Effects of local release of hepatocyte growth factor on peripheral nerve regeneration in acellular nerve grafts

Options for reconstructing peripheral nerve gaps after trauma are limited. The acellular nerve is a new kind of biomaterial used to reconstruct the peripheral nerve defect, but its use could be improved upon. We aimed to investigate the effect of adenoviral transfection with hepatocyte growth factor (HGF) on the functional recovery of transected sciatic nerves repaired by acellular nerve grafting. 30 Rats were divided into three groups (10/group) for autografting and acellular grafting, as well as acellular grafting with adenovirus transfection of HGF (1 × 10⁸ pfu) injected in muscles around the proximal and distal allograft coapation. Sciatic functional index (SFI) was evaluated every 4 weeks to week 16 by measuring rat footprints on walking-track testing. The three groups presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. Weight of the gastrocnemius and soleus muscles, histologic and morphometric study and neovascularization in the nerve grafts were evaluated at week 16. Autografting gave the best functional recovery, but HGF-treated acellular grafting gave better recovery than acellular grafting alone. Neovascularization was greater with HGF-treated acellular grafting than with autografting and acellular grafting alone. Axonal regeneration distance of autografting on the 20th postoperative day was the longest in the three groups,while that of acellular grafting alone was the smallest. Acellular nerve grafting may be useful for functional peripheral nerve regeneration, and with human HGF gene transfection may improve on acellular grafting alone in functional recovery.     

Postnatal development of conduction velocity and fibre size in the rat tibial nerve

The maximum conduction velocity (CV) and fibre diameters (D) were determined in the tibial nerve of developing rats. In 1-day-old rats CV of the fastest motor and sensory fibres (assessed separately) was 1.4 m/sec on the average and increased to 35 m/sec by postnatal day 30. The maximum conduction rate in adult rats ranged from 60 to 84 m/sec. Diameters of at least 100 nerve fibres in each age group were measured in electronmicrographs. The calibre of myelinating fibres in 1-day-old rats was 0.5–1.5 μm. By day 90 after birth the range of myelinated fibre size extended to 1.5–12.5 μm. The factor relating conduction rate and total fibre diameter of the largest fibres (i.e. the value of ) was found to vary with age, increasing from 1.1 to 6.2 between postnatal days 1 and 90. These results indicate that functional and morphological properties of peripheral nerve fibres in the rat undergo considerable changes during postnatal ontogeny until they reach adult values.     

Embryonal and postnatal development of mast cells in rat reripheral nerve

Summary A study was made on the embryonal and the postnatal development of mast cells in peripheral nerves of the rat. Mast cells containing a few metachromatic granules were first seen in the epineurium of the sciatic nerve in 19 day old fetuses. From the 20th day of pregnancy mast cells were also present in the endoneurium. The number of mast cells in the nerves increased considerably during the first postnatal weeks. No mast cells were seen in the spinal nerve roots or in the dorsal root ganglia. Radioautography showed that a high proportion of endoneural mast cells of 3 day old rat sciatic nerves incorporate³H-thymidine whereas only a few mast cells in 15 and 21 day old rat were labelled.It is known that the perineurium acts as a diffusion barrier to Compound 48/80 in adult rats. Therefore, this histamine liberator applied to the surface of adult rat sciatic nerves caused degranulation of epineural and perineural mast cells but not of endoneural mast cells. However, when the compound was applied to the surface of sciatic nerves in newborn rats also the endoneural mast cells became degranulated. Consequently, the function of the perineurium to act as a diffusion barrier to this compound is not fully developed in immature animals.

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Zusammenfassung Die embryonale und postantale Entwicklung der Mastzellen im peripheren Nerven der Ratten wurde untersucht. Mastzellen mit wenigen metachromatischen Granula wurden im Epineurium des N. ischiadicus frühestens bei 19 Tage alten Feten gesehen. Ab dem 20. Gestationstag waren Mastzellen auch in Endoneurium vorhanden. Die Zahl der Mastzellen in den Nerven nahm während der ersten postnatalen Wochen erheblich zu. In den spinalen Nervenwurzeln und Spinalganglien waren keine Mastzellen nachweisbar. Autoradiographische Untersuchungen ergaben, daß ein großer Anteil der endoneuralen Mastzellen im N. ischiadicus von 3 Tage alten Ratten³H-Thymidin aufnahm, während bei 15 und 21 Tage alten Tieren nur wenige Mastzellen markiert waren.Es ist bekannt, daß das Perineurium erwachsener Ratten als Diffusionsbarriere für die Substanz 48/80 wirkt. Deshalb verursachte die Aufbringung dieser Histamin-freisetzenden Substanz auf die Oberfläche des N. ischiadicus erwachsener Ratten eine Degranulation der epi- und perineuralen Mastzellen, nicht aber der endoneuralen Mastzellen. Hingegen wurden nach Aufbringung der Substanz auf die Oberfläche des N. ischiadieus neugeborener Ratten auch die endoneuralen Mastzellen degranuliert. Das weist darauf hin, daß die Barrierefunktion des Perineuriums bei unreifen Tieren noch nicht voll entwickelt ist.

     

Regeneration of a transected peripheral nerve

Summary The regeneration of transected peripheral nerves of mice was studied using autoradiographical and electron microscopical techniques. In general, maximal proliferation occurred between the 5th and 7th day after dissection and stopped when the cells emigrating from the proximal and distal stumps of the nerve started to contact one another. Special attention was paid to the reaction of the connective tissue cells of the endo-, epi- and peri-neurium. The perineurial cells seemed to dedifferentiate between the 3rd and 5th day after the transection and then started to proliferate into the defect. Labelled perineurial cells were completely absent, when the minifascicles were fully developed in the neuroma. The epineurial fibroblasts started to proliferate during the 1st day. Even 6 weeks after transection the multiplication rate was about ten fold that of the controls. The results are discussed with special reference to clinical nerve repair.     

Ultrastructure of peripheral nerve in Cockayne's syndrome

Summary The ultrastructure of sural nerve biopsies was studied in two sisters with Cockayne's syndrome. Both had severe physical and mental retardation and evidence of peripheral neuropathy. Striking alterations in the myelin sheath with relative preservation of the axis cylinder were noted in both. There were also electron dense bodies in the Schwann cells. These findings support the suggestion that Cockayne's syndrome may be a form of leukodystrophy.     

Retrograde transneuronal transfer of the C-fragment of tetanus toxin

The transport of the C-fragment of tetanus toxin after intramuscular injection was studied with immunohistochemical techniques. Brainstems and spinal cords of mice were examined after injections of C-fragment into the tongue or forearm. Labelled motorneurons were observed within 6 h. By two days after injection, the bulk of detectable C-fragment had passed out of motorneurons into the surrounding neuropil. C-fragment, like native tetanus toxin, appears to be transferred from motorneurons to presynaptic structures to an extent not observed with other proteins. It is useful in the study of retrograde transneuronal transfer since it lacks the toxicity of tetanus toxin.     

皮肤神经活体组织检查在周围神经病诊断中的应用

目的 探讨皮肤神经活体组织检查在周围神经病诊断中的作用,建立正常参考值范围,并比较临床表现、神经电生理检查与表皮神经纤维病变的一致性.方法 对51例有周围神经病症状和(或)体征的患者进行皮肤神经活体组织检查,计算表皮神经纤维密度(IENFD);同时收集10名健康志愿者作为对照.51例患者中,41例行常规肌电图及神经传导速度(NCV)检查.21例行皮肤交感反射(SSR),比较IENFD与NCV及SSR的一致性.结果 对照组与病例组相比,大腿IENFD(根/mm)分别为21.4±2.7及15.0±6.3(t=2.976,P=0.004);小腿IENFD分别为15.4±2.2及8.1±5.9(t=3.191,P=0.002).病例组与对照组相比大、小腿IENFD均有减少,差异有统计学意义.51例患者中,皮肤神经活体组织检查异常48例(94.1%),其中33例表现为长度依赖性周围神经病变;41例行常规肌电图检查,21例异常(51.2%);21例行SSR检查,异常17例(81.0%).仅表现为小纤维病变症状和(或)体征的29例患者中,27例(93.1%)皮肤神经活体组织检查异常;其中20例行NCV,异常6例(30.0%);14例行SSR,11例异常.结论 皮肤神经活体组织检查操作简单安全,对于以小神经纤维受累为主的周围神经病皮肤神经活体组织检查有较高的灵敏度.     

Effects of tetanus toxin on functional inhibition after injection in separate cortical areas in rat

Tetanus Toxin is widely used as a model of chronic focal epilepsy and is assumed to act by blocking neurotransmitter release with high selectivity for inhibitory synapses. However, the exact mechanisms are not fully understood, since, e.g., GABA release is only temporarily decreased although epileptiform activity persists pointing towards a change in the interplay of excitation and inhibition. Furthermore there have been reports about different effects of tetanus toxin after injection in separate brain areas. Therefore, we investigated the functional inhibition after injecting tetanus toxin either in the motor or sensory cortex of adult rats by using a paired-pulse paradigm as a measure of excitatory and inhibitory drive. Tetanus toxin injection into the motor cortex (n=10) induced a marked, long-lasting reduction in inhibition which was highly significant in most parts of the injected cortical area. Injections into the sensory cortex, however, showed less marked changes in inhibition which were more widespread and significant only in 3 of 14 animals injected. These results give further evidence for a prominent effect of tetanus toxin on functional inhibition and strengthen the idea of a differential effect in separate cortical areas. They may be accounted for by the different cytoarchitecture of cortical areas with variable inhibitory and excitatory intracortical connections.     

The effect of extraction of the intrafascicular contents of peripheral nerve trunks on perineurial structure

Summary The intrafascicular contents have been extracted from the tibial nerve of the rabbit through perineurial incisions. Within 6–8 days following this procedure, the perineurial cells separate from one another, become dissociated from their basement membranes and assume a fibroblast-like appearance. The intrafascicular space becomes populated with endoneurial fibroblasts. With the ingrowth of regenerating axons, bundles of axons and associated Schwann cells become surrounded by cells of fibroblastic appearance which undergo perineurial transformation resulting in the development of multiple small fascicles. The cells of the surrounding perineurium appear to reassume a lamellar organization and to reestablish contacts with each other with the formation of junctional complexes. It is therefore suggested that neural structures may be responsible for the development and maintenance of the structural organization of the perineurium.     

The effect of vincristine on nerve regeneration in the rat. An electrophysiological study

Weekly injections of vincristine to produce a dose-dependent delay in regeneration following sciatic nerve crush. With 20 micrograms/kg/wk recovery was similar to that in control animals. With 50 and 100 micrograms/kg/wk electrophysiological evidence of reinnervation of the foot muscles was significantly delayed and muscle action potential amplitude increased at a slower rate. However, once begun the increase in motor nerve conduction velocity was closer to that in control animals. With 200 micrograms/kg/wk no evidence of reinnervation of the foot muscles was found even after 6 months. These doses produced no abnormality of muscle action potential amplitude or of nerve conduction velocity on the opposite non-crushed side.     

Characterization of the tetanus toxin model of refractory focal neocortical epilepsy in the rat

PURPOSE: To characterize in detail a model of focal neocortical epilepsy. METHODS: Chronic focal epilepsy was induced by injecting 25-50 ng of tetanus toxin or vehicle alone (controls) into the motor neocortex of rats. EEG activity was recorded from electrodes implanted at the injection site, along with facial muscle electromyographic (EMG) activity and behavioral monitoring intermittently for up to 5 months in some animals. Drug responsiveness was assessed by using the antiepileptic drugs (AEDs) diazepam (DZP) and phenytoin (PHT) delivered systemically, while 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), a competitive antagonist at AMPA receptors, was administered directly to the brain to investigate the potential benefits of focal drug delivery. RESULTS: Tetanus toxin induced mild behavioral seizures that persisted indefinitely in all animals. EEG spiking activity, occurring up to 80% of the time, correlated with clinical seizures consisting of interrupted behavioral activity, rhythmic bilateral facial twitching, and periods of abrupt motor arrest. Seizures were refractory to systemic administration of DZP and PHT. However, focal delivery of NBQX to the seizure site reversibly reduced EEG and behavioral seizure activity without detectable side effects. CONCLUSIONS: This study provides a long-term detailed characterisation of the tetanus toxin model. Spontaneous, almost continuous, well-tolerated seizures occur and persist, resembling those seen in neocortical epilepsy, including cortical myoclonus and epilepsia partialis continua. The seizures appear to be similarly resistant to conventional AEDs. The consistency, frequency, and clinical similarity of the seizures to refractory epilepsy in humans make this an ideal model for investigation of both mechanisms of seizure activity and new therapeutic approaches.     

Substrate-bound nerve growth factor promotes neurite growth in peripheral nerve

Nerve growth factor (NGF), in addition to its well-known effects as a soluble neurite growth-promoting factor, also appears to promote the elongation of neurites when it is adsorbed to tissue culture substrates. Peripheral nerve Schwann cells appear to possess a receptor for NGF on their surfaces which is induced substantially after axotomy. We have found that the adsorption of NGF onto cryostat sections of the distal stump of previously severed sciatic nerve enhances neurite growth over this tissue. This finding, coupled with the two previous observations, suggests that Schwann cell surface NGF receptors serve to bind to NGF-like growth factors so as to provide favorable surfaces for regenerating peripheral nerve axons.     

损伤神经周围注射自体PRP对周围神经损伤的修复效果观察

目的 探讨损伤神经周围注射自体富血小板血浆(platelet-rich plasma,PRP)对周围神经损伤的修复效果。方法 选取2017年1月-2018年7月于本院接受治疗的周围神经损伤患者130例,随机分为手术组、PRP联合组各65例,手术组患者进行显微外科手术,PRP联合组患者在手术基础上注射自体PRP进行治疗; 用酶联免疫吸附实验法检测血清C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)水平,用Western blot法检测胰岛素样生长因子1(IGF-1)、血管内皮生长因子(VEGF)表达水平,并对2组患者神经传导速度、下肢运动功能、基本功能优良率以及肌电图疗效进行检测。结果 治疗1、3、6、12个月后PRP联合组患者CRP、TNF-α水平均低于手术组,IGF-1、VEGF表达水平均高于手术组(P<0.05); 治疗1、3、6、12个月后PRP联合组患者感觉、运动神经传导速度、下肢运动功能评分、基本功能恢复优良率、肌电图愈显率均高于手术组(P<0.05)。结论 在手术治疗的基础上注射自体PRP对周围神经损伤患者进行治疗,能够减轻患者炎性损伤程度,上调IGF-1、VEGF的表达水平,加快神经传导速度,促进患者下肢运动功能恢复     

神经传导速度检测诊断酒精性周围神经病的价值

目的探讨神经传导速度(NCV)对慢性酒精中毒性周围神经病(CAPN)的诊断价值。方法采用肌电图检测52例CAPN患者的正中神经、尺神经、腓神经和胫神经的NCV,并与26例健康者进行对照比较。结果CAPN患者的NCV异常率为73.12%,明显高于对照组;下肢NCV异常率(80.77%)高于上肢异常率(76.47%);感觉神经传导速度(SCV)异常率(82.73%)高于运动神经传导速度(MCV)异常率(75.26%)。结论NCV检测可作为酒精中毒性周围神经病的方法之一。     

含神经生长因子的壳聚糖导管桥接周围神经缺损的实验研究

目的 应用含有神经生长因子(NGF)的壳聚糖神经导引管作为神经再生室桥接大鼠坐骨神经缺损，观察其对神经再生的作用。方法 选用Wistat大鼠60只，手术造成右后肢坐骨神经长约15mm的缺损，A组以含有NGF的壳聚糖神经导引管桥接神经缺损，B组则单纯采用壳聚糖导管，分别于术后4、12、24周进行大体及显微解剖观察、组织学检查、电镜观察和神经电生理测定。结果 A组在促进神经再生、加快血管化进程、再生神经纤维排列规律化、提高再生神经髓鞘化、加速再生神经功能重建等方面均优于B组。结论 壳聚糖神经导引管可以为大鼠坐骨神经再生提供一个良好的再生微环境，NGF对神经再生有显促进作用。     

Afterdischarge and interactions among fibers in damaged peripheral nerve in the rat

Sensory fibers trapped in nerve-end neuromas become abnormally excitable, and produce an ectopic discharge which is believed to contribute to paresthesias and pain associated with chronic nerve injury in man. Here we report that stimulation of injured nerves can alter this discharge, directly by antidromic invasion of active neuroma fibers, and indirectly through interactions with neighboring fibers. Antidromic stimulation of spontaneously active fibers in experimental neuromas in the rat sciatic nerve, using single electrical stimulus pulses, produced time-locking of rhythmic spontaneous firing and of spontaneous impulse bursts. Some initially silent fibers generated a burst of rhythmic afterdischarge when stimulated in this way. Stimulation delivered in brief trains (tetani) produced more prolonged alterations in spontaneous neuroma discharge, including excitation, suppression and combinations of the two. In some cases initially silent fibers were activated for extended periods. These responses to tetanic stimulation occurred even when the active fibers were not themselves stimulated, and reflect a novel form of fiber-fiber interaction in neuromas that we term 'crossed afterdischarge'. This interaction probably results from the accumulation of potassium ions within the extracellular compartment adjacent to active neuroma fibers during activation of their neighbors. It differs fundamentally from the high safety factor ephaptic cross-talk seen in acutely cut nerves and in neuromas of 30 or more days standing.     

Pathology of local anesthetic-induced nerve injury

Summary Nerve fiber injury and endoneurial edema were induced by the injection of the local anesthetic 2-chloroprocaine, tetracaine, procaine, etidocaine or mepivacaine into the soft tissue and fascia surrounding the sciatic nerve of Sprague-Dawley rats. Light microscopy demonstrated that the perineurial barrier was not mechanically damaged by the surgical procedure but, at 48 h post-injection, perineurial permeability was increased. Previous observations of leakage of horseradish peroxidase and the present report of neutrophils and eosinophils in the endoneurium indicate a disruption of blood-nerve barrier systems. Endoneurial edema was observed in the subperineurial, interstitial and perivascular regions. Axonal degeneration and demyelination occurred; the latter associated with accumulation of large lipid droplets in Schwann cells. Degranulation of mast cells, proliferation of fibroblasts and macrophage activity were noteworthy in affected areas. The findings are remarkable in that this is the first model of endoneurial edema by a neurotoxin which penetrates the perineurium, disrupting barrier system and inducing nerve fiber injury.Supported in part by USPHS NS18715, NS14162 and NS07078 and the Veteran's Administration Research Service     

Chronic nerve compression induces local demyelination and remyelination in a rat model of carpal tunnel syndrome

In a previous study, we demonstrated that chronic compression of rat sciatic nerve, a model of compressive neuropathies, triggered dramatic Schwann cell proliferation and concurrent apoptosis. Importantly, this Schwann cell response occurred before there are signs of overt axonal pathology, raising the question of whether there are alterations in axonal myelination in the areas of the nerve in which Schwann cell apoptosis and proliferation occur. Here, we use nerve teasing techniques and unbiased stereology to assess myelination in nerves after 1 and 8 months of compression. Evaluations of myelin thickness and axonal diameter (AD) using design-based, unbiased stereology revealed alterations in myelin structure that indicate remyelination, specifically a dramatic decrease in the average internodal length (IL) and an increase in the proportion of axons with thin myelin sheaths. The mean IL was reduced after 1 month of chronic nerve injury with no further decrease in IL at 8 months. There was limited change in average axonal diameter at both 1 and 8 months. Measures of myelin thickness revealed not only a greater than 6-fold increase in the number of axons with very thin (<5 microm thickness) myelin sheaths, but also a proportional decrease in the number of axons with the thick myelin sheaths characteristic of normal nerve. These results confirm that an early consequence of chronic nerve compression (CNC) is local demyelination and remyelination, which may be the primary cause of alterations in nerve function during the early period post-compression.     

神经生长因子治疗周围神经损伤的疗效观察

目的 观察神经生长因子治疗周围神经损伤的临床疗效.方法 纳入60例周围神经损伤患者,采用随机数字表法分为2组.试验组30例采用鼠NGF肌内注射治疗,对照组30例采用维生素B12治疗,治疗4周后观察疗效.观察指标包括疼痛(VAS)、麻木等临床症状和体征,同时观察单神经的神经电生理情况.结果 疼痛改善:试验组总有效率93.33％,对照组为53.33％;麻木改善:试验组总有效率86.67％,对照组为66.67％;2组比较差异均有统计学意义(P＜0.05).试验组恢复神经的感觉及运动电位的潜伏期时间均明显短于对照组,差异有统计学意义(P＜0.05);而波幅则均显著高于对照组,差异有显著性意义(P＜0.05).结论 神经生长因子能有效改善患者的疼痛、麻木症状,而且能对神经纤维的修复、电生理功能有促进作用.     

Morphological study of peripheral nerve changes induced by chloroquine treatment

Summary Nerve biopsies were performed in four patients with suspected chloroquine induced neuromyopathy. Three of the patients were treated with high doses of chloroquine for connective tissue disease, while one patient was taking this drug as malaria prophylaxis. Morphological studies demonstrated the presence of segmental demyelination and remyelination in all cases. Cytoplasmic inclusions were observed in Schwann cells, in perineurial and endothelial cells, and in some interstitial cells. They were never observed within axons. Occasional curvilinear profiles were seen in perineurial and Schwann cells. Perineurial calcifications were observed in two cases. The results of this morphological study suggest that chloroquine neuropathy is essentially due primary involvement of Schwann cells.