匹罗卡品致(癎)大鼠海马中表达生长抑素的中间神经元数目变化及其轴突出芽

目的 探讨表达生长抑素(SS)的中间神经元在颞叶癫(癎)的发生和自我修复中的作用.方法 建立匹罗卡品致(癎)大鼠模型.免疫组织化学方法检测各时间点海马不同区域SS中间神经元的数目变化及其轴突出芽情况;结合Fluoro-Jade B(FJB)行免疫荧光双标方法特异性检测大鼠癫(癎)持续状态(status epilepticus,SE)后7 d和60 d海马不同区域SS中间神经元及其轴、树突的变性情况.结果 实验组大鼠海马各区Ss阳性神经元数目均在SE后7 d减至最低(门区11.1±3.3,CA1区2.8±0.9,CA1区1.8±0.7,t=13.519、9.644、8.808,均P<0.01),慢性期开始部分恢复,SE后60 d时CA1区SS神经元数目(12.8±1.5)超过对照组(8.8±1.3,t=-4.506,P<0.01),但门区和CA3区SS神经元数目(分别为25.5±4.6和4.8±0.8)仍明显低于正常水平(t=4.691、3.953,P<0.01);sE后30 d大鼠海马回腔隙.分子层(lacunosum-moleculare,lm)和齿状回外分子层出现大量SS染色阳性纤维,60 d时海马CA1区全层均可见大量增多的SS阳性纤维.实验组中SE后7 d的海马CA1区、60 d的CA1、CA1区和门区均可观察到少部分被FJB染色的SS中间神经元胞体及其轴、树突.结论 SS中间神经元的缺失在颞叶癫(癎)的发生中起重要作用,其缺失部分是由于神经元的变性死亡所致;慢性期CA,区SS阳性纤维大量增多,可能在颞叶癫(癎)的发生和自我修复中起重要作用.     

磁共振扩散张量成像对创伤性轴索损伤的诊断应用研究

目的探讨磁共振扩散张量成像（DTI）技术对脑外伤导致轴索结构损伤的诊断应用价值。方法对22例健康志愿者（对照组）和29例创伤性脑损伤患者（TBI组）进行DTI检查,在工作站上处理获得各向异性（FA）图,测量TBI组和对照组半卵圆中心、胼胝体膝部和压部、内囊前肢和后肢及脑桥的FA值。TBI组根据临床格拉斯哥昏迷评分（GCS）分为轻度组（≥13分）和中重度组（≤12分）,并分别与对照组进行FA值比较,将TBI组各兴趣区的FA值与临床GCS评分作相关性分析。结果轻度TBI组半卵圆中心FA值下降,中重度TBI组半卵圆中心、胼胝体膝部和压部及内囊前肢和后肢的FA值不同程度下降,差异均有统计学意义（P＜0．01）。TBI组半卵圆中心、胼胝体膝部和压部及内囊前肢和后肢的FA值与GCS评分呈正相关（P＜0．05）。结论DTI是诊断创伤性轴索损伤的敏感序列。FA值下降能反映神经轴索的损伤及程度,有助于评判TBI的伤情和改善预后。     

The role of motor proteins in establishing the microtubule arrays of axons and dendrites

Neurons generate two distinct types of processes called axons and dendrites, both of which rely on highly organized arrays of microtubules for their growth and maintenance. Axonal microtubules are uniformly oriented with their plus-ends distal to the cell body, while dendritic microtubules are nonuniformly oriented. In neither case are the microtubules attached to the centrosome or any detectable structure that could establish their distinct patterns of polarity orientation. Here I describe several lines of evidence from my laboratory that support a model for the establishment of these microtubule arrays based on microtubule transport by motor proteins. Microtubules destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and rapidly released. The released microtubules are then transported into the developing processes. Early in neuronal development, the microtubules are transported with their plus-ends-leading into the developing axon and into the immature processes that will develop into dendrites. In the case of the developing dendrites, the plus-end-distal microtubules are later joined by a population of microtubules that are transported into these processes with their minus-ends-leading. Implicit in this model is that there are molecular motor proteins that transport microtubules with the appropriate orientation into each type of process. There is precedent for molecular motor proteins transporting microtubules during mitosis, and our results suggest that the same or similar motors are utilized during the development of axons and dendrites after a neuroblast becomes terminally postmitotic.     

Pick’s disease: hyperphosphorylated tau protein segregates to the somatoaxonal compartment

Pick bodies and ballooned cells of Pick’s disease and the neurofibrillary lesions of Alzheimer’s disease are characterized by the presence of hyperphosphorylated microtubule-associated protein tau. Little is known about the mechanisms underlying tau hyperphosphorylation in Pick’s disease and the distribution of abnormal tau in affected neurons. We have used a panel of phosphorylation-dependent (AT270, AT8, AT180, 12E8, PHF-1, AT10 and Tau-1) and phosphorylation-independent anti-tau antibodies (N-tau 5 and 134) to stain brain tissue sections from subjects with Pick’s disease and Alzheimer’s disease. These antibodies labeled Pick bodies and neurofibrillary lesions in a similar way, with the exception of antibody 12E8, which stained a subset of neurofibrillary tangles, but no Pick bodies. Moreover, abundant AT8- and PHF-1-positive neuritic profiles were observed in cortical areas rich in Pick bodies, even in the complete absence of neurofibrillary lesions. Unlike the Gallyas-positive neuropil threads of Alzheimer’s disease, which were of variable diameter and covered by spiny appendages, neuritic profiles of Pick’s disease showed a regular diameter, appeared smooth and were Gallyas-negative. In contrast to Alzheimer’s disease, dendritic branches of neurons containing Pick bodies were not labeled by anti-tau antibodies. In the hippocampus, numerous tau-positive axon terminals were found along dendrites of the polymorphic layer of the dentate gyrus. Our results indicate that tau proteins in Pick’s disease and Alzheimer’s disease share similar phosphorylated residues, with the exception of serine 262, which is phosphorylated in Alzheimer tangles but not in Pick bodies or neuritic profiles. Furthermore, we show that hyperphosphorylated tau segregates to different neuronal compartments in the two diseases, with a somatoaxonal distribution in Pick’s disease and a somatodendritic distribution in Alzheimer’s disease. Received: 7 March 1996 / Revised, accepted: 22 May 1996     

慢病毒介导的shRNA干扰PTEN表达促皮层神经元轴突再生及脊髓损伤修复

 目的 观察慢病毒介导特异性短发夹RNA（short hairpin RNA,shRNA）干扰第l0号染色体同源丢失性磷酸酶张力蛋白基因（phosphatase and tensin homology deleted on chromosome ten,PTEN）表达对皮层神经元轴突再生及脊髓损伤修复的影响。方法 体内实验和体外实验两部分各分四组：阴性对照组（DMEM）、空载慢病毒组（Lenti��control）、空白载体组（Lenti��scramble）、慢病毒介导shRNA组（Lenti��shRNA）。体外神经元转染72 h后,Western blot检测各组PTEN表达情况,免疫荧光检测神经元轴突再生能力;体内载体注射1周后,Western blot检测各组PTEN表达情况;6周后荧光显微镜观察皮质脊髓束穿越脊髓损伤局部周围增强绿色荧光蛋白荧光强度及突触素表达情况。采用大鼠脊髓损伤评分评价大鼠后肢运动恢复情况。结果 慢病毒介导shRNA组体外转染神经元后PTEN表达水平比阴性对照组下降83.75%±2.85%,与其他组比较具有统计学意义（F=4277,P< 0.05）;轴突长度（249.70±10.70）μm,大于阴性对照组（95.71±20.24） μm、空载慢病毒组（97.00 ± 22.82）μm及空白载体组（87.57±19.34）μm,差异具有统计学意义（F=84.74,P< 0.05）;每个神经元一级突起数量（5.800±0.359）个,大于阴性对照组（2.800±0.678）个、空载慢病毒组（2.900±0.389）个及空白载体组（3.000±0.877）个,其差异具有统计学意义（F=16.47,P< 0.05）;神经元突起穿越硫酸软骨素蛋白多糖基质的百分比20.60%±1.80%,大于阴性对照组6.70%±1.45%、空载慢病毒组5.50%±1.69%、空白载体组5.60%±1.77%,其差异具有统计学意义（F=94.90,P<0.05）。慢病毒介导shRNA注射大脑皮层运动区后,第6周大鼠脊髓损伤评分达（13.29±0.42）分,高于阴性对照组（7.00±1.48）分,空载慢病毒组（6.43±1.43）分,空白载体组（6.29±1.22）分,其差异具有统计学意义（F=44.85,P< 0.05）。皮层组织PTEN表达水平下降84.57%±1.87%,损伤中心尾端见绿色荧光,突触素染色阳性面积明显增大。结论 慢病毒介导shRNA下调PTEN基因表达后可明显提高脊髓损伤后轴突再生能力,促进神经功能修复。     

Rho/ROCK Ⅱ特异抑制性小分子多肽对脊髓损伤微环境下新生大鼠背根节神经元轴突再生的实验研究

目的 观察Rho/ROCK Ⅱ特异抑制性小分子多肽在脊髓损伤(SCI)微环境下对新生大鼠背根节神经元(DRGNs)轴突生长的影响. 方法 取健康雌性SD成年大鼠5只,按WD法制成T9平面以下截瘫模型,术后7d取T8-10节段脊髓制作截瘫大鼠脊髓提取液.取新生SD大鼠背根神经节经酶解消化、机械吹打、离心、重悬、纯化,进行原代培养观察.DRGNs体外培养5d后随机分组加入不同物质共同培养:A组:DRGNs +60 μL PBS;B组:DRGNs+60 μL截瘫大鼠脊髓提取液;C组:DRGNs+60 μL截瘫大鼠脊髓提取液+20 μL脂质体;D组:DRGNs+ 60μL截瘫大鼠脊髓提取液+20 μL脂质体+不同量多肽(2、4、6、8、10、12 μg).不同环境下共同培养2d后行免疫荧光,测量神经轴突长度和轴突远端平均荧光密度. 结果 B、C组平均轴突长度和荧光密度均小于其他组,差异有统计学意义(P＜0.05),而B、C组间差异无统计学意义(P＞0.05).8μg多肽组平均轴突长度增长最明显,平均荧光密度最大,与其他多肽组比较差异均有统计学意义(P＜0.05);2 μg多肽组与4μg多肽组的平均轴突长度和荧光密度比较差异均无统计学意义(P＞0.05);6μg多肽组、10 μg多肽组、12μg多肽组的平均轴突长度和荧光密度比较差异均无统计学意义(P＞0.05). 结论 Rho/ROCKⅡ特异抑制性小分子多肽能促进SCI微环境中DRGNs轴突生长,当多肽含量为8μg时作用最明显.     

切断后轴突再生方式和速度的初步研究

目的 建立离体中枢神经系统轴突机械切断模型,观察机械切断后轴突的再生方式和速度. 方法 离体培养新生小鼠皮质神经块,免疫荧光特异染色标记轴突和树突.机械切断轴突建立模型.通过追踪黏附在轴突残段表面细胞点的移动来观察轴突再生方式,并测量再生轴突的生长速度. 结果 (1)在机械切断小鼠皮质神经块轴突后,可见再生轴突从断端长出,分布在轴突残段上的细胞点随着轴突的再生而越过切痕散布到远端.(2)切断组再生轴突在第24,48小时的生长速度分别为(118±32)μm/d、(72±41 )μm/d,而对照组未切断轴突生长速度分别为(41±17)μm/d、(32±19)μm/d. 结论 切断后轴突再生方式是轴突残段生长延伸,而非轴突残端出芽生长,并且切断后再生轴突生长速度快于未切断轴突.     

自体激活雪旺细胞移植治疗急性脊髓损伤的实验研究

目的 探讨自体激活雪旺细胞（autologous activated Schwann cells,AASCs）移植治疗急性脊髓损伤的疗效.方法 通过结扎单侧隐神经从而激活自体雪旺细胞并进行体外分离、培养及纯化,测定其不同培养时期培养基中神经生长因子、脑源性神经营养因子含量的变化.Wistar大鼠90只,以纽约大学脊髓损伤打击器建立T10急性脊髓损伤模型.随机分为3组,每组30只：单纯DMEM移植对照组、自体未激活雪旺细胞（autologous Schwann cells,ASCs）移植组和AASCs移植组.对各组实验动物脊髓损伤后肢体功能的恢复情况进行行为学评分（BBB评分）、体感诱发电位与运动诱发电位（somatosensory evoked potential and motor evoked potentials,SEP ＆ MEP）、生物素标记的葡聚糖胺示踪皮质脊髓束（corticospinal tract,CST）观察,比较各组差异.结果 BBB评分4周以后组间比较差异有统计学意义（P＜0.05）,其中细胞移植组明显高于对照组（P＜0.05）;SEP、MEP潜伏期和波幅值8、12周后组间比较差异有统计学意义（P＜0.05）;CST标记距离损伤中心向头端方向0.6、1.2、1.8、2.4mm和距离损伤中心向尾端方向0.6、1.2、1.8mm组间比较差异有统计学意义（P＜0.05）.结论 AASCs可分泌大量神经营养因子,并能明显促进急性脊髓损伤后的轴突再生和肢体功能恢复.     

Early stage of re-innervation of frog slow muscle fibres

Summary Pyriformis muscles of Rana temporaria were denervated by crushing the sciatic nerve. One to three months later most slow fibres were re-innervated by motor axons with conduction velocities above 5 m/sec. Since normal slow fibres were never found to have motor axons conducting faster than 5 m/sec it is concluded that, at an early stage, slow fibres are re-innervated non-selectively by axons of the fast type.Supported by the Deutsche Forschungsgemeinschaft, Bad-Godesberg (Sonderforschungsbereich 38 Membranforschung).     

A type of apparently axonless granule cell in the cat auditory cortex

Summary In the auditory areas (A I, A II and the posterior ectosylvian gyrus) of the cat, unusually small granule cells apparently lacking an axon are described. They are exclusively located in layer 2/3 and sometimes impregnate in groups of 4 or 5, being probably quite frequent. Some morphological differences between granule cells of young and adult cats are noted.