大鼠脊髓损伤后巢蛋白表达

目的探讨成年大鼠脊髓损伤后损伤区局部巢蛋白（nestin）的表达及意义。方法应用Allen＇s法建立大鼠脊髓拟伤模型，行为学评分采用BBB评分（Basso，Beattie＆Bresnahan locomotor rating scale，BBB scale），观察局部病理组织学改变及用免疫荧光组织化学方法检测局部脊髓在不同时段nestin的阳性表达变化。结果伤后1w BBB评分最低，随后增加，到4w以后达到最高并进入平台期。常规病理学检查显示拟伤模型类似于临床常见的脊髓损伤。损伤后1W，可见损伤区附近nestin表达升高，2W达高峰，4W后nestin表达明显下调。结论脊髓损伤可诱导损伤区周围短暂的nestin阳性表达，后者可能存在脊髓损伤后的再生与修复中起重要作用。     

Proprioceptive neuropathy affects normalization of the H-reflex by exercise after spinal cord injury

The H-reflex habituates at relatively low frequency (10 Hz) stimulation in the intact spinal cord, but loss of descending inhibition resulting from spinal cord transection reduces this habituation. There is a return towards a normal pattern of low-frequency habituation in the reflex activity with cycling exercise of the affected hind limbs. This implies that repetitive passive stretching of the muscles in spinalized animals and the accompanying stimulation of large (Group I and II) proprioceptive fibers has modulatory effects on spinal cord reflexes after injury. To test this hypothesis, we induced pyridoxine neurotoxicity that preferentially affects large dorsal root ganglia neurons in intact and spinalized rats. Pyridoxine or saline injections were given twice daily (IP) for 6 weeks and half of the spinalized animals were subjected to cycling exercise during that period. After 6 weeks, the tibial nerve was stimulated electrically and recordings of M and H waves were made from interosseous muscles of the hind paw. Results show that pyridoxine treatment completely eliminated the H-reflex in spinal intact animals. In contrast, transection paired with pyridoxine treatment resulted in a reduction of the frequency-dependent habituation of the H-reflex that was not affected by exercise. These results indicate that normal Group I and II afferent input is critical to achieve exercise-based reversal of hyper-reflexia of the H-reflex after spinal cord injury.     

慢性压迫性脊髓损伤后巢蛋白mRNA表达的观察

目的观察成年大鼠慢性压迫性脊髓损伤后及减压后早期巢蛋白与巢蛋白mRNA的相关性表达。方法选用健康wistar大鼠50只，体重280～320g，制备慢性压迫性脊髓损伤中度、重度及重度损伤减压后3d、10d模型，取自距胜迫边缘5mm段脊髓组织切片。正常成年大鼠作为对照组。行巢蛋白免疫组织化学染色，巢蛋白mRNA原位杂交实验，计算机罔像分析仪定量分析，观察巢蛋白、巢蛋白mRNA在脊髓中央管、灰质和白质中表达的变化，探讨巢蛋白与巢蛋白mRNA表达的相关性。结果成年大鼠慢性膻迫性脊髓损伤中、重度及重度压迫损伤减压后3d，巢蛋白在自、灰质及脊髓中央管室管膜细胞中均有明显表达（P〈0．05），以重度压迫组最为显著（P〈0．01）。减压后10d组灰质与正常对照组比较，差异无显著性意义（P=0．483）。重度压迫组及减压后3d组，巢蛋白mRNA在脊髓灰质、白质及中央管室管膜细胞中均有显著性表达（P〈0．05），以灰质前角第Ⅸ板层、后角和室管膜下区最为显著。中度压迫组，巢蛋白mRNA在灰质前角第Ⅸ板层及中央管室管膜细胞中有显著性表达（P〈0．05），其余区域仅有微弱表达，而白质内巢蛋白mRNA表达于软脊膜下星形胶质细胞的足突中。减压后10d组灰质内巢蛋白mRNA的表达与正常对照组比较，无显著性差异（P=0．375）。正常对照组中无表达。结论成年大鼠慢性压迫性脊髓损伤及减压后早期存在神经前体细胞的增殖。增殖的神经前体细胞巢蛋白与巢蛋白mRNA表达的相关性具有与胚胎发育期脊髓相似的特征。     

大鼠急性脊髓损伤后细胞凋亡以及相关蛋白表达的实验研究

目的 观察脊髓损伤（spinalcordinjury，SCI）后细胞凋亡和相关调控基因（FasL和Caspase．3）的表达情况。方法 通过建立静压型SCI模型（30g的重量，压迫T10节段10min），并用TUNEL染色（terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling）和免疫组化染色方法，了解Sprague-Dawley大鼠SCI后（伤后6、12、24、48和72h以及伤后7、14、21d）细胞凋亡以及相关调控基因（FasL与Caspase-3）表达的情况。结果 大鼠SCI后损伤节段（T10节段）6h出现TUNEL阳性细胞数增加，12h达到高峰，持续3天～1周后显著减少；损伤相邻节段（T9、T11节段）伤后12h出现TUNEL阳性细胞，伤后3dTUNEL阳性细胞数达峰值；SCI后FasL与Cas-pase-3表达均有不同程度的增高。结论 大鼠脊髓损伤后多节段、长时间存在着大量的细胞凋亡；细胞凋亡可能与FasL-Caspase-3途径有关。     

降纤酶对脊髓损伤后VEGF的表达及细胞凋亡的作用

目的 观察降纤酶对大鼠脊髓损伤后血管内皮生长因子(VEGF)的表达及细胞凋亡的影响.方法 SD大鼠随机分为假手术组、对照组、干预组,每组各30只.各组分别于伤后各个时间点进行运动功能(BBB)评分,并随机处死5只,损伤部位取材进行HE染色和免疫组化染色,在光镜下观察、计算VEGF阳性细胞数及凋亡细胞数.结果 假手术组的BBB评分为21分,对照组、干预组在各时间点评分明显降低,干预组在5d及之后的各时间点BBB评分明显高于对照组(P＜0.05),尤其7d、14 d、28 d时间点两组有显著性差异(P＜0.01).脊髓损伤后对照组VEGF在脊髓损伤及损伤周边区高表达,5d达高峰,7d、14d表达仍较明显,28 d见少量表达.干预组各时间点VEGF的表达和对照组相比明显增加(P＜0.05),其中3d、5d、7d、14 d时间点差异显著(P＜0.01).凋亡细胞在脊髓损伤后逐渐增多,1d达高峰,此后逐渐减少;干预组各个时间点凋亡细胞数较对照组明显减少(P＜0.05),其中3d、5d、7d时间点差异显著(P＜0.01).相关性分析发现对照组1～5d时间点VEGF阳性细胞数和细胞凋亡数呈负相关(r=-0.90052,P＜0.05).结论 降纤酶可能通过促进VEGF的表达减少细胞凋亡,从而对急性脊髓损伤起治疗作用.     

西维来司他钠在大鼠急性脊髓损伤中的保护作用

目的探讨脊髓损伤后早期使用西维来司他钠(Sivelestat sodium)抑制细胞凋亡,对大鼠脊髓损伤的保护作用及机制。方法将60只SD大鼠随机分成5组,每组12只,Allen's脊髓损伤模型后,立即腹腔注射Sivelestat sodium 1.6 mg/kg,4.8 mg/kg,10 mg/kg,50 mg/kg,并连续7 d(1次/d),对照组给等量生理盐水,并采用脊髓运动功能评分(BBB scale)对大鼠进行双下肢神经功能评分。原位末端标记法(TUNEL)观察脊髓损伤后脊髓神经细胞凋亡情况。酶联免疫吸附法(ELASA)检测炎症因子。免疫印迹法检测甘油醛-3-磷酸脱氢_E3泛素连接酶-1(GAPDH-Siah1)细胞凋亡序列。结果以BBB标准评价大鼠的双下肢运动功能,治疗组的下肢活动功能明显好于对照组。4.8 mg/kg和10 mg/kg剂量组双下肢神经功能学评分与对照组比较具有明显差异(P0.01);50 mg/kg剂量组未见明显保护作用,因此未加入统计结果。TUNEL染色显示,sivelestat sodium明显减少了脊髓损伤后神经细胞凋亡,抑制脊髓损伤后炎症因子表达。sivelestat sodium显著抑制GAPDH-Siah1凋亡序列的表达。结论 sivelestat sodium通过抑制GAPDH-Siah1凋亡序列,减少脊髓损伤后脊髓神经细胞的凋亡,从而改善脊髓损伤大鼠后肢运动神经功能。     

细胞外三磷酸腺苷对大鼠脊髓损伤后胶质纤维酸性蛋白表达的影响

目的 研究细胞外三磷酸腺苷(ATP)对大鼠脊髓损伤后胶质纤维酸性蛋白(GFAF)表达和运动功能恢复的影响.方法 健康成年Wistar大鼠66只按照随机数字表法取6只作为正常对照组,余60只制作成脊髓打击伤动物模型,并再按照随机数字表法分为两组:ATP组(A组,给予ATP注射)和对照组(B组,给予等量生理盐水注射),每组30只大鼠.伤后1、3、7、14和28 d取材,应用免疫组织化学方法观察GFAP的表达,采用计算机图像分析系统进行半定量分析;并用改良的Tarlov评分观察大鼠脊髓损伤后运动功能的恢复情况.结果 大鼠脊髓损伤后GFAP的表达呈进行性升高,损伤后14 d达高峰;在损伤后7、14和28 d,A组大鼠GFAP的表达明显强于B组;脊髓损伤后14 d和28 d,A组大鼠改良的Tarlov评分明显大于B组;以上差异均有统计学意义(P<0.05).结论 细胞外ATP能促进大鼠损伤脊髓表达GFAP,并有助于大鼠脊髓损伤后运动功能的恢复.     

慢性渐进性压迫对大鼠行为功能及脊髓运动神经递质表达的影响

目的 :观察脊髓慢性受压后实验动物的行为功能与运动神经递质表达的变化情况。方法 :采用大鼠后路渐进性脊髓压迫动物模型 ,观察联合行为评分 (CBS) ,常规病理及免疫组化检测胆碱乙酰转移酶 (ChAT)的变化。结果 :免疫组织化学染色显示 ,在正常大鼠脊髓前角运动神经元及大小神经元ChAT均表达阳性 ,脊髓损伤后ChAT阳性细胞数减少 ,CBS升高 ,二者具有相关关系。结论 :脊髓受压抑制大鼠脊髓神经元合成ChAT ,从而影响实验动物的行为功能     

Potentiating paired corticospinal-motoneuronal plasticity after spinal cord injury

Background

Paired corticospinal-motoneuronal stimulation (PCMS) increases corticospinal transmission in humans with chronic incomplete spinal cord injury (SCI).Objective/Hypothesis: Here, we examine whether increases in the excitability of spinal motoneurons, by performing voluntary activity, could potentiate PCMS effects on corticospinal transmission.

促甲状腺释放激素类似物YM-14673对大鼠脊髓损伤后水肿的影响

目的研究促甲状腺释放激素（ＴＲＨ）类似物,ＹＭ－１４６７３大鼠脊髓损伤后水肿的影响。方法用改良Ａｌｌｅｎ氏法建立大鼠脊髓损伤模型,分设正常组、对照组和治疗组,治疗组在损伤后１５分钟注射ＹＭ－１４６７３,用称重法测量脊髓的水含量,公式：（湿重－干重）÷湿重×１００％。结果对照组示伤后２４小时脊髓水肿,治疗组显示在２４小时脊髓水肿减轻。结论早期应用ＴＲＨ类似物,ＹＭ-１４６７３可减轻脊髓损伤后的脊随水肿。     

Rapid changes in expression of glutamate transporters after spinal cord injury

Glutamate is a major excitatory neurotransmitter in the mammalian CNS. After its release, specific transporter proteins rapidly remove extracellular glutamate from the synaptic cleft. The clearance of excess extracellular glutamate prevents accumulation under normal conditions; however, CNS injury elevates extracellular glutamate concentrations to neurotoxic levels. The purpose of this study was to examine changes in expression and in spatial localization of glial glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2) and the neuronal glutamate transporter EAAC1 (EAAT3) after spinal cord contusion injury (SCI). The levels of all three transporters significantly increased at the epicenter of injury (T10) and in segments rostral and caudal to the epicenter as determined by Western blot analysis. Quantitative immunohistochemistry demonstrated an increase in GLAST staining in laminae I-V and lamina X both rostral and caudal to the epicenter of injury. Staining for GLT-1 increased significantly in lamina I rostral to the injury site and in the entire gray matter caudal to the injury site. A significant increase in EAAC1 staining was observed in laminae I-IV rostral to the epicenter of injury and throughout the gray matter caudal to the injury site. The results suggest that upregulation of these high affinity transporters occurs rapidly and is important in regulating glutamate homeostasis after SCI.     

香芹酚对大鼠急性脊髓损伤的保护作用

目的 探讨香芹酚对大鼠脊髓损伤（SCI）后神经功能的影响及其机制。方法 将60只雄性SD大鼠（200~250 g）随机分为5组:假手术组（n=12）、SCI组（n=12）、香芹酚组（n=36）,香芹酚组根据香芹酚剂量分为低、中、高剂量3个亚组,每亚组12只。低、中、高剂量香芹酚组SCI后30 min腹腔注射香芹酚,剂量分别为10、20、40 mg/kg,每日一次;假手术组和SCI组每日腹腔注射等量生理盐水。采用Allen法建立大鼠SCI模型;假手术组只行椎板切除手术。SCI后24、48、72 h,采用BBB评分评估大鼠神经功能;SCI后72 h,采用ELISA法检测损伤脊髓组织丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、过氧化氢酶（CAT）、caspase-3活性;Western-blot法检测损伤脊髓组织Bax,Bcl-2蛋白表达水平。结果 SCI后,大鼠BBB评分均明显降低（P<0.05）,损伤脊髓组织水肿指数以及MDA、caspase-3和Bax水平均明显增高（P<0.05）,而SOD、GSH、CAT、Bcl-2水平均明显降低（P<0.05）;香芹酚能明显改善大鼠BBB评分（P<0.05）,明显降低水肿指数以及MDA、caspase-3和Bax水平（P<0.05）,而显著增加CAT、SOD、GSH、Bcl-2水平（P<0.05）。结论 香芹酚可通过减轻脊髓水肿、抑制氧化应激反应以及抗凋亡作用而对SCI大鼠发挥神经保护作用。     

Spontaneous long-term remyelination after traumatic spinal cord injury in rats

The capability of the central nervous system to remyelinate axons after a lesion has been well documented, even though it had been described as an abortive and incomplete process. At present there are no long- term morphometric studies to assess the spinal cord (SC) remyelinative capability. With the purpose to understand this phenomenon better, the SC of seven lesionless rats and the SC of 21 rats subjected to a severe weight-drop contusion injury were evaluated at 1, 2, 4, 6, and 12 months after injury. The axonal diameter and the myelination index (MI=axolemmal perimeter divided by myelinated fiber perimeter) were registered in the outer rim of the cord at T9 SC level using a transmission electron microscope and a digitizing computer system. The average myelinated fiber loss was 95.1%. One month after the SC, 64% of the surviving fibers were demyelinated while 12 months later, only 30% of the fibers had no myelin sheath. The MI in the control group was 0.72±0.07 (X±S.D.). In the experimental groups, the greatest demyelination was observed two months after the lesion (MI=0.90±0.03), while the greatest myelination was observed 12 months after the injury (MI=0.83±0.02). There was a statistical difference (p<0.02) in MI between 2 and 12 months which means that remyelination had taken place. Remyelination was mainly achieved because of Schwann cells. The proportion of small fibers (diameter=0.5 μm or less) considered as axon collaterals, increased from 18.45% at 1 month to 27.66% a year after the contusion. Results suggest that remyelination is not an abortive phenomenon but in fact a slow process occurring parallel to other tissue plastic phenomena, such as the emission of axon collaterals.     

Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury

Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in inflammation, wound healing and other pathological processes after neurological disorders. MMP-2 promotes functional recovery after spinal cord injury (SCI) by regulating the formation of a glial scar. In the present study, we aimed to investigate the expression and/or activity of several MMPs, after SCI and human umbilical cord blood mesenchymal stem cell (hUCB) treatment in rats with a special emphasis on MMP-2. Treatment with hUCB after SCI altered the expression of several MMPs in rats. MMP-2 is upregulated after hUCB treatment in spinal cord injured rats and in spinal neurons injured either with staurosporine or hydrogen peroxide. Further, hUCB induced upregulation of MMP-2 reduced formation of the glial scar at the site of injury along with reduced immunoreactivity to chondroitin sulfate proteoglycans. Blockade of MMP-2 activity in hUCB cocultured injured spinal neurons reduced the protection offered by hUCB which indicated the involvement of MMP-2 in the neuroprotection offered by hUCB. Based on these results, we conclude that hUCB treatment after SCI upregulates MMP-2 levels and reduces the formation of the glial scar thereby creating an environment suitable for endogenous repair mechanisms.     

FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury

After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.     

急性大鼠脊髓损伤模型的建立与评估

目的探查大鼠椎骨解剖学特点,为制作大鼠脊髓损伤模型定位提供参考和解剖学依据;建立一种更可靠的急性大鼠脊髓损伤模型。方法将10只体重为200~250 g Wistar大鼠脊柱区进行解剖,对椎骨位置和形态特点进行观察。另取36只成年雌性Wistar大鼠随机分成3组,每组12只:脊髓损伤(SCI)组、假手术组、正常组。各组定期行为学观察(BBB评分),术后30 d进行组织学观察和神经电生理检测。结果第9、10、11胸椎棘突之间距离最为靠近;第一腰椎棘突与脊柱两侧银白色腱膜第一个相交接处对应。HE染色:SCI组可见组织结构不完整,损伤区可见大片坏死灶。BBB评分:SCI组术后第2周开始恢复,最终BBB评分未超过6分。神经电生理(SEP,MEP)检测:SCI组可以明显看到SEP与MEP的峰-峰值急剧降低,且潜伏期明显延长。结论本实验模型制作方法操作简便、重复性好,是较为理想的方法,为大鼠脊髓损伤模型的制作提供了解剖学依据和有力保证。     

Production of tumor necrosis factor in spinal cord following traumatic injury in rats

Production of tumor necrosis factor (TNF) in the spinal cord following traumatic injury has been studied. In these experiments, the level of TNF was examined in the homogenate of the spinal cord, cerebrospinal fluid (CSF) and serum (n = 56). TNF could be detected in the injured spinal cord but not in the normal spinal cord. The TNF level increased in the spinal cord after the injury. At the lesion site, a maximal TNF concentration was observed 1 h after the injury, and the TNF concentration remained at this level until 8 h after the injury. Thereafter, it decreased gradually. However, TNF still could be detected 72 h after the injury. No TNF could be detected in the CSF and serum, collected from rats both with and without spinal cord injury (SCI). This study thus suggests that TNF is produced locally in the spinal cord following traumatic injury, and this TNF production is caused by the injury. The present results also demonstrate that TNF production is an acute and rapid reaction in the spinal cord following traumatic injury.