大鼠脊髓损伤后神经营养素及受体表达的变化

目的:观察神经营养素及受体在脊髓损伤后的表达变化。方法:SD大鼠30只,设正常组、假伤组、脊髓损伤组。Allen's法复制5g×10cm脊髓损伤模型。用免疫组化和免疫电镜方法观察各组神经生长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养素-3(NT-3)及其受体TrkA,TrkB,TrkC表达的变化。结果:正常大鼠脊髓神经营养素及其受体表达较少,损伤后表达明显增加。NGF和BDNF伤后1～7d持续高表达,NT-3、TrkA、TrkB和TrkC伤后1～3d表达较高,与正常组和假伤组相比,差异有显著性意义。结论:神经营养素及受体在急性脊髓损伤中表达增加,起保护性作用。     

减压脊髓神经恢复分子机制的研究

目的探讨手术减压对慢性脊髓损伤的治疗分子机制。方法采用大鼠后路渐进性脊髓压迫模型,然后行手术减压。经观察行为评分(BBB),常规病理及胆碱乙酰转移酶(ChAT)和脑源性神经营养因子(BDNF)及其受体TrkB的免疫组织化学检测。结果脊髓损伤后ChAT免疫组织化学阳性细胞数减少,BDNF和TrkB表达增加,BBB下降;减压后,ChAT阳性细胞数增多,BDNF和TrkB表达恢复,BBB改善,减压组与压迫组比较差异有统计学意义(P<0.05)。结论手术减压作用的分子机制可能是其促进了慢性压迫性损伤脊髓运动神经元合成ChAT和运动神经递质乙酰胆碱,且减压可加速BDNF和TrkB的转运,从而加速实验动物行为功能的恢复。     

脊髓腹侧压迫损伤后MP、Iloprost和Riluzole对Bcl—2表达的影响

目的：观察脊髓腹侧迫损伤后Bcl-2表达的动态变化及神经保护剂对Bcl-2表达的影响。方法：大鼠胸脊髓腹侧压迫损伤后,将受试运动分别用甲基强的松龙（MP）、Iloprost,Riluzole及以上三种药物合用联合治疗。应用免疫组织化学及原位杂交方法检测治疗组及损伤组中Bcl-2的表达。结果：脊髓伤后1h Bcl-2蛋白增加,伤后3h达高峰,伤后2周表达接近于正常水平。Bcl-2 mRNA的表达也有相似的变化过程。。MP、Iloprost和Riluzole能增加Bcl-2蛋白表达,但对Bcl-2 mRNA的表达增加不明显。结论：作用方式不同的几种神经保护剂均能增加脊髓损伤组织中的Bcl-2蛋白的表达。     

氯化锂对脊髓损伤模型大鼠运动功能的作用和机制

[目的]观察氯化锂对脊髓损伤后BDNF/TrkB信号表达的作用,探讨其对脊髓损伤大鼠肢体运动功能恢复的作用和机制。[方法]36只SD大鼠随机分为假手术组(12例)、对照组(12例)、氯化锂组(12例),假手术组仅行椎板切除术,对照组和氯化锂组采用NYU脊髓打击器建立脊髓损伤模型,术后第1、3、5 d和第7 d行BBB肢体运动功能评分。干预7 d后取材,采用尼氏染色光镜观察神经元形态,免疫荧光定位BDNF阳性细胞,并计数进行半定量分析,Western blot检测BDNF蛋白、TrkB蛋白和p-TrkB蛋白表达。[结果]在脊髓损伤后肢体运动功能方面,自干预后第3 d起,氯化锂组BBB评分高于对照组,差异具有统计学意义(P<0.05),至术后第5 d,两组间BBB评分差异更加明显(P<0.01)。尼氏染色提示氯化锂组脊髓损伤后神经元的形态优于对照组。氯化锂组脊髓损伤后脊髓前角运动神经元BDNF阳性细胞数高于对照组,差异具有统计学意义(P<0.01);Western blot检测表明氯化锂组的BDNF蛋白和p-TrkB蛋白表达高于对照组,两组间差异有统计学意义(P<0.05),但两组间TrkB蛋白表达差异无统计学意义(P>0.05)。[结论]氯化锂能促进脊髓损伤对照大鼠肢体运动功能恢复,其机制可能与其促进脊髓损伤后脊髓前角运动神经元分泌BDNF,促进TrkB受体磷酸化,从而上调BDNF/TrkB信号表达,促进脊髓损伤后神经元存活、再生和轴突再生、再髓鞘化有关。     

功能训练促进颈脊髓损伤大鼠功能恢复的机制研究

目的：探讨大鼠颈脊髓不完全性损伤后前肢功能训练促进大鼠前肢功能恢复的机制.方法：在立体定位仪的引导下,致伤大鼠双侧红核和皮质脊髓背侧束后,对大鼠行前肢功能训练6周.免疫组化检测损伤脊髓节段脑源性神经生长因子（brain-derived neurotrophic factor,BDNF）的表达,皮质脊髓束投射神经元（corticospinal neurons,CSNs）中生长相关蛋白43（growth-associated protein 43,CAP43）和神经营养素共同受体P75（P75NTR）的表达,荧光金逆行示踪CSNs存活情况.结果：大鼠不完全性颈脊髓损伤后,前肢功能训练可上调脊髓前角神经元BDNF与CSNs中GAP43和P75NTR的表达,减少CSNs死亡.结论：大鼠颈脊髓不完全性损伤后,前肢功能训练通过上调脊髓前角神经元BDNF与CSNs中GAP43和P75NTR的表达以及减少CSNs的死亡等机制增加未损伤皮质脊髓腹侧束（vCST）的出芽,进而促进大鼠前肢功能恢复.     

神经营养素-3基因修饰嗅鞘细胞移植对急性脊髓损伤作用的实验研究

目的：观察神经营养素-3（NT-3）基因转染嗅鞘细胞（OEG）移植对急性大鼠脊髓损伤的作用。方法：将自行构建的质粒DEGFP-NT3应用脂质体介导的方法导人体外培养的嗅鞘细胞，并移植入急性脊髓损伤大鼠体内．连续观察12周．与接受单纯OEG、空白质粒转染OEG移植的脊髓损伤大鼠进行比较。结果：移植转染DEGFP-NT3后的OEG能在体内长期存活，表达NT-3基因，与对照组比较能更好地促进脊髓损伤区轴突的再生和后肢功能的恢复。结论：OEG是脊髓损伤基因治疗较好的受体细胞。转染NT-3基因的OEG移植后可以在体内较长时间存活．能明显促进急性脊髓损伤神经纤维的再生和功能恢复，为基因修饰嗅鞘细胞在脊髓损伤治疗中的应用提供了实验和理论依据。     

实验性脊髓损伤后腺苷A1受体的变化

目的：观察大鼠脊髓损伤后腺苷A1受体的变化。方法：腹侧压迫法造成T13脊髓中度损伤,分别于伤前,伤后10min,1h,4h,24h取伤段脊髓制备成膜受体,采用特异性A1受体放射性配基（[3H]CHA)结合,Scatchard分析法测定A1受体的最大结合数（Bmax)及解离常数Kd值,结果：脊髓损伤后,代表A1受体亲和力的解离常数Kd值无明显改变,而最大结合数在伤后1h显著升高,4h达最高峰,24h仍显著高于伤前水平,结论：脊髓损伤后A1受体结合数的增高可能是脊髓损伤后的一种保护反应。     

脊髓损伤后神经营养素家族及其受体表达的研究进展

脊髓损伤所致的截瘫至今仍缺乏有效的治疗方法。哺乳动物中枢神经系统再生能力有限,其轴突的再生由神经元固有特性和所处的环境所决定,部分归因为缺乏足够的营养支持．这种营养支持来源于自身,而神经营养素家族是这种营养支持之一。现对脊髓损伤后神经营养素家族及其受体表达的研究进展综述如下。     

腺病毒介导的脑源性神经营养因子基因治疗大鼠神经根损伤

目的：观察腺病毒介导的脑源性神经营养因子(BDNF)基因脊髓内转移对神经根损伤后的治疗效果。方法：制作大鼠神经根切断吻合模型，分为两组：治疗组通过显微注射法在立体定位仪上将2μl重组腺病毒BDNF载体(AxCA—BDNF)直接注入大鼠神经根损伤部相应的脊髓腹角；损伤组注射病毒缓冲液。术后观察霍乱毒素-辣根过氧化物酶(CT-HRP)逆行标记的神经元数目及坐骨神经功能指数(SFI)的改变。结果：与损伤组比较，治疗组神经根伤后CT—HRP标记神经元的数目明显增加，坐骨神经功能指数(SFI)的恢复率明显升高。结论：AxCA—BDNF基因治疗对大鼠脊髓水平的神经根修复是一种有效的方法。     

神经干细胞移植促进脊髓损伤后脑源性神经营养因子的表达

目的：探讨神经干细胞（NSCs）移植对大鼠脊髓损伤后脑源性神经营养因子（BDNF）表达的影响及其意义。方法：NSCs提取自新生Wistar大鼠的海马区，经培养、鉴定。制作大鼠脊髓损伤（SCI）模型，于伤后第7天移植NSCs。实验分为3组：NSCs移植组（A组），DMEM填充组（B组），正常对照组（C组）。应用RT—PCR法和免疫组化法观察细胞移植后BDNF基因表达的变化。结果：RT—PCR结果分析，移植术后第1、3、5天，A组BDNF mRNA的表达量明显高于B组，差异有统计学意义（P〈0．05）。组化结果分析，移植术后第7、14、28天BDNF的表达量A组明显高于B组，差异有统计学意义（P〈0．05）。结论：NSCs在移植后可上调脑源性神经营养因子BDNF基因的表达，是其修复脊髓损伤的机制之一。     

Delayed transplantation with exogenous neurotrophin administration enhances plasticity of corticofugal projections after spinal cord injury

Functional deficits following spinal cord injury (SCI) result from a disruption of corticofugal projections at the lesion site. Not only direct regeneration of the severed axons but also anatomical re-organization of spared corticofugal pathways can reestablish connections between the supraspinal and spinal motor centers. We have previously shown that delayed transplantation of fetal spinal cord tissue and neurotrophin administration by two weeks after SCI supported recovery of forelimb function in adult rats. The current study determined whether the same intervention enhances plasticity of corticofugal fibers at the midbrain and spinal cord level. Anterograde tracing of the left corticorubral fibers revealed that the animals with transplants and neurotrophins (BDNF or NT-3) increased the extent of the traced fibers crossing to the right red nucleus (RN), of which the axons are spared by a right cervical overhemisection lesion. More neurons in the left motor cortex were recruited by the treatment to establish connections with the right RN. The right corticorubral projections also increased the density of midline crossing fibers to the axotomized left RN in response to transplants and neurotrophins. Transplants plus NT-3, but not BDNF, significantly increased the amount of spared corticospinal fibers in the left dorsolateral funiculus at the spinal level both rostral and caudal to the lesion. These results suggest that corticofugal projections retain the capacity until at least two weeks after injury to undergo extensive reorganization along the entire neuraxis in response to transplants and neurotrophins. Targeting anatomical plasticity of corticofugal projections may be a promising strategy to enhance functional recovery following incomplete SCI.     

Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study

The goal of the present study was to develop a porcine spinal cord injury (SCI) model, and to describe the neurological outcome and characterize the corresponding quantitative and qualitative histological changes at 4-9 months after injury. Adult Gottingen-Minnesota minipigs were anesthetized and placed in a spine immobilization frame. The exposed T12 spinal segment was compressed in a dorso-ventral direction using a 5-mm-diameter circular bar with a progressively increasing peak force (1.5, 2.0, or 2.5?kg) at a velocity of 3?cm/sec. During recovery, motor and sensory function were periodically monitored. After survival, the animals were perfusion fixed and the extent of local SCI was analyzed by (1) post-mortem MRI analysis of dissected spinal cords, (2) qualitative and quantitative analysis of axonal survival at the epicenter of injury, and (3) defining the presence of local inflammatory changes, astrocytosis, and schwannosis. Following 2.5-kg spinal cord compression the animals demonstrated a near complete loss of motor and sensory function with no recovery over the next 4-9 months. Those that underwent spinal cord compression with 2 kg force developed an incomplete injury with progressive partial neurological recovery characterized by a restricted ability to stand and walk. Animals injured with a spinal compression force of 1.5?kg showed near normal ambulation 10 days after injury. In fully paralyzed animals (2.5?kg), MRI analysis demonstrated a loss of spinal white matter integrity and extensive septal cavitations. A significant correlation between the magnitude of loss of small and medium-sized myelinated axons in the ventral funiculus and neurological deficits was identified. These data, demonstrating stable neurological deficits in severely injured animals, similarities of spinal pathology to humans, and relatively good post-injury tolerance of this strain of minipigs to spinal trauma, suggest that this model can successfully be used to study therapeutic interventions targeting both acute and chronic stages of SCI.     

Differential effects of low versus high amounts of weight supported treadmill training in spinally transected rats

Intensive weight-supported treadmill training (WSTT) improves locomotor function following spinal cord injury. Because of a number of factors, undergoing intensive sessions of training may not be feasible. Whether reduced amounts of training are sufficient to enhance spinal plasticity to a level that is necessary for improving function is not known. The focus of the present study was to assess differences in recovery of locomotor function and spinal plasticity as a function of the amount of steps taken during WSTT in a rodent model of spinal cord injury. Rats were spinally transected at 5 days of age. When they reached 28 days of age, a robotic system was used to implement a weight-supported treadmill training program of either 100 or 1000 steps/training session daily for 4 weeks. Antibodies for brain-derived neurotrophic factor (BDNF), TrkB, and the pre-synaptic marker, synaptophysin, were used to examine the expression of these proteins in the ventral horn of the lumbar spinal cord. Rats that received weight-supported treadmill training performed better stepping relative to untrained rats, but only the rats that received 1000 steps/training session recovered locomotor function that resembled normal patterns. Only the rats that received 1000 steps/training session recovered normal levels of synaptophysin immunoreactivity around motor neurons. Weight-supported treadmill training consisting of either 100 or 1000 steps/training session increased BDNF immunoreactivity in the ventral horn of the lumbar spinal cord. TrkB expression in the ventral horn was not affected by spinal cord transection or weight-supported treadmill training. Synaptophysin expression, but not BDNF or TrkB expression was correlated with the recovery of stepping function. These findings suggested that a large amount of weight-supported treadmill training was necessary for restoring synaptic connections to motor neurons within the locomotor generating circuitry. Although a large amount of training was best for recovery, small amounts of training were associated with incremental gains in function and increased BDNF levels.     

Upregulation of EphA receptor expression in the injured adult rat spinal cord

After spinal cord injury (SCI), the inability of supraspinal neurons to regenerate or reform functional connections is likely due to proteins in the surrounding microenvironment restricting regeneration. EphAs are a family of receptor tyrosine kinases that are involved in axonal guidance during development. These receptors and their ligands, the Ephrins, act via repulsive mechanisms to guide growing axons towards their appropriate targets and allow for the correct developmental connections to be made. In the present study, we investigated whether EphA receptor expression changed after a thoracic contusion SCI. Our results indicate that several EphA molecules are upregulated after SCI. Using semiquantitative RT-PCR to investigate mRNA expression after SCI, we found that EphA3, A4, and A7 mRNAs were upregulated. EphA3, A4, A6, and A8 receptor immunoreactivity increased in the ventrolateral white matter (VWM) at the injury epicenter. EphA7 had the highest level of immunoreactivity in both control and injured rat spinal cord. EphA receptor expression in the white matter originated from glial cells as coexpression in both astrocytes and oligodendrocytes was observed. In contrast, gray matter expression was localized to neurons of the ventral gray matter (motor neurons) and dorsal horn. After SCI, specific EphA receptor subtypes are upregulated and these increases may create an environment that is unfavorable for neurite outgrowth and functional regeneration.     

腺苷在继发性脊髓损伤中的作用及其机制

目的 通过观察大鼠脊髓损伤后内源性腺苷含量的变化,以及外源性腺苷地脊髓损伤后神经功能的影响,探讨腺苷在髓继发性损伤中的作用。方法 采用大鼠Ｔ１３脊髓腹侧压迫模型,用微透析技术每２０分钟连续收集脊髓损伤后脊髓组织细胞上液,用高效液相色谱仪紫外检测法检测细胞外液腺式的含量;伤前１４分钟蛛网膜下腔给予非特异性腺苷受体激动剂２－氯腺苷,观察伤后神经功能评分、倾斜平面临界角和组织学变化。结果 脊髓损伤后腺苷     

Spinal activation of serotonin 1A receptors enhances latent respiratory activity after spinal cord injury

BACKGROUND/OBJECTIVE: Hemisection of the cervical spinal cord results in paralysis of the ipsilateral hemidiaphragm. Removal of sensory feedback through cervical dorsal rhizotomy activates latent respiratory motor pathways and restores hemidiaphragm function. Because systemic administration of serotonin 1A receptor (5HT1A) agonists reversed the altered breathing patterns after spinal cord injury (SCI), we predicted that 5HT1A receptor activation after SCI (C2) would activate latent crossed motor pathways. Furthermore, because 5HT1 A receptors are heavily localized to dorsal horn neurons, we predicted that spinal administration of 5HT1A agonists should also activate latent motor pathways. METHODS: Hemisection of the C2 spinal cord was performed 24 to 48 hours, 1 week, or 16 weeks before experimentation. Bilateral phrenic nerve activity was recorded in anesthetized, vagotomized, paralyzed Sprague-Dawley rats, and 8-OH-DPAT (5HT1A agonist) was applied to the dorsal aspect of the cervical spinal cord (C3-C7) or injected systemically. RESULTS: Systemic administration of 8-OH-DPAT led to a significant increase in phrenic frequency and amplitude, whereas direct application to the spinal cord increased phrenic amplitude alone. Both systemic and spinal administration of 8-OH-DPAT consistently activated latent crossed phrenic activity. 8-OH-DPAT induced a greater respiratory response in spinal injured rats compared with controls. CONCLUSION: The increase in crossed phrenic output after application of 8-OH-DPAT to the spinal cord suggests that dorsal horn inputs, respiratory and/or nonrespiratory, may inhibit phrenic motor output, especially after SCI. These findings support the idea that the administration of 5HT1A agonists may be a beneficial therapy in enhancing respiratory neural output in patients with SCI.     

Social and environmental enrichment improves sensory and motor recovery after severe contusive spinal cord injury in the rat

Neuropathic pain and motor dysfunction are difficult problems following spinal cord injury (SCI). Social and environmental enrichment (SEE), which models much of the clinical rehabilitation environment for post-SCI persons, is the focus of the current investigation which examines the effects of multiple-housing and the addition of climbing spaces, improved bedding and crawl toys on the sensory and motor recovery following a severe contusive SCI. Efficacy was determined with sensory testing, open-field motor behavioral testing, lesion volume analysis and quantification of brain-derived neurotrophic factor (BDNF) in the lumbar spinal cord with and without SEE provided during the recovery period. Sensory and motor testing were performed weekly for 12 weeks following SCI. SEE significantly and permanently reversed cutaneous allodynia, but not thermal hyperalgesia, to near normal levels. The gross locomotor performance (BBB [Basso, Beattie, and Bresnahan] motor scores) significantly improved about two points. In addition, the BBB subscale scores were significantly improved nearly seven points by the end of the study. SEE also significantly improved foot rotation to normal levels and reduced gridwalk footfall errors nearly 50%, but had no effect on stride length or base of support dysfunctions. SEE significantly increased the total volume of a thoracic segment of cord encompassing the injury site at 12 weeks, by reducing cavitation and increasing both the volume of grey and white matter spared, compared to SCI alone. When BDNF levels were examined in the injured lumbar spinal cord, SEE significantly returned BDNF levels to near-normal. These data suggest that immediate use of SEE after contusive SCI is able to improve overall spinal cell survival and prevent much of the sensory and motor dysfunction that accompanies contusive SCI.     

The influence of spinal canal narrowing and timing of decompression on neurologic recovery after spinal cord contusion in a rat model.

STUDY DESIGN: The effect of spinal canal narrowing and the timing of decompression after a spinal cord injury were evaluated using a rat model. OBJECTIVE: To evaluate whether progressive spinal canal narrowing after a spinal cord injury results in a less favorable neurologic recovery. Additionally, to evaluate the effect of the timing of decompression after spinal cord injury on neurologic recovery. SUMMARY OF BACKGROUND DATA: Results in previous studies are contradictory about whether the amount of canal narrowing or the timing of decompression after a spinal cord injury affects the degree of neurologic recovery. METHODS: Forty adult male Sprague-Dawley rats were equally divided into a control group, in which spacers of 20%, 35%, and 50% were placed into the spinal canal after laminectomy, and an injury group in which the spacers were placed after a standardized incomplete spinal cord injury. After spacer removal, neurologic recovery in both was monitored by Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale (Ohio State University, Columbus, OH) motor scores and transcranial magnetic motor evoked potentials for 6 weeks followed by histologic examination of the spinal cords. Subsequently, 42 rats were divided into five groups in which, after spacer placement, the time until decompression was lengthened 0, 2, 6, 24, and 72 hours. Again, serial BBB motor scores and transcranial magnetic motor evoked potentials were used to assess neurologic recovery for 6 weeks until the animals were killed for histologic evaluation. RESULTS: Spacer placement alone in the control animals resulted in no neurologic injury until canal narrowing reached 50%. All of the control groups (spacer only) exhibited significantly better (P < 0.05) motor scores compared with the injury groups (injury followed by spacer insertion). Within the injury groups the motor scores were progressively lower as spacer sizes increased from the no-spacer group to the 35% group. The results in the 35% and 50% groups were not statistically different. The results of the time until decompression demonstrated that the motor scores were consistently better the shorter the duration of spacer placement (P < 0.05) for each of the time groups (0, 2, 6, 24, and 72 hours) over the 6-week recovery period. Histologic analysis showed more severe spinal cord damage as both spinal canal narrowing and the time until decompression increased. CONCLUSION: The results in this study present strong evidence that the prognosis for neurologic recovery is adversely affected by both a higher percentage of canal narrowing and a longer duration of canal narrowing after a spinal cord injury. The tolerance for spinal canal narrowing with a contused cord appears diminished, indicating that an injured spinal cord may benefit from early decompression. Additionally, it appears that the longer the spinal cord compression exists after an incomplete spinal cord injury, the worse the prognosis for neurologic recovery.