抗BDNF血清对小鼠坐骨神经损伤后脊髓前角运动神经元内GAP-43表达的影响

小鼠坐骨神经压榨损伤后 ,腹腔注射抗 BDNF血清 ,动物存活 2周。用组织原位杂交技术与免疫组织化学方法观察生长相关蛋白 ( GAP-4 3)在脊髓腰骶膨大部前角运动神经元的表达 ,并对实验结果进行图像分析。结果发现 ,注射抗 BDNF血清后坐骨神经损伤侧脊髓前角 GAP-4 3m RNA的阳性神经元与 GAP-4 3免疫反应阳性神经元的数目减少 ,阳性神经元的光密度也降低 ,上述改变在统计学上均有显著意义。结果提示 ,小鼠坐骨神经损伤后内源性 BDNF可能参与脊髓前角运动神经元 GAP-4 3的表达     

牛脊髓前角匀浆皮下注射损伤豚鼠脊髓运动神经元

目的探讨牛脊髓前角匀浆对豚鼠脊髓、前根及坐骨神经的影响。方法采用新鲜牛脊髓前角匀浆免疫豚鼠。观察脊髓、前根、坐骨神经光镜及超微结构的变化。结果豚鼠体重在第4次免疫后明显下降。脊髓前角运动神经元变性和丢失,有卫星、噬节及墓穴现象形成。电镜下最主要的表现是线粒体异常;其次神经元核周质内异常神经丝聚集,形成包涵体;轴突内异常神经丝聚集形成轴索球。前根及坐骨神经的变化,表现为轴索变性及继发的髓鞘改变。结论牛脊髓前角匀浆可以作为抗原引起豚鼠脊髓、前根及坐骨神经免疫所介导的损伤,为进一步研究运动神经元病的发病机制提供了可靠的方法。     

锌对坐骨神经损伤小鼠脊髓运动神经元的保护作用

目的研究锌对坐骨神经损伤(sciatic nerve injury,SNI)模型小鼠脊髓前角运动神经元的保护作用。方法 48只CD1小鼠随机分4组,SNI小鼠模型,假手术组,低锌组(SNI后每天腹腔注射氯碘羟喹10mg/kg·d);高锌组(SNI后每天腹腔注射氯化锌溶液20 mg/kg·d)。应用原子吸收光谱技术、免疫组织化学技术和图像分析技术检测SNI后第7天锌含量变化对模型动物脊髓前角运动神经元caspase-3表达的影响。结果损伤组脊髓的锌含量降低,脊髓前角运动神经元caspase-3表达上调,阳性细胞百分比增大,光密度增加(0.01)。高锌组能增加脊髓的锌含量,下调caspase-3表达,降低阳性细胞百分比和光密度(0.01);而P低锌组则使脊髓的锌含量更少,caspase-3表达更多,阳性细胞百分比和光密度更高(0.01)。结论锌能抑制SNI模型小鼠脊髓前角运动神经元caspase-3的表达,锌对运动神经元具有保护作用。     

小鼠坐骨神经损伤后内源性BDNF对脊髓前角运动神经元内突触素Ⅰ mRNA表达的调节

本研究旨在探讨小鼠坐骨神经损伤后内源性BDNF是否参与调节脊髓前角运动神经元内突触素ImRNA的表达。在小鼠坐骨神经压榨损伤后,腹腔注射BDNF抗体中和内源性BDNF,动物存活1～2周,用组织原位杂交技术观察突触素ImRNA在脊髓腰骶膨大部前角运动神经元内的表达。结果显示:注射BDNF抗体后坐骨神经损伤侧脊髓前角突触素ImRNA阳性运动神经元的数目和平均光密度与实验对照组相比显著下降(P<0.01)。本研究结果提示,小鼠坐骨神经损伤后内源性BDNF可参与脊髓前角运动神经元内突触素ImRNA表达的调节。     

臂丛损伤后脊髓前角运动神经元超微结构改变

目的探讨大鼠臂丛损伤导致脊髓前角运动神经元死亡的机制。方法成年雄性SD大鼠24只,其中对照组6只,损伤组18只。建立3种臂丛损伤模型:右C7前根撕脱(A组);右C7前根撕脱+同侧C5～T1后根离断(B组);右C7前根撕脱+右C5与C6之间脊髓半横断(C组)。术后14d取C7节段脊髓,采用尼氏染色方法和透射电镜技术,观察脊髓前角运动神经元的存活率及其超微结构改变。结果术后2周A组脊髓前角运动神经元的存活率最高,B组居中,C组最低。3个臂丛损伤组C7前角均可见凋亡特征性改变:运动神经元内核染色质聚集靠边,核固缩、碎裂、核膜皱褶并内陷,并有染色质团块形成的凋亡小体。细胞体积缩小,胞浆内细胞器密集,线粒体轻度肿胀,核周粗面内质网减少,游离核糖体增多,胞浆内可见较多的空泡。神经元胞体周围的有髓神经纤维和无髓神经纤维呈轻度肿胀,髓鞘的板层结构消失。结论臂丛损伤诱导脊髓运动神经元死亡途径中存在凋亡和坏死两种机制,运动神经元可形成凋亡小体。     

小鼠脊髓前角运动神经元的分离、培养和鉴定

目的:探讨一种分离小鼠脊髓前角运动神经元的方法,为运动神经元相关的研究提供依据。方法:分离13 d的C57BL/6胎鼠脊髓,利用Optiprep分离液经密度梯度离心获得脊髓前角运动神经元,培养后观察神经元细胞形态的变化,免疫荧光双标法对分离运动神经元的纯度进行判定。结果:将分离到的运动神经元进行培养,能够观察到典型的神经元细胞形态及轴突生长。免疫荧光双标证明培养的细胞为运动神经元细胞,纯度约95%。结论:Optiprep分离液用于小鼠运动神经元的分离,可获得良好的分选效果。Optiprep分离液可用于运动神经元相关的研究。     

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     

运动对小鼠脊髓前角运动神经元树突结构可塑性的影响

HRP—CB标记结合sholl分析方法分析研究5、13、24月龄三个年龄组C57BL/6J小鼠脊髓前角α—运动神经元树突结构的可塑性变化,以及不同时间的长期适量运动(跑转笼)对运动神经元树突结构可塑性变化的可能作用。以同年龄对照组心重/体重比率均值的二倍标准差(X+2SD)做为运动有效标准。结果在三个年龄对照组中,老年鼠(对照组Ⅱ)神经元树突野缩小,树突分支数和总长度明显减少(P<0.05),以远离胞体的分支丢失为主。经过8和19个月运动训练后,脊髓前角运动神经元树突野扩大,分支数和树突总长度明显增加(P<0.01),甚至超过青年对照,以近胞体分支的增生更明显。结果表明,长期的适量运动能够延缓衰老过程中小鼠脊髓前角运动神经元树突的丢失,促进树突的可塑性增生。     

联合应用神经生长因子和神经节苷脂1对坐骨神经损伤大鼠脊髓神经元的保护作用

目的：了解联合应用神经生长因子(NGF)和神经节苷脂1(GMl)对大鼠周围神经损伤后脊髓神经元的保护作用。方法：选用SD大鼠,分为生理盐水(NS)组、NGF组、GM1组和NGF GM1组,将大鼠坐骨神经造成5mm缺损,术中硅胶管内局部加药、术后大鼠损伤侧小腿肌注药物。术后定期光、电镜观察L4～I6脊髓前角神经元结构变化,测定损伤远段和近段神经传导速度。结果：脊髓运动神经元数目以及神经传导速度4周时,NGF组和GM1组均多于或快于NS组,NGF GM1组则多于或快于NS组、NGF组和GM1组,8周时NGF GM1组、NGF组、GM1组组间无显著性差异但均多于或快于NS组。结论：NGF GM1对周围神经损伤后脊髓运动神经元退变的保护作用与单用NGF或GM1相比,能更早期地发挥作用,并且效果优于单用NGF或GM1。     

Regulatory expression of Neurensin-1 in the spinal motor neurons after mouse sciatic nerve injury

Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.     

大鼠坐骨神经损伤后脊髓前角神经元死亡数量的研究

目的：研究周围神经损伤后，脊髓前角运动神经元胞体的死亡数量变化。方法：选择10只正常SD大鼠，先计算两侧的脊髓前角运动神经元胞体数量是否对称；再选择35只SD大鼠，切断并原位吻合其右侧坐骨神经，左侧不作任何处理、作为对照，于术后不同时间取L4～6节段脊髓作HE染色，计算脊髓前角运动神经元胞体数量的变化。结果：正常SD大鼠两侧的脊髓前角运动神经元胞体数量呈对称分布；右侧坐骨神经损伤后，其脊髓前角运动神经元胞体数量较左侧减少。结论：大鼠坐骨神经损伤后，脊髓前角运动神经元的胞体有死亡，其死亡具有一定的时间特征。     

大鼠周围神经移植修复后脊髓前角运动神经元变化的动态观察

目的　观察大鼠移植神经远侧吻合口再通术后脊髓前角细胞形态及功能变化,为长段神经移植后行远侧吻合口再通术提供理论支持。 方法　实验采用SD大鼠120只,随机分为6组,分别为：(1)神经单纯切断组;(2)神经切断缝合组;(3)神经移植修复组;(4)游离神经移植修复后远侧缝合口切除组;(5)游离神经移植修复后远侧缝合口切除再缝合组; (6)假手术组。术后1、2、4、8、12和16周取出脊髓腰膨大,进行组织学观察和免疫组化检测。 结果　实验组术后1周胞浆轻度水肿,线粒体肿胀,空泡化。术后2~4周,神经元超微结构的改变明显加重,术后4~8周进入恢复期。第3组术后1周,BDNF在运动神经元内表达开始增加,2周达到了高峰,8周下降。NGF的表达在术后2周开始上升, 8周达到高峰,之后持续下降。第5组BDNF的表达在术后8~16周期间呈上升趋势,NGF的表达在术后8周一直维持平稳状态,而对照组各时间段无明显变化。 结论　长段神经移植修复术后,适时行远侧吻合口切除重新吻合,既去除了远侧吻合口的瘢痕,同时再次激活神经元功能的状态,有利于功能恢复。     

高压氧前处理脊髓损伤大鼠前角运动神经元的凋亡*☆

背景：脊髓损伤后难以修复,损伤后保护残存的神经元是促进神经再生的关键。 目的：验证高压氧预处理可以通过抑制早期的细胞凋亡来保护脊髓前角运动神经元。 方法：随机将26只雄性Wistar大鼠等分成模型组和实验组。实验组在给予高压氧5 d后与模型组同时制作脊髓T9~10全横断模型。 结果与结论：尼氏染色显示脊髓T9~T10全横断后8 h及1 d,脊髓前角的浓染的细胞多见,与模型组相比,实验组脊髓前角浓染的细胞较少。TUNEL染色也显示脊髓T9~T10全横断后脊髓损伤后8 h~1 d,2组大鼠脊髓前角内均可见大量的凋亡神经元,3 d时凋亡神经元数量减少。相比于模型组,高压氧预处理8 h,1 d后大鼠脊髓前角凋亡神经元较少(P < 0.05,        P < 0.01)。说明高压氧预处理能对脊髓损伤后前角运动神经元起保护作用。       

Altered expression of nectin-like adhesion molecules in the peripheral nerve after sciatic nerve transection

Following axotomy several processes involving cell-cell interaction occur, such as loss of synapses, axon guidance, and remyelination. Two recently discovered families of cell-cell adhesion molecules, nectins and nectin-like molecules (necls) are involved in such processes in vitro and during development, but their roles in nerve injury have been largely unknown until recently. We have previously shown that axotomized motoneurons increase their expression of nectin-1 and nectin-3 and maintain a high expression of necl-1. We here investigate the expression of potential binding partners for motoneuron nectins and necls in the injured peripheral nerve. In situ hybridization (ISH) revealed a decreased signal for necl-1 mRNA in the injured nerve, whereas no signal for necl-2 was detected before or after injury. The signals for necl-4 and necl-5 mRNA both increased in the injured nerve and necl immunoreactivity displayed a close relation to axon and Schwann cell markers. Finally, signal for mRNA encoding necl-5 increased in axotomized spinal motoneurons. We conclude that peripheral axotomy results in altered expression of several necls in motoneurons and Schwann cells, suggesting involvement of the molecules in regeneration.     

Histopathological investigation of neuroprotective effects of Nigella sativa on motor neurons anterior horn spinal cord after sciatic nerve crush in rats

The aim of this study was designed to evaluate the possible protective effects of Nigella sativa (NS) on the neuronal injury in the sciatic nerve of rats. The rats were randomly allotted into one of the three experimental groups: A (control), B (only trauma) and C (trauma and treated with NS); each group contain 10 animals. Sciatic nerve injury was performed by placing an aneurysm clip on the left leg. Rats were neurologically tested over 24 h after trauma. The rats in NS-treated group was given NS (in a dose of 400 mg/kg body weight) once a day orally for 30 days starting just after trauma. Control and untreated (only trauma) rats were injected with the same volume of isotonic NaCl as the treated animals that received NS. Tissue samples were obtained for histopathological investigation. To date, no histopathological changes of neurodegeneration in the sciatic nerve after trauma in rats by NS treatment have been reported. Results showed in the group B (only trauma), the neurons of sciatic nerve tissue became extensively dark and degenerated with picnotic nuclei. Treatment of NS markedly reduced degenerating neurons after trauma and the distorted nerve cells were mainly absent in the NS-treated rats. The morphology of neurons in groups treated with NS was well protected, but not as neurons of the control group. The number of neurons in sciatic nerve tissue of group B (only trauma) was significantly less than both control and treated with NS groups. The morphology of neurons revealed that the number of neurons were significantly less in group B compared to control (P < 0.001) and group C (P < 0.01) rats’ motor neurons anterior horn spinal cord tissue. We conclude that NS therapy causes morphologic improvement on neurodegeneration in sciatic nerve after trauma in rats.     

Interleukin-1 beta promotes sensory nerve regeneration after sciatic nerve injury

Nerve injury brings about axonal disconnection, and thus axonal extension is one of the important steps for nerve regeneration. Expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1beta) is increased at the early stage of nervous system injury, and previously IL-1beta has been reported to promote neurite outgrowth by inhibiting RhoA activity in vitro. However, the effect of IL-1beta on axonal extension in vivo has not been obvious. Now we examine whether IL-1beta takes advantages on sciatic nerve regeneration. Sciatic nerves of rats are transected and sutured, and IL-1beta or PBS is locally administered for 2 weeks. Although IL-1beta does not influence on motor functional recovery, it promotes sensory functional recovery, estimated by toe pinch test, and increases the number and the area of neurofilament-positive axons at 12 weeks compared with PBS. Moreover IL-1beta, which promotes Schwann cell proliferation and thus may inhibit myelination, does not impair remyelination, estimated by myelin basic protein. These findings suggest that IL-1beta may contribute to sensory nerve regeneration following sciatic nerve injury by promoting axonal extension.     

人参皂苷Rb1促进小鼠坐骨神经损伤修复的作用及机制研究

目的 构建小鼠坐骨神经损伤（SNI）模型,探讨人参皂苷Rb1对小鼠坐骨神经损伤修复的促进作用及机制。 方法 选取成年雄性昆明小鼠78只,随机分为假手术组（26只）、模型组（26只）、治疗组（26只）。模型制作采用钳夹损伤法,假手术组仅游离坐骨神经。模型制作后30 min,治疗组腹腔注射10 mg/kg人参皂苷Rb1,模型组与假手术组给予同等体积的生理盐水。采用坐骨神经功能指数（SFI）对小鼠坐骨神经功能进行追踪评价;利用HE染色及生长相关蛋白43(GAP43)免疫荧光染色检测损伤14 d后坐骨神经再生修复情况;利用透射电子显微镜观察损伤节段髓鞘的结构改变。损伤后14 d,检测脑源性神经营养因子(BDNF)、神经生长因子(NGF)mRNA水平的表达变化。模型制作后3 d、7 d,利用Ki67、S100β免疫荧光染色检测施万细胞的增殖、迁移能力;利用Real-time PCR检测炎症相关因子以及施万细胞激活相关转录因子mRNA表达水平;通过Western blotting对Sox10在蛋白水平的变化进行验证。 结果 治疗组SFI评分高于模型组小鼠,神经近端、远端HE染色较均一。透射电子显微镜提示,治疗组髓鞘结构、厚度较模型组明显改善,髓鞘内神经纤维排列较为整齐。免疫荧光染色结果显示,治疗组坐骨神经GAP43+轴突伸长明显优于模型组,BDNF、NGF等营养因子表达升高。进一步检测发现,给予Rb1后损伤节段附近Ki67+细胞增殖、S100β+施万细胞迁移明显增多。Real-time PCR结果显示,治疗组肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β mRNA表达水平明显下降,施万细胞激活相关转录因子表达水平上升。 结论 10 mg/kg Rb1能降低再生组织环境中炎症因子表达水平,增加BDNF、NGF等营养因子的表达水平,促进施万细胞激活,从而促进小鼠坐骨神经损伤后的神经再生。     

腺病毒介导的神经营养素-3基因能够在大鼠脊髓前角运动神经元内过表达神经营养素-3

目的观察腺病毒介导的神经营养素-3(NT-3)基因在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元的过表达。方法在坐骨神经内直接注射含有绿色荧光蛋白(GFP)基因(报告基因)的NT-3基因重组腺病毒(Ad-NT-3-GFP),7d后应用免疫荧光组织化学染色技术,在荧光显微镜下观察脊髓前角运动神经元的NT-3过表达。结果 GFP表达组(对照组)和NT-3加GFP表达组两组动物的L4和L5脊髓段横切片上,有绿色荧光蛋白阳性标记的细胞。在NT-3加GFP表达组,还可以观察到NT-3阳性标记的细胞,这种细胞能与绿色荧光蛋白阳性标记的细胞重合,是过表达NT-3的前角运动神经元。与GFP表达组的前角运动神经元形态比较,NT-3加GFP表达组的过表达NT-3的前角运动神经元呈现更富有分支的突起。结论腺病毒介导的NT-3基因能够在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元内过表达NT-3,这为下一歩应用NT-3基因治疗策略修复实验性脊髓损伤提供初歩的实验资料。