异种脊髓前角匀浆对豚鼠前角运动神经元的影响

目的：观察豚鼠脊髓前角运动神经元在异种脊髓前角匀浆免疫后的变化。方法：猪脊髓前角匀浆免疫豚鼠后,豚鼠脊髓前角以苏木精-伊红、甲苯胺蓝及IgG免疫组化染色,同时电镜下观察前角运动神经元的超微变化。结果：豚鼠脊髓前角运动神经元存在变性和丢失,以神经元固缩、胶质细胞围绕破坏的神经元形成卫星现象及小墓穴为主,脊髓前角运动神经元胞质内IgG沉积呈颗粒状分布。运动神经元胞质内内质网扩张、线粒体肿胀。结论：猪脊髓前角匀浆作为抗原可引起豚鼠下运动神经元损伤,说明猪与豚鼠运动神经元存在共同抗原,自身免疫机制可能参与了运动神经元变性过程。     

免疫介导运动神经元损伤豚鼠模型的建立

目的建立免疫介导运动神经元损伤动物模型。方法取成年雄性白化病远交系Hartley豚鼠,用新鲜牛脊髓前角匀浆、弗氏佐剂和PBS制成油包水乳剂,于豚鼠背部皮下/皮内接种。观察动物的行为学表现及肌电图改变。取材进行组织学和血清抗豚鼠脊髓前角抗体检查。结果牛脊髓前角匀浆免疫后,豚鼠17/19只出现肢体和/或颈部肌肉无力、肌肉萎缩;肌电图示失神经改变及运动单位电位波幅增高、时限增长;运动皮质大锥体细胞及大脑脚有髓神经纤维均有不同程度的改变;脊髓颈腰膨大前角运动神经元变性、丢失及胶质细胞增生。免疫组豚鼠血清内抗豚鼠脊髓前角抗体滴度升高,脊髓前角运动神经内可见IgG沉积。结论本实验成功建立了免疫介导的运动神经元损伤动物模型。     

线粒体在免疫介导的神经元损伤过程中的特征

目的：观察免疫介导的神经元损伤的发病机制。方法：利用猪的脊髓前角匀浆免疫Lewis大鼠,通过透射电镜对Lewis大鼠的脊髓前角运动神经元及突起进行观察。结果：Lewis大鼠脊髓前角运动神经元有不同程度的变性丢失,残存的神经元内可见大量异常的线粒体及神经丝的异常聚集、Golgi器及内质网扩张,基质水合过度或基质脱水,核及核仁相对正常。结论：免疫介导的神经元变性过程中,线粒体可能是抗体攻击的目标,导致细胞能量代谢障碍,继发神经元变性死亡。     

免疫介导的脊髓运动神经元损伤过程中细胞周期素依赖蛋白激酶5的表达及意义

目的:研究免疫介导的神经元损伤过程中细胞周期素依赖蛋白激酶5(CDK5)表达的特征,探讨其损伤机制。方法:建立免疫介导的脊髓前角运动神经元损伤的模型,利用免疫组化方法观察脊髓前角运动神经元CDK5及p35/ p25的表达特征。结果:对照组豚鼠脊髓运动神经元CDK5及p35/p25主要在细胞核及细胞膜下表达,胞质着色淡;注射牛脊髓前角匀浆而无肢体瘫痪的豚鼠,脊髓运动神经元CDK5及p35/p25表达在数量上增加,强度上增强;有明显肢体瘫痪的豚鼠CDK5及p35/p25免疫反应阳性神经元数量明显减少,部分胞质内无着色。结论:CDK5及p35/ p25在神经元胞质内的易位表达,在免疫介导的神经元损伤过程中具有重要的作用。     

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     

慢性压迫性颈脊髓损害的超微结构观察

目的:对颈脊髓慢性压迫性损伤的超微结构进行观察研究,探讨其病理生理机制。方法:短毛豚鼠12只,8只用于模型制备,4只为正常对照。建立颈脊髓慢性压迫动物模型,分别于术后4周、8周对实验动物进行运动功能及电生理评价,并分批处死动物取样切片,以日本东芝H-600型透射电镜(每次动物4只),与正常组进行对照。结果:术后4周脊髓前角运动神经元胞体肿胀,胞浆内粗面内质网排列尚规则,核糖体仅部分脱落,部分线粒体肿胀,线粒体嵴清晰。白质前索呈原发性脱髓鞘改变,髓鞘变薄排列松散,胶质细胞水肿,部分有脱髓鞘改变,轴索结构基本正常。脊髓内毛细血管腔变细,内皮细胞肿胀。术后8周脊髓前角神经元胞体缩小或固缩,核内染色质成团块状,粗面内质网排列紊乱,线粒体广泛肿胀,嵴模糊。白质侧索髓鞘排列松散,出现原发性脱髓鞘改变,前索广泛脱髓鞘改变,部分轴索出现变性、坏死等改变。脊髓内毛细血管腔闭塞,内皮细胞凋亡变性。结论:来自前方的颈脊髓慢性压迫性损害,早期主要引起脊髓前索的原发性脱髓鞘改变。随着压迫程度的加重和时间的延长,逐渐出现脊髓内血供障碍、脊髓前角运动神经元退变、前索轴突变性等不可逆性改变。     

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。     

大鼠坐骨神经损伤急性期缓激肽在相应脊髓前角的变化

本文应用免疫细胞化学方法，结合图像分析仪，研究了缓激肽在脊髓腰段及Ｌ４－６前根节的分布，以及坐骨神经切断后，它在相应的前角运动神经元的相对含量的变化规律。研究发现：缓激肽免疫阳性反应物分布于Ｌ４－Ｌ６背根节及腰骶髓灰质的第Ⅱ、Ⅲ、Ⅳ、Ⅸ层的神经元及脊髓白质的神经胶质细胞和神经纤维。在神经损伤的研究中，相应脊髓的前角运动神经元的缓激肽含量，在损伤后第１５ｈ减少，以后逐渐增多，在损伤后２４ｈ基本恢复到     

脱细胞支架联合电针对坐骨神经损伤大鼠脊髓前角运动神经元的保护作用

目的 :探讨脱细胞支架(AS)联合电针对坐骨神经损伤(SNI)大鼠脊髓前角运动神经元的保护作用。方法 :首先制备AS,用于桥接损伤的神经。其次切除大鼠右侧坐骨神经10 mm,建立大鼠SNI模型。将SNI模型大鼠随机分为模型组(M)、AS桥接组(AS)和AS联合电针治疗组(AST)。模型组不予任何干预,AS组将支架桥接于两断端处,AST组在支架桥接术后2 d给予电针进行治疗,采用20 Hz、1 mA疏密波相间的电流,针刺穴位为环跳和阳陵泉,每次电针15 min,7 d 1个疗程。电针4周后,用电生理记录仪检测各组大鼠坐骨神经传导速度和波幅,用尼氏染色观察各组大鼠脊髓前角运动神经元的形态结构,用免疫印迹检测各组大鼠脊髓脑源性神经营养因子(BDNF)和神经生长因子(NGF)蛋白的表达。结果 :AST组大鼠坐骨神经传导速度和波幅明显高于AS组;尼氏染色显示AST组脊髓前角运动神经元胞体形态较完整,尼氏体呈蓝紫色、斑块状,偶见部分核移位现象,尼氏体的数量明显多于AS组和模型组;免疫印迹结果显示AST组脊髓内BDNF和NGF蛋白表达量均高于AS组和模型组。结论 :脱细胞支架联合电针不仅可增加大鼠坐骨神经传导速度及波幅,还可阻止脊髓前角运动神经元中尼氏体肿胀与溶解,并可上调脊髓内BDNF和NGF蛋白的表达,对SNI所致的脊髓前角运动神经元损伤有保护作用。     

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。     

脊髓小胶质细胞反应性在大鼠周围神经再生中的作用(英文)

为探讨大鼠坐骨神经损伤后脊髓小胶质细胞反应性、脊髓腹角运动神经元脱失与坐骨神经再生之间的关系,制备了SD大鼠右侧坐骨神经钳夹损伤模型,术后3d和7d测定相应脊髓节段小胶质细胞免疫反应性、腹角运动神经元数量,4周时于光镜和电镜下评价坐骨神经变性和再生。结果显示:(1)坐骨神经损伤后3d,脊髓腹角小胶质细胞OX-42免疫反应性开始明显增强(P<0.05);(2)脊髓腹角损伤同侧与对侧运动神经元数量比明显降低(P<0.05),说明同侧运动神经元存活数量减少;(3)组织学评价显示损伤神经再生不良;(4)simvastatin(一种降胆固醇药物,具有潜在的免疫调节作用)干预组较非simvastatin干预组小胶质细胞进一步激活,运动神经元存活数量增加,坐骨神经再生良好。本研究结果提示,脊髓腹角小胶质细胞的激活可能在大鼠周围神经损伤后的再生中发挥重要的保护作用。     

Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions,with special reference to motoneurons

Summary In situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexed's lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord. The upregulation of GAP-43 mRNA found in contralateral, presumably uninjured motoneurons after peripheral nerve transection, as well as in dorsal horn neurons after a dorsal root transection, indicates that GAP-43 levels are altered not only as a direct consequence of a lesion, but also after changes in the synaptic input to the neurons.     

脊神经前根吻合重建脊髓损伤大鼠的股四头肌功能

背景：如何重建脊髓损伤肢体运动功能对截瘫患者具有重要的意义。 目的：探索利用脊髓损伤平面以上健存的脊神经前根与支配股四头肌的腰神经建立神经通路,恢复脊髓损伤后股四头肌的神经支配和肌收缩功能。 方法：对清洁级SD大鼠L₁神经根的前根与L3神经根的前根进行显微吻合。经一段时间(6个月)的轴突再生后,期望建立新的肌肉收缩功能。神经缝合后6个月,在破坏L₂ 脊髓节段前后,分别进行神经电生理检测,观察股四头肌神经支配情况。 结果与结论：在同侧L₂半切脊髓前后,电刺激移植神经干时可记录到股四头肌的收缩肌电图。在同侧L₂半切脊髓前后,电刺激L₁感觉根时可同样在股四头肌记录到肌电图。说明利用脊髓损伤平面以上健存的L₁神经根前根与L₃神经前根移植吻合能重建新的股四头肌神经支配反射通路,并使股四头肌低级反射中枢上移。     

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。     

Impact of acute inflammation on spinal motoneuron synaptic plasticity following ventral root avulsion

Background  

Ventral root avulsion is a proximal nerve root lesion in which ventral motor nerve rootlets are torn from surface of the spinal cord, resulting in extensive death of motoneurons. It has been previously shown that if such lesioning is performed in an animal with experimental autoimmune encephalomyelitis (EAE), a significant number of motoneurons can be rescued despite an intense inflammatory reaction. This rescue effect has been attributed to production of a number of neurotrophic factors by invading T cells. Synaptological changes may be involved in neuronal degeneration, and a better understanding of the role of these changes may be of importance for developing new strategies to promote neuronal survival. The objective of the present work was to evaluate neuronal survival, astroglial reaction and synaptic input changes in spinal cord anterior horn motor nuclei after ventral root avulsion in animals with EAE, both during peak disease and after remission.     

The organization of serotonin fibers in the anterior column of the mammalian spinal cord. An immunohistochemical study

Detailed comparative analysis of the organization of serotonin fibers in the anterior column of the mammalian spinal cord (rat, guinea pig, cat, dog and monkey) was carried out by use of the indirect antibody peroxidase-antiperoxidase (PAP) method. The plexus formation of serotonin-containing varicose fibers around the alpha-motoneurons in the monkey anterior horn was in much closer apposition to the cell bodies in comparison with the spinal cords of the rodents and carnivores. The results may suggest that anterior horn motoneurons in the simian spinal cord are intimately innervated by serotonin fibers in a manner different from that of rodents and carnivores. Furthermore, the small cell groups endowed with particularly dense networks of serotonin fibers were demonstrable in the anterior horn of L1-L2 segments of rats, and L3-L4 of guinea pigs and monkeys; however, in the lumbar levels of the carnivores this was not the case. Hence it seems doubtless that there exists in the lumbar anterior horn of the rodent and primate spinal cords a cell group with an unknown specialized function.     

Glatiramer acetate positively influences spinal motoneuron survival and synaptic plasticity after ventral root avulsion

Avulsion of ventral roots induces degeneration of most axotomized motoneurons. At present there are no effective strategies to prevent such neuronal loss and to preserve the affected spinal circuits. Interestingly, changes in the spinal cord network also occur during the course of the experimental model of multiple sclerosis (experimental autoimmune encephalomyelitis—EAE). Glatiramer acetate (GA) significantly reduces the seriousness of the symptoms during the exacerbation of EAE. However, little is known about its effects on motoneurons. In the present study, we investigated whether GA has an influence on synapse plasticity and glial reaction after ventral root avulsion (VRA). Lewis rats were subjected to the avulsion of lumbar ventral roots and treated with GA. The animals were sacrificed after 14 days of treatment and the spinal cords processed for immunohistochemistry. A correlation between the synaptic changes and glial activation was obtained by performing immunolabeling against synaptophysin, GFAP and Iba-1. GA treatment preserved synaptophysin labeling, and significantly reduced the glial reaction in the area surrounding the axotomized motoneurons. After ventral root avulsion, GA treatment was also neuroprotective. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which may in turn contribute to future treatment strategies after proximal lesions to spinal motoneurons.