大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的:探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系.方法:用免疫组织化学技术、HE染色和Tunnel法,观察坐骨神经切断后1,6,12,24 h及3,7和14 d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化.结果:坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化,星形胶质细胞的活化早于小胶质细胞;后期运动神经元发生凋亡,HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕.结论:研究结果提示,坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切.     

大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的：探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系。方法：用免疫组织化学技术、HE染色和Tunnel法，观察坐骨神经切断后1，6，12，24h及3，7和14d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化。结果：坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化，星形胶质细胞的活化早于小胶质细胞；后期运动神经元发生凋亡，HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕。结论：研究结果提示，坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切。     

脊髓损伤模型大鼠神经胶质细胞增殖与胶质纤维酸性蛋白的表达

背景：星形胶质细胞可以通过细胞裂解释放各种神经营养因子,并可促进损伤脊髓的修复。 目的：观察脊髓损伤模型大鼠神经胶质纤维酸性蛋白的表达及对其后肢功能恢复的影响。 方法：将SD大鼠采用Allen's法撞击T9~10节段致脊髓损伤,造模成功后蛛网膜下腔移植骨形态发生蛋白7,并设置仅蛛网膜下腔移植His蛋白的正常SD大鼠做对照。用BBB评分法评估两组大鼠后肢的运动功能,用免疫组织化学染色法和Western-blot法观察各组神经胶质纤维酸性蛋白的表达。 结果与结论：BBB评分结果显示,模型组大鼠脊髓损伤后下肢功能自行恢复率达68%。模型组脊髓损伤3和7 d,损伤区域神经胶质纤维酸性蛋白表达逐渐增加(P < 0.05),随后逐渐下降,于脊髓损伤28 d后逐渐恢复到对照组水平(P > 0.05)。脊髓损伤后1~14 d两组胶质纤维酸性蛋白表达逐渐升高(P > 0.05)。结果证实,脊髓损伤后蛛网膜下腔移植骨形态发生蛋白7可诱导星形胶质细胞增殖,神经胶质纤维酸性蛋白的表达增强,进而促进脊髓损伤大鼠后肢功能的恢复。     

大鼠坐骨神经修复后重组睫状神经营养因子对相关神经元生长相关蛋白43、生长抑素表达的影响

目的观察周围神经修复后，重组睫状神经营养因子(CNTF)对相关神经元中生长相关蛋白表达的调控作用。方法用硅管套接切断的成年大鼠坐骨神经，在神经切断处给予重组CNTF，用免疫组织化学和原位杂交组织化学方法结合计算机图像分析观测L4脊髓和L4、L5脊神经节中生长相关蛋白43(GAP43)和生长抑素(SOM)mRNA的相对含量。结果在CNTF组修复侧脊髓前角外侧核，大、中型神经元胞质中神经元GAP43阳性物质的面积百分率显著高于生理盐水组，SOM mRNA杂交信号阳性的大、中型神经元的数量少于生理盐水组，但两组脊神经节的相应指标无显著差别。结论坐骨神经修复后，外加重组CNTF能上调相关运动神经元表达GAP43，下调其表达SOM mRNA，但对感觉神经元的相应作用不明显。     

急性脊髓损伤模型大鼠与白细胞介素1受体拮抗剂的保护作用**

背景：白细胞介素1受体拮抗剂对大鼠急性脊髓损伤后脊髓功能修复具有保护作用,但具体机制不明。 目的：观察白细胞介素1受体拮抗剂对大鼠急性脊髓损伤组织神经丝蛋白质200和胶质纤维酸性蛋白的影响。 方法：SD大鼠随机分成假手术组,生理盐水对照组和白细胞介素1受体拮抗剂治疗组。采用改良Allen氏打击法建立急性脊髓损伤大鼠模型。分别在建模后1,48和72 h获取损伤段8 mm脊髓标本。 结果和结论：免疫组织化学染色检测,白细胞介素1受体拮抗剂治疗组神经丝蛋白质200和胶质纤维酸性蛋白的表达较假手术组和生理盐水组高,差异有显著性意义(P < 0.05)。提示白细胞介素1受体拮抗剂可使急性脊髓损伤大鼠模型损伤段脊髓神经丝蛋白质200和胶质纤维酸性蛋白表达增加,对急性脊髓损伤发挥保护作用。     

胶质细胞源性神经营养因子缓释微球及NogoA, ChABC缓释微球联合应用损伤脊髓再生的形态学变化*☆

摘要 背景：促进轴突再生的原则是改善抑制再生的环境和提高轴突生长能力,措施主要有轴突生长抑制因子阻滞剂和神经营养因子应用。用可降解微球加载药物是一种在局部提供持续药物释放的方法。 目的：探讨胶质细胞源性神经营养因子、NogoA、ChABC 缓释微球联合应用促进大鼠损伤脊髓再生病理形态学修复的作用。 方法：建立SD大鼠T10 脊髓完全横断伤模型,分别在损伤局部给予生理盐水、胶质细胞源性神经营养因子、胶质细胞源性神经营养因子缓释微球、NogoA缓释微球、ChABC 缓释微球及3种微球联合治疗,并设立未造模的正常组及假手术组。损伤后10周,每组行四甲基若丹明葡聚糖胺顺行示踪,及神经丝蛋白200、生长相关蛋白43、胶质细胞源性神经营养因子免疫组化检查,并采用免疫组化图像分析系统进行定量分析。 结果与结论：胶质细胞源性神经营养因子、NogoA、ChABC 缓释微球联合能提高脊髓损伤局部神经丝蛋白200、生长相关蛋白43、胶质纤维酸性蛋白的表达水平,显示局部脊髓再生修复加强,其效果优于单用胶质细胞源性神经营养因子缓释微球。提示,胶质细胞源性神经营养因子缓释微球及NogoA,ChABC 缓释微球联合促大鼠损伤脊髓再生修复其效果优于单用胶质细胞源性神经营养因子缓释微球。 关键词：胶质细胞源性神经营养因子;微球;聚乳酸-聚羟基乙酸共聚物;脊髓损伤;神经再生 doi:10.3969/j.issn.1673-8225.2011.03.014     

RNAi靶向沉默预处理星形胶质细胞胶质细胞源性神经营养因子对神经元凋亡的影响

研究表明外源性的胶质细胞源性神经营养因子可对脑缺血损伤时的神经元有保护作用,但关于内源性的胶质细胞源性神经营养因子的神经元保护作用目前机制不清。鉴于此,实验以正常培养的星形胶质细胞培养基,胶质细胞源性神经营养因子高表达星形胶质细胞培养基和采用RNAi技术沉默胶质细胞源性神经营养因子表达的星形胶质细胞培养基,作用于缺血神经元,观察不同条件培养基对神经元凋亡的影响。结果验证RNAi靶向沉默预处理星形胶质细胞胶质细胞源性神经营养因子的表达可促进神经元凋亡,氧糖剥夺预处理可上调星形胶质细胞的胶质细胞源性神经营养因子的表达,能明显降低神经元的凋亡。     

常氧及低氧条件下胶质源性神经营养因子体外诱导中脑神经干细胞向多巴胺能神经元的分化

背景：神经干细胞的定向诱导分化和扩增受细胞自身基因和外来信号的调控。 目的：观察中脑源性神经干细胞在常氧、低氧和胶质源性神经营养因子诱导下向多巴胺能神经元的分化情况。 方法：无菌条件下分离E12小鼠胚胎腹侧中脑组织,胰酶消化和机械吹打制成单细胞悬液,在无血清培养基中培养扩增;Nestin免疫细胞化学染色方法鉴定神经干细胞。在有血清培养基中对纯化神经干细胞自然分化;神经元特异性烯醇化酶和胶质纤维酸性蛋白免疫细胞化学染色方法分别鉴定神经元和星形胶质细胞。建立常氧和低氧环境,设置常氧组、常氧+胶质源性神经营养因子组、低氧组、低氧+胶质源性神经营养因子组,按实验分组在有血清条件下诱导分化。 结果与结论：在低氧条件下,中脑神经干细胞向多巴胺能神经元分化均高于常氧组;尤其是低氧环境和胶质源性神经营养因子诱导下向多巴胺能神经元分化比例更高,表型更成熟。说明低氧环境下胶质源性神经营养因子可明显促进中脑神经干细胞分化为数量足够、形态及功能成熟的多巴胺能神经元。     

胶质纤维酸性蛋白与中枢神经系统疾病

<正>胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP) 是分子量为50-52kD的酸性蛋白,是星形胶质细胞的主要成分 之一,富含谷氨酸和天冬氨酸,以中间微丝蛋白和可溶性蛋白 两种形式存在于胶质细胞的胞浆中,是星形胶质细胞的骨架蛋 白。GFAP在星形胶质细胞受到刺激引起反应时,其表达发生 变化,因此GFAP能够用来特异性地标记星形胶质细胞、并被 认为是星形胶质细胞活化的标志。活化的星形胶质细胞 内GFAP的增高,可能起保护神经元的作用;星形胶质细胞在 损伤区分泌多种神经营养因子,以促进中枢神经和周围神经轴     

异体骨髓间充质干细胞移植治疗大鼠脊髓损伤

背景：脊髓损伤的修复目前尚无良好的治疗手段,细胞移植能促进神经轴突再生及脊髓功能恢复,为治疗脊髓损伤提供了可能,但因脊髓损伤模型及移植方式不同,其治疗效果并不相同。 目的：验证异体骨髓间充质干细胞移植对大鼠脊髓损伤的治疗作用。 方法：全骨髓贴壁法分离大鼠骨髓间充质干细胞。健康SD大鼠随机分为3组,细胞移植组、对照组和假手术组。细胞移植组和对照组采用改良Allen重物打击法制造大鼠脊髓损伤模型,假手术组仅暴露脊髓。术后4周,每周进行运动功能评分,ELISA检测脊髓损伤组织中脑源性神经营养因子、神经生长因子表达;免疫荧光染色检测脊髓组织中NF200和胶质纤维酸性蛋白表达。 结果与结论：与对照组比较,细胞移植组大鼠运动功能明显改善,脊髓组织中脑源性神经营养因子、神经生长因子蛋白含量明显增高(P < 0.05);移植组大鼠脊髓囊腔较小,NF200表达明显增加,胶质纤维酸性蛋白表达减少。提示异体骨髓间充质干细胞移植能增加损伤脊髓神经生长因子含量,抑制胶质瘢痕形成,促进神经轴突再生,改善大鼠脊髓损伤后运动功能恢复。     

不同年龄大鼠坐骨神经损伤后脊髓中神经生长导向因子Slit-1及Robo-2受体的表达变化

目的观察不同年龄大鼠坐骨神经损伤后,轴突导向因子Slit-1及其Robo-2受体在脊髓中的表达,以探讨不同年龄大鼠外周神经损伤后再生神经具有靶向性差异的可能机制。方法老年、成年和幼年大鼠各20只,建立左侧坐骨神经横断、硅胶管桥接模型。通过免疫荧光染色观察Slit-1蛋白和Robo-2受体在腰段脊髓中表达的变化,计算其荧光强度值,并进行统计学分析。结果伤后2周和4周,3组大鼠脊髓前角Slit蛋白均有较高表达,但各组间无显著差异。伤后2周和4周各组Robo-2受体表达均升高,其中老年鼠脊髓前角Robo-2受体表达明显高于成年和幼年组,差异有统计学意义(P0.05)。结论大鼠坐骨神经损伤后能刺激脊髓前角Slit-1高表达,不同年龄大鼠脊髓组织中Robo-2受体表达的差异可能决定了Slit-1在再生神经中的靶向性调节作用。     

HRP逆行示踪评价复合骨髓间充质干细胞的无细胞神经移植物

背景：作者前期将无细胞神经移植物与骨髓间充质干细胞复合培养,成功构建了组织工程人工神经。 目的：应用辣根过氧化物酶(HRP)神经逆行示踪技术对无细胞神经移植物复合骨髓间充质干细胞构建的神经移植复合体桥接大鼠坐骨神经缺损后运动神经元的保护作用进行评价。 方法：成年清洁级健康雄性SD大鼠,随机分成3组：①实验组：采用复合骨髓间充质干细胞的无细胞神经移植物桥接大鼠坐骨神经缺损。②空白对照组：采用无细胞神经移植物桥接大鼠坐骨神经缺损。③自体神经对照组：采用自体神经移植桥接大鼠坐骨神经缺损。术后12周应用辣根过氧化物酶神经逆行示踪技术对脊髓前角运动神经元的再生进行评价。 结果与结论：术后12周脊髓前角运动神经元再生评价结果显示：实验组优于无细胞神经移植物组,而与自体神经移植物组相比差异无显著性意义。证实无细胞神经移植物复合骨髓间充质干细胞构建组织工程人工神经修复大鼠坐骨神经缺损,对大鼠脊髓运动神经元具有保护作用,可能达到与自体神经移植相似的效果。 关键词：无细胞神经移植物;骨髓间充质干细胞;辣根过氧化物酶;神经移植;大鼠     

Modification of astrocytes in the spinal cord following dorsal root or peripheral nerve lesions

Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to monitor the response of astrocytes in the rat spinal cord to either dorsal root or sciatic nerve lesions. Image analysis methods were used to provide a quantitative correlate of the reactive gliosis. Multiple dorsal root section elicited a rapid increase in GFAP immunoreactivity of astrocytes unilaterally within the spinal cord along the pathway of the degenerating dorsal root axons in the dorsal and ventral horns and this gliosis persisted in the dorsal horn beyond the time at which active phagocytosis of degenerative debris occurred. Labeling of proliferating cells using [3H]thymidine revealed that none of the dividing cells contained detectable GFAP, suggesting that the increased GFAP labeling represents primarily a hypertrophy rather than a proliferation of astrocytes. Comparison of animals that had been deafferented in the early neonatal period with those deafferented as adults indicated that the GFAP immunoreactive response persisted following neonatal lesions but that it was markedly less intense than after adult lesions. Sciatic nerve section in adults does not result in extensive frank degeneration but it does evoke a rapid and marked increase in staining of astrocytes both in the dorsal horn and in the ventral horn. Transganglionic changes in GFAP staining in the dorsal horn occur by 3 days post-operatively, which is much earlier than the time of dorsal root ganglion neuron death caused by the sciatic nerve lesion. These experiments indicate that astrocytes can respond to signals from a variety of changes in neurons, including not only Wallerian degeneration, but also retrograde and transganglionic changes.     

Expression of ciliary neurotrophic factor is maintained in spinal motor neurons of amyotrophic lateral sclerosis

Ciliary neurotrophic factor (CNTF) was originally identified as a potent survival factor for a variety of neuronal cell types in vitro and in vivo and in particular in spinal motor neurons of embryonic chick and rat. Using a monoclonal antibody against CNTF (clone 4–68) we analysed the expression of CNTF in paraffin sections of seven human brains and spinal cords immunocytochemically using the ABC method and compared these results with sections of the spinal cords of patients suffering from amyotrophic lateral sclerosis (ALS). In normal human tissue of the central nervous system CNTF immunoreactivity was found in most of the motor neurons of the motor cortex and ventral horn, neurons of the nucleus oculomotorius, intermediolateralis, thoracicus, ependymal cells as well as in smooth muscle cells and endothelial cells of small arteries. A reduced number of astrocytes showed a positive immunocytochemical reaction. In peripheral nerves and nerve roots of the spinal cord we also found a positive staining of Schwann cells and some axons. These immunoreactions could be confirmed by Western blot analyses. Next we analysed postmortem paraffin sections of the spinal cord of seven patients suffering from ALS (age range 30–76 years, median age 46 years, female/male = 4:3). We found CNTF immunoreactivity in most of the motor neurons of the ventral horn in 5 cases. In two cases the number of positively stained motor neurons was less. From these results we conclude that CNTF is expressed in a high number of upper and lower motor neurons in the human CNS and that its expression is maintained in ALS patients.     

Ciliary neurotrophic factor activates spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival.

At focal CNS injury sites, several cytokines accumulate, including ciliary neurotrophic factor (CNTF) and interleukin-1beta (IL-1beta). Additionally, the CNTF alpha receptor is induced on astrocytes, establishing an autocrine/paracrine loop. How astrocyte function is altered as a result of CNTF stimulation remains incompletely characterized. Here, we demonstrate that direct injection of CNTF into the spinal cord increases GFAP expression and astroglial size and that primary cultures of spinal cord astrocytes treated with CNTF, IL-1beta, or leukemia inhibitory factor exhibit nuclear hypertrophy comparable to that observed in vivo. Using a coculture bioassay, we further demonstrate that CNTF treatment of astrocytes increases their ability to support ChAT(+) ventral spinal cord neurons (presumably motor neurons) more than twofold compared with untreated astrocytes. Also, the complexity of neurites was significantly increased in neurons cultured with CNTF-treated astrocytes compared with untreated astrocytes. RT-PCR analysis demonstrated that CNTF increased levels of FGF-2 and nerve growth factor (NGF) mRNA and that IL-1beta increased NGF and hepatocyte growth factor mRNA levels. Furthermore, both CNTF and IL-1beta stimulated the release of FGF-2 from cultured spinal cord astrocytes. These findings demonstrate that cytokine-activated astrocytes better support CNS neuron survival via the production of neurotrophic molecules. We also show that CNTF synergizes with FGF-2, but not epidermal growth factor, to promote DNA synthesis in spinal cord astrocyte cultures. The significance of these findings is discussed by presenting a new model depicting the sequential activation of astrocytes by cytokines and growth factors in the context of CNS injury and repair.     

Bridging sciatic nerve gap using tissue-engineered nerves constructed with neural tissue-committed stem cells derived from bone marrow

BACKGROUND: Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation.OBJECTIVE: To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells (NTCSCs) derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves.DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed in the Anatomical laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006.MATERIALS: Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawiey rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs.METHODS: All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group: the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group: a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group: a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap.MAIN OUTCOME MEASURES: Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase (HRP, 20%) was injected into the gastrocnemius muscle to retrogradely label the L4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method.RESULTS: NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeted neurons in L4-5 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group (P>0.05), but significantly different from the blank nerve scaffold transplantation group (P<0.05). CONCLUSION: NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograff nerve transplantation.     

Effect of nerve section on the spinal distribution of neighboring nerves

The spinal cord distribution of axonal terminals of peripheral nerves that innervate the skin of the upper medial thigh was examined in rats using transganglionic transport of horseradish peroxidase (HRP) and wheat-germ agglutinin-conjugated HRP (WGA-HRP). Chronic transection of the sciatic nerve or both the sciatic and saphenous nerves did not alter this distribution. Therefore, long-distance sprouting of intact 'thigh nerve' afferents in the dorsal horn is apparently not the mechanism whereby spinal dorsal horn neurons deafferented by sciatic and saphenous neurectomy, gain novel receptive fields in the cutaneous distribution of neighbouring intact nerves of the thigh.     

Midkine expression in rat spinal motor neurons following sciatic nerve injury

Midkine (MK), a heparin-binding growth factor, is produced in the developing and damaged nervous system. However, the role of MK in peripheral nerve injury has not been clarified. Here, we investigated MK expression in lumbar spinal motor neurons after rat sciatic nerve injury by immunohistochemical, in situ hybridization, and Western blot analyses. The rat sciatic nerve showed complete degeneration after local freezing. Numerous regenerated myelinated and thin nerve fibers were observed 3 weeks after injury. Intense MK immunoreactivity was detected in the ipsilateral spinal motor neurons of the anterior horn of the lumbar spinal cord after 1 day and in ipsilateral and contralateral spinal motor neurons from 4 days to 1 week after injury. It decreased after 2 weeks and again transiently increased in spinal motor neurons after 3 weeks. MK was found in the motor neurons and axon of the sciatic nerve. However, it was not detected in normal neurons and axon. In situ hybridization showed the expression of MK mRNA in lumbar spinal motor neurons of the anterior horn, but it was not present in Schwann cells or non-neuronal cells. Low-density lipoprotein receptor-related protein (LRP) immunoreactivity, a cell membrane receptor of MK, was observed in anterior horn motor neurons, but receptor-type protein tyrosine phosphatase zeta (PTPzeta) immunoreactivity as a signaling receptor complex of MK was not observed. LRP and PTPzeta immunoreactivities were observed in Schwann cells of the injured and uninjured sciatic nerve. Our findings suggest that MK is synthesized, released, and taken up in anterior horn motor neurons in an autocrine fashion with LRP. MK may have a role in degeneration and regeneration after peripheral nerve injury.