension of whole grafts is due to a 10-20% decrease in the maximum tetanic tension of individual motor units, whereas standard grafts also show a 40-45% decrease in the number of motor units. Compared with control values, the fatigability of 100-mg grafts in rats is decreased, whereas larger 3-g grafts in cats show an increased fatigability. The deficits observed in large grafts can be reduced, but not eliminated, by grafting with neurovascular anastomoses.     

Transplantation and transposition of skeletal muscles into the faces of monkeys

Restoration of normal facial movement after long-term facial paralysis with muscle atrophy has not yet been achieved reliably by either free grafts, in which fibers degenerate and regenerate, or by grafts made with microneurovascular repair, in which most fibers survive. Our purpose was to compare the structural and functional properties of free muscle grafts and continuously perfused muscle flaps transplanted into the faces of monkeys. In adult monkeys, the facial muscles were replaced by either a free graft of a donor muscle from the lower limb or a denervated flap of ipsilateral temporalis muscle. Each graft or flap was reinnervated with the preserved buccal branch of the facial nerve. The control muscles, grafts, and flaps were examined 90 days later for gross appearance, contractile properties, and fiber areas. Compared with muscle flaps, free grafts showed greater adaptability to the new location and innervation and a closer approximation to the structural and functional properties of the original facial musculature.     

Plasticity of skeletal muscle: regenerating fibers adapt more rapidly than surviving fibers

The properties of mammalian skeletal muscle demonstrate a high degree of structural and functional plasticity as evidenced by their adaptability to an atypical site after cross-transplantation and to atypical innervation after cross-innervation. We tested the hypothesis that, regardless of fiber type, skeletal muscles composed of regenerating fibers adapt more readily than muscles composed of surviving fibers when placed in an atypical site with atypical innervation. Fast muscles of rats were autografted into the site of slow muscles or vice versa with the donor muscle innervated by the motor nerve to the recipient site. Surviving fibers in donor muscles were obtained by grafting with vasculature intact (vascularized muscle graft), and regenerating fibers were obtained by grafting with vasculature severed (free muscle graft). Our hypothesis was supported because 60 days after grafting, transposed muscles with surviving fibers demonstrated only a slight change from the contractile properties and fiber typing of donor muscles, whereas transplanted muscles with regenerating fibers demonstrated almost complete change to those of the muscle formerly in the atypical site.     

The effect of two episodes of denervation and reinnervation on skeletal muscle contractile function.

Sensory or motor "baby-sitting" has been proposed as a clinical strategy to preserve muscle integrity if motion-specific axons must regenerate over a long distance to reach denervated target muscles. Denervated muscles are innervated temporarily by using axons from nearby sensory or motor nerves. After motion specific motor axons have reached the target, the baby-sitter nerve is severed and motion-specific axons are directed to the target. Although this strategy minimizes denervation time, the requisite second episode of denervation and reinnervation might be deleterious to muscle contractile function. This study was designed to test the hypothesis that two sequential episodes of skeletal muscle denervation and reinnervation result in greater force and power deficits than a single peripheral nerve injury and repair. Adult Lewis rats underwent either transection and epineurial repair or sham exposure of the left peroneal nerve. After a 4-month recovery period, the contractile properties of the extensor digitorum longus muscle of the sham exposure group (control, n = 9) and one of the nerve division and repair groups (repair group 1, n = 9) were evaluated with measurements of the maximum tetanic isometric force, peak power, and maximal sustained power. A third group of rats underwent a second cycle of nerve division and repair (repair group 2, n = 9) at this same time point. Four months postoperatively, contractile properties of the extensor digitorum longus muscles were evaluated. Maximum tetanic isometric force and peak power were significantly reduced in repair group 2 rats as compared with repair group 1 and control rats. Maximal sustained power was not significantly different between the groups. These data support our working hypothesis that skeletal muscle contractile function is adversely affected by two cycles of denervation and reinnervation as compared with a single episode of nerve division and repair.     

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.     

The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: II. Functional evaluation of reinnervated muscle.

Isometric contractile function was evaluated in primates receiving peripheral nerve allografts and autografts. Twelve adult male cynomolgus monkeys received both sural nerve allografts and autografts to the ulnar nerve in opposite forearms. Half the animals received Cyclosporin A (CsA) immunosuppression (25 mg/kg per day); the remaining animals received placebo. One year following nerve engraftment, isometric contractile muscle function was evaluated in reinnervated abductor digiti quinti and intact abductor pollicis brevis muscles. Maximal twitch tension (Pt), tetanic tension (P(o)), time to peak tension (tpt), rate of rise of twitch tension (DP/dt), and muscle fatigue were evaluated at optimal muscle length (L(o)). All reinnervated muscles distal to nerve autografts and allografts in both Cyclosporin A-immunosuppressed and placebo-treated animals generated equivalent maximal twitch tension, tetanic tension, and time to peak tension, with no significant difference between groups (p > 0.05 by ANOVA). There was a tendency toward increased muscle fatiguability in Cyclosporin A-treated animals (p > 0.05). However, the rate of rise of twitch tension was significantly faster in the reinnervated and intact muscles of Cyclosporin A-treated primates (p < 0.05). Evidence of excellent functional reinnervation across nerve allografts and autografts similar to that seen in histologic and electrophysiologic studies was noted. Cyclosporin A immunosuppression did not significantly enhance recovery of muscle function distal to nerve allografts in this model.     

Regeneration and transplantation of muscles in old rats and between young and old rats.

In order to compare the regenerative ability of skeletal muscle between young (5 month) and old (26 month) rats, sliced or intact extensor digitorum longus muscles were freely autografted into young and old rats and also reciprocally grafted from young to old inbred animals and vice versa. Sixty days after grafting, the transplants were analyzed for contractile and histochemical properties. There was a relative similarity between the contraction times of both normal control muscles and of all groups of transplants, although the contraction time tended to be prolonged and histochemical fiber pattern was more often found to be uniform in grafts of senescent animals. All groups of transplants possessed histochemically heterogeneous fiber types at 60 days. The experiments demonstrate that skeletal muscle in old rats possesses a substantial degree of regenerative ability and that the free tranpllantation of entire muscles in old animals is feasible.     

The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: I. Histologic and electrophysiologic assessment.

Nerve regeneration across peripheral nerve allografts and control autografts in primates immunosuppressed with Cyclosporin A was quantitatively evaluated by electrophysiologic and histologic methods. Twelve cynomolgus monkeys received 3-cm autografts and allografts in contralateral ulnar nerves. They were immunosuppressed with Cyclosporin A at 25 mg/kg per day or placebo vehicle. Morphometric analysis of nerve graft and distal nerve segments was assessed at 1 year after engraftment. Quantitative electrophysiologic studies were performed percutaneously at 6 and 12 months, and compound action potentials were measured directly across the nerve grafts at 1 year. Excellent regeneration was seen across autografts and allografts in Cyclosporin A-treated and placebo-treated recipients.     

Ipsilateral and cross-over elongation of the motor nerve by nerve grafting: an experimental study in sheep

In difficult reconstructions, ipsilateral or cross-over nerve grafting is sometimes necessary to achieve reinnervation and motor function. This experimental study in sheep was to answer the question of limitation of elongation of a motor nerve by grafting, the question of the optimal time for suturing the nerve graft to the muscle nerve, and the question of the successful application of this surgical technique in extremities. In 18 sheep, the vastus nerve was elongated by a saphenous nerve graft as long as possible up to 30 cm (step 1). In 10 animals the nerve graft was applied ipsilaterally, and in 8 animals it was used as a cross-over nerve graft to the contralateral limb. The time between nerve grafting and connection of the distal end of the nerve graft to the freshly cut rectus nerve supplying the rectus femoris muscle (step 2) was variable: 0, 3, 6, 9, and 12 months. In all animals, the final experiments (step 3) were performed 6 months after the last operation (step 2). Muscle force measurements in the rectus femoris muscle and quantitative analysis of the number and diameter of myelinated nerve fibers in cross sections of the nerve biopsies at different levels showed that elongation of a motor nerve by nerve grafting is principally not limited. The functional results were rather inhomogeneous and therefore unpredictable (ipsilateral group: maximum tetanic tension = 27 to 172 N; cross-over group: 0 to 227.5 N). Nevertheless, crossover nerve grafting is recommended for selected cases even in extremities. There was no correlation between the time interval between the two operations and the functional or morphologic results, although better functional results were obtained when the distal nerve suture (step 2) was performed some months after nerve grafting (step 1). A clear correlation was found only between the number of regenerated axons in the rectus nerve behind the second suture line and the muscle function.     

Functional reinnervation of skeletal muscle in the adult rat by means of a peripheral nerve graft introduced into the spinal cord by dorsal approach

In the adult Mammal, different types of neurons, whose processes have been damaged in the CNS, may regrow lengthy axons along autologous PNS grafts. In the present study, PNS bridges were used to join the spinal cord (C5 level) to a nearby skeletal muscle (m. longissimus atlantis) which was denervated prior to direct graft insertion into an aneural region. From 3 to 5 months later, the following results were obtained: in situ and in vitro electrical stimulation of the graft determined partial or full contraction of the reconnected muscle. Intracellular recordings showed miniature endplate potentials (mepps). Endplate potentials (epps), evoked by stimulating the nerve graft, were recorded at the same points after partial blockade of the transmission with low doses of curare. They were suppressed with higher concentrations. The overall appearance of cross semithin and thin sections of the grafts was typical of regenerating nerves. EM observations of reinnervated muscles revealed typical neuromuscular junctions located either around the site of grafting or in the site of original endplates. In situ HRP application to the transected PNS bridges led to extensive labeling of neuronal somata located, close to the site of grafting, in the spinal grey matter and in adjacent spinal ganglia. When HRP was injected into the recommended muscle, neuronal labeling was almost restricted to typical motoneurons of the ventral horn. These results indicate that spinal neurons, and especially motoneurons, are probably involved in the formation, through PNS grafts, of new functional cholinergic connections with denervated skeletal muscles.     

Free flap from the flexor aspect of the wrist for resurfacing defects of the hand and fingers

The distal portion of the flexor aspect of the forearm has been used as the donor site of full-thickness skin grafts, venous skin grafts, and Chinese forearm flaps. This article describes the use of a free flap harvested from the flexor aspect of the wrist and based on the superficial palmar branch of the radial artery to repair skin defects of the hand and fingers. The advantages of this flap are as follows: (1) the operative field is the same; (2) the radial artery is preserved; (3) it is thin, pliable, and hairless and thus can supply a gliding surface for tendons beneath it; (4) when it involves a palmaris longus tendon and/or the palmar cutaneous branch of the median nerve, it can be used as a vascularized tendon or nerve graft; and (5) in view of the flow-through type of the pedicle of the flap, the digital artery can be reconstructed simultaneously. However, it should be noted that a hypesthesia in the proximal central carpal area remains when the palmar cutaneous branch of the median nerve is harvested as a vascularized nerve graft. The scar of the donor site should be left in the distal wrist crease. If it is not lying in the distal wrist crease, it may suggest that the patient has tried to commit suicide.     

Adaptations of skeletal muscle grafts to chronic changes of physical activity

After grafting, many structural and functional characteristics of skeletal muscle change with time until reaching a stable value. For several characteristics, these stable values are less than those observed in control skeletal muscle. Characteristics such as mass and fiber cross-sectional area are influenced by chronic changes in physical activity. The extent of our understanding of the dimensions and mechanisms of activity-induced adaptations of grafts is limited and based solely on experiments with rats. Improvements in mass, protein content, oxidative capacity, and glycogen concentration have been documented with conditioning by running of sufficient intensity and duration. The growth and development of soleus muscle grafts are severely impaired when normal weight-bearing activity is removed. The mass and maximum tension development of grafts are increased with ablation of muscles that function synergistically to the grafts, but are diminished after chronic electrical stimulation. Chronic electrical stimulation does increase oxidative capacity, capillarity, and the resistance to fatigue. Much remains to be learned about the mechanisms by which activity-related adaptations occur in skeletal muscle grafts.     

Tissue engineering of peripheral nerves: A comparison of venous and acellular muscle grafts with cultured Schwann cells

Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

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.     

The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine-alpha-bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP-less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra-junctional AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP-less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP-less grafts as early as 20 days postgrafting. The results of this study support the existence of still-unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well-differentiated endplates.     

Plasticity in mammalian skeletal muscle.

While it has been recognized for many years that different limb muscles belonging to the same mammal may have markedly differing contractile characteristics, it is only comparatively recently that it has been demonstrated that these differences depend upon the motor innervation. By appropriately changing the peripheral nerve innervating a mammalian skeletal muscle, it is possible to change dramatically the contractile behaviour of the reinnervated muscle. The manner by which the motor innervation determines the nature of a muscle fibre's contractile machinery is not completely understood, but it appears that the number and pattern of motor nerve impulses reaching the muscle play an important role. The biochemical changes occurring within muscle fibres whose contractile properties have been modified by altered motor innervation include the synthesis of different contractile proteins.     

Effect of foreign innervation on contractile and histochemical properties of the transplanted levator ani muscle of the rat.

Heterotopic transplantation of the levator ani (LA) muscle into the bed of the fast tibialis anterior (TA) or slow soleus (SOL) muscle respectively results in transformation of contractile and histochemical properties of the muscle dependent on the new "foreign" innervation. This transformation is observed after transplantation of minced muscle tissue and of free grafts. The result of transformation is more pronounced in the case of free LA-TA grafts which show progressive shortening of contractile response, whereas the LA-SOL shows slight shortening. The heterotopically transplanted free LA-SOL and the LA-TA grafts become relatively faster than the respective original muscle, suggesting operation of myogenic factors related to the fast LA muscle. Maximal tetanic tension output of the free heterotopic grafts 60 days after transplantation recovers to only about a quarter of the correspondong control muscles. Recovery of speed of contraction in the transplanted LA muscle is similar to that observed after selfreinnervation after crushing the pudendal nerve close to its entry into the muscle. In the heterotopically transplanted muscles the reversal of the originally uniform histochemical fibre pattern to a mixed fibre pattern in respect to ATPase and SDH activity is dependent on the type of innervation. After selfreinnervation of the LA muscle by the pudendal nerve a uniform fibre pattern is maintained with regeneration of the nerve.     

Total lower lip reconstruction with a composite radial forearm-palmaris longus tendon flap: a clinical series

Large, full-thickness lip defects after head and neck surgery continue to be a challenge for reconstructive surgeons. The reconstructive aims are to restore the oral lining, the external cheek, oral competence, and function (i.e., articulation, speech, and mastication). The authors' refinement of the composite radial forearm-palmaris longus free flap technique meets these criteria and allows a functional reconstruction of extensive lip and cheek defects in one stage. A composite radial forearm flap including the palmaris longus tendon was designed. The skin flap for the reconstruction of the intraoral lining and the skin defect was folded over the palmaris longus tendon. Both ends of the vascularized tendon were laid through the bilateral modiolus and anchored with adequate tension to the intact orbicularis muscle of the upper lip. This procedure was used in 12 patients. Six patients had cancer of the lower lip, five patients had a buccal cancer involving the lip, and one patient had a primary gum cancer that extended to the lower lip. Total to near-total resection (more than 80 percent) of the lower lip was indicated in six patients. In two other patients, the cancer ablation included more than 80 percent of the lower lip and up to 40 percent of the upper lip. A radial forearm palmaris longus free flap was used in all cases for reconstruction of the defect. Free flap survival was 100 percent. At the time of final evaluation, which was 1 year after the operation, all patients had good oral continence at rest (static suspension) and had achieved sufficient oral competence when eating. Ten patients were able to resume a regular diet, and two patients could eat a soft diet. All patients regained normal or near-normal speech and had an acceptable appearance. The described refinement of the composite radial palmaris longus free flap technique allows the reconstruction of the lower lip with a functioning oral sphincter; the technique can be recommended for patients who need large lower lip resection. It provides functional recovery of the reconstructed lower lip synchronizing with the remaining upper lip.     

Cultured epithelial autografts for giant congenital nevi

Eight pediatric patients with giant congenital nevi confluent over 21 to 51 percent body surface area were treated by excision and grafting. The nevus was excised to the muscle fascia, and the open wound was grafted with cultured epithelial autografts and split-thickness skin grafts. The patients have been followed from 17 to 56 months. Seventeen operations were performed in the eight patients, excising a mean of 6.9 percent body surface area at each procedure. The mean duration of anesthesia was 3.7 hours, and the mean operative blood loss was 12.3 percent estimated blood volume. The mean "take" for the cultured epithelial autografts was 68 percent, and for the split-thickness skin grafts, 84 percent. Epithelialization of open wound areas adjacent to the grafts was somewhat slower for the cultured epithelial autografts than for the split-thickness skin grafts, but it led to a healed wound in all patients except one. Ten of the 17 areas grafted with cultured epithelial autografts resulted in small open wounds that required regrafting. Wound contraction under the cultured epithelial autografts and under split-thickness skin grafts was similar and depended more on the anatomic site grafted than on the type of graft employed. in 16 of 17 operations, the cultured epithelium remained as a permanent, durable skin coverage. The use of cultured epithelial autografts allowed a larger area of excision than would have been possible with split-thickness skin grafts alone and, therefore, a more rapid removal of nevus. Cultured epithelial autograft are an important new technique in the care of patients with giant congenital nevi.