Growth hormone treatment enhances the functional recovery of sciatic nerves after transection and repair

Introduction: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration. Methods: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance. Results: GH‐treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH. Conclusion: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair. Muscle Nerve, 2012     

Remnant neuromuscular junctions in denervated muscles contribute to functional recovery in delayed peripheral nerve repair

Schwann cell proliferation in peripheral nerve injury(PNI)enhances axonal regeneration compared to central nerve injury.However,even in PNI,long-term nerve damage without repair induces degeneration of neuromuscular junctions(NMJs),and muscle atrophy results in irreversible dysfunction.The peripheral regeneration of motor axons depends on the duration of skeletal muscle denervation.To overcome this difficulty in nerve regeneration,detailed mechanisms should be determined for not only Schwann cells but also NMJ degeneration after PNI and regeneration after nerve repair.Here,we examined motor axon denervation in the tibialis anterior muscle after peroneal nerve transection in thyl-YFP mice and regeneration with nerve reconstruction using allografts.The number of NMJs in the tibialis anterior muscle was maintained up to 4 weeks and then decreased at 6 weeks after injury.In contrast,the number of Schwann cells showed a stepwise decline and then reached a plateau at 6 weeks after injury.For regeneration,we reconstructed the degenerated nerve with an allograft at 4 and 6 weeks after injury,and evaluated functional and histological outcomes for 10 to 12 weeks after grafting.A higher number of pretzel-shaped NMJs in the tibialis anterior muscle and better functional recovery were observed in mice with a 4-week delay in surgery than in those with a 6-week delay.Nerve repair within 4 weeks after PNI is necessary for successful recovery in mice.Prevention of synaptic acetylcholine receptor degeneration may play a key role in peripheral nerve regeneration.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University on 5 July 2017,30 March 2018,and 15 May 2019(A2017-311C,A2018-297A,and A2019-248A),respectively.     

周围神经损伤修复及功能恢复评价

摘要 目的：评价修复周围神经缺损的各种生物型人工材料的性能、应用以及功能恢复评定方法,寻找适宜的周围神经替代物。 方法：以“神经导管,周围神经损伤修复,生物材料,许旺细胞”为关键词,采用计算机检索2004-01/2010-11相关文章。纳入与生物材料以及组织工程神经相关的文章;排除重复研究或Meta分析类文章。以28篇文献为主,重点讨论周围神经修复生物型人工材料的种类、性能以及适宜的功能恢复评定方法。 结果：以脱细胞神经基质以及人工合成可降解材料为主体的复合型生物工程材料可作为较理想的支架材料应用于周围神经组织工程。脱细胞神经支架解决了自体神经来源受限、移植物排斥反应等问题,韧性与可塑性接近自体神经,微环境更利于周围神经再生。人工合成可降解材料具有生物降解、可塑性、一定的通透性等优势,且已有商品化成品出现。若将上述材料分别合理构建复合材料,有可能得到性能良好的组织工程神经移植物。周围神经修复后功能恢复评定方法主要以大体与形态学观察、组织学、神经肌肉机能学评定为主,辅以分子生物学技术。各类评定方法的应用有利于筛选出最适宜的周围神经损伤修复材料与构建方案。 结论：周围神经损伤修复生物型人工材料研究发展迅速,但仍没有超越自体神经移植的支架材料。脱细胞神经基质以及人工合成可降解材料复合构建支架可作为较好的周围神经支架,但仍需要与种子细胞、神经营养因子等联合构建,以取得良好的促进再生效果。当前,对周围神经损伤修复效果的评定更加注重于神经肌肉功能的恢复,迫切需要筛选出最佳的修复材料以及构建方案以满足组织工程神经移植以及功能康复的要求,达到对周围神经损伤后形态、结构修复与功能重建的目的。 关键词：神经组织工程;周围神经;功能恢复;生物型人工材料;神经移植物 doi:10.3969/j.issn.1673-8225.2011.08.030     

BDNF/TrkB signaling regulates HNK-1 carbohydrate expression in regenerating motor nerves and promotes functional recovery after peripheral nerve repair

Functional recovery after peripheral nerve injury is often poor despite high regenerative capacity of peripheral neurons. In search for novel treatments, brief electrical stimulation of the acutely lesioned nerve has recently been identified as a clinically feasible approach increasing precision of axonal regrowth. The effects of this stimulation appear to be mediated by BDNF and its receptor, TrkB, but the down-stream effectors are unknown. A potential candidate is the HNK-1 carbohydrate known to be selectively reexpressed in motor but not sensory nerve branches of the mouse femoral nerve and to enhance growth of motor but not sensory axons in vitro. Here, we show that short-term low-frequency electrical stimulation (1 h, 20 Hz) of the lesioned and surgically repaired femoral nerve in wild-type mice causes a motor nerve-specific enhancement of HNK-1 expression correlating with previously reported acceleration of muscle reinnervation. Such enhanced HNK-1 expression was not observed after electrical stimulation in heterozygous BDNF or TrkB-deficient mice. Accordingly, the degree of proper reinnervation of the quadriceps muscle, as indicated by retrograde labeling of motoneurons, was reduced in TrkB+/- mice compared to wild-type littermates. Also, recovery of quadriceps muscle function, evaluated by a novel single-frame motion analysis approach, and axonal regrowth into the distal nerve stump, assessed morphologically, were considerably delayed in TrkB+/- mice. These findings indicate that BDNF/TrkB signaling is important for functional recovery after nerve repair and suggest that up-regulation of the HNK-1 glycan is linked to this phenomenon.     

Antibodies to the neural cell adhesion molecule disrupt functional recovery in injured nerves

Tubes containing specific monoclonal antibodies to the neural cell adhesion molecule (N-CAM) were applied to transected sciatic nerves to attempt to perturb the recovery of muscle function. Physiological recordings were used to estimate the return of function. The decline of implanted antibody over 28 days was estimated and negatively correlated with the degree of functional recovery. No significant immune responses were detected in response to the implanted material. The data implicated N-CAM as a significant component of nerve regeneration.     

fMRI of delayed albumin treatment during stroke recovery in rats: implication for fast neuronal habituation in recovering brains.

Accumulating experimental and clinical data suggest that albumin may be neuroprotective for stroke. Here, we use functional magnetic resonance imaging (fMRI) to evaluate the therapeutic efficacy of albumin and its effects on the recovery of stimuli-induced cerebral hemodynamics. For this purpose, fMRI activity in the ipsilesional somatosensory (SS) cortex was assessed using a well established rat model of transient 90 min focal ischemia and electrical forelimb stimulation. Rats were treated with either saline or albumin via intracerebroventricular injections at 12 h post-stroke onset. Despite this delayed treatment time, when compared to the saline-treated rats (n=7), there were significant enhancements of the fMRI activation in the albumin-treated rats (n=6) for both blood oxygenation level dependence (BOLD) and functional cerebral blood volume (fCBV) responses. Interestingly, the temporal characteristics of the ipsilesional SS BOLD responses in the albumin-treated rats appeared considerably altered compared to those of contralesional responses while such temporal alterations were not pronounced for the fCBV responses. These characteristic fMRI temporal profiles of the albumin-treated brains may be due to altered neuronal responses rather than altered integrity of neurovascular coupling, which implies an unusually fast habituation of neuronal responses in the lesional SS cortex. The correlation between various MRI-derived structural parameters and the fMRI response magnitude was also characteristic for albumin and control groups. Taken together, these data suggest that restoration of fMRI response magnitudes, temporal profiles, and correlations with structure may reveal the extent and specific traits of albumin treatment associated stroke recovery.     

Spinal cord repair in neonatal rats: a correlation between axonal regeneration and functional recovery

The present study aimed to analyse how anatomical regeneration contributes to functional recovery after experimental spinal cord repair. Thoracic spinal cord of neonatal rats was completely transected to make a gap and repaired by grafting a section of embryonic spinal cord. Six weeks after surgery, outcome of locomotor performance was assessed using an open field locomotor scale (BBB scale). Axonal regeneration across the repaired site was quantitatively assessed in the raphe, vestibular, and red nuclei and the sensorimotor cortex by a retrograde tracing method. The rats that had no labelled neurons in any of the supraspinal nuclei showed no hind-forelimb coordination. The rats that had labelled neurons in the brainstem nuclei but not in the sensorimotor cortex showed hind-forelimb coordination of varying grades depending on the amount of regeneration. The rats that had labelled neurons in all of the examined nuclei showed almost normal locomotion. In addition to a relationship between distribution of the labelled neurons and functional recovery, a positive correlation was observed between number of the labelled neurons in each of the supraspinal nuclei and locomotor performance of the rat. Thus the grade of restored function appeared to be regulated by distribution and number of fibres regenerated across the repaired site and into the target region. These results suggest that accurate reconstruction of neural connections is essential for significant functional recovery after spinal cord repair.     

The effect of nerve growth factor on functional recovery after injury to the chorda tympani and lingual nerves

Nerve growth factor (NGF) is known to ameliorate central changes and enhance the regeneration of damaged axons in the early stages after peripheral nerve injury. We have assessed the long-term outcome of placing NGF at a nerve repair site by determining the functional characteristics of several groups of sensory afferent and autonomic efferent fibres in the cat lingual nerve. Six months after entubulation repair, with or without the incorporation of NGF, the recovery of secretomotor and vasomotor efferents was determined by recording salivary flow from the submandibular gland and temperature changes on the tongue surface, each evoked by stimulation of the repaired nerve. Electrophysiological recordings from the lingual and chorda tympani nerves proximal to the repair allowed characterisation of mechanosensitive, thermosensitive and gustatory afferents. When compared with data from uninjured control animals, both repair groups showed changes in spontaneous discharge and persistent reductions in conduction velocity, receptor sensitivity, proportion of gustatory units, and rate of salivary secretion. Comparisons between the outcome of repair with or without NGF revealed few differences. In the NGF group the conduction velocity of afferents in the lingual nerve was lower, and the level of spontaneous activity was higher. However, NGF appeared to preferentially enhance the regeneration of thermosensitive afferents, suggesting that it may play a role in determining the phenotypic profile of the regenerating axonal population. This suggests that future therapeutic enhancement of regeneration after peripheral nerve injury may require a combination of factors to encourage regeneration of specific fibre groups.     

Normal proximal and delayed distal conduction in the pudendal nerves of patients with idiopathic (neurogenic) faecal incontinence.

The latency of the response in the external anal sphincter muscle following transcutaneous stimulation of the cauda equina at the L1 vertebral level was measured in nine women with neurogenic faecal incontinence (mean 7.3 SD 0.7 ms) and 11 normal subjects (mean 5.6 SD 0.6 ms) (p = 0.01). There was no difference in conduction velocity between the L1 and L4 vertebral levels thus supporting the suggestion that conduction delay in faecal incontinence occurs distally.     

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3 V, 20 Hz, 20 min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24 weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5 mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro‐Gold retrograde‐labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24 weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain‐derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF‐mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.     

A magnetic evaluation of peripheral nerve regeneration: II. The signal amplitude in the distal segment in relation to functional recovery

Motor and sensory function in a healthy nerve is strongly related to the number of neuronal units connecting to the distal target organs. In the regenerating nerve the amplitudes of magnetically recorded nerve compound action currents (NCACs) seem to relate to the number of functional neuronal units with larger diameters regenerating across the lesion. The goal of this experiment was to compare the signal amplitudes recorded from the distal segment of a reconstructed nerve to functional recovery. To this end, the peroneal nerves of 30 rabbits were unilaterally transected and reconstructed. After 6, 8, 12, 20, and 36 weeks of regeneration time the functional recovery was studied based on the toe-spread test, and the nerve regeneration based on the magnetically recorded NCACs. The results demonstrate that the signal amplitudes recorded magnetically from the reconstructed nerves increase in the first 12 weeks from 0% to 21% of the amplitudes recorded from the control nerves and from 21% to 25% in the following 23 weeks. The functional recovery increases from absent to good between the 8th and the 20th week after the reconstruction. A statistically significant relation was demonstrated between the signal amplitude and the functional recovery (P < 0.001). It is concluded that the magnetic recording technique can be used to evaluate the quality of a peripheral nerve reconstruction and seems to be able to predict, shortly after the reconstruction, the eventual functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:750–755, 1998.     

Stressful life events predict delayed functional recovery following treatment for mania in bipolar disorder

Identifying predictors of functional recovery in bipolar disorder is critical to treatment efforts to help patients re-establish premorbid levels of role adjustment following an acute manic episode. The current study examined the role of stressful life events as potential obstacles to recovery of functioning in various roles. 65 patients with bipolar I disorder participated in a longitudinal study of functional recovery following clinical recovery from a manic episode. Stressful life events were assessed as predictors of concurrent vs. delayed recovery of role functioning in 4 domains (friends, family, home duties, work/school). Despite clinical recovery, a subset of patients experienced delayed functional recovery in various role domains. Moreover, delayed functional recovery was significantly associated with presence of one or more stressors in the prior 3 months, even after controlling for mood symptoms. Presence of a stressor predicted longer time to functional recovery in life domains, up to 112 days in work/school. Interventions that provide monitoring, support, and problem-solving may be needed to help prevent or mitigate the effects of stress on functional recovery.     

Fractionated radiation facilitates repair and functional motor recovery after spinal cord transection in rat

Previous studies suggest that motor recovery does not occur after spinal cord injury because reactive glia abort the natural repair processes. A permanent wound gap is left in the cord and the brain-cord circuitry consequently remains broken. Single-dose x-irradiation destroys reactive glia at the damage site in transected adult rat spinal cord. The wound then heals naturally, and a partially functional brain-cord circuitry is reconstructed. Timing is crucial; cell ablation is beneficial only within the third week after injury. Data presented here point to the possibility of translating these observations into a clinical therapy for preventing the paralysis following spinal cord injury in the human. The lesion site (at low thoracic level) in severed adult rat spinal cord was treated daily, over the third week postinjury, with protocols of fractionated radiation similar to those for treating human spinal cord tumors. This resulted, as with the single-dose protocol, in wound healing and restoration of some hindquarter motor function; in addition, the beneficial outcome was augmented. Of the restored hindlimb motor functions, weight-support and posture in stance was the only obvious one. Recovery of this motor function was partial to substantial and its incidence was 100% instead of about 50% obtained with the single-dose treatment. None of the hindlimbs, however, regained frequent stepping or any weight-bearing locomotion. These data indicate that the therapeutic outcome may be further augmented by tuning the radiation parameters within the critical time-window after injury. These data also indicate that dose-fractionation is an effective strategy and better than the single-dose treatment for targeting of reactive cells that abort the natural repair, suggesting that radiation therapy could be developed into a therapeutic procedure for repairing injured spinal cord.     

Hepatocyte growth factor promotes endogenous repair and functional recovery after spinal cord injury

Many therapeutic interventions using neurotrophic factors or pharmacological agents have focused on secondary degeneration after spinal cord injury (SCI) to reduce damaged areas and promote axonal regeneration and functional recovery. Hepatocyte growth factor (HGF), which was identified as a potent mitogen for mature hepatocytes and a mediator of inflammatory responses to tissue injury, has recently been highlighted as a potent neurotrophic and angiogenic factor in the central nervous system (CNS). In the present study, we revealed that the extent of endogenous HGF up-regulation was less than that of c-Met, an HGF receptor, during the acute phase of SCI and administered exogenous HGF into injured spinal cord using a replication-incompetent herpes simplex virous-1 (HSV-1) vector to determine whether HGF exerts beneficial effects and promotes functional recovery after SCI. This treatment resulted in the significant promotion of neuron and oligodendrocyte survival, angiogenesis, axonal regrowth, and functional recovery after SCI. These results suggest that HGF gene delivery to the injured spinal cord exerts multiple beneficial effects and enhances endogenous repair after SCI. This is the first study to demonstrate the efficacy of HGF for SCI.     

Survival, regeneration and functional recovery of motoneurons after delayed reimplantation of avulsed spinal root in adult rat

We have established that extensive reinnervation and functional recovery follow immediate reimplantation of avulsed ventral roots in adult rats. In the present study, we examined the consequences of reimplantation delayed for 2 weeks after avulsion of the C6 spinal root. Twelve and 20 weeks after delayed reimplantation, 57% and 53% of the motoneurons in the injured spinal segment survived. More than 80% of surviving motoneurons regenerated axons into the reimplanted spinal root. Cholinesterase-silver staining revealed axon terminals on endplates in the denervated muscles. The biceps muscles in reimplanted animals had atrophied less than those in animals with avulsion only, as indicated by muscle wet weight and histological appearance. After electrical stimulation of the motor cortex or the C6 spinal root, typical EMG signals were recorded in biceps of reimplanted animals. The latency of the muscle potential at 20 weeks was similar to that of sham-operated controls. Behavioral recovery was demonstrated by a grooming test and ipsilateral forepaw movements were well coordinated in both voluntary and automatic activities. These results demonstrate that ventral root reimplantation can protect severed motoneurons, enable the severed motoneurons to regenerate axons, and enhance the recovery of forelimb function even when it is delayed for 2 weeks after avulsion.     

Local and distal responses to injury in the rapid functional recovery from spinal cord contusion in rat pups

Young rats display an accelerated rate of locomotor recovery after contusive spinal cord injury (SCI) compared to adults subjected to a similar standardized injury. We examined possible differences in the responses to SCI at the injury site and in the distal cord that might contribute to this rapid recovery. P14-15 rats were studied at 1, 3, 5, 7, and 28 days after injury at T8 produced with a weight drop device (10 g x 2.5 cm). We used immunohistochemistry to investigate distal plasticity of serotonergic and noradrenergic pathways that have been shown to modulate locomotion. After SCI, pups exhibited an expected decrease in monoaminergic innervation of the lumbosacral cord, consistent with partial loss of these descending pathways. Unlike published results for the adult, we found no evidence of partial reinnervation with time after injury. On the other hand, oligodendrocytes at and near the lesion epicenter of the young rats appeared unexpectedly resilient to the insult. No evidence of oligodendrocyte loss in spared white matter was detected at 24 h after injury, as compared to the 50% loss reported in adults. Rather, there was a significant increase in the density of oligodendrocytes by 5 days after injury that was associated with a dramatic upregulation of markers for glial progenitor cells after pup SCI. Our results suggest that an altered glial response near the injury epicenter as compared to that in adults is likely to contribute to the more rapid rate of recovery in hindlimb locomotor function in young rats after SCI.     

Characterization of tests of functional recovery after median and ulnar nerve injury and repair in the rat forelimb

The majority of human peripheral nerve injuries occur in the upper limb but the majority of studies in the rat are performed in the hindlimb. The upper and lower limbs differ in dexterity and control by supraspinal systems, so an upper limb model is a better representation of the common form of human injury. The purpose of this study was to further develop a rat model involving lesions of the median and ulnar nerves. To produce different degrees of misdirection of axons following nerve repair, we studied nerve crush, cut and repair of the two nerves, and cut and repair with crossover. Assessment of functional recovery was performed using a battery of motor and sensory tests: the staircase test, which assesses skilled forepaw reaching; grip strength meter, which assesses grip strength; pawprint analysis, which assesses toe spread and print length; horizontal ladder, which assesses forepaw placement during skilled locomotion; modified Randall-Selitto device and electronic von Frey probes, which assess fine touch; and cold probes, which assess temperature sensation. All tests revealed deficits in forepaw function after nerve injury except the print length and modified Randall-Selitto device. The time course of functional recovery was observed over 15 weeks. The final degree of functional recovery achieved was related to the misdirection of axon regeneration. The tests that most clearly revealed the effects of axon misdirection on function were the skilled paw reaching and grip strength tests. The lesion model and functional tests that we have developed will be useful in testing therapeutic strategies for treating the consequences of inaccurate axon regeneration following peripheral nerve injury in humans.