大鼠羊膜上皮细胞移植对损伤脊髓生长相关蛋白43表达的影响

目的:探讨羊膜上皮细胞(AECs)移植对大鼠损伤脊髓生长相关蛋白43(GAP-43)表达和神经功能恢复的影响.方法: 取E12d～14d的SD大鼠AECs体外培养;改良-Allen撞击法制备大鼠脊髓损伤(SCI)模型,并随机分成假手术组、SCI+生理盐水对照组和SCI+AECs移植组;分别于术后第1、3、7、14和28天通过BBB 神经行为学评分法对神经功能进行评估,观察其恢复状况,并采用免疫组织化学和免疫印迹法检测脊髓组织GAP-43表达的变化.结果: GAP-43的表达于脊髓损伤后第7天显著增加,AECs移植后损伤脊髓中的GAP-43持续高表达至第28天,而盐水对照组的表达于术后14d左右逐渐恢复至正常水平.与盐水对照组相比较,AECs移植后2～4周脊髓损伤大鼠的后肢运动功能得到一定的改善.结论: AECs移植后可使损伤大鼠脊髓GAP-43的表达增高,并在一定程度上促进了大鼠后肢运动功能的恢复.     

银杏酮酯对大鼠坐骨神经损伤后生长相关蛋白43表达的影响

目的:研究银杏酮酯对大鼠坐骨神经损伤后生长相关蛋白43(GAP-43)表达的影响。方法:SD大鼠78只,随机分成正常组、损伤对照组与实验组,给予不同处理,后两组切断右侧坐骨神经并缝合。实验组给予银杏酮酯200mg·kg-1.d-1溶于1ml生理盐水中灌胃,损伤对照组给予生理盐水1ml灌胃,正常组不做处理。分别于术后1、3、7、14、21及28d取吻合口远段的神经、相应节段的脊神经节及脊髓,应用免疫组织化学和图像分析的方法研究所取组织中GAP-43蛋白的表达并进行定量分析。结果:实验组坐骨神经、脊神经节及脊髓中GAP-43蛋白免疫阳性区域面积和平均光密度值在术后7、14和21d明显高于对照组。结论:大鼠坐骨神经损伤后用银杏酮酯治疗,在早期可促使坐骨神经及相应节段脊神经节和脊髓组织中的GAP-43蛋白表达增加。     

α-硫辛酸对大鼠全横断脊髓损伤后GAP-43和Caspase-3表达的影响

目的:研究α-硫辛酸对脊髓全横断损伤(SCI)大鼠损伤部位神经生长相关蛋白(GAP-43)、神经细胞凋亡相关蛋白(Caspase-3)的表达,探讨α-硫辛酸对大鼠脊髓全横断损伤功能恢复的作用。方法:制作并评价SD大鼠SCI模型后,64只大鼠随机分为假手术组、脊髓损伤组(SCI组)、SCI+游泳训练组(游泳组)和SCI+α-硫辛酸组(硫辛酸组)。各组按手术后7、14、21、28 d等4个时间点收集标本,每个时间点大鼠均为4只。各组大鼠进行BBB评分。各组4个时间点的GAP-43和Caspase-3表达用免疫组化测定,同时用Western Blot检测GAP-43表达。结果:游泳训练和用硫辛酸均能提高脊髓损伤大鼠的BBB评分(P0.05)。与SCI组比较,游泳组和硫辛酸组GAP-43表达显著增加,Caspase-3表达则明显降低(P0.05),且硫辛酸组比游泳组的GAP-43表达增加和Caspase-3表达降低更为明显(P0.05)。结论:游泳训练和α-硫辛酸可以上调脊髓损伤大鼠GAP-43表达和抑制Caspase-3表达促进损伤神经修复,对改善大鼠运动功能有一定疗效。     

GAP-43治疗大鼠脊髓横断后神经中丝NF200表达的变化

通过制备成年SD大鼠完全性脊髓损伤模型,研究生长相关蛋白(GAP-43)治疗大鼠脊髓损伤后神经中丝(NF200)表达的变化,探讨GAP-43在再生修复中的作用,为临床治疗提供实验依据。实验采用雌性8周龄SD大鼠75只,制成脊髓损伤模型后随机分为三组:GAP-43抗体组、GAP-43抗原组和对照组,每组25只。使用直接注射法将GAP-43抗原和GAP-43多克隆抗体分别注入抗原组和抗体组的大鼠脊髓的断端,对照组仅切断脊髓而不给药,最后观察各组大鼠肢体功能的恢复情况。用BBB评分法对不同时段大鼠的行为学表现进行评分,用HE染色及免疫组化染色观察NF200的表达,并对其进行相关性分析。结果发现对照组在不同的时间段行为学评分最低和抗原组评分最高,脊髓损伤区病理改变明显好转,NF200的表达呈进行性增高,且前角神经元NF200的表达早于后角神经元。抗体组早期恢复出现明显的停滞状态,但停药后能很快恢复。说明GAP-43能促进损伤脊髓的恢复,而抗体对损伤脊髓恢复的影响是可逆的,这对于脊髓再生的研究是一种值得探讨的新方法,对进一步探索脊髓损伤的治疗具有重要的意义。     

臂丛损伤脊髓运动神经元与神经根GAP-43 mRNA表达

目的：探讨臂丛根性撕脱伤后脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达变化及其影响因素,为臂丛损伤的修复治疗提供理论依据.方法：本实验创立三种臂丛根性撕脱伤模型：C7前根撕脱（Ⅰ组）;C7前根撕脱＋切断同侧C5～T1后根（Ⅱ组）;C7前根撕脱＋C5和C6之间作同侧脊髓半横断（Ⅲ组）.术后2周按CBS评分标准检查动物神经缺失症状,用SYBR Green荧光定量RT-PCR方法检测脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达改变.结果：根据CBS评分标准,对照组计为0分,Ⅰ组计分较低、Ⅲ组计分最高.对照组C7神经元胞体和C7神经根中GAP-43 mRNA表达量相近,但三种损伤组术后2周神经元胞体内GAP-43 mRNA表达均上调,而神经根内表达却下调.结论：（1）臂丛根性撕脱伤后脊髓腹角运动神经元胞体GAP-43 mRNA表达受突触前机制的调控;（2）臂丛损伤2周时神经元胞体内GAP-43 mRNA表达呈现高峰期,此时进行神经移位术将显著提高神经修复的效果.     

依赖性膜磷脂结合蛋白AnnexinⅡ对大鼠脊髓横断损伤的修复作用

目的在体内实验中观察钙依赖性膜磷脂结合蛋白AnnexinⅡ对大鼠脊髓损伤以及神经干细胞移植的作用。方法在成年大鼠全横断脊髓损伤的局部微量注射AnnexinⅡ,观察其对损伤的脊髓神经细胞存活、神经纤维生长和损伤后的功能恢复的影响,以及对联合移植的胚胎神经干细胞移植后存活和迁移的影响。结果在动物脊髓横断损伤6周,神经干细胞移植4周后,损伤局部微量注射AnnexinⅡ＋NSC移植组实验动物的运动评分结果较空白对照组和单独进行NSC移植组都有明显增高（JP〈0．01）,脊髓损伤处的空洞面积也较上述两组显著减小（P〈0．01）;注射AnnexinⅡ＋NSC组损伤节段以上荧光金标记神经细胞数目较空白对照组明显增多（P〈0．01）。结论AnnexinⅡ蛋白可能具有减小局部损伤空洞面积,保护损伤的神经元和神经纤维并防止其走向变性和死亡的效应,并对神经干细胞的移植效应有一定的促进和协同作用,使脊髓损伤大鼠的功能得到一定程度的恢复。     

糖尿病大鼠海马生长相关蛋白GAP-43表达的研究

目的:确定链脲佐菌素(STZ)诱导的糖尿病大鼠早期海马区生长相关蛋白(GAP-43)的表达,研究GAP-43在糖尿病时海马组织中的变化并讨论其意义。方法:SD雄性大鼠16只分为对照组和糖尿病组。用STZ制作糖尿病大鼠模型,饲养5 d,然后测血糖以确定模型大鼠成功。4周后进行灌注固定,按要求进行组织学病理检查和免疫组织化学方法观察糖尿病组和对照组的大鼠海马CA1、CA2、CA3和CA4区GAP-43的表达情况。结果:与对照组大鼠比较,糖尿病大鼠血糖明显升高(P0.05),部分锥体细胞的胞体明显增大,核显著着色;海马各区GAP-43的表达显著,特别是CA1和CA3区的表达增高更为显著(P0.05),同时海马各区GAP-43阳性颗粒平均灰度值明显降低(P0.05)。结论:结果表明,在糖尿病大鼠早期海马中GAP-43的表达发生了改变,提示大脑出现了实质性损伤并伴有再生,而这些改变主要发生在记忆相关的CA1和CA3区,这对研究糖尿病性神经病变具有重要意义。     

微囊化异体坐骨神经组织细胞移植对大鼠脊髓损伤后c-fos表达的影响

目的探讨微囊化异体坐骨神经组织细胞移植对大鼠脊髓损伤后c-fos表达的影响。方法95只SD大鼠随机分为4组,A组为微囊化坐骨神经细胞移植组（30只）;B组为坐骨神经细胞移植组（30只）;C组为单纯损伤对照组（30只）,仅用明胶海绵填塞SCI腔隙;D组为正常对照组（5只）,仅打开椎管,暴露脊髓不作移植。用免疫组织化学的方法观察移植术后6h-14d脊髓中c-fos基因的表达情况。结果脊髓半横断损伤后对照组脊髓中6h、12h内表达c-fos阳性的神经元数增多,平均光密度值升高,至24h又明显增强。而A组脊髓后角内的c-fos表达明显低于同时间点的B组和C组（P〈0．05）。结论微囊化坐骨细胞移植可能降低c-fos的表达,并在一定程度上减轻脊髓的继发性损伤。     

督脉电针与神经干细胞移植联合应用促进大鼠受损伤脊髓组织产生神经生长活性物质

目的探讨督脉电针与神经干细胞移植联合应用能否促进受损伤脊髓组织产生神经生长活性物质。方法成年大鼠分为正常组、对照组、神经干细胞移植组（NSCs组）、督脉电针组（电针组）和督脉电针＋神经干细胞移植组（电针NSCs组）。除正常组外均实施T10段脊髓全横断手术,其中电针组和电针NSCs组于术后5d进行电针治疗。分别于术后14d和28d取材,进行受损伤脊髓组织环-磷酸腺苷（cAMP）含量的放射免疫检测;用Westen blottlng检测神经营养素-3（NT-3）、神经营养因子受体（Trk）以及生长相关蛋白-43（GAP-43）的水平。结果 1．在术后14d,NSCs组、电针组和电针NSCs组的脊髓损伤区cAMP含量较正常组有增加。但除电针组外,脊髓损伤区的上段或下段组织cAMP含量低于正常组。在术后28d,电针NSCs组脊髓损伤区上段、下段组织cAMP含量仍保持较高水平。NSCs组和电针NSCs组的脊髓损伤区cAMP含量较正常组有所增加。2．NT-3、Trk及GAP-43的表达以电针组和电针NSCs组较为明显,对照组和NSCs组的3种蛋白表达均低于电针组和电针NSCs组。结论督脉电针能促进受损伤的脊髓组织产生cAMP;督脉电针与NSCs移植联合应用能保持受损伤的脊髓组织较高水平的cAMP,而且NT-3、Trk及GAP-43的含量也有增高的趋势。     

腺病毒介导CNTF基因转染嗅鞘细胞移植对视神经损伤大鼠GAP-43表达影响

目的:通过检测大鼠视神经损伤后生长相关蛋白(GAP-43)的表达变化观察腺病毒介导睫状神经营养因子(ciliary neurotrophic factor,CNTF)基因转染的嗅鞘细胞(olfactory ensheathing cells,OECs)移植对颅内段视神经损伤后神经纤维长距离再生的促进作用.方法:建立额下显微外科入路大鼠颅内段视神经损伤动物模型并分组,构建腺病毒介导CNTF基因转染的嗅鞘细胞并移植入动物眼玻璃体内,检测GAP-43表达的积分光密度值.结果:成功培养分离出稳定分泌腺病毒介导CNTF基因的嗅鞘细胞,CNTF治疗组和OECs-CNTF治疗组GAP-43表达较损伤对照组显著增加(P<0.01), OECs(嗅鞘细胞)-CNTF组较单纯CNTF治疗组GAP-43表达明显增加,两组间差异明显(P<0.05).结论:腺病毒介导CNTF转染嗅鞘细胞移植对大鼠颅内段视神经损伤后神经纤维长距离再生具有明显的促进作用.     

Effect of cervical spinal cord hemisection on the expression of axon growth markers

To evaluate the plasticity processes occurring in the spared and injured tissue after partial spinal cord injury, we have compared the level of axon growth markers after a C2 cervical hemisection in rats between the contralateral (spared) and ipsilateral (injured) cervical cord using western blotting and immunohistochemical techniques. In the ipsilateral spinal cord 7 days after injury, although GAP-43 levels were increased in the ventral horn caudal to the injury, they were globally decreased in the whole structure (C1–C6). By contrast, in the contralateral intact side 7 days and 1 month after injury, we have found an increase of GAP-43 and βIII tubulin levels, suggesting that processes of axonal sprouting may occur in the spinal region contralateral to the injury. This increase of GAP-43 in the contralateral spinal cord after cervical hemisection may account, at least partially, to the spontaneous ipsilateral recovery observed after a cervical hemisection.     

Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity

We have investigated potential mechanisms by which exercise can promote changes in neuronal plasticity via modulation of neurotrophins. Rodents were exposed to voluntary wheel running for 3 or 7 days, and their lumbar spinal cord and soleus muscle were assessed for changes in brain-derived neurotrophic factor (BDNF), its signal transduction receptor (trkB), and downstream effectors for the action of BDNF on synaptic plasticity. Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord. Synapsin I, a synaptic mediator for the action of BDNF on neurotransmitter release, increased in proportion to GAP-43 and trkB mRNA levels. CREB mRNA levels increased in proportion to BDNF mRNA levels. In separate experiments, the soleus muscle was paralyzed unilaterally via intramuscular botulinum toxin type A (BTX-A) injection to determine the effects of reducing the neuromechanical output of a single muscle on the neurotrophin response to motor activity. In sedentary BTX-A-treated rats, BDNF and synapsin I mRNAs were reduced below control levels in the spinal cord and soleus muscle. Exercise did not change the BDNF mRNA levels in the spinal cord of BTX-A-treated rats but further reduced the BDNF mRNA levels in the paralyzed soleus relative to the levels in sedentary BTX-A-treated rats. Exercise also restored synapsin I to near control levels in the spinal cord. These results indicate that basal levels of neuromuscular activity are required to maintain normal levels of BDNF in the neuromuscular system and the potential for neuroplasticity.     

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.     

Differential distribution of growth associated protein (GAP-43) in the motor nuclei of the adult rat conus medullaris

GAP-43 is normally produced by neurons during developmental growth and axonal regeneration, but it is also expressed in specific regions of the normal adult nervous system. We studied the protein expression of GAP-43 within the conus medullaris portion of the spinal cord in adult male rats. Immunohistochemistry for choline acetyltransferase (ChAT) was first performed to identify specific efferent autonomic and motor nuclei in lumbosacral segments of the spinal cord. Adjacent sections were then processed for GAP-43 immunoreactivity (IR). We show GAP-43 IR in the superficial portion of the dorsal horn, the intermediolateral nucleus, and the dorsal commissural tract. We also demonstrate a differential distribution of GAP-43 IR between different motor nuclei of the conus medullaris. Using densitometry, the most prominent GAP-43 IR was detected in the dorsolateral and dorsomedial motor nuclei, which represent the human Onufs nucleus homologue. Confocal microscopy of double immunofluorescent labeling for ChAT and GAP-43 demonstrate GAP-43 IR in the neuropil of the autonomic and motor nuclei, and many of the GAP-43 IR arbors are in close apposition with the efferent cholinergic neurons. We note that the efferent neurons of both the autonomic and somatic nuclei, which are ultimately responsible for the integrated normal control of the lower urinary tract, bowel and sexual functions, are heavily innervated by GAP-43 enriched projections. We speculate that these functionally related neurons retain a physiological GAP-43-associated synaptic plasticity throughout adult life.     

臂丛损伤再生中GAP-43 mRNA及其蛋白的表达

目的：分析臂丛损伤后脊髓前角运动神经元表达GAP-43 mRNA及其蛋白的变化规律，探讨神经损伤再生的机制。方法：建立3种臂丛损伤模型：右C₇前根撕脱（A组）；右C₇前根撕脱＋同侧C5-T₁后根断离（B组）；右C₇前根撕脱+右C₅C₆间脊髓半横断（C组）。用荧光定量RT-PCR方法检测术后14 d时 C₇前角GAP-43 mRNA的表达量。用免疫组化方法检测术后1、 3、 7、14 d脊髓前角GAP-43免疫阳性运动神经元的表达。结果：对照组C₇前角GAP-43 mRNA呈低表达，损伤组GAP-43 mRNA表达显著上调。损伤组术后1 d、3 d时均未见C₇前角 GAP-43免疫阳性神经元，术后7 d各损伤组GAP-43免疫阳性神经元开始出现，14 d时免疫阳性神经元数目达到高峰。3组间比较，C组表达量最高，B组最低，A组居中。结论：臂丛损伤诱导运动神经元GAP-43 mRNA及其蛋白表达上调，GAP-43合成增加是神经元蛋白重组所致，与轴索再生和神经功能重建有关。     

Continuous brain-derived neurotrophic factor (BDNF) infusion after methylprednisolone treatment in severe spinal cord injury

Although methylprednisolone (MP) is the standard of care in acute spinal cord injury (SCI), its functional outcome varies in clinical situation. Recent report demonstrated that MP depresses the expression of growth-promoting neurotrophic factors after acute SCI. The present study was designed to investigate whether continuous infusion of brain-derived neurotrophic factor (BDNF) after MP treatment promotes functional recovery in severe SCI. Contusion injury was produced at the T10 vertebral level of the spinal cord in adult rats. The rats received MP intravenously immediately after the injury and BDNF was infused intrathecally using an osmotic mini-pump for six weeks. Immunohistochemical methods were used to detect ED-1, Growth associated protein-43 (GAP-43), neurofilament (NF), and choline acethyl transferase (ChAT) levels. BDNF did not alter the effect of MP on hematogenous inflammatory cellular infiltration. MP treatment with BDNF infusion resulted in greater axonal survival and regeneration compared to MP treatment alone, as indicated by increases in NF and GAP-43 gene expression. Adjunctive BDNF infusion resulted in better locomotor test scores using the Basso-Beattie-Bresnahan (BBB) test. This study demonstrated that continuous infusion of BDNF after initial MP treatment improved functional recovery after severe spinal cord injury without dampening the acute effect of MP.     

Evaluation of cellular organization and axonal regeneration through linear PLA foam implants in acute and chronic spinal cord injury

There are few studies of neural implants in spinal cord injury (SCI) focused on supporting directed axon growth. In this study, we fabricated a macroporous poly (lactic acid) (PLA) foam with oriented inner channels. Amorphous foam without linear channels served as a control in an acute SCI injury model, and the effectiveness of foam with linear channels was further investigated in a chronic SCI model. Implants were placed into a 2 mm hemisection lesion cavity at the T8 spinal cord level in adult rats. Two weeks post-implantation, tissue sections including the implants were examined using antibodies against GFAP, p75, ED-1, laminin, GAP-43, and CGRP. Foam implants were well-integrated with the host spinal cord. In linear foams, numerous DAPI-stained cells were found within the inner channels. Schwann cells but not astrocytes had migrated within the channels. Intense laminin staining was observed throughout the extracellular matrix substrate. GAP-43- and CGRP-positive axons grew through the implants following the linear channels. In the amorphous control foams, DAPI staining distributed evenly through the pores. However, the growth of GAP-43 or CGRP-positive axons was misguided and impeded at the entrance area of the foam. Higher numbers of GAP-43 and CGRP-positive axons grew into linear foam implants after chronic SCI than acute SCI. These results suggest the potential application of linear foam implants in cell and axon guidance for SCI repair, especially for chronic SCI.