人胚胎脑提取液的运动神经营养活性研究

目的 从人胚胎脑中提取对运动神经元具有生物活性的神经营养因子。方法 用人胚胎脑组织制备提取液,以成年Wistar大鼠脑提取液及生理盐水为对照,在切断坐骨神经的乳鼠动物模型上,检测运动神经营养活性。结果 在损伤侧局部注射提取液,术后30天损伤侧腰髓运动神经元存活率;第2月人胚脑提取液组为81.4％;第4～5月胎儿脑提取液组为80.7％;成年大鼠脑提取液为50.7％;生理盐水空白对照组为25％。结论 人胚胎脑提取液与两对照组相比具有明显的运动神经营养活性。     

胎鼠肢芽提取液生物活性研究

目的：从胎鼠肢芽中提取运动神经元具有生物活性的神经营养因子，方法：用Ｗｉｓｔａｒ大鼠第１５天胎鼠肢芽制备提取液，以成年Ｗｉｓｔａｒ大鼠骨骼肌提取液及生理盐水对对照，在切断坐骨神经的乳鼠动物模型上，检测其运动神经营养活性，用ＳＤＳ－聚丙烯酰胺凝胶电脉对两种提取液组份进行对比。结果：术后３０天损伤侧相应的腰髓运动神经元存活率，注射肢芽提取液组为７９．２％，成体骨骼肌提以液组为４２．１％，生理盐水组为２     

运动神经营养因子给药途径研究

给药途径问题是神经营养因子临床应用所临的许多尚待解决的问题之一，本实验用自制具有显著运动神经营养活性的胚胎肢芽提取法，在乳鼠坐骨神经怕动物模型上，研究对比了损伤远隔部位肌肉注射和损伤局部注射两种给药途径地创伤性运动神经元死亡的保护效果，结果是：损伤侧脊髓前角运动神经元存活率，远隔肌内注射组为４８．６％，损伤局部注射组为７９．２％，生理盐水组为２３．７％，此结果提示：神经营养因子作为药和用于防止创伤     

人胚骨骼肌提取液中运动神经元营养活性物质的研究

<正>神经营养因子(NTFs)在神经系统发育、正常功能维持和损伤修复中起着极其重要的作用.本研究用纯化培养的运动神经元鉴定了人胚骨骼肌提取液的运动神经元营养活性,用分子生物学等方法从人胚骨骼肌提取液中初步分离纯化出运动神经营养成分.研究结果表明:人胚骨骼肌抽取液能维持运动神经元存活约1～2周,促进突起生长,而对照组的运动神经元在3天左右迅速死亡.人胚骨骼肌提取液和脊髓非运动神经元对运动神经元的营养作用有协同性,共同维持运动神经元存活2—3周.通过用鼠胚骨骼肌抽取液对照培养,表明人隔骨骼肌提取液中也含有运动神经元营养物质,其作用受种属限制不明显,最佳效应浓度为100μg/ml.人胚骨骼肌提取液经硫酸铵分级盐析、Sephcry S-200分子筛等蛋白纯化过程.每步纯化所得样品经多     

胎脑提取液对坐骨神经损伤大鼠脊髓运动神经元酶活性的影响

目的：研究大鼠坐骨神经损伤后，胎脑提取液对脊髓运动神经元乙酰胆碱酯酶(AChE)和琥珀酸脱氢酶(SDH)活性的影响。方法：Wistar大鼠，随机分为实验组、对照组和正常组，前两组再随机分成术后3个时间组。无菌条件下制作坐骨神经钳夹损伤模型，酶组织化学方法结合显微图像分析，观察各组大鼠脊髓运动神经元AChE和SDH活性的变化。结果：术后1w实验组及对照组均比正常组明显降低；术后2w实验组开始升高，对照组进一步降低；术后3w实验组与正常组相近，对照组开始升高。结论：胎脑提取液对脊髓前角运动神经元的溃变有保护作用，可促进大鼠受损神经元的恢复。     

限定骨骼肌提取液（ME,10—50kDa）影响培养的大鼠脊髓腰段运动神经元生长的

用胎鼠的限定骨骼肌提取液（ＭＥ，１０￣１５ｋＤａ）作用于无血清培养的胚胎大鼠脊髓腰段腹角细胞后，用四唑盐（ＭＴＴ）微量自动比色定量法和ＬＤＨ活性测定检测了骨骼肌提取液对大鼠脊髓运动神经元生长存活的生物活性，结果显示：细胞培养４ｄｉｖ后，ＭＴＴ微量比色的ＯＤ值随加入骨骼肌提取液浓度的增加而升高，含骨骼肌提取液２００和４００ｕｇ／ｍｌ蛋白的实验孔与单纯无血清的对照孔比较有极显著差异（Ｐ〈０．００１），     

限定骨骼肌提取液（ME，10～50kDa）影响培养的大鼠脊髓腰段运动神经元生长的定量研究

用胎鼠的限定骨骼肌提取液（ＭＥ，１０～５０ｋＤａ）作用于无血清培养的胚胎大鼠脊髓腰段腹角细胞后，用四唑盐（ＭＴＴ）微量自动比色定量法和ＬＤＨ活性测定法检测了骨骼肌提取液对大鼠脊髓运动神经元生长存活的生物活性，结果显示：细胞培养４ｄｉｖ后，ＭＴＴ微量比色的ＯＤ值随加入骨骼肌提取液浓度的增加而升高，含骨骼肌提取液２００和４００μｇ／ｍｌ蛋白的实验孔与单纯无血清的对照孔比较有极显著性差异（Ｐ＜０．００１），说明２００μｇ／ｍｌ以上浓度的骨骼肌提取液对脊髓腹角神经元的生长有明显的促进作用。细胞培养１４ｄｉｖ后，ＬＤＨ活性测定ＯＤ值的线性回归斜率亦随加入骨骼肌提取液浓度的增加而升高，说明存活细胞数的增加。实验说明培养神经细胞的ＭＴＴ和ＬＤＨ的检测方法有可能成为评估运动神经营养因子活性的指标。     

雪旺细胞和层粘蛋白对脊髓前角运动神经元损伤的保护作用

选３０只成年Ｗｉｓｔａｒ大鼠，坐骨神经切断后，分别应用体外培养的雪旺细胞，层粘蛋白和生理盐水于神经侧断端，４周后，观察损伤侧腰４、５节段脊髓前角运动神经元的存活率，神经元酸性磷酸酶和胆碱脂酶活性变化。结果：生理盐水组脊髓前角运动神经元存活率为５９％，酸性磷酸酶活性明显增强，胆碱脂酶活性明显降低；雪旺细胞组和层粘蛋白组脊髓前角运动神经元存活率分别为８２．３％和８１．１％，酸性磷酸酶和胆碱脂酶活性较对     

GDNF及dvHSV-GDNF对坐骨神经损伤大鼠脊髓前角运动神经元的影响

为了探讨胶质细胞源性神经营养因子及单纯疱疹病毒载体介导的胶质细胞源性神经营养因子 (dv HSV-GDNF)对坐骨神经损伤大鼠脊髓前角运动神经元的作用 ,本实验对成年大鼠造成双侧坐骨神经损伤后 ,于右侧损伤处分别施加胶质细胞源性神经营养因子和 dv HSV-GDNF;左侧损伤处施加生理盐水作为对照。分别取损伤后 4、7、14和 2 8d大鼠的脊髓 L4 ～ L6 节段 ,经石蜡包埋切片后行 Nissl染色 ,计数前角运动神经元数量并进行统计学分析。结果发现 :坐骨神经损伤后 4、7、14和 2 8d,右侧脊髓前角运动神经元的数量明显高于左侧。提示 :胶质细胞源性神经营养因子和 dv HSV-GDNF可减少坐骨神经损伤大鼠脊髓前角运动神经元的死亡     

失神经支配肌肉提取液对创伤性运动神经元胞体死亡的保护作用

周围神经损伤后的功能恢复常不尽如人意，原因之一是周围神经损伤会导致一定数量的神经元胞体死亡。对于感觉神经元胞体的退变死亡，于损伤局部连续施用神经生长因子即可取得较好的保护效应，但对于运动神经元胞体的损伤性死亡，目前尚无一种有效的保护因子。作者从失神经支配肌肉能诱导其邻近的正常运动神经纤维发芽生长这一现象受到启发，研究了失神经支配肌肉提取液（ＤＭＥ）对周围神经损伤所导致的运动神经无胞体死亡的保护作用。结果表明，用失去神经支配后５ｄ的骨骼肌制成的提取液最具运动神经元营养活性。经乳鼠坐骨神经损伤实验模型检测，这种自制的ＤＭＥ能保护８７．６％的脊髓腰段前角运动神经元在坐骨神经损伤后一个月继续存活，而正常骨骼肌提取液的保护率仅为３７．３％。如不加以保护，则神经元胞体的存活率仅为８％。本研究结果提示骨骼肌的失神经支配可能会引起肌肉的某些代谢的改变，从而合成和分泌一些靶源性运动神经营养因子。     

Effects of growth hormone on skeletal muscle. II. Studies on regeneration and denervation in adult rats

The effects of human recombinant growth hormone (rhGH) on regenerating skeletal muscle after ischaemic necrosis and on denervated skeletal muscle were studied in normal adult rats. One group of rats was treated with 4 IE rhGH daily by subcutaneous injections, while control rats were injected with saline. The treatment with rhGH resulted in increased levels of insulin-like growth factor-I (IGF-I) in serum. Ischaemic necrosis was achieved in the extensor digitorum longus (EDL) muscle by cutting the supplying vessels and nerve fascicles at the entrance into the muscle. The wet weight and DNA: protein ration in the regenerating muscle were determined 2 and 4 weeks after the operation. The weight of the regenerating muscles in the rats treated with rhGH during the period of study was larger than in the control rats, while the DNA:protein ratio did not differ significantly between the groups. Denervation of the EDL and soleus muscles followed by subsequent reinnervation was obtained by freezing the sciatic nerve with a forceps chilled in liquid nitrogen. Rats treated with rhGH during the period of denervation and reinnervation, i.e. during the 4 weeks after the freezing of the sciatic nerve, revealed increased weight of both the reinnervated and normal muscles compared to corresponding muscles of control rats. Denervation of the EDL and soleus muscles without subsequent reinnervation was achieved by cutting the sciatic nerve at the level of the thigh. Four weeks after denervation the muscles showed atrophy, mainly affecting type 2 fibres in the EDL muscle and both type 1 and type 2 fibres in the soleus muscle.     

The immunophilin ligand FK506, but not the P38 kinase inhibitor SB203580, improves function of adult rat muscle reinnervated from transplants of embryonic neurons

Injury to the adult CNS often involves death of motoneurons, resulting in the paralysis and progressive atrophy of muscle. There is no effective therapy to replace motoneurons in the CNS. Our strategy to replace neurons and to rescue denervated muscles is to transplant dissociated embryonic day 14-15 (E14-15) ventral spinal cord cells into the distal stump of a peripheral nerve near the denervated muscles. Here, we test whether long-term delivery of two pharmacological inhibitors to denervated muscle, FK506 or SB203580, enhances reinnervation of muscle from embryonic cells transplanted in the tibial nerve of adult Fischer rats. FK506, SB203580 (2.5 mg/kg) or saline was delivered under the fascia of the medial gastrocnemius muscle for 4 weeks, beginning when muscles were denervated by section of the sciatic nerve. After 1 week of nerve degeneration, one million E14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of each rat in the three treatment groups. Ten weeks later, all cell transplants had neuron-specific nuclear protein (NeuN) positive neurons. Neuron survival and axon regeneration were similar across treatments. An average (+/-S.E.) of 210+/-66, 100+/-36 and 176+/-58 myelinated axons grew distally from the cell transplants of rats with muscles treated with FK506, SB203580 or saline, respectively. Regenerating axons in muscles of all three treatments groups were detected with antibodies against phosphorylated neurofilaments and synaptophysin, and motor end plates were labeled with alpha-bungarotoxin. Muscles of rats that received transplants of media only had no axon growth, indicating that the muscles were denervated. The mean muscle fiber areas of rats that received cell transplants and had long-term delivery of FK506, SB203580 or saline to muscles were significantly larger than those of denervated muscle fibers. Thus, cell transplantation reduced muscle atrophy. Transplantation of embryonic cells also resulted in functional muscle reinnervation. Electromyographic activity and force were evoked from >90% of the muscles of rats with cell transplants, but not from denervated muscles. FK506-treated muscles were significantly more fatigue resistant than naive control muscles. FK506-treated muscles also had significantly stronger motor units than those in SB203580 or saline-treated muscles. These data suggest that a pathway regulated by FK506 improves the function of muscles reinnervated by embryonic neurons placed in peripheral nerve.     

Nerve injury in adult rats causes abnormalities in the motoneuron dendritic field that differ from those seen following neonatal nerve injury

Disruption of neuromuscular contact by nerve-crush during the early postnatal period causes increased activity and abnormal reflex responses in affected motoneurons, but such changes are not found after nerve-crush in adult animals. We found previously that neonatally lesioned cells develop an abnormal dendritic field, which may explain the functional changes. Here we have studied the dendritic morphology of the same motoneuron pool after nerve-crush at maturity in order to correlate the observed alterations in morphology with physiological findings. One to two months after sciatic nerve-crush in adult animals, motoneurons supplying the extensor hallucis longus muscles of the rat were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase. The dendritic tree of labelled cells was then analysed. Following adult nerve-crush, the dendritic tree of the motoneurons was smaller but did not display the localised increase in dendritic density seen after neonatal nerve-crush. These findings support the view that such specific morphological changes contribute to the physiological abnormalities seen only after neonatal nerve injury.     

The effect of peripheral nerve injury on disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Around 20% of familial cases of amyotrophic lateral sclerosis have been shown to carry mutations in Cu/Zn superoxide dismutase 1 (Cu/Zn SOD1). Transgenic mice over-expressing human mutant SOD1 genes have been developed and in this study we examined the effect of nerve injury on disease progression in these mice. Firstly, disease progression in uninjured mice was characterised using physiological methods. Muscle force, contractile characteristics and motor unit survival was established at 90 days, an early symptomatic stage and also at the end-stage of the disease, at 130 days. In addition, muscle histochemistry was examined and the extent of motoneuron survival established morphologically. By 90 days of age, there is a significant reduction in muscle force, and nearly 40% of motoneurons within the sciatic motor pool have already died. By 130 days, the muscles are significantly weaker, and there is a dramatic change in the phenotype of extensor digitorum longus (EDL), which changes from a fast fatigable muscle, to a fatigue resistant muscle with a high oxidative capacity. By this stage of the disease, only 40% of motor units in EDL survive, with only 29% of motoneurons surviving within the sciatic motor pool. Following injury to the sciatic nerve in SOD1^(G93A) mice, there is an acceleration in disease progression so that 90 day old mice show deficits that are only seen at the end stage in uninjured SOD1^(G93A) mice. It is therefore possible that mutant SOD1 toxicity increases the vulnerability of motoneurons and muscles to stressful stimuli such as nerve injury.     

Morphological changes in the sciatic nerve,skeletal muscle,heart and brain of rabbits receiving continuous sciatic nerve block with 0.2% ropivacaine

Objective: To investigate the morphological changes in various tissues of rabbits receiving sciatic nerve block with 0.2% ropivacaine for 48 h. Methods: Twenty healthy were randomly assigned to normal saline group (N group) and ropivacaine group (R group). The right sciatic nerve was exposed, and a nerve-blocking trocar cannula embedded. Animals received an injection of 0.5% ropivacaine hydrochloride at a dose of 0.75 ml/kg. Rabbit was then connected to an infusion pump containing 50 ml of normal saline in N group, or to a infusion pump containing 0.2% ropivacaine hydrochloride in R group at 0.25 ml/kg•h-1. Results: In both R group and N group, a small number of nerve cells exhibited pyknotic degeneration. More nerve cells with pyknotic degeneration were found in R group than in N group (P<0.001). At 48 h after surgery, there was a significant correlation between the abnormality of right hind limb and the degree of edema in sciatic nerve (P<0.01). Conclusion: Pyknotic degeneration of sciatic nerve increased after an infusion of 0.2% ropivacaine hydrochloride for 48 h, suggesting the neurotoxicity of ropivacaine. An infusion of 0.2% ropivacaine hydrochloride for 48 h may cause necrosis of skeletal muscle cells. The sciatic nerve edema would greatly affect the hindlimb motor while both pyknotic degeneration of sciatic nerve and skeletal muscle have little influence on the hindlimb movement. After an infusion of 0.2% ropivacaine hydrochloride for 48 h, the morphology of right atrium and brain tissues around the ventriculus tertius and medulla oblongata remained unchanged.     

骨骼肌提取液对大鼠坐骨神经损伤所致腰段脊髓腹角和背根节神经细胞溃变的影响

用ＣＴ－ＨＲＰ逆行追踪法及ＣｈＡＴ单克隆抗体的ＡＢＣ免疫组织化学技术研究了新生大鼠骨骼肌提取液（ＭＥ，２０～５０ｋＤ）对钳夹大鼠坐骨神经所致的腰骶部脊髓腹角和背根节神经细胞溃变的影响。实验结果显示：注射ＭＥ实验组的脊髓腹角运动神经元和背根节细胞的存活均数与注射盐水的对照组相比，其比值为６：０，有显著性差异（Ｐ＜０．０１）。提示骨骼肌提取液（２０～５０ｋＤ）对脊髓腹角运动神经元和背根节感觉神经细胞的演变有明显的保护作用。     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L_3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。