BDNF和NT—3在鸡胚脊髓发育中的表达——免疫组织化学研究

探讨脑源性神经营养因子（BDNF）和神经营养因子3（NT－3）在脊髓发育中的表达。取Hamburger 30期和40期鸡胚腰段脊髓制作20μm厚冰冻切片,分别用BDNF和NT－3抗体行ABC免疫组化染色。观察BDNF、NT－3样免疫阳性反应物（BDNF－IR、NT－3－IR）在两时相脊髓的分布。结果显示：30期组,BDNF－IR主要分布于脊髓腰段腹角神经元的核周质及神经突起内。NT－3－IR则仅限于腹角前群神经元,胞核、胞浆均梁色;40期组,BDNF－IR除腹角染色外,还扩展至整个脊髓肖解神经元及神经突起。另外,亦见一些BDNF－IR的胶质细胞。与之类似,NT－3－IR除分布于腹角前群神经元外,中间带、背角内亦出现了些NT－3阳性神经元。结果表明：BDNF和NT－3在脊髓的表达随发育进程由腹角向背角扩展。提示：BDNF和NT－3的生理作用与脊髓的发育有关。     

背根切断对脊髓后角Ⅱ板层BDNF,NT-3表达的影响——免疫组织化学研究

目的　采用免疫组织化学技术探讨切断背根 (L1)后脊髓Ⅱ板层脑源性神经营养因子 (BDNF)和神经营养因子 3 (NT 3)表达的变化。方法　将成年雄猫 5只行单侧L1背根切断术 (对侧为非手术侧 )。术后 5天取L1脊髓制作2 0 μm厚冰冻切片 ,用BDNF及NT 3抗体分别进行免疫组化染色。观察BDNF、NT 3免疫阳性反应物在脊髓的分布 ,计数单位面积内Ⅱ板层BDNF阳性膨体密度及NT 3阳性细胞数。结果用t检验进行统计分析。结果　BDNF样免疫反应物在Ⅱ板层主要分布于神经膨体 ,NT 3样免疫反应物在神经元及胶质细胞均有分布。背根切断后 ,手术侧Ⅱ板层BDNF阳性膨体数量明显较非手术侧者减少 (P <0 0 1)。而手术侧Ⅱ板层NT 3阳性神经元及胶质细胞数量则较非手术侧者明显增加(P <0 0 1)。结论　背根切断后脊髓Ⅱ板层BDNF ,NT 3的表达发生不同变化。BDNF减少 ,而NT 3表达增多。提示BD NF和NT 3在脊髓损伤修复中的不同作用。     

神经生长因子家族成员NGF、BDNF、NT-3和NT-4在猫L6脊髓的分布

采用免疫组织化学方法观察神经生长因子家庭成员NGF,BDNF、NT3和NT4在成年猫L6脊髓的分布。结果;在L6脊髓灰质均可见四种生长因子的免疫阳性细胞,这些细胞主要是腹角及凝角深部的大神经元及背角浅层的小神经元,灰质内亦内NGF,NT3及NT4阳性的胶质细胞,但数量多少不等,其中NT3者最多,其次是NGF,NT4者最少,此外,Ⅱ板层内还可见较多BDNF及少量NT-3阳性神经膨体,本文结果表明,在成年猫脊髓存在NGF,BDNF、NT3和NT4,但其分布有差异。     

神经生长因子家族成员NGF、BDNF、NT-3和NT-4在猫L6脊髓的分布

采用免疫组织化学方法观察神经生长因子家庭成员NGF、BDNF、NT3和NT4在成年猫L6脊髓的分布.结果在L6脊髓灰质均可见四种生长因子的免疫阳性细胞,这些细胞主要是腹角及背角深部的大神经元及背角浅层的小神经元.灰质内亦内NGF、NT3及NT4阳性的胶质细胞,但数量多少不等.其中NT3者最多,其次是NGF,NT4者最少.此外,Ⅱ板层内还可见较多BDNF及少量NT-3阳性神经膨体.本文结果表明,在成年猫脊髓存在NGF、BDNF、NT3和NT4,但其分布有差异.     

晚妊人胎脊髓内一氧化氮合酶阳性神经元和阳性纤维的分布及其生物学意义

取７例人胎脊髓标本,用还原型辅酶Ⅱ（β－ＮＡＤＰＨ）组织化学方法对人胎脊髓内一氧化氮合酶（ＮＯＳ）阳性神经元和阳性纤维的分布进行了观察。ＮＯＳ阳性神经元在妊娠３２周至３９周胎龄人胎脊髓内的分布和细胞形态无明显差异,主要位于后角深层（Ⅲ、Ⅳ层）、中央管周围灰质和中间带外侧核（ＩＭＬ）;前角内可见少数散在的ＮＯＳ阳性神经元;在脊髓白质内有密集的ＮＯＳ阳性的胶质样细胞分布。ＮＯＳ阳性纤维主要见于后角浅层（Ⅰ、Ⅱ层）和中间带。脊髓内ＮＯＮ阳性神经元和阳性纤维的分布,提示脊髓内ＮＯＳ可能与内脏活动的调节和躯体感觉传入的调制有关;ＮＯＳ阳性的胶质样细胞可能参与白质内神经纤维的髓化过程。     

大鼠脊髓挤压伤后NT-3、NT-4在腹角运动神经元表达的变化

我们前面的研究已证实,神经生长因子和脑源性神经营养因子与成年大鼠挤压性脊髓损伤修复有关。在本研究中,通过免疫组织化学ABC法,我们探讨了挤压伤后不同时间脊髓腹角神经元NT-3和NT-4的表达。结果显示,在对照组,NT-3和NT-4的阳性反应主要分布在脊髓腹角神经元,挤压性脊髓损伤后7天和21天,NT-3阳性神经元的数量较对照组和24小时组明显增加,比较之,损伤后24小时和7天,NT-4阳性神经元的数量已较正常者增多,且NT-T的反应强度21天者较24小时和7天者有增多。结果表明NT-3和NT-4的表达在挤压性损伤后的脊髓腹角神经元被不同程度地上调,提示NT-3和NT-4可能与挤压性脊髓损伤修复有关。     

代谢型谷氨酸受体五种亚型在猫脊髓内的分布—免疫组织化学方法研究

为研究代谢型谷氨酸受体各亚型在猫脊髓内的定位分布特征,本实验采用免疫组织化学方法调查了代谢型谷氨酸受体五种亚型（ｍＧｌｕＲ１,ｍＧｌｕＲ２,ｍＧｌｕＲ３,ｍＧｌｕｒ５及ｍＧｌｕＲ７）在猫脊髓（颈２、颈６、胸５、腰６、骶１－２）内的分布状况。结果如下：（１）在背角深层（Ⅲ－Ⅳ层）和腹角内分布有大量中小型（＜３０μｍ）ｍＧｌｕＲ１样免疫反应阳性神经元,背角浅层（Ⅰ、Ⅱ层）为阴性;（２）ｍＧｌｕＲ２／３样免疫反应阳性产物仅见于背角Ⅱ层内侧部,其它部位为阴性;（３）致密的ｍＧｌｕＲ５样免疫反应阳性产物主要分布于猫脊髓背角Ⅱ层的神经毯内,背角Ⅰ层及深层呈中等密度染色;（４）致密的ｍＧｌｕＲ７样免疫反应阳性产物分布于背角Ⅱ层的神经毯内,背角Ⅰ层及深层（Ⅲ－Ⅳ层）呈中等密度染色。此外,腹角运动神经元和骶髓副交感核内的神经元亦呈现ｍＧｌｕＲ７样免疫反应阳性。单侧切断背根,发现术侧背角浅层内ｍＧｌｕＲ７样免疫反应阳性产物密度略有降低,而ｍＧｌｕＲ５样免疫反应阳性产物密度与对侧基本相同。本研究结果显示代谢型谷氨酸受体五种亚型在所调查的猫脊髓各节段内（颈２、颈６、胸５、腰６、骶１－２）的分布存在差异,提示它们在介导谷氨酸传递的信     

大鼠脊髓后角内神经降压素对一级传入C纤维突触前抑制效应的形态学证据(英文)

本研究的目的是为了揭示大鼠脊髓后角内的神经降压素是否对一级传入C纤维进行突触前调节,荧光显微镜下观察到,在脊髓Ⅰ－Ⅲ层内,和压素样免疫反应性（NTLI）的分布与来源于西非单豆素的同工凝集素Ⅰ－B4（Ⅰ－B）的结合位点到分布有部分重叠,在其聚集激光扫描显微镜下进一步观察显微镜下,在蛛网膜下腔注射辣椒素的大鼠脊髓后角浅层内,一些NTLI阳性终末与变性终末形成突触性和／或非突触性接触。以上结果表明,NT可通过轴轴突触和／或非突触性轴轴接触的一级传入C纤维的传入进行突触前抑制。此外,脊髓后角内还存在变性终末与含NTLI树突形成的轴树突触。     

亨廷顿蛋白相关蛋白1在成年大鼠脊髓中的分布

目的观察亨廷顿蛋白相关蛋白1(huntingtin-associated protein 1, HAP1)在成年大鼠脊髓中的分布特点.方法采用免疫组织化学ABC法和免疫印迹(Western blotting)方法.结果免疫组织化学结果显示,在成年大鼠脊髓中,以背角灰质浅层(Rexed Ⅰ,Ⅱ层)的HAP1免疫反应性最强,阳性细胞最密集,免疫反应产物除分布在胞体外,还大量弥散分布于胞体间的神经毡内;背角深层有部分HAP1免疫反应阳性细胞呈散在分布,中央管周围灰质(Rexed X)内阳性胞体密度和免疫反应性强度仅次于后角浅层,而在脊髓腹角,偶见HAP1免疫反应阳性神经元.此外, Western blotting分析显示,脊髓背角内HAP1表达水平明显高于脊髓前角.结论 HAP1主要分布于大鼠脊髓背角灰质浅层和中央管周围灰质神经元内,提示其可能与痛觉信息一级传入和/或调控有关.     

体外培养大鼠海马细胞白细胞介素－1的表达及缺氧的影响

采用免疫组化方法,观察了体外培养大鼠海马细胞对人重组白细胞介素１β（ｒｈＩＬ－１β）抗血清的免疫反应以及缺氧的影响。结果可见,体外培养的大鼠海马神经元和神经胶质细胞对抗ｒｈＩＬ－１β抗血清均呈弱阳性免疫染色反应,缺氧后免疫染色反应明显增强。经图像分析表明,缺氧后神经元和神经胶质细胞的平均光密度均较缺氧前增加,尤以缺氧２ｈ时增加最明显,继续缺氧４─１２ｈ,神经元和神经胶质细胞的平均光密度又逐渐减弱。以上结果表明,大鼠海马脑区存在抗ｒｈＩＬ－１β抗血清的免疫反应细胞;缺氧使对抗ｒｈＩＬ－１β抗血清的免疫染色反应增强,提示ＩＬ－１β与脑缺氧损伤的调控有关。     

扬子鳄颈髓一氧化氮合酶(NOS)和乙酰胆碱酯酶(AChE)阳性神经元的分布

本实验分别应用还原型尼克酰胺嘌呤二核苷酸脱氢酶（ＮＡＤＰＨ－ｄ）和乙酰胆碱酯酶（ＡＣｈＥ）方法,对扬子鳄颈髓ＮＯＳ和ＡＣｈＥ阳性神经元的分布进行了研究。结果表明：颈髓前角、中央灰质均含有ＮＯＳ和ＡＣｈＥ阳性神经元,颈髓后角有较为丰富的ＮＯＳ和ＡＣｈＥ阳性纤维和终末以及显色淡的ＮＯＳ阳性神经元。     

Effect of Electroacupuncture on Neurotrophin Expression in Cat Spinal Cord after Partial Dorsal Rhizotomy

Neuroplasticity of the spinal cord following electroacupuncture (EA) has been demonstrated although little is known about the possible underlying mechanism. This study evaluated the effect of EA on expression of neurotrophins in the lamina II of the spinal cord, in cats subjected to dorsal rhizotomy. Cats received bilateral removal of L1–L5 and L7–S2 dorsal root ganglia (DRG, L6 DRG spared) and unilateral EA. They were sacrificed 7 days after surgery, and the L6 spinal segment removed and processed by immunohistochemistry and in situ hybridization histochemistry, to demonstrate the expression of neurotrophins. Significantly greater numbers of nerve growth factor (NGF) and neurotrophin-3 (NT-3) positive neurons, brain-derived neurotrophic factor (BDNF) immunoreactive varicosities and NT-3 positive neurons and glial cells were observed in lamina II on the acupunctured (left) side, compared to the non-acupunctured, contralateral side. Greater number of neurons expressing NGF mRNA was also observed on the acupunctured side. No signal for mRNA to BDNF and NT-3 was detected. The above findings demonstrate that EA can increase the expression of endogenous NGF at both the mRNA and protein level, and BDNF and NT-3 at the protein level. It is postulated that EA may promote the plasticity of the spinal cord by inducing increased expression of neurotrophins.     

大鼠脊髓诱发电位与脊髓损伤的关系——Ⅲ.脊髓局部损毁后电位的变化及电位来源分析

本文描述了大鼠脊髓L_1节段后柱、后索、侧索和前角的诱发电位及其损伤后的变化,并观察了切断L_4、L_5脊神经背、腹根与横断高位颈髓对电位的影响,以进行行电位来源分析。结果可见,上述四个区域的诱发电位基本由早反应三相波和晚反应组成。分别电解损毁这些部位后,电位波幅均普遍降低,晚期反应较早反应降低明显。后柱或后索受损对电位影响最大。局部损毁后可见L_1及T_(13)水平的硬膜上电位改变明显,尤其晚反应减弱、波峰平坦。反应时值与潜伏时未见明显改变。切断L_4脊神经背、腹根后、电位基本消失。去大脑对电位未见明显影响。结果表明,刺激坐骨神经诱发的脊髓电位起源于低位腰段传入神经和脊髓内多通路的兴奋传导,在一定程度上受腹根逆行活动的影响,与大脑及脊髓下行传导束活动无直接联系。脊髓诱发电位的幅度与波形改变可作为脊髓损伤的判断指标之一。     

Spatiotemporal Changes of NGF,BDNF and NT-3 in the Developing Spinal Cords of Embryonic Chicken

Spatiotemporal changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the spinal cords of chick embryonic stage day 7 (E7) and day 14 (E14) were examined by using immunohistochemistry and Western blot. Intensive NGF immunoreaction (IR) was detected in the white matter of the spinal cords, while BDNF-IR in perikaryon and neurite, and NT-3-IR in the nucleus and cytoplasm were seen in the neurons of the ventral horn in the gray matter. Comparatively, the expressions for three growth factors have expanded largely into the dorsal horn at E14, and the level of proteins for these growth factors increased significantly in the spinal cords from E7 to E14. Morphological observation showed that the lumbar spinal cords of E7 appeared rectangular, whereas it gave a butterfly shape in the gray matter consisting of the typical ventral horn, dorsal horn and intermediate zone at E14. The present findings indicated that the spatiotemporal changes of NGF, BDNF and NT-3 could be associated to the morphological changes of developing spinal cords, suggesting the possible roles of three growth factors in the development of spinal cords.     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

Expression of neurotrophin receptors trkB and trkC and their ligands in rat adrenal gland and the intermediolateral column of the spinal cord

Neurotrophins and their trk receptors constitute major classes of signaling molecules with important actions in the developing and adult nervous system. With regard to the sympathoadrenal cell lineage, which gives rise to sympathetic neurons and chromaffin cells, neurotrophin-3 (NT-3) and nerve growth factor (NGF) are thought to influence developing sympathetic neurons. Neurotrophin requirements of chromaffin cells of the adrenal medulla are less well understood than those for NGF. In order to provide the bases for understanding of putative functions of neurotrophins for the development and maintenance of chromaffin cells and their preganglionic innervation, in situ hybridization has been used to study the expression of brain-derived neurotrophic factor (BDNF) and NT-3, together with their cognate receptors trkB and trkC, in the adrenal gland and in the intermediolateral column (IML) of the spinal cord. BDNF is highly expressed in the embryonic adrenal cortex and later in cells of the cortical reticularis zone. Adrenal medullary chromaffin cells fail to express detectable levels of mRNAs for BDNF, NT-3, and their cognate receptors trkB and trkC. Neurons in the IML express BDNF and trkB, and low levels of NT-3 and trkC. Our data make it unlikely that BDNF and NT-3 serve as retrograde trophic factors for IML neurons but suggest roles of BDNF and NT-3 locally within the spinal cord and possibly for sensory nerves of the adrenal cortex.     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.