Neural nature of the time-dependent factor(s) involved in motor reinnervation

The influence of a time interval between nerve transection and reimplantation into a foreign muscle on the effectiveness of reinnervation was studied in the rat. We evaluated the weight loss and the maximal twitch and tetanic tensions developed by the reinnervated muscle upon nerve stimulation 60 days after reimplantation (i) of an acutely severed nerve onto an acutely denervated muscle; (ii) of a chronically (20 days) severed nerve onto an acutely denervated muscle, and (iii) of an acutely severed nerve onto a chronically denervated muscle. The best recovery was obtained when a chronically severed nerve was implanted into an acutely denervated muscle. We conclude that a neural time-dependent factor(s) is involved in motor reinnervation.     

Influence of atrophy on the efficiency of muscle reinnervation

The effectiveness of motor reinnervation carried out after an interval of 20 days between nerve transection and reimplantation onto a foreign muscle was studied in the rat. Sixty days after reimplantation the compound action potential, the maximal indirect twitch and tetanic tensions, and weight loss were evaluated. The functional data demonstrated an incomplete recovery which differed from the complete functional restoration observed in an acutely denervated muscle reinnervated with a chronically severed nerve. Time-dependent changes induced in the muscle by denervation seem therefore to influence the efficacy of motor reinnervation.     

Enhanced reinnervation after neurotization with Schwann cell transplantation

We investigated the feasibility of using Schwann cell transplantation to enhance reinnervation after direct nerve-to-muscle neurotization (NMN). The denervated anterior tibial muscle was neurotized by tibial nerve implantation, and Schwann cell suspension (transplantation group) or an equivalent volume of culture medium (control group) was injected at the implantation site. In the control group, few axons invaded the muscle, demonstrating that skeletal muscle was poorly permissive to the advancement of axons. In the transplantation group, a large number of regenerating axons grew for a longer distance throughout the muscle, and reinnervated motor endplates were significantly more abundant. Enhanced reinnervation and functional recovery of the muscle in the transplantation group was confirmed by a significant increase in the compound muscle action potential and in muscle weight. These results suggest that intramuscular Schwann cell transplantation has potential as a cell therapy to improve functional recovery after NMN.     

Effects of tenotomy on muscles reinnervated by a foreign nerve

Tenotomy of the rat soleus (SOL) and gastrocnemius (MG) muscles produces a central degeneration in slow fatigue-resistant fibers, but not in similar fibers of muscles in the extensor and peroneal compartments. To investigate the part that innervation plays in rendering a particular fiber type in a particular muscle susceptible to this degeneration, the SOL, extensor digitorum longus (EDL), and MG muscles were experimentally reinnervated by foreign nerves and tenotomized. When the SOL was reinnervated by the common peroneal nerve, slow fatigue-resistant fibers showed lesions, but when the EDL was reinnervated by the nerve to the SOL, no lesions were found after tenotomy. When the MG was reinnervated by the nerve to the SOL, slow fatigue-resistant fibers that had differentiated in regions normally occupied almost entirely by fast fatigable fibers showed characteristic lesions. These results show that the failure of tenotomy to produce lesions in the EDL is not due to the nature of its innervation and that a fiber type not normally susceptible to the degenerative change will become susceptible when transformed to the slow fatigueresistant type.     

Effect of tenotomy on self-reinnervated and randomly reinnervated soleus muscle of rat.

The time course and degree of atrophic changes caused by tenotomy were compared in normal, self-reinnervated and randomly reinnervated soleus muscle 6 months after transsection and reunion of the nerve at different distances from the muscle. Comparison was made between the behaviour of Type I and Type II fibers, distinguished on the basis of histochemical myofibrillar ATPase and succinic dehydrogenase reactions. Cross-sectional areas of individual muscle fibers were measured using Quantimet 720 image analyser. Selective atrophy of Type I muscle fibers as determined by structural and histochemical changes was observed after tenotomy of normal, self-reinnervated and randomly reinnervated soleus muscles after transsection of the muscular branch of the tibial nerve, Type II muscle fibers in randomly reinnervated muscles were found to be relatively insensitive to tenotomy, as in normal muscle. In randomly reinnervated muscles after transsection and reunion of the sciatic nerve, tenotomy did not cause any visible structural and histochemical abnormalities although a decrease of muscle weight and cross-sectional surface area of fibers was noted. Since in these muscles Type II fibers increased to about 70% of the muscle fiber population, it is suggested that the increased percentage of Type II fibers seemed to prevent the atrophic changes in Type I fibers after tenotomy.     

Recovery of muscle after different periods of denervation and treatments

Three aspects of reinnervation and recovery of skeletal muscle following various periods of denervation were investigated: (1) the effect of duration of denervation; (2) the effect of hyperthyroidism on recovery; and (3) whether the muscle or the nerve limits recovery. The rat medial gastrocnemius (MG) nerve was cut and then resutured after 0, 3, 7, 21, or 56 days. In a second group of animals, the MG muscle was denervated and, in addition, the animal received triiodothyronine (T₃) supplementation during reinnervation. The third group of animals had the denervated MG muscle reinnervated by a larger number of newly transected foreign axons. The force produced by the reinnervated muscle depends on the period that the muscle was denervated. Recovery was impaired when the period of denervation exceeded 7 days. T₃ treatment did not benefit the return of force production, nor did providing the muscle with a larger number of newly transected axons.     

Effect of neurotrophin-3 on reinnervation of the larynx using the phrenic nerve transfer technique

Current techniques for reinnervation of the larynx following recurrent laryngeal nerve (RLN) injury are limited by synkinesis, which prevents functional recovery. Treatment with neurotrophins (NT) may enhance nerve regeneration and encourage more accurate reinnervation. This study presents the results of using the phrenic nerve transfer method, combined with NT-3 treatment, to selectively reinnervate the posterior cricoarytenoid (PCA) abductor muscle in a pig nerve injury model. RLN transection altered the phenotype and morphology of laryngeal muscles. In both the PCA and thyroarytenoid (TA) adductor muscle, fast type myosin heavy chain (MyHC) protein was decreased while slow type MyHC was increased. These changes were accompanied with a significant reduction in muscle fibre diameter. Following nerve repair there was a progressive normalization of MyHC phenotype and increased muscle fibre diameter in the PCA but not the TA muscle. This correlated with enhanced abductor function indicating the phrenic nerve accurately reinnervated the PCA muscle. Treatment with NT-3 significantly enhanced phrenic nerve regeneration but led to only a small increase in the number of reinnervated PCA muscle fibres and minimal effect on abductor muscle phenotype and morphology. Therefore, work exploring other growth factors, either alone or in combination with NT-3, is required.     

Reinnervation of denervated skeletal muscle by central neurons regenerating via ventral roots implanted into the spinal cord

The reinnervation of denervated skeletal muscle by central axons regenerating via a ventral root implanted into the spinal cord was examined in rats. The 8th thoracic ventral root was severed and its distal end implanted into the ventro-lateral column of the spinal cord via a stab incision. In control animals the root was severed, but was not implanted into the stab incision. After 12-14 months the animals were examined electrophysiologically to determine the presence or absence of motor units in the 8th intercostal muscle which were reinnervated by centrally derived axons regenerating via the implant. Such units were found in implanted animals, but in none of the controls. Evidence that the motor units were reinnervated by central axons included the facts that the units could be activated either, (1) reflexly (i.e. trans-synaptically) by electrical stimulation of the dorsal roots or spinal cord, or (2) pharmacologically by either the intraspinal injection of glutamate or acetycholine, or by the systemic administration of strychnine. Great care was taken to ensure that the only feasible connection between the spinal cord and the 8th intercostal muscle was via the site of implantation. The EMG signals from the motor units were of large amplitude, typical of reinnervated muscle, and their individual activation resulted in discernible contractions of regions of the T8 intercostal muscle. We conclude that regenerating CNS neurons can be guided to innervate denervated skeletal muscle by the implantation of severed ventral roots into the spinal cord. The neuromuscular synapses formed are functional and persistent. The findings may be relevant to the restoration of function after nervous injuries, such as the avulsion of ventral roots.     

Spontaneous reinnervation of a free muscle flap

A latissimus dorsi muscle flap was used to repair a severe traumatic avulsion defect of the dorsum of the foot in a 3-year-old girl. The severed peroneal nerve apparently regenerated across a large gap and spontaneously reinnervated the denervated muscle flap. This resulted in a functional flap as demonstrated clinically and electromyographically. Surgical methods of muscle reinnervation and the influence of neurotrophic factors are discussed.     

Regeneration and functional recovery in the upper extremity of rats after various types of nerve injuries

The aim was to establish an accurate, reproducible, and simple method to evaluate functional recovery after different types of nerve injuries to the brachial plexus of rats. To that end, pawprints, measured as distance between the first and fourth and second and third digits, were used for evaluation of injuries including crush injury, transection/repair, or graft repair of the median, ulnar, and radial nerves. Immunocytochemistry of the C-terminal flanking peptide of neuropeptide Y (CPON) and neurofilaments was used to investigate the cell body response and axonal outgrowth, respectively. Functional recovery was dependent on the severity as well as on the level of the lesion. Neither a single injury to the median nerve nor an injury to the ulnar nerve affected the pawprint, while an injury to both these nerves or a single injury to the radial nerve caused impairment of pawprints. There was a rapid recovery after crush injury to these nerves compared to previous reports of a similar injury to the sciatic nerve. The pattern of axonal outgrowth was related to the severity of the lesion. A conditioning lesion, i.e., an initial lesion of the same nerve preceding a test injury by a few days, of both motor/sensory fibers led to a quicker functional recovery. Surprisingly, conditioning of only sensory fibers had nearly the same effect. The cell body response was dependent on the level of the nerve lesion. The upper extremity of rats might be useful to evaluate the effects of new repair methods after nerve injuries using functional evaluation with pawprints as a simple and accurate method.     

One hour electrical stimulation accelerates functional recovery after femoral nerve repair

The clinical outcome of peripheral nerve injuries requiring surgical repair is usually poor and efficient therapies do not exist. Recent work has suggested that low-frequency electrical stimulation of the severed nerve which produces repeated discharges of the parent motoneuron perikarya positively influences axonal regeneration, even if applied once for a period of only 1 h. Here we provide the first evidence for locomotor functional benefits of such stimulation. We transected the femoral nerve of adult C57BL/6J mice proximal to the bifurcation of the quadriceps and saphenous branches and electrically stimulated the proximal nerve stump for 1 h at 20-Hz frequency prior to nerve repair with a silicone cuff. Three months later, the ability of the quadriceps muscle to extend the knee in sham-stimulated mice had recovered to 63% of the preoperative values as estimated by single-frame motion analysis. After electrical stimulation, the outcome was only slightly better (73%) but the rate of functional recovery was considerably accelerated. Near-maximum recovery was achieved 6 weeks earlier than in the control group. The beneficial effects were associated with larger motoneuron cell bodies and increased diameters of regenerated axons in the quadriceps nerve branch, but not with enhanced preferential reinnervation by motoneurons of muscle as opposed to skin. The observed acceleration of functional restoration and the positive effects on motoneurons and regenerated axons indicate the potential of a clinically feasible approach for improvement of nerve repair outcome in human patients in which delayed target reinnervation is a factor limiting recovery.     

The effect of a conditioning lesion on sudomotor axon regeneration

The effects of a conditioning lesion on the rate of sudomotor axon regeneration were judged by the recovery of sweat gland (SG) secretion after cholinergic stimulation. Three groups of mice were given a conditioning lesion by crushing the sciatic nerve at mid-thigh 4, 7, and 14 days before a test lesion. A 4th group received a conditioning crush of the tibial nerve at the ankle 7 days before the test lesion. Control mice had a single test lesion. SG reinnervation in control mice began 19 days after the test lesion, and was functionally complete by 41 days. In groups with the conditioning lesion 4, 7, and 14 days before the test operation, the first reactive SGs reappeared at 16, 15, and 16 days respectively after the test lesion, and maximal recovery occurred by 33, 32, and 39 days. In mice with the distal conditioning lesion, reinnervation began at 19 days and was maximal by 36 days. In summary, a nerve conditioning lesion placed from 4 to 14 days prior to and at the same site as a test lesion significantly accelerated the growth rate of the fastest regenerating unmyelinated sudomotor axons and reduced the time until most SGs were reinnervated. A more distally placed test lesion reduced the interval for recovery.     

Electrophysiological study of conditioning lesion effects on rat sciatic nerve regeneration following either prior section or freeze. II. Blocking by prior tenotomy

The conditioning lesion effects refer to the earlier formation and the accelerated regeneration of axonal sprouts following two successive axotomies. In a previous study, we observed that a prior freeze or a prior cut of rat sciatic nerve resulted in differences in the enhancement of the regeneration rate and the reduction of the initial delay. These differences were interpreted as a possible non-neuronal cells influence on the intrinsic regulation of the conditioning lesion phenomenon. In the present study, we attempted to modify the status of the muscles using tenotomy before the prior nerve injury to determine the respective influence of the muscular cells on conditioning lesion effects. Thus, the conditioning lesion, which was either a cut or a freeze of the tibial nerve at the ankle, was performed 14 days after foot sole muscles were tenotomized, close to their insertion into the calcaneus bone. The test lesion was always a freeze of the sciatic nerve at midthigh performed 7 days following the prior lesion. The elongation of the regenerating sprouts was electrophysiologically evaluated and the regeneration rate as well as the initial delay were calculated by means of regression analysis. Tenotomy did not influence the regeneration as was demonstrated in a group with a single sciatic nerve lesion. In contrast, when prior lesion was performed, the tenotomy prevented both the enhancement of the rate of regeneration and the reduction of the initial delay, whatever was the type of the conditioning lesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of recovery following a novel partial nerve lesion in a rat model

Partial nerve lesions with a varying degree of retained function and often a painful neuroma pose a dilemma for the clinician. Surgical treatment of partial nerve lesion is perilous because of possible damage to intact axons and subsequent loss of retained function. We present a new rat model of a partial nerve lesion, allowing further study to improve treatment for this condition. A partial (50%) lesion of the tibial portion of the rat sciatic nerve was created and compared to standard crush and neurectomy control lesions. The extent of lost function and the progress of postoperative recovery following the three lesions were compared using serial walking track analyses and end-point muscle weight ratios for atrophy as outcome measures. All groups had tibial functional indices (TFI) significantly different from one another after 1 week. TFIs for the crush group returned to normal by 4 weeks, whereas the neurectomy group showed no recovery. The partial lesion group gradually improved, reaching a plateau of 44% by 7 weeks. Gastrocnemius muscle weight ratios for the partial, crush, and neurectomy lesions at 9 weeks were 0.63, 0.87, and 0.32, respectively. There was a strong correlation between the TFI and muscle weight ratios (r(2) = 0.89; P < 0.001) suggesting that these outcome measures are highly predictive of function. In conclusion, the partial lesion showed a gradual but incomplete functional recovery with a complementary degree of muscle atrophy. The model may prove useful in the evaluation of proposed treatments for partial nerve lesions and the associated painful state.     

Factors limiting motor recovery after facial nerve transection in the rat: combined structural and functional analyses

It is believed that a major reason for the poor functional recovery after peripheral nerve lesion is collateral branching and regrowth of axons to incorrect muscles. Using a facial nerve injury protocol in rats, we previously identified a novel and clinically feasible approach to combat axonal misguidance--the application of neutralizing antibodies against neurotrophic factors to the injured nerve. Here, we investigated whether reduced collateral branching at the lesion site leads to better functional recovery. Treatment of rats with antibodies against nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factor, insulin-like neurotrophic factor I, ciliary neurotrophic factor or glial cell line-derived neurotrophic factor increased the precision of reinnervation, as evaluated by multiple retrograde labelling of motoneurons, more than two-fold as compared with control animals. However, biometric analysis of vibrissae movements did not show positive effects on functional recovery, suggesting that polyneuronal reinnervation--rather than collateral branching --may be the critical limiting factor. In support of this hypothesis, we found that motor end-plates with morphological signs of multiple innervation were much more frequent in reinnervated muscles of rats that did not recover after injury (51% of all end-plates) than in animals with good functional performance (10%). Because polyneuronal innervation of muscle fibres is activity-dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies could be soon designed and tested.     

Sensory reinnervation of muscle spindles after repair of tibial nerve defects using autogenous vein gratis

Motor reinnervation after repair of tibial nerve defects using autologous vein grafts in rats has previously been reported, but sensory reinnervation after the same repair has not been fully investigated. In this study, partial sensory reinnervation of muscle spindles was observed after repair of lO-mm left tibial nerve defects using autologous vein grafts with end-to-end anasto- mosis in rats, and functional recovery was confirmed by electrophysiological studies. There were no significant differences in the number, size, or electrophysiological function of reinnervated muscle spindles between the two experimental groups. These findings suggest that repair of short nerve defects with autologous vein grafts provides comparable results to immediate end-to-end anastomosis in terms of sensory reinnervation of muscle spindles.     

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3‐treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine α‐bungarotoxin and neurofilament antibody. Four weeks after surgery, most end‐plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS‐treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission. © 2010 Wiley‐Liss, Inc.     

Misdirection of regenerating axons and functional recovery following sciatic nerve injury in rats

Poor functional recovery found after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. We applied brief electrical stimulation (ES) to the common fibular (CF) but not the tibial (Tib) nerve just prior to transection and repair of the entire rat sciatic nerve, to attempt to influence the misdirection of its regenerating axons. The specificity with which regenerating axons reinnervated appropriate targets was evaluated physiologically using compound muscle action potentials (M responses) evoked from stimulation of the two nerve branches above the injury site. Functional recovery was assayed using the timing of electromyography (EMG) activity recorded from the tibialis anterior (TA) and soleus (Sol) muscles during treadmill locomotion and kinematic analysis of hindlimb locomotor movements. Selective ES of the CF nerve resulted in restored M-responses at earlier times than in unstimulated controls in both TA and Sol muscles. Stimulated CF axons reinnervated inappropriate targets to a greater extent than unstimulated Tib axons. During locomotion, functional antagonist muscles, TA and Sol, were coactivated both in stimulated rats and in unstimulated but injured rats. Hindlimb kinematics in stimulated rats were comparable to untreated rats, but significantly different from intact controls. Selective ES promotes enhanced axon regeneration but does so with decreased fidelity of muscle reinnervation. Functional recovery is neither improved nor degraded, suggesting that compensatory changes in the outputs of the spinal circuits driving locomotion may occur irrespective of the extent of misdirection of regenerating axons in the periphery.     

Shock wave treatment improves nerve regeneration in the rat

Introduction: The aims of the experiments were to: (1) determine whether low‐energy shock wave treatment accelerates the recovery of muscle sensitivity and functionality after a nerve lesion; and (2) assess the effect of shock waves on the regeneration of injured nerve fibers. Methods: After compression of a muscle nerve in rats the effects of shock wave treatment on the sequelae of the lesion were tested. In non‐anesthetized animals, pressure pain thresholds and exploratory activity were determined. The influence of the treatment on the distance of nerve regeneration was studied in immunohistochemical experiments. Results: Both behavioral and immunohistochemical data show that shock wave treatment accelerates the recovery of muscle sensitivity and functionality and promotes regeneration of injured nerve fibers. Conclusion: Treatment with focused shock waves induces an improvement of nerve regeneration in a rodent model of nerve compression. Muscle Nerve 47: 702–710, 2013     

Trophic reactions of crayfish muscle fibers and neuromuscular synapses after denervation,tenotomy, and immobilization

Severed motor nerve terminals remain morphologically intact and functionally competent for several months following transection of the motor nerve to the claw opener muscle of crayfish (Procambarus clarki). However, severed motor axons are not entirely normal in that excitatory synapses produce junctional potentials that are somewhat smaller than control values. Tenotomy or immobilization often produce little change in synaptic potentials for at least 180 days. Opener muscle fibers with severed motor axons or immobilized muscle fibers show little ultrastructural change for at least 100 days, provided the nerve terminals remain intact. However, after tenotomy, muscle fibers atrophy within 20 to 30 days. This atrophy is more rapid and more severe if nerve terminals on that muscle fiber have degenerated. These atrophic changes in muscle fine structure include disorganization of myofibrils and disruption or loss of the sarcomere Z bands. In summary, our data show that drastic changes in crayfish muscle structure can proceed after tentomy without concomitant changes in function of the motor nerve terminals; and conversely, although transecting the motor nerve has some effect on motor nerve terminals, decentralization has little effect on muscle fiber structure as long as nerve terminals remain intact. These data are in agreement with the hypothesis that crustacean muscle fibers are trophically dependent on the presence of functional nerve terminals and on passive fiber tension (or resting length).