大鼠脊髓半横断损伤后神经营养因子bFGF、GDNF的表达变化

目的探讨脊髓半横断损伤后早期不同时间碱性纤维母细胞生长因子(bFGF)、胶质细胞源性神经营养因子(GDNF)的表达变化. 方法在成年SD大鼠脊髓T9～T10间半横断,取损伤位点尾侧段T10节段制作冰冻切片,运用bFGF、GDNF兔抗血清以免疫组化亲合素-生物素-过氧化物酶复合物法(ABC法)染色.观察并计数腹角bFGF、GDNF的阳性神经元数.结果 bFGF、GDNF主要分布于正常大鼠脊髓腹角神经元细胞浆,损伤后腹角bFGF、GDNF阳性神经元数在3 d组(n=6)、7 d组(n=6)、21 d组(n=6)均较假手术组(n=6)明显增加(P<0.01),bFGF阳性神经元数在术后3 d时达高峰,随伤后时间的延长进行性减少(P<0.01).而GDNF阳性神经元数在术后7 d时达高峰,3 d组与21 d组比较没有显著差异.结论脊髓半横断损伤(hSCI)后bFGF、GDNF表达明显增加,提示它们可能在hSCI早期修复中发挥作用.     

3-硝基丙酸预处理对大鼠局灶性脑缺血Bcl-2和Bax mRNA表达的影响

目的探讨3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血半暗带Bc l-2和Bax mRNA表达的影响。方法将大鼠腹腔注射3-NPA 20 mg/kg,3 d后制作局灶性脑缺血再灌注模型;采用逆转录聚合酶链反应,观察3-NPA预处理对脑缺血再灌注1 h、6 h、12 h、24 h及48 h额顶部皮质Bc l-2和Bax mRNA表达的影响,并与假手术组和缺血再灌注组比较。结果与假手术组比较,缺血再灌注组和3-NPA预处理组各时间点Bc l-2和Bax mRNA表达极显著增强(均P<0.01);与缺血再灌注组比较,3-NPA预处理组各时间点Bc l-2mRNA表达显著增强(均P<0.05),再灌注12~48 h Bax mRNA的表达显著降低(均P<0.05)。结论增强Bc l-2的表达、抑制Bax的表达,可能是3-NPA预处理抑制细胞凋亡、诱导脑缺血耐受的机制之一。     

大鼠脊髓全横断损伤模型的建立

目的建立一种实用、可靠的大鼠脊髓全横断损伤模型。方法55只SD大鼠随机分为假手术组及脊髓全横断损伤组,横断组选择T12节段横断大鼠脊髓。分别于术后24h、7d及21d取L2节段制成20μm冰冻切片行HE染色,并记录其后肢运动功能评分(BBB评分)。结果脊髓全横断大鼠双下肢运动功能BBB评分在同一时间点之间相比明显低于假手术组,差别有统计学意义(P<0.01)。而且,脊髓横断以下节段出现大量空泡,神经元数量明显减少,胶质细胞明显增生,以腹角为甚;全横断组腹角神经元计数与假手术组在同一时间点比较有明显差异(P<0.01)。大鼠的30天存活率达72%。结论本方法为一种稳定可靠,操作简单的大鼠脊髓全横断模型制作法。     

孕酮对兔脊髓急性缺血再灌注损伤后细胞凋亡的影响

目的探讨孕酮对兔脊髓急性缺血再灌注损伤后细胞凋亡的影响。方法将90只日本大耳白兔随机分为假手术组(n=30)、脊髓损伤组(n=30)与孕酮治疗组(n=30),各组又进一步分为12 h、24 h、36 h、48 h、72 h、14 d等6个亚组,每亚组5只。假手术组只行腹部切开术但不阻断腹主动脉。脊髓损伤组和孕酮治疗组均制作缺血再灌注模型,制模成功后孕酮治疗组每24h注射孕酮1次(将孕酮溶于玉米油,浓度10 mg/ml,注射剂量为8 ml/kg),脊髓损伤组在相同时间点注射等量生理盐水。采用TUNEl染色评估细胞凋亡,采用BBB评分评估运动功能。结果 HE染色结果显示,假手术组脊髓形态正常;脊髓损伤组可见脊髓结构被破坏,正常神经元数目减少;孕酮治疗组较脊髓损伤组正常神经元数目多。TUNEL染色结果显示,脊髓损伤组和孕酮治疗组各时间点凋亡细胞数均明显高于假手术组(P0.05);孕酮治疗组各时间点凋亡细胞的数目均明显少于脊髓损伤组(P0.05)。假手术组术后12 h BBB评分为17.3分,术后14 d时升高到21.0分;脊髓损伤组术后12 h BBB评分为0.6分,随后缓慢升高,术后14 d时评分为9.2分。孕酮治疗组BBB评分变化与脊髓损伤组相似,但术后36 h开始,各时间点评分均明显高于相应脊髓损伤组(P0.05)。结论孕酮治疗可改善脊髓急性缺血再灌注损伤兔运动功能,可能与抑制细胞凋亡有关。     

骨髓基质干细胞静脉移植治疗大鼠脊髓损伤模型后Bcl-2、Bax的表达

目的 通过静脉移植绿色荧光蛋白转基因小鼠骨髓基质干细胞治疗大鼠脊髓挫伤模型,研究骨髓基质干细胞的移植对Bcl-2、Bax表达变化的影响,探讨静脉移植骨髓基质干细胞治疗脊髓损伤的可行性.方法 SD成年雌性大鼠,随机分成正常组、对照组、静脉移植组(后2组再分为术后1、3、7、14、21、28d组).用自制改良的Allen 装置使不锈钢杆自由落下,造成T12脊髓节段挫裂伤.在术前及术后对大鼠进行BBB评分和爬网格试验.术后第2d移植绿色荧光蛋白转基因小鼠的MSCs.之后,分别于1、3、7、14、21、28d取损伤(T12脊髓节段)节段上下约3cm固定,制作15～20μm厚的连续冰冻切片,每隔5片取一片组织进行荧光细胞观察及Bcl-2、Bax抗体免疫组织化学ABC染色.在荧光显微镜下观察细胞移植组灾光细胞,在Olympus光学显微镜下对脊髓灰质前角进行阳性细胞计数,利用HPLAS-1000高清晰图文分析系统检测Bcl-2、Bax免疫阳性反应物的平均灰度值.应用SPSS11.0软件包进行组间多个样本均数比较的单因素方差分析、q检验.结果 在正常组脊髓灰质的前角可见Bcl-2、Bax阳性神经元的表达,Bcl-2免疫阳性产物主要定位于核膜中.Bax主要分布于位于神经元胞浆中.Bcl-2和Bax阳性细胞伤后7d达高峰,随后开始下降.细胞移植后该规律不变.对照组Bcl-2阳性神经元表达与各移植组比较有显著性差异(P＜0.05).结论 与损伤对照组相比细胞移植治疗组7、14d组Bcl-2阳性细胞数上升、Bax阳性细胞数下降,灰度值升高2组间有显著性差异,说明移植骨髓基质干细胞下调,7d及14d组Bax而上调Bcl-2的表达.     

蛛网膜下腔移植骨髓基质干细胞治疗大鼠脊髓损伤模型Fas、Fas-L表达变化

目的 通过蛛网膜下腔移植绿色荧光蛋白转基因小鼠骨髓基质干细胞治疗大鼠脊髓挫伤模型,观察凋亡因子Fas和Fas-L在移植后不同时点的变化情况,为骨髓基质干细胞治疗脊髓损伤及机制提供实验室依据.方法 (1)成年健康SD雌鼠66只(体重200～250g)随机分成11组,每组6只,即空白对照组、损伤对照组、细胞移植治疗组(后2组再分为术后1、3、7、14、21d组).(2)在手术前、后对大鼠进行BBB评分和爬网格试验.术后第2d移植MSCs.之后,制作15～20μm厚的连续冰冻切片,间隔取片进行荧光细胞观察及Fas和Fas-L抗体免疫组织化学ABC染色.并在光学显微镜下对脊髓灰质前角进行阳性细胞计数,用图文分析系统检测Fas和Fas-L免疫阳性反应物的平均灰度值.应用SPSS11.0软件包进行组间多个样本均数比较的单因素方差分析、q检验.结果 3组脊髓组织中均可见Fas和Fas-L阳性细胞.损伤对照组和细胞移植治疗组Fas和Fas-L 阳性细胞数出现先升高后降低的趋势,1d组就出现表达,持续到14d组,21d组表达接近正常对照组,高峰出现在7d.21d接近正常对照组.损伤对照组与细胞移植治疗组相比较,阳性细胞数在7d、14d组出现差异(P＜0.05) 细胞移植治疗组Fas和Fas-L 1、3、7d组灰度值低于正常对照组(P＜0.05) .结论 与损伤对照组相比细胞移植治疗组7 、14d组Fas、Fas-L阳性细胞数下降,灰度值升高,2组间有显著性差异,说明移植骨髓基质干细胞下凋7d及14d组Fas、Fas-L的表达.     

生物素葡聚糖胺皮质脊髓束顺行示踪技术在大鼠脊髓损伤模型中的应用

目的探讨生物素葡聚糖胺(BDA)神经示踪技术及脊髓半横断损伤模型在大鼠脊髓损伤修复的实验研究中应用。方法采用成年Sprague-Dawley大鼠,分为脊髓致伤组(n=10)和致伤对照组(n=10)。致伤组动物在相当于T7椎板水平横行剪断脊髓的后2/3;对照组动物术中仅切除椎板,不切断脊髓。术后第15d,右侧开颅,用10?A示踪剂注入右侧的感觉运动区皮质内。2周后取出大脑和脊髓组织,采用自由漂乳法行BDA染色显影。术后实验动物功能测评采用BBB运动功能评分,所得数据采用Student'st-test进行统计学原理。结果(1)脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);(2)BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在同侧中脑、桥脑及延髓的腹侧面行走,在锥体交叉后皮质脊髓束主要在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;对照组皮质脊髓束BDA染色可一直延伸至L1水平。结论大鼠半脊髓切断结合应用BDA顺行示踪技术可以对脊髓损伤后的神经修复状况进行可靠的形态学评判,是研究脊髓损伤后中枢神经纤维再生修复较为理想的动物模型     

硫酸镁对大鼠脑弥漫性轴索损伤海马区Bcl-2和Bax蛋白表达影响的研究

目的研究脑弥漫性轴索损伤后神经细胞迟发性死亡的机制,探讨镁离子对大鼠脑弥漫性轴索损伤后神经元的保护作用。方法采用Marmarou方法制备大鼠重度脑弥漫性轴索损伤模型,分为创伤组(n=25)、生理盐水组(n=40)和硫酸镁组(n=40)。硫酸镁组伤后半小时给予25%硫酸镁(750μmol/kg),生理盐水组在相同时间给予等量的生理盐水腹腔注射,于伤后6小时、24小时、3天、5天及7天5个时相点处死。采用HE染色、免疫组织化学技术动态观察大鼠海马区的组织病理改变,Bcl-2和Bax蛋白的表达情况,以及使用硫酸镁干预后对上述表达的影响。结果①Bcl-2蛋白的表达:假手术组大鼠海马区仅见极少量Bcl-2阳性细胞,着色淡。在伤后6小时即有大量的Bcl-2阳性细胞,随时间渐增,24小时达到高峰,3～7天逐渐减少。②Bax蛋白的表达:在DAI后大鼠海马区有大量Bax阳性细胞,在伤后6小时就有所增加,24小时显著增加,3天达到高峰。Bcl-2/Bax值在损伤后随时间逐渐上升。③硫酸镁组中,海马区的Bax表达与对照组相比有所减少,而Bcl-2相应增加,Bcl-2/Bax比值也是上调的,均有统计学意义(P<0.05)。结论大鼠脑弥漫性轴索损伤后,海马区神经元存在有迟发性细胞死亡即凋亡现象。Bax与Bcl-2参与细胞凋亡过程。硫酸镁可通过抑制Bax蛋白,上调Bcl-2蛋白,减少神经细胞凋亡,对促进神经细胞修复和功能重塑有益。     

脊髓损伤过程中β-1,4半乳糖基转移酶的表达研究

目的 探讨β-1,4半乳糖基转移酶(β-1,4-GalT)在横断性脊髓损伤后的时空表达变化以及细胞定位情况.方法 将42只成年SD大鼠随机分为假手术组和T9横断伤8 h、1 d、3 d、5 d、7 d、14 d组,每组6只.采用实时定量PCR测定损伤后各时间段β-1,4-GalT在脊髓中的表达变化;采用原位杂交与免疫荧光标记方法检测β-1,4-GalT在脊髓中的分布以及伤后的定位改变.结果 脊髓横断损伤后,β-1,4-GalT-Ⅴ在损伤上、下段表达高峰分别出现在损伤后8 h和1 d,之后逐渐下降,至伤后14 d降低至假手术组水平.增高的mRNA主要分布于损伤周围的细胞中及脊髓后角浅层的感觉神经元中.荧光原位杂交结果显示,β-1,4-GalT-Ⅴ主要分布于巨噬细胞和小胶质细胞以及病理状态下的少突胶质细胞.同时β-1,4-GalT-Ⅴ mRNA在损伤后大量表达于富含P物质和IB-4的脊髓后角浅层的感觉神经元中.结论 脊髓损伤后β-1,4-GalT在基因和细胞水平呈现明显的时空变化,并且与巨噬细胞和小胶质细胞以及病理状态下的少突胶质细胞存在共定位.该酶可能参与了损伤后的继发性损伤,即伤后早期的炎性反应,并可能与脊髓损伤后的神经病理性疼痛有关.     

不同潜伏期致痫大鼠海马神经元线粒体损伤及Fas和Bax表达

目的观察不同潜伏期致癫痫持续状态(SE)大鼠海马区神经元线粒体超微结构损伤及Fas和Bax的表达。方法分别采用海人酸腹腔注射(A组)和尾静脉注射(B组)诱发不同潜伏期的大鼠SE。于SE终止后3、6、24、48、72点取海马,电镜观察线粒体的超微结构,半定量RTPCR检测Fas、Bax的mRNA表达。另取10只不作任何处理的大鼠作为正常对照组。结果A组潜伏期为97±11min,线粒体肿胀,神经元呈凋亡征;B组潜伏期为48±13min,线粒体肿胀且伴膜的崩解,神经元呈坏死表现。B组Fas及BaxmRNA的表达与对照组相比无明显差异(P>0.05);与对照组相比A组Fas及BaxmRNA表达均于SE后6h增加(P<0.05),48h达高峰(P<0.001),并持续至72h(P<0.001)。结论不同潜伏期的SE导致了不同程度的线粒体损伤,进而决定了神经元死亡的分子机制。     

Establishment and evaluation of a rat model of complete transected spinal cord injury*☆

BACKGROUND： The establishment of a rat model of complete transected spinal cord injury lacks technological specifications. The current models lack concordance and reliability, and the death rate of the experimental animals is high. Therefore, there is a great need for a reliable model to apply clinical applications of therapy. OBJECTIVE： To construct a rat model of complete transected spinal cord injury characterized by stability, reproducibility, and a high animal survival rate. DESIGN： Completely randomized controlled study. SETTING： Department of Neurosurgery, Xiangya Hospital of Central South University. MATERIALS： Fifty-five healthy specific pathogen free grade adult female Sprague Dawley rats were provided by the Experimental Animal Department, Xiangya Medical College, Central South University. Olympus BX51 imaging collecting analytic system was provided by Olympus Company, Japan; and SEN-7203 Nihon-Kohden electrical stimulator by Nihon Kohden, Japan. METHODS： This study was performed at the Laboratory of Neurosurgery, Xiangya Hospital of Central South University from April to June 2006. Experimental grouping： 55 rats were randomly divided into model group （n = 40） and sham surgery group （n = 15）. In the model group, a self-made sliver hook was passed through the ventral side to support the spinal cord at the T12 segment and to shear it off. A complete transected spinal cord, 2 mm in length, was resected. In the sham surgery group, the spinal cord was identically exposed. The dura mater of the spinal cord was cut open, but the spinal cord was not damaged. MAIN OUTCOME MEASURES： Histopathological changes after spinal cord injury at L2 segment were observed subsequent to hematoxylin and eosin staining under optical microscopy. Olympus BX51 imaging collecting analytic system was used to count spinal cord ventral horn neurons. Motor function of rat hindlimb was evaluated with the Basso, Beattie and Bresnahan （BBB） scale. Paraplegia was evaluated as 0 point, and complete normality as     

大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的：探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系。方法：用免疫组织化学技术、HE染色和Tunnel法，观察坐骨神经切断后1，6，12，24h及3，7和14d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化。结果：坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化，星形胶质细胞的活化早于小胶质细胞；后期运动神经元发生凋亡，HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕。结论：研究结果提示，坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切。     

Effect of interleukin-2 on neurogliocyte and neuron apoptosis in rat models of acute spinal cord injury

BACKGROUND： lnterleukin-2 （IL-2） may influence the growth and survival of nerve cells following spinal cord injury and resuscitate the proliferation and maturation of oligodendrocytes. OBJECTIVE： To observe the effect of IL-2 on neuronal apoptosis of neurogliocytes at different times following acute spinal cord injury in rats. DESIGN, TIME AND SETTING： A randomized grouping trial based on cellular morphology was performed at the Institute of Traumatic Orthopedics of Shandong Province between October 2004 and January 2006. MATERIALS： A total of 72 adult, male, Sprague Dawley rats were included in this study and were divided into a control group and an IL-2 group. The Bcl-2 monoclonal antibody and TUNEL kit were purchased from Wunan Boster Biological Technology Corporation. METHODS： Spinal cord injury was induced in all the rats by dropping a weight from a height of 25 cm onto the exposed spinal cord at vertebral levels T7-11, thus producing a mild lesion. Immediately following the modeling, the rats were injected with daily IL-2 （10 uL） intramuscularly （the IL-2 group）. Other rats received an injection of physiological saline 0.5 mL/d （the control group）. MAIN OUTCOME MEASURES： Bcl-2 immunohistochemistry was applied to detect the Bcl-protein and positive cell expression. The TUNEL method was used to count the number of apoptotic cells. RESULTS： The expression level of Bcl-2 proteins increased significantly in spinal cord tissues during the first day after acute spinal cord injury, reaching a peak on days 3 and days 8 in the control and IL-2 groups, respectively. They were more prevalent in neurogliocytes than in neurocytes, and then began to decrease on day 14. From then until day 21, less expression was detected （P 〈 0.05）. In the control group, many apoptotic cells existed after 24 hours, and most of them were gliocytes; apoptotic cells reached a peak after 3-8 days. They then decreased gradually until day 21, when a small number of cells were still available. In the IL-2 grou     

Fas receptor and neuronal cell death after spinal cord ischemia.

Cell death from spinal cord injury is mediated in part by apoptotic mechanisms involving downstream caspases (e.g., caspase-3). Upstream mechanisms may involve other caspases such as procaspase-8, a 55 kDa apical caspase, which we found constitutively expressed within spinal cord neurons along with Fas. As early as 1.5 hr after transient ischemia, activated caspase-8 (p18) and caspase-8 mRNA appeared within neurons in intermediate gray matter and in medial ventral horn. We also detected evidence for an increase in death receptor complex by co-immunoprecipitation using Fas and anti-procaspase-8 after ischemia. At early time points, Fas and p18 were co-expressed within individual neurons, as were activated caspase-8 and caspase-3. Moreover, we detected p18 in cells before procaspase-3 cleavage product (p20), suggesting sequential activation. The appearance of cytosolic cytochrome c and gelsolin cleavage after ischemia was consistent with mitochondrial release and caspase-3 activation, respectively. Numerous terminal deoxynucleotidyl transferase-mediated DNA nick end-labeling-positive neurons contained p18 or p20 (65 and 80%, respectively), thereby supporting the idea that cells undergoing cell death contain both processed caspases. Our data are consistent with the idea that transient spinal cord ischemia induces the formation of a death-inducing signaling complex, which may participate in caspase-8 activation and sequential caspase-3 cleavage. Death receptors as well as downstream caspases may be useful therapeutic targets for limiting the death of cells in spinal cord.     

Protective effects of prostaglandin E1 perfusion againstspinal cord ischemia-reperfusion injury in a rabbit model

BACKGROUND: Prostaglandin E1 (PGE1) is known to be protective in ischemia-reperfusion of heart, lung, renal, and liver tissue. It still remains to be determined whether PGE1 exhibits similar protection against spinal cord ischemia-reperfusion injury in a rabbit model. OBJECTIVE: To observe the large, ventral horn, motor neurons of the spinal cord, as well as limb function, and to investigate whether perfusion of PGE1 exhibits protective effects against spinal cord ischemia-reperfusion injury in a rabbit model. DESIGN, TIME AND SETTING: Controlled observation. The experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University between June and October 2007. MATERIALS: Twenty male, New Zealand white rabbits, weighing 2.0 kg and of mixed gender, were used in the present study. The following chemicals and compounds were used: prostaglandin El injectable powder, as well as malondialdehyde and ATPase kits. Animal intervention was in accordance with animal ethical standards. METHODS: We separated rabbits into control and experimental groups randomly, with 10 rabbits in each group. Rabbits were used as spinal cord ischemia models by segmentally cross-clamping the infrarenal aorta. The control group was subsequently perfused for five minutes with blood and saline solution, and the experimental group was perfused for 5 minutes with blood and saline solution containing PGE1 (100 ng/kg/min). MAIN OUTCOME MEASURES: The neurological function of the hind limbs was assessed 12, 24, and 48 hours after model establishment. All animals were sacrificed and spinal cords were harvested for histological analyses. The large motor neurons in the ventral horn of L1-7 were observed by inverted microscope. RESULTS: All 20 rabbits were included in the final analysis, without any loss. In the ventral horn of the L5-7 segments, there were more large motor neurons that appeared viable in the experimental group than the control group (P<0.05). The scores of hind limb functions were greater in the experimental group after 12, 24, and 48 hours (P<0.01). CONCLUSION: Perfusion of PGEI reduced the amount of neuronal damage in the spinal cord ischemia-reperfusion injury rabbit model. These results correlated with increased numbers large motor neurons in the ventral horn of the spinal cord, as well as improved hind limb function.     

Astrocyte-conditioned medium attenuates glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats

Abstract

Objectives:

To better understand the neuroprotective role of astrocytes in spinal cord injury (SCI), we investigated whether astrocyte-conditioned medium (ACM) can attenuate glutamate-induced apoptotic cell death in primary cultured spinal cord neurons.

Methods:

Spinal cord neurons were pretreated with ACM for 24?hours. Subsequently, they were exposed to glutamate (125?μM) for 1?hour. The neurons were then incubated for 24?hours. Following that, measurements assessing cell viability and lactate dehydrogenase (LDH) release were performed. Apoptosis was confirmed through cell morphology using Hoechst 33342 staining and terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay. Assessment for expression of apoptotic enzymes, including Caspase-3, Bcl-2 and Bax, was performed using Western Blot Analysis.

Results:

Astrocyte-conditioned medium pretreatment of neurons showed both an increase in spinal cord neuron viability and a decrease in LDH release in a dose-dependent pattern. Moreover, pretreatment seems to attenuate glutamate-induced apoptotic cell death, antagonise glutamate-induced up-regulation of Caspase-3 expression and downregulate Bcl-2/Bax protein expression ratio.

Conclusions:

By attenuating glutamate-induced apoptotic cell death in primary cultured spinal cord neurons of rats, ACM seems to provide a neuroprotective effect by regulating apoptosis-related protein expression. Our results provide an experimental basis for clinical applications and potential therapeutic use of ACM in SCI.     

Expressions of caspase-3, Tunel, and Hsp72 immunoreactivities in cultured spinal cord neurons of rat after exposure to glutamate, nitric oxide, or peroxynitrite

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanisms are not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) or 72kDa heat shock protein (HSP72). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immu-noreactivity for caspase-3 became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 24h. TUNEL positive nuclei were found in spinal large motor neurons from 24 h, and the positive cell proportion greatly increased at 48 h in contrast to the vehicle. On the other hand, the immunoreactivity of HSP72 in the ventral horn was already positive at 0 h, and gradually decreased in the course of time with glutamate, NOC18 or PN than vehicle treatment. In the dorsal horn, the proportion of caspase-3 positive small neurons greatly increased at 6 to 48 h after addition of glutamate. The present results suggest that both excitotoxic and oxidative stress play important role in the apoptotic pathway in cultured rat spinal neurons.     

神经干细胞移植影响脊髓全横断大鼠大脑运动皮质相关凋亡基因的表达

背景：多项研究已证实神经干细胞能促进脊髓损伤大鼠神经功能的恢复,但其分子机制还不清楚。 目的：观察神经干细胞移植对脊髓全横断损伤大鼠大脑运动皮质相关凋亡基因Bax,Bcl-2和Caspase-3 mRNA表达的影响。 设计、时间及地点：随机对照动物实验,于2007-07/2008-12在昆明医学院神经科学研究所完成。 材料：孕14~15 d绿色荧光蛋白转基因鼠5只,取其胚胎用于神经干细胞培养。清洁级健康成年雌性SD大鼠88只,随机分成3组：假手术组8只、模型组40只、细胞移植组40只。 方法：模型组、细胞移植组大鼠建立T9脊髓全横断脊髓损伤模型,假手术组只行T8椎板切除。用DMEM/F12调整胎鼠神经干细胞密度为2×1010 L-1,吸取细胞悬液15 μL滴加到约2 mm3大小的明胶薄片上,细胞移植组将此明胶薄片植入大鼠脊髓两横断面之间的间隙处。分别于细胞移植后3,7,14,21,28 d取材进行指标检测。 主要观察指标：RT-PCR法检测大脑运动皮质Bax,Bcl-2和Caspase-3 mRNA表达的变化。 结果：与假手术组比较,模型组各时间点Bax的表达均无明显差异(P > 0.05),术后14,28 d Bcl-2的表达明显减少(P < 0.05),术后3 d Caspase-3的表达明显升高(P < 0.05)。与模型组比较,细胞移植组在神经干细胞移植后3 d Bax的表达明显减少(P < 0.05),移植后14,21 d Bcl-2的表达明显增高(P < 0.05),移植后3,7 d Caspase-3的表达明显减少(P < 0.05)。 结论：神经干细胞移植后,可能通过调控大脑运动皮质相关凋亡基因 Bax,Bcl-2和Caspase-3 mRNA的表达促进大鼠全横断脊髓损伤修复。