坐骨神经近侧端损害的神经电生理诊断意义

目的:探讨坐骨神经近侧端损害的神经电生理诊断意义.方法:对68例坐骨神经损害患者进行坐骨神经近侧端和远侧端的运动神经传导速度(MCV)检测比较,同时检测54例患肢H-反射以及坐骨神经支配肌肉的肌电图(EMG).结果:坐骨神经近侧端损害54例(79%),坐骨神经远侧端损害16例(23.5%),两者比较差异有显著意义(P相似文献   

周围神经损伤后Tropic 1808基因表达蛋白的免疫组织化学研究

目的 研究Tropic 1808基因表达蛋白在受损大鼠坐骨神经中的表达与分布。方法 用酶联间接法和双重免疫荧光法进行免疫组织化学染色。并通过计算机图像处理技术，测量阳性区域的强度与面积。结果 损伤神经的近侧端和远侧端中施万细胞均有Tropic 1808基因表达蛋白的阳性免疫反应，阳性免疫反应物主要分布于施万细胞膜上，远侧端的阳性反应物强度和面积强于和大于近侧端。结论 周围神经损伤后，Tropic 1808基因表达蛋白分布于损伤近侧端和远侧端的施万细胞膜上，并且远侧端中该蛋白的表达强于近侧端。     

大鼠坐骨神经干内S—100蛋白检测

用Ｗｅｓｔｅｍ Ｂｌｏｔ方法确认周围神经于内含有Ｓ－１００，然后运用免疫组化ＰＡＰ方法对Ｗｉｓｔａｒ大鼠坐骨神经的近侧和远侧进行Ｓ－１００检测，染色结果用电子图像进行系统分析处理。结果显示：Ｓ－１００分布于周围神经束间膜，雪旺氏细胞浆内及细胞膜上，神经轴内未见有Ｓ－１００存在，图像处理结果证实，正常大鼠坐骨神经干内的Ｓ－１００呈均匀分布。     

生长相关蛋白在损伤坐骨神经中的表达

目的：研究大鼠周围神经损伤与再生过程中生长相关蛋白的表达。方法：取正常SD大鼠坐骨神经和离断损伤后不同时间近侧端和远侧端神经组织，采用Anti-GAP－43抗体以Western blot方法检测GAP－43的表达变化。结果：NC膜上43kDa位置出现阳性反应条带，在正常坐骨神经中反应微弱，损伤后明显加强，随损伤时间延长，神经近侧端的反应无明显改变，神经元侧端的反应逐渐加强，以第3周为最强，之后逐渐减弱，结论：GAP－43在正常坐骨神经中低表达，损伤后近侧端与远侧端表达均增加，远侧端的表达增加以第3周为高峰。     

大鼠神经垂体催产素能神经元内神经分泌颗粒的免疫电镜观察

目的　观察大鼠神经垂体催产素（ＯＴ）能神经元轴突及终末内颗粒囊泡形态学及免疫细胞化学特点。方法　应用电镜包埋后免疫细胞化学方法,对大鼠神经垂体进行ＯＴ免疫染色。结果　在ＯＴ能神经元轴突终末内,可见ＯＴ免疫反应性的致密核芯大颗粒囊泡和无ＯＴ免疫反应性的清亮小泡存在。部分囊泡与轴膜靠近,并可见颗粒囊泡与轴膜融合形成的胞吐现象;在轴突非终末部分,也可见ＯＴ免疫反应性的颗粒囊泡,散在分布轴浆内,还可见无ＯＴ免疫反应性的颗粒囊泡,呈散在或聚集分布,两种囊泡均可位于微管之间。结论　位于轴突和轴突终末内的囊泡,在形态学和ＯＴ免疫染色方面有明显差异,推测它们在功能上也有不同。     

神经垂体多肽激素分泌颗粒释放形式和转运途径的形态学研究

目的探讨神经垂体多肽激素分泌颗粒的释放形式和在细胞外正常转运途径的结构基础。方法对大鼠神经垂体进行光镜、透射电镜和扫描电镜观察。结果神经垂体主要由无髓神经纤维、垂体细胞及富含毛细血管的结缔组织组成。毛细血管内皮为有孔型(50nm)，外有基膜与血管周隙分隔。完整的大型膜包分泌颗粒(100～300nm)不仅大量存在于无髓神经纤维内，而且也见于血管周隙内。垂体囊由单层扁平上皮细胞和上皮下结缔组织构成，囊上皮细胞间存在许多不规则的囊上皮孔(2～5μm)，上皮孔附近经常可见分泌颗粒。结论这些结构特征提示，神经垂体多肽激素或分泌颗粒的释放形式，存在一种连同颗粒被膜的整体释放；释放入血管周隙内的多肽激素或分泌颗粒，更易经组织间隙、囊上皮孔进入脑脊液发挥旁分泌作用，而非直接通过毛细血管壁进入血液。     

pro-BDNF在初级感觉神经元内的顺行和逆行输送

摘　要　研究表明脑源性神经营养因子(BDNF)在神经元内可顺行运输至末梢并释放到下一级神经元参与突触可塑性等功能。而成熟的BDNF由其前体分子(pro -BDNF)经蛋白酶水解形成。体外研究表明pro -BDNF可以激活神经营养因子受体TrkB并导致TrkB的磷酸化,但pro -BDNF在体内的作用目前仍不清楚。本文拟探讨内源性pro -BDNF在周围神经的运输。将大鼠坐骨神经结扎或压榨背根制成模型,动物存活不同时间后取坐骨神经、背根神经节和脊髓进行pro -BDNF免疫组织化学染色检测。结果显示:pro- BDNF免疫阳性产物沉积于坐骨神经和背根损伤处的近侧端和远侧端, 24h达高峰,近侧端可持续达 7d而远侧端可达3d;在坐骨神经的近侧端和远侧端、背根神经节和脊髓可检测到全分子量大小的完整pro -BDNF;在转染pro- BDNF质粒后,PC12细胞内可见pro -BDNF免疫阳性产物分布于胞体和突起。这些结果提示pro- BDNF可以象成熟BDNF一样在感觉神经内顺行和逆行运输,并可由神经元分泌、释放而发挥生理作用。     

神经元来源细胞外囊泡促进神经干细胞的神经生成

背景：已有研究表明，神经干细胞能够分化为神经元、星形胶质细胞和少突胶质细胞等。间充质干细胞来源细胞外囊泡被证实能够透过血脑屏障到达中枢神经损伤部位，促进神经修复。然而，神经元来源细胞外囊泡是否促进神经干细胞向有益于神经生成的方向分化，帮助神经修复，还不是很明确。目的：探究神经元来源细胞外囊泡是否有利于神经干细胞向神经生成的方向分化。方法：用胰酶消化法从新生SD大鼠大脑皮质中提取神经元和神经干细胞。收集培养神经元的细胞上清，提取神经元来源细胞外囊泡。将培养10 d的神经干细胞与神经元来源细胞外囊泡或PBS共培养7 d，采用免疫印迹、免疫荧光和RT-qPCR技术分别检测神经元、神经干细胞、少突胶质细胞和星形胶质细胞特异性标志蛋白的表达。结果与结论：神经干细胞与神经元来源细胞外囊泡共培养后，高表达神经元特异性蛋白β3微管蛋白、神经丝蛋白200和少突胶质细胞特异性蛋白髓鞘碱性蛋白，低表达星形胶质细胞特异性标志蛋白胶质纤维酸性蛋白。这些结果说明，神经元来源细胞外囊泡能够促进神经干细胞向神经元和少突胶质细胞分化，抑制其向星形胶质细胞分化。     

大鼠坐骨神经钳伤后变性和再生过程中S—100蛋白的变化

选用Ｗｉｓｔａｒ雌性大鼠４０只，分设正常，神经损伤，损伤对照组，运用免疫组化ＰＡＰ方法对神经干内的Ｓ－１００蛋白检测。染色结果经形态学观察及电子计算机图像处理系统分析结果表明：坐骨神经干内Ｓ－１００蛋白含量与损伤神经再生时间有密切关系，并观察到损伤第５，７天，损伤神经侧Ｓ－１００轴浆转运现象，推测Ｓ－１００可能参于了周围神经再生微环境的变化，从而影响损伤神经元的存活和神经纤维的再生。     

应激性溃疡胃壁胆碱能神经超微结构变化

目的：探讨脑血管病发生时应激性溃疡的发病机理及防治。方法：采用透射电镜和组化电镜技术，观察了１０例应激性胃溃疡病人及４例正常人胃壁的胆碱能神经的超微结构，用多功能医学图像分析系统检测了神经终末的囊泡数目和大小，进行统计学处理。结果：组化电镜下乙酰胆碱酯酶（ＡＣｈＥ）阳性反应物以散在颗粒形式出现在神经纤维内，其大小不等，直径约６０ｎｍ。神经终末有３种囊泡：无颗粒、有颗粒和大暗颗粒囊泡。检测无颗粒囊泡数目，病人的（９９％）高于正常人的（７６％），Ｐ＜０．０１。结论：应激性溃疡发病与胆碱能神经密切相关     

Real-time imaging of Rab3a and Rab5a reveals differential roles in presynaptic function

We investigated the roles of two Rab-family proteins, Rab3a and Rab5a, in hippocampal synaptic transmission using real-time fluorescence imaging. During synaptic activity, Rab3a dissociated from synaptic vesicles and dispersed into neighbouring axonal regions. Dispersion required calcium-dependent exocytosis and was complete before the entire vesicle pool turned over. In contrast, even prolonged synaptic activity produced limited dispersion of Rab5a. A GTPase-deficient mutant, Rab3a (Q81L), dispersed more slowly than wild-type Rab3a, and decreased the rate of exocytosis and the size of the recycling pool of vesicles. While overexpression of Rab3a did not affect vesicle recycling, overexpression of Rab5a reduced the recycling pool size by 50%. We propose that while Rab3a preferentially associates with recycling synaptic vesicles and modulates their trafficking, Rab5a is largely excluded from recycling vesicles.     

On the noradrenaline loading in axonal amine storage granules in rat crushed sciated nerves.

The increase of NA in rat sciatic nerve above a crush was investigated. The transported amounts of NA did not increase quite linearly with time, but more NA was found above the crush at 6 and 12 h than would be expected from the 3 h value. One possible reason for this phenomenon--an increased NA loading of the accumulated axonal granules--was investigated by 2 types of double crush experiments. One type involved simultaneous double crushes 1-1.5 cm apart. The increase in NA in the isolated segment 6 h after crushing indicated that the axonal amine storage granules had increased their NA load by about 70%. In the second type ("delayed double crushes") a distal crush was made 6 h before a second crush, 1-1.5 cm proximal to the first crush. 1-12 h after the second high crush the NA content of the isolated segment was assayed. The results indicated an increased NA content in the axonal granules of 75% already 3-4 h after the isolation of the segment, remaining constant up to 9 h after the second crush. The results indicate that axonal storage granules may increase their NA content by a factor of about 2 (1.7) while being transported distally in the axons. This information together with the information from the preceding article of a mobile NA fraction of 45%, was used to calculate the rate of transport of NA granules proximal to a crush. The value obtained was 9 mm/h which is in good agreement with the value obtained for transport distal to a crush (8 mm/h) in the preceding article.     

Axonal transport of synapsin I- and cholinergic synaptic vesicle-like material; further immunohistochemical evidence for transport of axonal cholinergic transmitter vesicles in motor neurons

The axonal transport of organelles in motor axons in the sympathectomized rat sciatic has been studied using two antisera which recognize specific components of synaptic vesicles. Anti-synapsin I recognizes synapsin I (SYN I) which is affiliated with the external membrane of synaptic vesicles, while rabbit-anti-synaptic vesicle antiserum (RASVA) recognizes integral membrane glycoproteins in cholinergic synaptic vesicles. Immunofluorescence studies, including cytofluorimetric scanning, show that immuno-reactive (IR) material recognized by both antisera: (1) rapidly accumulate proximal to a crush; (2) the material has a granular appearance in the microscope; (3) is redistributed in an isolated segment, and (4) that the transport of the material is sensitive to vinblastine. Thus the proximodistal transport has the characteristics of fast axonal transport. Furthermore, recycling organelles, accumulating on the distal side of a crush are recognized by RASVA, but carry only very little SYN I-IR. The results give further support to the hypothesis that motor cholinergic axons transport axonal cholinergic vesicles towards the motor endplates.     

On the Noradrenaline Loading in Axonal Amine Storage Granules in Rat Crushed Sciated Nerves1

The increase of NA in rat sciatic nerve above a crush was investigated. The transported amounts of NA did not increase quite linearly with time, but more NA was found above the crush at 6 and 12 h than would be expected from the 3 h value. One possible reason for this phenomenon—an increased NA loading of the accumulated axonal granules—was investigated by 2 types of double crush experiments. One type involved simultaneous double crushes 1-1.5 cm apart. The increase in NA in the isolated segment 6 h after crushing indicated that the axonal amine storage granules had increased their NA load by about 70%. In the second type (“delayed double crushes”) a distal crush was made 6 h before a second crush, 1-1.5 cm proximal to the first crush. 1–12 h after the second high crush the NA content of the isolated segment was assayed. The results indicated an increased NA content in the axonal granules of 75% already 3–4 h after the isolation of the segment, remaining constant up to 9 h after the second crush. The results indicate that axonal storage granules may increase their NA content by a factor of about 2 (1.7) while being transported distally in the axons. This information together with the information from the preceding article of a mobile NA fraction of 45%, was used to calculate the rate of transport of NA granules proximal to a crush. The value obtained was 9 mm/h which is in good agreement with the value obtained for transport distal to a crush (8 mm/h) in the preceding article.     

Axonal transport of ribonucleoprotein particles (vaults).

RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the beta-subunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure-in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general.     

Synapsin dispersion and reclustering during synaptic activity.

Presynaptic modulation of synaptic transmission provides an important basis for control of synaptic function. The synapsins, a family of highly conserved proteins associated with synaptic vesicles, have long been implicated in the regulation of neurotransmitter release. However, direct physiological measurements of the molecular mechanisms have been lacking. Here we show that in living hippocampal terminals, green fluorescent protein (GFP)-labeled synapsin Ia dissociates from synaptic vesicles, disperses into axons during action potential (AP) firing, and reclusters to synapses after the cessation of synaptic activity. Using various mutated forms of synapsin Ia that prevent phosphorylation at specific sites, we performed simultaneous FM 4-64 measurements of vesicle pool mobilization along with synapsin dispersion kinetics. These studies indicate that the rate of synapsin dispersion is controlled by phosphorylation, which in turn controls the kinetics of vesicle pool turnover. Thus synapsin acts as a phosphorylation-state-dependent regulator of synaptic vesicle mobilization, and hence, neurotransmitter release.     

The axonal transport motor ‘kinesin’ is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat

Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4–12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38–IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon.     

The axonal transport motor 'kinesin' is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat.

Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4-12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38-IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon.     

Endocytic vacuoles formed following a short pulse of K+ -stimulation contain a plethora of presynaptic membrane proteins

It is now well established that the membrane of synaptic vesicles is recycled following exocytosis. However, little is known concerning the identity of the primary or secondary endocytic structures and their molecular composition. Using cultured rat cerebellar granule cells we combined uptake of horseradish peroxidase as a fluid phase marker and immunogold labeling for a variety of presynaptic proteins to assess the molecular identity of the stimulation-induced endocytic compartments. Short periods (5 or 30 s) of stimulation with 50 mM KCl were followed by periods of recovery for up to 30 min. Stimulation resulted in the formation of horseradish-peroxidase-filled vacuoles in the axonal varicosities as the apparent primary endocytic compartment. Horseradish peroxidase-filled synaptic vesicles were formed when stimulated cells were allowed to recover in horseradish peroxidase-free culture medium. Horseradish peroxidase-filled vacuoles as wells as vesicles contained the synaptic vesicle membrane proteins VAMP II, synaptotagmin, SV2, and synaptophysin, the vesicle-associated proteins rab 3A and synapsin I, and in addition SNAP-25. No incorporation of vesicle proteins into the plasma membrane was observed. Horseradish peroxidase-filled vesicles and vacuoles generated on incubation of unstimulated granule cells with horseradish peroxidase for prolonged periods of time were equally immunolabeled. Renewed stimulation of prestimulated granule cells with either 100 mM KCl or 30 microM Ca2+ ionophore A23187 resulted in a reduction of horseradish peroxidase-filled vacuoles suggesting that the vacuolar membrane compartment was exocytosis-competent. Our results suggest that varicosities of cultured cerebellar granule cells possess a fast stimulation-induced pathway for recycling the entire synaptic vesicle membrane compartment. The primary endocytic compartment represents not a synaptic vesicle but a somewhat larger vesicle protein-containing vacuolar entity from which smaller vesicles of identical protein composition may be regenerated. Endocytic vacuoles and synaptic vesicles share membrane and membrane-associated proteins and presumably also major functional properties.