Effects of Epidural Spinal Cord Stimulation and Treadmill Training on Locomotion Function and Ultrastructure of Spinal Cord Anterior Horn after Moderate Spinal Cord Injury in Rats

Objective:To investigate the effects of epidural spinal cord stimulation(ESCS) and treadmill training on the locomotion function and ultrastructure of spinal cord anterior horn after moderate spinal cord injury in rats.Method:Nine adult female Sprague-Dawley rats were randomly distributed into three groups:①spinal cord injury group(SI,n=3).②spinal cord injury plus ESCS group(SE,n=3).③spinal cord injury plus treadmill training group(TT,n=3).All rats received a moderate spinal cord injury surgery.Four weeks after surgery,rats in SE group received an electrode implantation procedure,with the electrode field covering spinal cord segments L2-S1.Four weeks after electrode implantation,rats received subthreshold ESCS for 30 min/d.Rats in TT group received 4cm/s treadmill training for 30min/d.Rats in SI group received no intervention,as a control group.All procedures in these three groups lasted four weeks.The open field Basso,Beattie and Bresnahan(BBB) scale was used before and after intervention to evaluate rats' hindlimb motor function.Result:After four weeks intervention,rats in TT group improved their open field locomotion scores to 20.In contrast,no significant improvement was observed in groups SI and SE.The morphology of synapses and neurons were similar regardless of whether rats had undergone ESCS,treadmill training or not.Conclusion:ESCS alone was not sufficient to improve the walking ability of spinal cord injured rats.ESCS or treadmill training alone might not contribute to the changes of ultrastructure in anterior horn of spinal cord that underlie the recovery of walking ability.Further research is needed to understand the contributions of combination of ESCS and treadmill training to the rehabilitation of spinal cord injured rats.

p75 neurotrophin receptor signal pathway influence on apoptosis in anterior horn neurons of the spinal cord in a rat model of cauda equina compression injury

BACKGROUND： Studies have demonstrated that cauda equina compression results in apoptosis of motor neurons in the spinal cord. The combination of p75 neurotrophin receptor （p75NTR） and precursor of nerve growth factor （pro-NGF） expression initiates the apoptotic pathway and induces neuronal apoptosis. However, few reports have focused on the p75-mediated mechanism of neuronal apoptosis following cauda equine compression injury OBJECTIVE： To determine apoptosis of spinal cord neurons and activation of the pro-NGF-p75NTR-JNK（c-Jun N-terminal kinase） signal pathway in rats following cauda equina compression, and to verify experimental outcomes. DESIGN, TIME AND SETTING： A randomized, controlled, in vivo experiment was performed at the Medical Experimental Center of Xi＇an Jiaotong University between April and November in 2008. MATERIALS： Streptavidin-perosidase kit was purchased from Wuhan Boster, China; in situ end labeling detection kit was provided by Promega, USA; type AEG-220G electron microscope was purchased from Hitachi, Japan. METHODS： A total of 48 healthy, adult, female, Sprague Dawley rats were randomly assigned to three groups： normal （n = 6）, sham-surgery （n = 6）, and compression （n = 36）. The compression group was randomly assigned to six subsets at 1,3, 5, 7, 14, and 28 days, respectively, with 6 rats in each subset. A cylindrical silica gel stick was implanted into the rats to compress 75% of the vertebral canal in the compression group; in the sham-surgery group, only vertebral resection was performed; and no procedures were performed in the normal group. MAIN OUTCOME MEASURES： At 1,3, 5, 7, 14, and 28 days following compression, L2-3 spinal cord segments were processed for immunohistochemistry, in situ cell apoptosis detection, and transmission electron microscopy observation. Nissl staining was used to observe neuronal survival in the L2 spinal cord segment. Immunohistochemistry was applied to detect expressions of pro-NGF, p75NTR, and JNK in the L2 segment. TUNEL fluorometric method was used to observe apoptosis of neurons in the L2 segment. RESULTS： In the normal and sham-surgery groups, little neuronal apoptosis was observed in the L2-3 spinal cord segment. At 3 days after compression injury, pro-NGF, p75NTR and JNK expression was observed in the spinal cord. Expression levels reached a peak at 7 days, and then gradually decreased. In the compression and sham-surgery groups, neurons primarily expressed pro-NGF and p75NTR. The number of JNK-positive neurons in the compression group was dramatically increased compared with the sham-surgery group （P〈 0.05）. A few neurons were apoptotic in the spinal cord 1 day after compression injury. The number of apoptotic neurons gradually increased and reached a peak at 7 days, and subsequently decreased. Apoptosis was still detectable at 28 days. There was a positive correlation between p75NTR expression and neuronal apoptosis （r= 0.75, P〈 0.05）. CONCLUSION： Following cauda equina compression injury, apoptosis of spinal cord neurons was observed. The compression-induced neuronal apoptosis was associated with p75NTR expression in the L2-3 spinal cord segment.

Sciatic nerve injury alters the spatial arrangement of neurons and glial cells in the anterior horn of the spinal cord

The spatial arrangement of the cell is important and considered as underlying mechanism for mathematical modeling of cell to cell interaction.The ability of cells to take on the characteristics of other cells in an organism,it is important to understand the dynamical behavior of the cells.This method implements experimental parameters of the cell-cell interaction into the mathematical simulation of cell arrangement.The purpose of this research was to explore the three-dimensional spatial distribution of anterior horn cells in the rat spinal cord to examine differences after sciatic nerve injury.Sixteen Sprague-Dawley male rats were assigned to control and axotomy groups.Twelve weeks after surgery,the anterior horn was removed for first-and second-order stereological studies.Second-order stereological techniques were applied to estimate the pair correlation and cross-correlation functions using a dipole probe superimposed onto the spinal cord sections.The findings revealed 7% and 36% reductions in the mean volume and total number of motoneurons,respectively,and a25% increase in the neuroglial cell number in the axotomized rats compared to the control rats.In contrast,the anterior horn volume remained unchanged.The results also indicated a broader gap in the pair correlation curve for the motoneurons and neuroglial cells in the axotomized rats compared to the control rats.This finding shows a negative correlation for the distribution of motoneurons and neuroglial cells in the axotomized rats.The cross-correlation curve shows a negative correlation between the motoneurons and neuroglial cells in the axotomized rats.These findings suggest that cellular structural and functional changes after sciatic nerve injury lead to the alterations in the spatial arrangement of motoneurons and neuroglial cells,finally affecting the normal function of the central nervous system.The experimental protocol was reviewed and approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences(approval No.IR.SBMU.MSP.REC1395.375) on October 17,2016.

Early electrical field stimulation prevents the loss of spinal cord anterior horn motoneurons and muscle atrophy following spinal cord injury

Our previous study revealed that early application of electrical field stimulation（EFS） with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury（SCI）. The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T_（10）. SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov＇s motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.

Visualizing Wallerian degeneration in the corticospinal tract after sensorimotor cortex ischemia in mice

Stroke can cause Wallerian degeneration in regions outside of the brain,particularly in the corticospinal tract.To investigate the fate of major glial cells and axons within affected areas of the corticospinal tract following stroke,we induced photochemical infarction of the sensorimotor cortex leading to Wallerian degeneration along the full extent of the corticospinal tract.We first used a routine,sensitive marker of axonal injury,amyloid precursor protein,to examine Wallerian degeneration of the corticospinal tract.An antibody to amyloid precursor protein mapped exclusively to proximal axonal segments within the ischemic cortex,with no positive signal in distal parts of the corticospinal tract,at all time points.To improve visualization of Wallerian degeneration,we next utilized an orthograde virus that expresses green fluorescent protein to label the corticospinal tract and then quantitatively evaluated green fluorescent protein-expressing axons.Using this approach,we found that axonal degeneration began on day 3 post-stroke and was almost complete by 7 days after stroke.In addition,microglia mobilized and activated early,from day 7 after stroke,but did not maintain a phagocytic state over time.Meanwhile,astrocytes showed relatively delayed mobilization and a moderate response to Wallerian degeneration.Moreover,no anterograde degeneration of spinal anterior horn cells was observed in response to Wallerian degeneration of the corticospinal tract.In conclusion,our data provide evidence for dynamic,pathogenic spatiotemporal changes in major cellular components of the corticospinal tract during Wallerian degeneration.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.

神经根撕脱后脊髓运动神经元的病理表现

目的 :观察神经根撕脱后脊髓前角运动神经元的病理表现 ,探讨在该条件下运动神经元死亡的神经生物学机制。方法 :选择成年SD雌性大鼠 2 0只 ,体重 2 0 0 - 30 0g ,撕脱右侧臂丛C5-T8神经根 ,术后动物存活 3d、5d、1周后取C5-C8节段脊髓 ,对冰冻切片行NADPH -d组化、c -jun免疫组化、中性红和HE染色。观察神经元的形态 ,计数脊髓前角NOS阳性运动神经元及存活运动神经元数目 ,以非损伤侧的前角运动神经元数目为 10 0 % ,计算百分比。结果 :神经根撕脱 3d、5d、1周后 ,损伤侧脊髓前角NOS运动神经元平均阳性率分别为 :0 74%± 0 5 9%、2 4 83%± 6 73%、5 1 16 %± 8 6 7%。神经元平均存活率分别为 93 0 0 %± 4 32 %、93 6 7%± 5 2 7%、89 83%±2 6 5 % ;c -Jun从撕脱术后 3d就有表达 ,5d后表达开始减少。运动神经元形态变化不明显。 1周时偶见前角运动神经元的胞核偏位 ,但核膜清晰 ,核仁尚存 ,染色体固缩。结论 :脊神经根撕脱 1周内 ,脊髓前角运动神经元NOS表达递增 ,c-Jun表达递减 ,运动神经元开始死亡。NO/NOS可能通过抑制神经元损伤后的再生反应 ,促进脊髓前角运动神经元的死亡。

不同强度训练后大鼠脊髓前角细胞线粒体的定量研究

目的 :研究不同强度耐力训练对大鼠脊髓前角细胞线粒体超微结构的不同影响 ,探讨适合神经系统发展的最佳训练强度。方法 :2 4只雄性SD大鼠随机分为 4组 ,即对照组、小强度运动组、大强度运动组、大强度运动力竭组各 6只。训练后对各组大鼠脊髓前角细胞线粒体进行电镜观测并用体视学方法做定量分析。结果 :各训练组脊髓前角细胞线粒体数量增多 ,嵴多而致密 ,基质电子密度增高。但大强度运动力竭组出现线粒体嵴断裂、空泡变 ,甚至线粒体裂解现象。体视学测量结果表明 ,各训练组与对照组之间以及各训练组之间线粒体Vv、Sv、Nv、δ均发生不同程度的改变。结论 :不同强度耐力训练可引起大鼠脊髓前角细胞线粒体形态结构的不同改变 ,小强度运动训练通过线粒体形状改变和膜面积增加即可达到能量代谢的需求 ;大强度运动可引起线粒体的总体积、数量及膜面积等形态结构发生适应性代偿 ,为有效的训练强度 ;大强度力竭运动则可引起线粒体形态结构的不可逆损害 ,不利于机体健康。

大鼠坐骨神经损伤后脊髓前角神经元死亡数量的研究

目的 :研究周围神经损伤后 ,脊髓前角运动神经元胞体的死亡数量变化。方法 :选择 10只正常SD大鼠 ,先计算两侧的脊髓前角运动神经元胞体数量是否对称 ;再选择 3 5只SD大鼠 ,切断并原位吻合其右侧坐骨神经 ,左侧不作任何处理、作为对照 ,于术后不同时间取L4～ 6节段脊髓作HE染色 ,计算脊髓前角运动神经元胞体数量的变化。结果 :正常SD大鼠两侧的脊髓前角运动神经元胞体数量呈对称分布 ;右侧坐骨神经损伤后 ,其脊髓前角运动神经元胞体数量较左侧减少。结论 :大鼠坐骨神经损伤后 ,脊髓前角运动神经元的胞体有死亡 ,其死亡具有一定的时间特征。

猫脊髓前角运动神经元区的P物质样及胆碱乙酰化酶样免疫反应

用ABC法对猫脊髓前角运动神经元区(Ⅸ层)的P物质样及胆碱乙酰化酶样免疫反应(SP-LI和ChAT-LI)的分布作了观察与比较。结果发现:(1)Ⅸ层的大、中型神经元几乎均为ChAT-LI阳性,部分ChAT-LI神经元胞体及近端树突表面有少量ChAT-LI终扣。(2)SP-LI出现于Ⅸ层多数大、中型神经元的胞体和突起根部,约半数以上SP-LI神经元显示较强呈色反应。较多SP-LI串珠状终末纤维散在于Ⅸ层,SP-LI神经元表面可见SP-LI终扣。以上提示:(1)P物质(SP)和乙酰胆碱(ACh)共存于前角运动神经元,SP可能参与调制神经肌接头的胆碱能传递。(2)前角运动神经元尚接受SP能及胆碱能神经支配。

龟龄集对大鼠小脑皮质和脊髓前角内突触小泡蛋白变化的影响

目的研究龟龄集对大鼠小脑皮质和脊髓前角内突触小泡蛋白表达的影响,探讨龟龄集抵抗中枢神经系统衰老的机制。方法采用神经行为结合免疫组织化学和图像分析的方法,检测了小脑皮质和脊髓颈膨大、腰膨大前角内突触小泡蛋白的含量及其意义。结果喂药组小脑皮质和脊髓颈、腰膨大前角内突触小泡蛋白的含量均明显高于对照组（P〈0.01~P〈0.001）。结论龟龄集可延缓神经元的衰老,防止中枢神经系统内突触小泡蛋白的丢失,维持动物正常的运动及其运动的灵活性。

针刺对实验性脊髓损伤前角运动神经元酶学的影响

目的:探讨针刺对大鼠脊髓损伤前角运动神经元酶学的影响。方法:采用酶组织化学染色方法定性分析针刺治疗前后脊髓前角运动神经元乙酰胆碱酯酶(ACHE)、琥珀酸脱氢酶(SDH)、酸性磷酸酶(ACP)的含量变化;同时,运用全自动图像分析仪系统进行定量分析。结果:脊髓损伤后ACHE和SDH含量减少(P<001);ACP含量增加(P<001)。而针刺组针刺后均较对照组同时段点ACHE、SDH酶含量高;ACP酶含量低(P<005,P<001)。结论:针刺能调节脊髓损伤后前角运动神经元酶含量,具有抑制或延缓神经元变性程度并能促进其恢复。

大鼠骨骼肌35kD运动神经诱向因子单克隆抗体的产生和鉴定

为探究脊髓运动神经元诱向因予,用大鼠腓骨肌提取液(PMe)作Phast电泳,分离PMe中能促进脊髓前角神经元生长的35kD蛋白带免疫动物。细胞融合,经MTT微量比色筛选,获得2株抗大鼠骨骼35kD蛋白的单克隆抗体。命名为ME10,MG9。2株单抗对体外培养的脊髓前角运动神经元的生长活性显示抑制作用,MTT OD值分别为0.023±0.003,0.030±0.005,以ME10抑制效应更强。与单独加35kD蛋白凝胶时促进细胞生长的OD值0.050±0.002比较,差别有显著意义(ME10 P<0.01,G9P<0.05)。为鉴定ME10单抗的特异性,进行中和试验。结果发现:ME10对运动神经元生长的抑制作用被35kD蛋白特异性地中和,说明ME10是3kD蛋白特异性抗体。用ME10单抗进行免疫组化反应,在骨骼肌细胞浆内,运动神经末稍都有免疫反应阳性物质,表明从腓肠肌组织提取液中分离的35kD蛋白是由骨骼肌细胞产生的神经诱向因子。

运动对小鼠寿命和脊髓前角神经元形态和数量的影响

用C_(57)BL/3J小鼠29只,分成运动组(12只),对照组(12只)和青年组(5只)三组.运动组从第6个月起隔天跑转笼2小时,对照组不跑转笼.当这两组小鼠死亡总数达50%时(经过16个月零8天),将两组余下的小鼠(22月龄)全部杀死,取出脊髓腰膨大(L_2)与青年组(6月龄)的相比较.结果表明:运动组与对照组的存活率无显著性差异,P>0.05;老年的小鼠脊髓前角神经元总数比青年组的少,但运动组神经元减少较轻,P<0.05,减少的主要的是小神经元,大、中神经元并没有减少;而对照组神经元减少明显,P<0.01,减少的主要是大、中神经元.在三组随机抽出的大神经元胞体切面面积中,运动组最大,青年组居中,对照组最小,两两比较P均<0.01.实验结果提示:长期适量的运动对小鼠的寿命影响不大,但可能会减轻脊髓前角神经元在衰老过程中的死亡程度,最明显的影响是将运动神经元保存下来.

运动对发育期小鼠脊髓前角细胞形态学的影响

选用 1 8～ 2 0日龄昆明种雄性同源小鼠 ,经配对 ,随机分配于运动组与对照组各 4只。按计划 ,运动组活动 8周后 ,同时处死两组小鼠。在脊髓腰骶膨大处取材 ,Niss/法染色 ,光学显微镜观察。结果表明 ,运动组前角细胞的胞核、核仁显著增大 (P <0 0 0 1 ) ;核质比增大 2 5 3 %。

耐力训练对脊髓前角细胞超微细胞的影响

雄性SD大鼠19只,分为游泳耐力训练组(13只)和对照组(6只).用体视学方法研究耐力训练8周后脊髓前角细胞线粒体超微结构的变化.电镜显示训练组大鼠前角细胞线粒体的数量明显地增多,嵴多而致密,基质电子密度增高.体视学方法测量表明,训练组大鼠前角细胞线粒体体密度和面密度均比对照组的有很显著的增大,P<0.001.实验结果提示:耐力训练对脊髓前角细胞有一定的影响,体视学方法能检测出前角细胞早期的超微结构改变.

大鼠脊髓前角神经元内维生素D依赖性钙结合蛋白与小白蛋白共存

目的 :观察大鼠脊髓前角神经元内维生素D依赖性钙结合蛋白 (CB)与小白蛋白 (PV)的共存情况 .方法 :免疫荧光组织化学双重标记技术 .结果 :脊髓前角内的部分中、小型神经元呈CB或PV阳性 ;绝大部分CB阳性的前角神经元亦呈PV阳性 ,即CB与PV共存于这些神经元 .结论 :CB与PV共存于部分前角神经元 ,这些神经元可能为Renshaw细胞并参与对前角α运动神经元的反馈抑制调控 .

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。

坐骨神经损伤后神康灵对脊髓前角神经元的保护作用的初步观察

目的 探讨坐骨神经损伤后神康灵对脊髓前角神经元的保护作用。方法 采用 2 8只成年体重为 2 0 0g的Wistar大鼠 ,随机分成 2组 (实验组和对照组 ) ,分别于术后 6、8周通过扫描电镜检测脊髓前角神经元的形态和超微结构的变化 ;脊髓前角切片HE染色 ,从而探讨神康灵对坐骨神经损伤后脊髓前角神经元的保护作用。结果 6和 8周时 ,实验组脊髓前角神经元变性的程度明显小于对照组 ;存活的数量明显多于对照组。

以胸髓前角先受累的肌萎缩侧索硬化的临床特点

目的探讨胸髓前角先受累的肌萎缩侧索硬化(ALS)患者的临床特征。方法回顾性分析3例以胸髓前角先受累的ALS患者的临床资料。结果3例患者均为男性,发病年龄分别为52岁、66岁、62岁;均以呼吸困难为首发表现,有明显的肋间肌和腹直肌萎缩,而肢体肌无力及肌萎缩的出现相对较轻、较迟;肌电图显示上肢及椎旁肌的神经源性损害。结论以胸髓前角先受累的ALS患者以男性多见,起病年龄晚于ALS的平均发病年龄,呼吸困难明显,呼吸肌萎缩早于肢体肌无力及肌萎缩,肌电图检查可以确诊。