脊髓创伤后施万细胞神经生长因子受体的表达及在轴突再生中…

为了探讨人脊髓损伤后再生过程，用免疫组织化学及电镜等研究了２１例脊髓创伤后存活２小时至５４年患者的脊髓尸检材料。结果表明：创伤后４天，损伤的脊髓组织内便可见再生的轴突支芽。创伤４．５个月以后，损伤的脊髓出现许多轴突再生巢。人含有定向排列 突及施万细胞，部分轴突已被施万细胞的髓鞘所包绕。巢内施万细胞表达了神经生长因子受体（ＮＧＦＲ）。此外，受损平面脊神经根内，施万细胞的ＮＧＦＲ表达增强，以上结果表明     

实验性脊髓损伤的治疗进展

本文综述了皮质类固醇、促甲状腺释放激素、神经节苷脂、阿片受体拮抗剂,钙通道阻滞剂、一氧化氮合成酶抑制剂、促细胞生长因子等药物以及大网膜脊髓移植、神经组织脊髓内移植的作用机制、实验及临床应用情况。并分析了实验及临床治疗效果差异的原因。     

大网膜脊髓移植治疗实验性脊髓损伤的研究

目的：为验证大网膜是否对脊髓损伤有修复作用。方法：本车实验采用SD雌性大白鼠20只，分实验、对照两组。在同等条件及体感诱发电位监测下造成截瘫。实验组施行带蒂大网膜脊髓移植．对照组截瘫后不作任何处理，动物饲养存活3～8周后进行辣根过氧化酶逆行追踪法研究。结果：发现实验组红核内标记细胞总数为1846个，对照组为556个。P值<0．05，提示差别显。结论：结果表明脊髓损伤后．大网膜脊髓移植对红核脊髓束神经纤维轴浆运输的恢复有促进作用，从形态学的角度进一步肯定了大网膜脊髓移植治疗外伤性截瘫的临床价值．     

骨髓基质细胞移植促进脊髓全横断损伤区神经元轴突的再生变化☆○

背景：近年来,部分学者证明骨髓基质细胞移植可促进轴突再生,改善脊髓损伤引起的运动功能障碍,但目前关于移植骨髓基质细胞如何促进轴突再生,移植细胞与再生轴突的关系尚不清楚。 目的：通过免疫荧光组织化学和免疫电镜的方法,探讨移植骨髓基质细胞促进脊髓全横断损伤区轴突再生的机制。 设计、时间及地点：随机对照动物实验,细胞学体内观察,于2006-03/2007-06在新加坡国立大学解剖系完成。 材料：清洁级Wistar新生大鼠1只,用于骨髓基质细胞培养。清洁级成年雌性Wistar大鼠36只,无菌条件下显露、切断脊髓T10,制备脊髓全横断损伤模型。 方法：通过传代法培养、纯化骨髓基质细胞。36只成年Wistar雌性大鼠随机投币法分为移植组和对照组,每组18只。移植组大鼠脊髓全横断损伤9 d后以1×1011 L-1的密度移植骨髓基质细胞,缺损区5 μL,损伤区上、下1 mm处各2.5 μL,对照组动物在相同部位注射等量DMEM完全培养基,注射速度1 μL/min。 主要观察指标：①移植骨髓基质细胞存活、分化情况。②轴突再生情况。③移植组和对照组宿主自身的nestin、NF200、GFAP和CNP阳性细胞在脊髓损伤区存活情况。④内源性CNP阳性细胞和再生纤维关系。 结果：骨髓基质细胞移植2周时,脊髓损伤区可见大量CFDA-SE标记的移植细胞,随时间延长,存活的移植细胞数目逐渐降低,考虑脊髓损伤区内大量的OX42阳性吞噬细胞/激活小胶质细胞及空洞可能影响移植细胞的存活。虽然骨髓基质细胞数目逐渐降低,骨髓基质细胞移植可促进损伤区轴突的再生,而且还可促进宿主自身的nestin、NF200、GFAP和CNP阳性细胞在脊髓损伤区存活。宿主自身CNP和许旺细胞促进损伤轴突的再生和髓鞘形成。 结论：移植骨髓基质细胞移植可促进宿主自身CNP和许旺细胞在脊髓损伤区存活,后者具有促进损伤轴突再生和髓鞘形成的作用。     

低温保存神经干细胞复苏后移植对脊髓损伤大鼠轴突再生的影响：逆行示踪标记观察**★

目的：关于神经干细胞移植治疗脊髓损伤的研究已有一些报道，对细胞移植的时间、方式以及检测的指标各有不同观点。实验观察了低温保存的神经干细胞复苏后移植对大鼠脊髓损伤后轴突再生的影响。 方法：实验于2005-06/2006-06在中国医科大学实验动物中心完成。①实验材料：选取Wistar成年大鼠36只，雌雄不限，体质量250~300 g，由中国医科大学实验动物部提供。新生大鼠10只，用作神经干细胞的分离与培养。实验过程中对动物处置符合动物伦理学标准。②实验方法：将获得的神经干细胞在处于对数生长期阶段-70 ℃冻存2周，复温后用5-溴脱氧尿嘧啶核苷法标记。制备大鼠脊髓损伤模型，伤后立即进行移植干预，实验分为3组：神经干细胞移植组、DMEM培养液填充组、空白对照组。③实验评估：应用免疫组织化学法观察移植细胞存活及迁移情况，行辣根过氧化物酶逆行示踪法观察脊髓损伤处轴浆运输的重建。 结果：36只脊髓横断损伤模型因麻醉过量死亡4只，感染死亡5只，予补足。复苏的神经干细胞经Brdu核标记后移植到脊髓损伤区，在损伤脊髓区域可检测到标记的阳性细胞。第7天可见，第14天增多，第28天逐渐减少并消失。辣根示踪技术显示神经干细胞移植组较DMEM培养液填充组阳性细胞明显增多，组间差异有统计学意义。 结论：低温保存的神经干细胞移植到脊髓损伤区域后可存活，并参与脊髓损伤处轴浆通路的结构重建。     

小发夹RNA抑制Nogo基因表达促进脊髓损伤修复的实验研究

目的 利用RNA干涉（RNAi）技术使特定的基因（Nogo）沉默，探索脊髓损伤的治疗方法。方法 设计有小发夹结构的两条DNA序列，PCR扩增带有U6启动子的小发夹，腺病毒包装重组体，转染少突胶质细胞，采用Westernblot法分析Nogo-66的蛋白表达水平。结果成功地构建了靶向Nogo基因RNAi的带有U6启动子的小发夹重组体，Nogo-66蛋白表达水平明显下降。结论 靶向RNAi小发夹重组体经腺病毒包装后，成功转染少突胶质细胞并能有效抑制Nogo-66基因的表达。     

嗅鞘细胞修复脊髓损伤轴突髓鞘化的研究与进展

髓鞘的完整性是保证其包裹的轴突具备正常生理功能的前提，脊髓的损伤往往伴有不同程度的髓鞘损伤，因此如何使损伤的轴突重新获得髓鞘化成为损伤轴突再生的关键。早期对嗅鞘细胞的研究表明，嗅鞘细胞可以促进并参与损伤脊髓的髓鞘再生，从而达到使损伤轴突再生，恢复其生理功能目的。然而随研究的广泛深入，近期得出了不同的结论，即嗅鞘细胞并不参与损伤轴突的再次髓鞘化，其促进轴突髓鞘化并生长的机制需进一步研究。     

神经生长因子对创伤性脊髓损伤大鼠膀胱功能和轴突损伤修复的作用研究

目的 探讨神经生长因子(Nerve growth factor, NGF)对创伤性脊髓损伤(Traumatic spinal cord injury, t-SCI)大鼠膀胱功能和脊髓神经轴突损伤修复的影响及其分子机制。方法 取30只雄性Sprague-Dawley(SD)大鼠,通过改良Allen’s击打法构建创伤性脊髓损伤模型,随机分为假手术组、损伤组和NGF组,每组各10只;采用血脑屏障(Blood-brain barrier, BBB)评分观察术前、术后大鼠的后肢运动功能;BL-420生物仪实验系统检测尿动力学;甲苯胺蓝染色吻合口远端截取的左侧腰6前根,计算有髓轴突数量;采用苏木精-伊红(Hematoxylin eosin, HE)染色大鼠膀胱组织;原位末端标记法(TdT-mediated dUTP nick and labeling, TUNEL)染色大鼠损伤严重的脊髓,观察脊髓神经细胞的凋亡率;蛋白免疫印迹法(Western blot)检测脊髓组织中原癌基因丝氨酸/苏氨酸蛋白激酶(proto-oncogene serine/threonine-protein kinase, R...     

大鼠脊髓损伤后Nogo-A表达变化的免疫组织化学研究

目的观察大鼠脊髓损伤后不同时间点Nogo—A蛋白在脊髓的表达变化。方法大鼠分脊髓损伤组、假手术组和正常对照组。损伤动物存活1d、3d、7d后，分别进行Nogo—A抗体的免疫组织化学染色。结果损伤部位的灰质神经元和白质少突胶质细胞均呈明显的Nogo—A免疫阳性反应，随着损伤后存活时间的延长，两者的阳性反应细胞相对染色强度及数量均逐渐下降。结论脊髓损伤后，Nogo—A在灰质神经元有表达，其表达相对强度和表达细胞数量先明显增加，随后逐渐减少，与少突胶质细胞的表达变化相似。     

Axonal regrowth through collagen tubes bridging the spinal cord to nerve roots

The capacity of central nervous system (CNS) axons to elongate from the spinal cord to the periphery throughout a tubular implant joining the ventral horn of the spinal cord to an avulsed root was investigated in a model of brachial plexus injury. The C5-C7 roots were avulsed by controlled traction and the C6 root was bridged to the spinal cord over a 3 mm gap by the use of a collagen cylinder containing or not containing an autologous nerve segment, or an autologous nerve graft. Nine months later, the functionnality and the quality of the axonal regrowth was evaluated by electrophysiology, retrograde labelling of neurons, and histological examination of the gap area. A normal electromyogram of the biceps was observed in all animals where the C6 root was bridged to the spinal cord. The mean average amplitude of the motor evoked potentials was comprised between 17.51 ± 12.03 μV in animals repaired with a collagen cylinder, and 27.83 ± 22.62 μV when a nerve segment was introduced in the tube. In nonrepaired animals spontaneous potentials reflecting a muscle denervation were observed at electromyography. Retrograde labelling indicated that a mean number of 58.88 ± 37.89 spinal cord neurons have reinnervated the biceps in animals repaired with a tube versus 78.38 ± 62.11 when a nerve segment was introduced in the channel, and 97.25 ± 56.23 in nerve grafting experiments. Analyses of the repair site showed the presence of numerous myelinated regenerating axons. In conclusion, our results indicate that spinal cord neurons can regenerate through tubular implants over a 3 mm gap, and that this axonal regrowth appeared as effective as in nerve grafting experiments. The combination of an implant and a nerve segment did not significantly increase the regeneration rate. J. Neurosci. Res. 49:425–432, 1997. © 1997 Wiley-Liss, Inc.     

Axonal regrowth through a collagen guidance channel bridging spinal cord to the avulsed C6 roots: Functional recovery in primates with brachial plexus injury

Intraspinal implantation of a collagen guidance channel (CGC) to promote axon regeneration was investigated in marmosets with brachial plexus injury. After avulsion of the right C5, C6 and C7 spinal roots, a CGC containing (group B) or not (group A) a nerve segment, or a nerve graft (group C), was ventro-laterally implanted into the cord to bridge the ventral horn and the avulsed C6 roots. No spinal cord dysfunction was observed following surgery. Two months later, the postoperative flaccid paralysis of the lesioned arm improved. In five months, a normal electromyogram of the affected biceps muscle was recorded in all repaired animals. Motor evoked potentials were obtained with a mean amplitude of 13.37 ± 13.66 μV in group A, 13.21 ± 5.16 μV in group B and 37.14 ± 35.16 μV in group C. The force of biceps muscle contraction was 27.33 ± 20.03 g (group A), 24.33 ± 17.03 g (group B) and 37.38 ± 21.70 g (group C). Retrograde tracing by horseradish peroxidase showed labelled motoneurons ipsilaterally located in the C5 and C6 ventral horn, nearby the implantation site. The mean labelled neurons was 32.33 ± 21.13, 219.33 ± 176.29 and 64.33 ± 23.54 in group A, B and C respectively. Histological analysis presented numerous myelinated and unmyelinated regenerating axons in the implant of these animals. Statistical analysis did not show significant difference among the three repaired groups. Our results indicate that spinal neurons can regenerate through a CGC to avulsed nerve roots and induce motor recovery in primates. J. Neurosci. Res. 51:723–734, 1998. © 1998 Wiley-Liss, Inc.     

Axonal regeneration from injured dorsal roots into the spinal cord of adult rats.

Injury to the central processes of primary sensory neurons produces less profound changes in the expression of growth-related molecules and less vigorous axonal regeneration than does injury to their peripheral processes. The left L4, L5, and L6 dorsal roots of deeply anaesthetized adult Sprague-Dawley rats were severed and reanastomosed, and in some animals, the ipsilateral sciatic nerve was crushed to increase the expression of growth-related molecules. After between 28 days and three months, the sciatic nerve of most animals was injected with transganglionic tracers and the animals were killed 2-3 days later. Other animals were perfused for electron microscopy. Very few regenerating axons entered the spinal cord of the rats without sciatic nerve injuries. Labelled axons, however, were always found in the spinal cord of rats with sciatic nerve injuries. They often entered the cord around blood vessels, ran rostrally within the superficial dorsal horn, and avoided the degenerating white matter. The animals with a conditioning sciatic nerve crush had many more myelinated axons around the dorsal root entry zone (DREZ) and on the surface of the cord. Thus, a conditioning lesion of their peripheral processes increased the ability of the central processes of myelinated A fibres to regenerate, including to sites (such as lamina II) they do not normally occupy. Astrocytes, oligodendrocytes, and meningeal fibroblasts in and around the DREZ may have inhibited regeneration in that region, but growth of the axons into the deep grey matter and degenerated dorsal column was also blocked.     

Axonal regeneration from the spinal cord to peripheral nerve induced by end-to-side neurorrhaphy Evidence from acetylcholinesterase staining and Fluorogold retrograde tracing

BACKGROUND： In recent years, surgeons have advocated root or trunk repair of avulsed nerve roots for overall recovery. However, donor nerves pose a major problem, because they do not contain adequate numbers of axons. Moreover, the procedures lead to nerve deficits in the donor nerve following transplantation. OBJECTIVE： To observe whether axonal regeneration occurs by end-to-side neurorrhaphy in the peripheral nerve and spinal cord. DESIGN, TIME AND SETTING： A neuroanatomical, randomized, controlled, animal study was performed at Functional Anatomy Lab in Nagoya University School of Medicine from May 2002 to July 2003. MATERIALS： Fluorogold was purchased from Fluorochrome, LLC, USA. BX50 light microscope and fluorescent microscope were purchased from Olympus, Japan. METHODS： A total of 21 rats were randomly divided into three groups, and the posterior avulsion injury model （C6-8） of the brachial plexus was performed. In the ventral root graft group, the avulsed C7 ventral roots were reanastomosed to the small anterior lateral aspect window of the spinal cord via nerve grafts. In the dorsal root graft group, the C7 dorsal roots were reanastomosed at the small pia mater window of the posterior lateral aspect of the spinal cord via nerve grafts. In the control group, the avulsed nerve roots were not repaired. MAIN OUTCOME MEASURES： The nerve grafts were collected from the ventral and dorsal root graft groups, and the C7 proximal nerve end was collected from the control group. Acetylcholinesterase staining was performed on the tissue. Fluorogold retrograde tracing technique was applied to determine the origin of the regenerating axons. RESULTS： Results showed that acetylcholine-positive axons existed in nerve grafts of the ventral and dorsal root graft groups. However, axons were not found in the avulsed nerve roots of the control group. Fluorogold retrograde tracing confirmed the presence of fluorogold-containing neurons in the ventral and dorsal horn of the ventral and dorsal root graft groups. Fluorogold-positive neurons were not observed in the control group. CONCLUSION： End-to-side neurorrhaphy induced axonal regeneration from the spinal cord to the peripheral nervous system.     

慢性压迫性脊髓损伤后巢蛋白mRNA表达的观察

目的观察成年大鼠慢性压迫性脊髓损伤后及减压后早期巢蛋白与巢蛋白mRNA的相关性表达。方法选用健康wistar大鼠50只，体重280～320g，制备慢性压迫性脊髓损伤中度、重度及重度损伤减压后3d、10d模型，取自距胜迫边缘5mm段脊髓组织切片。正常成年大鼠作为对照组。行巢蛋白免疫组织化学染色，巢蛋白mRNA原位杂交实验，计算机罔像分析仪定量分析，观察巢蛋白、巢蛋白mRNA在脊髓中央管、灰质和白质中表达的变化，探讨巢蛋白与巢蛋白mRNA表达的相关性。结果成年大鼠慢性膻迫性脊髓损伤中、重度及重度压迫损伤减压后3d，巢蛋白在自、灰质及脊髓中央管室管膜细胞中均有明显表达（P〈0．05），以重度压迫组最为显著（P〈0．01）。减压后10d组灰质与正常对照组比较，差异无显著性意义（P=0．483）。重度压迫组及减压后3d组，巢蛋白mRNA在脊髓灰质、白质及中央管室管膜细胞中均有显著性表达（P〈0．05），以灰质前角第Ⅸ板层、后角和室管膜下区最为显著。中度压迫组，巢蛋白mRNA在灰质前角第Ⅸ板层及中央管室管膜细胞中有显著性表达（P〈0．05），其余区域仅有微弱表达，而白质内巢蛋白mRNA表达于软脊膜下星形胶质细胞的足突中。减压后10d组灰质内巢蛋白mRNA的表达与正常对照组比较，无显著性差异（P=0．375）。正常对照组中无表达。结论成年大鼠慢性压迫性脊髓损伤及减压后早期存在神经前体细胞的增殖。增殖的神经前体细胞巢蛋白与巢蛋白mRNA表达的相关性具有与胚胎发育期脊髓相似的特征。     

血循环因素在鼠周围神经移植修复脊髓损伤中的作用

在建立带血管神经（ＶＮ）干移植修复脊髓损伤的基础上，对自体ＶＮ与游离神经（ＦＮ）移植修复脊髓损伤的研究进行了比较。结果表明：ＶＮ与脊髓连接处无明显空洞反应，瘢痕组织少，大量神经纤维长入移植神经内，ＶＮ组在脑干、脊髓及神经根背节有２９２２±７５４．３ＨＲＰ神经元，而ＦＮ组仅只有２０±６．５ＨＲＰ神经元（Ｐ值＜０．０１）。有髓轴突的数目、直径及截面积ＶＮ组非常明显地大于ＦＮ组（Ｐ值＜０．０１）认为：带血管神经移植后，改善移植物及其周围组织的血液供应；保证雪旺氏细胞成活，产生多种高浓度神经营养因子；促使成纤维细胞释放大剂量载脂蛋白Ｄ，有利于髓鞘的形成。     

大鼠急性脊髓损伤后细胞凋亡以及相关蛋白表达的实验研究

目的 观察脊髓损伤（spinalcordinjury，SCI）后细胞凋亡和相关调控基因（FasL和Caspase．3）的表达情况。方法 通过建立静压型SCI模型（30g的重量，压迫T10节段10min），并用TUNEL染色（terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling）和免疫组化染色方法，了解Sprague-Dawley大鼠SCI后（伤后6、12、24、48和72h以及伤后7、14、21d）细胞凋亡以及相关调控基因（FasL与Caspase-3）表达的情况。结果 大鼠SCI后损伤节段（T10节段）6h出现TUNEL阳性细胞数增加，12h达到高峰，持续3天～1周后显著减少；损伤相邻节段（T9、T11节段）伤后12h出现TUNEL阳性细胞，伤后3dTUNEL阳性细胞数达峰值；SCI后FasL与Cas-pase-3表达均有不同程度的增高。结论 大鼠脊髓损伤后多节段、长时间存在着大量的细胞凋亡；细胞凋亡可能与FasL-Caspase-3途径有关。     

Peripheral nerve grafts after cervical spinal cord injury in adult cats

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.     

Reinnervation of a denervated skeletal muscle by spinal axons regenerating through a collagen channel directly implanted into the rat spinal cord

In the present study, the continuity between the central nervous system (CNS) and the peripheral nervous system (PNS) was restored by mean of a collagen channel in order to reinnervate a skeletal muscle. Three groups of animals were considered. In the first group, one end of the collagen channel was implanted in the cervical spinal cord of adult rats. The other end was connected to a 30-mm autologous peripheral nerve graft (PNG) implanted into the denervated biceps brachii muscle. The gap between the spinal cord and the proximal nerve stump varied from 3 to 7 mm. In the second group of animals, the distal end of the PNG graft was ligatured in order to compare the survival of the growing axons in the presence and in the absence of a muscular target. In the third group of animals, the extraspinal stump of the collagen channel was ligatured. Our study demonstrates that spinal neurons and dorsal root ganglion (DRG) neurons can grow long axons through the collagen channel over a 7-mm gap and reinnervate a denervated skeletal muscle. The results also indicate that the presence of a PNG at the extraspinal stump of the collagen channel is essential for axonal regrowth and that the muscle target contributes to the long-term maintenance of the regenerating axons. These data might be interesting for clinical application when the continuity between the CNS and PNS is interrupted such as in root avulsion.     

Resistance to anoxic injury in the dorsal columns of adult rat spinal cord following demyelination

In order to examine the relationship between myelination and sensitivity to anoxia in adult white matter, we studied action potential conduction in the spinal cord dorsal column of adult rats in which focal demyelinating lesions had been produced using ethidium bromide/X-irradiation. Acutely isolated spinal cords from control rats and following demyelination were maintained in vitro at 36°C and compound action potentials were studied following supramaximal stimulation. The compound action potential was totally abolished within 12 min of the onset of anoxia in normal dorsal columns, but was not abolished until 50 min following the onset of anoxia in demyelinated dorsal columns. Compound action potentials showed significantly greater recovery (to 58.1±12.2% of control amplitude) in demyelinated dorsal columns compared to controls (30.8±5.3%) following 120 min of reoxygenation. These results show that focal demyelination is associated with reduced sensitivity to anoxia within white matter of the adult spinal cord.