Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND: Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells.OBJECTIVE: To investigate the effects of valproic acid on proliferation of endogenous neural sterm cells in a rat model of spinal cord injury.DESIGN, TIME AND SETTING: A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007.MATERIALS: A total of 45 adult, Wistar rats were randomly divided into sham surgery (n=5), injury(n=20), and valproic acid (n=20) groups. Valproic acid was provided by Sigma, USA.METHODS: Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours (twice in total).MAIN OUTCOME MEASURES: Nestin expression (5 mm from injured center) was detected using immunohistochemistry at 1, 3 days, 1, 4, and 8 weeks post-injury.RESULTS: Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week (P<0.05).Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was nosignificant difference in nestin expression between the valproic acid and injury groups. At 1 week,there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group (P<0.05). Expression reached a peak by 4 weeks, and it was still present at 8 weeks.CONCLUSION: Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.     

吗啡预处理与脑缺血再灌注损伤后细胞凋亡：抑制效应的途径？

[摘要] 背景：大量研究已经证明凋亡是脑缺血再灌注损伤后神经元损伤的重要形式,且这一过程可以人为干预以改善预后。药物预处理对缺血再灌注损伤后神经元凋亡的影响是脑缺血研究的热点。吗啡是临床常用药,几项研究显示其对某种形式的脑损伤有保护作用,但吗啡预处理对脑缺血再灌注损伤后神经元凋亡的影响尚未见报道。 目的: 探讨吗啡预处理对大鼠全脑缺血再灌注损伤后神经元凋亡及相关基因表达的影响。 设计、时间及地点：2008年6月-2009年8月在青岛大学医学院脑血管病研究所完成分子生物学水平的随机对照实验。 材料：神经元凋亡及免疫组化检测试剂盒均由武汉博士德公司提供 方法: 健康雄性成年Wistar大鼠72只,随机分成4组：假手术组;脑缺血/再灌注组;吗啡预处理1mg/kg组;吗啡预处理7mg/kg组,18只/组。依再灌注时间不同,各组又分为再灌注1d、3d、7d 三个亚组,6只/亚组。以Pusinelli方法为标准建立四动脉阻断法全脑缺血模型,假手术组仅暴露第一颈椎双侧翼孔和双侧颈总动脉而不烧灼,不夹闭动脉;脑缺血组缺血前60min腹腔注射生理盐水2mg/kg;吗啡预处理1mg/kg组及吗啡预处理7mg/kg组分别在脑缺血前60min腹腔内注射吗啡1mg/kg及7mg/kg。在脑缺血8分钟后恢复血流,再灌注1d、3d、7d后断头取脑制作石蜡切片。 主要观察指标： HE染色观察海马CA1区组织病理学改变,TUNEL法检测海马CA1区神经元凋亡,免疫组化检测海马CA1区Casepase-3蛋白表达。 结果：HE染色：假手术组海马CA1区神经元结构正常;脑缺血组则出现大量的肿胀、核固缩及胞浆空泡样变异常细胞并神经元数量显著减少;吗啡预处理组细胞肿胀、核皱缩及细胞缺失的病理学改变显著轻于脑缺血再灌注组。凋亡细胞计数：与假手术组比较,缺血组和吗啡预处理组海马CA1区神经元凋亡数明显增加(P<0.01) ;与缺血再灌注组比较, 吗啡预处理组神经元的凋亡数明显减少(P<0.01);与吗啡预处理1mg/kg组比较,吗啡预处理7mg/kg组神经元凋亡数显著降低(P< 0.05 或P< 0.01)。Casepase-3蛋白表达：缺血组和吗啡预处理组Casepase-3表达明显高于假手术组(P<0.01);吗啡预处理组Casepase-3表达显著低于缺血再灌注组(P<0.01);吗啡预处理7mg/kg组Casepase-3表达明显低于吗啡预处理1mg/kg组(P< 0.05 )。应用吗啡后,在1d、3d、7d三个时点,神经元凋亡的减少趋势与Casepase-3降低的趋势一致。 结论： 吗啡预处理可减轻缺血性脑损伤,提高脑缺血耐受性,且大剂量吗啡效果优于小剂量;吗啡抗凋亡作用机制与Casepase-3密切有关。     

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。     

Apoptotic and anti-apoptotic mechanisms following spinal cord injury

A number of studies have provided evidence that cell death from moderate traumatic spinal cord injury (SCI) is regulated, in part, by apoptosis that involves the caspase family of cysteine proteases. However, little or no information is available about anti-apoptotic mechanisms mediated by the inhibitors of apoptosis (IAP) family of proteins that inhibit cell death pathways. In the present study, we examined caspase and IAP expression in spinal cords of rats subjected to moderate traumatic injury. Within 6 h after injury, caspase-8 and-9 (2 initiators of apoptosis) were predominantly present in gray matter neurons within the lesion epicenter. By 3 days following spinal cord injury (SCI), caspase-8 and-9 immunoreactivity was localized to gray and white matter cells, and by 7 days following SCI, both upstream caspases were expressed in cells within white matter or within foamy macrophages in gray matter. Caspase-3, an effector caspase, was evident in a few fragmented cells in gray matter at 24 h following injury and then localized to white matter in later stages. Thus, distinct patterns of caspase expression can be found in the spinal cord following injury. XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the cord. Immunoblots of spinal cord extracts revealed that the processed forms of caspases-8 and-9 and cleavage of PARP are present as early as 6 h following trauma. The expression of caspases corresponded with the detection of cleavage of XIAP into 2 fragments following injury. cIAP-1 and cIAP-2 expression remained constant during early periods following SCI but demonstrated alterations by 7 days following SCI. Our data are consistent with the idea that XIAP may have a protective role within the spinal cord, and that alteration in cleavage of XIAP may regulate cell death following SCI.     

tetrandrine神经元凋亡的影响,bcl-2和bax表情证明急性脊髓损伤* *

观察汉防己甲素（Tetrandrine ,Tet）对大鼠急性脊髓损伤后组织结构神经细胞凋亡和运动功能恢复的影响并探讨其作用机制。方法：选用100只成年大鼠,随机分为四组,即假手术组10只（A组）、损伤对照组（B组）30只、甲基强的松龙治疗组（CD组）30只、Tet治疗组（DC组）30只。胸8、9椎板切除后B、C、D组用加速压迫型Allen’s打击法制成脊髓损伤模型。C组和D组动物于制模前、伤后24h、伤后48h尾静脉注射甲基强的松龙(MP)和Tet。各组大鼠于术后8h,1d,3d,7d,14d行BBB评分,分别于术后8h,1d,3d,7d,14d取损伤段脊髓行石蜡切片HE染色,观察脊髓组织的形态结构变化和免疫组织化学染色检测细胞凋亡因子bcl-2、bax 。结果： 伤后7d、14dC组与D组大鼠运动功能评分(BBB评分)显著高于B组,各时间点C组与D组评分无统计学意义;A组的脊髓组织HE染色正常 ,C组与D组脊髓组织损害较B组轻,术后8h-14d动态观擦,3d—7d损伤表现最为严重,达到损伤高峰期;A组中bax、bcl-2表达较少,C组与D组bax 表达较B组少,而bcl-2表达较B组多。结论：汉防己甲素可通过增加bcl-2表达、降低bax 表达,抑制急性脊髓损伤后神经细胞的凋亡,能有益于脊髓组织的保护,促进运动功能的恢复。     

丙戊酸对脊髓损伤大鼠内源性神经干细胞增殖的影响

BACKGROUND： Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells.
OBJECTIVE： To investigate the effects of valproic acid on proliferation of endogenous neural stem cells in a rat model of spinal cord injury.
DESIGN, TIME AND SETTING： A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007.
MATERIALS： A total of 45 adult, Wistar rats were randomly divided into sham surgery （n = 5）, injury （n = 20）, and valproic acid （n = 20） groups. Valproic acid was provided by Sigma, USA. METHODS： Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours （twice in total）.
MAIN OUTCOME MEASURES： Nestin expression （5 mm from injured center） was detected using immunohistochemistry at 1,3 days, 1, 4, and 8 weeks post-injury.
RESULTS： Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week （P 〈 0.05）. Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was no significant difference in nestin expression between the valproic acid and injury groups. At 1 week, there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group （P 〈 0.05）. Expression reached a peak by 4 weeks, and it was still present at 8 weeks.
CONCLUSION： Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.     

Electrical fields and the promotion of endogenous angiogenesis in a rat spinal cord injury model

BACKGROUND:Previous studies have shown that direct current electrical fields affect development and growth of human microvascular endothelial cells,but the role of electrical fields on promoting angiogenesis in tissues following spinal cord injury remains poorly understood.OBJECTIVE:To determine the effects of electrical fields on angiogenesis and spinal cord repair following traumatic spinal cord injury in rats.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Chongqing Key Laboratory of Neurology,Affiliated Hospital of Chongqing Medical University,China from September 2007 to August 2008.MATERIALS:Hydrogen blood flow detector(Soochow University Medical Instrument,China),Power Lab System(AD Instruments,Colorado Springs,CO,USA)and mouse anti-vascular endothelial growth factor(VEGF)monoclonal antibody(Sigma-Aldrich,St.Louis,MO,USA)were used in this study.METHODS:A total of 60 healthy,adult,Sprague Dawley rats were equally and randomly assigned to sham-surgery,model,and electrical field groups.The Allen's weight-drop method was used to induce complete spinal cord injury in the model and electrical field groups.Rats in the electrical field group were implanted with silver needles and electrical fields(350 V/m)were applied following traumatic injury.MAIN OUTCOME MEASURES:Latency of somatosensory-evoked potential was detected and spinal cord blood flow was measured by hydrogen blood flow detector.Microvascular density was determined by histological analysis.VEGF expression in the spinal cord was observed by immunohistochemical staining.RESULTS:Recovery of spinal cord blood flow was significantly increased in the electrical field group(at 1,2,4,8,and 24 days after injury)compared with the model group(P < 0.05 or P <0.01).Latency of P1 waves in somatosensory-evoked potential of electrical field group(at 1,2,4,8,and 24 days after injury)was significantly shorter than the model group(P < 0.05 or P < 0.01).Microvascular density and VEGF expression were greater in the electrical field group compared with the model group at 24 days after injury(P< 0.01).CONCLUSION:Electrical fields(350 V/m)promoted angiogenesis within injured rat tissue following spinal cord injury and improved spinal cord function.Electrical fields could help to ameliorate spinal cord injury.The mechanisms of action could be related to increased VEGF expression.     

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。     

神经干细胞移植修复鼠脊髓损伤的实验研究

目的 :观察神经干细胞移植治疗对鼠脊髓损伤后神经结构修复和功能恢复的作用并探讨其作用机制。方法 :制备鼠T10 脊髓损伤模型 ,体外培养、诱导鼠神经干细胞 ,定量评价神经干细胞移植对脊髓损伤后神经结构修复和功能恢复的影响。结果 :与对照组相比 ,神经干细胞移植组明显的增强了GAP 43mRNA的表达 ,促进了脊髓ChAT阳性的运动神经元的再生、结构的修复和下肢运动功能的恢复。结论 :神经干细胞移植促进了脊髓损伤后神经结构的修复和功能的恢复 ,是急性脊髓损伤一种有效的治疗方案     

Time representation of mitochondrial morphology and function after acute spinal cord injury

Changes in mitochondrial morphology and function play an important role in secondary damage after acute spinal cord injury. We recorded the time representation of mitochondrial morphology and function in rats with acute spinal cord injury. Results showed that mitochondria had an irregular shape, and increased in size. Mitochondrial cristae were disordered and mitochondrial membrane rupture was visible at 2–24 hours after injury. Fusion protein mitofusin 1 expression gradually increased, peaked at 8 hours after injury, and then decreased to its lowest level at 24 hours. Expression of dynamin-related protein 1, amitochondrial fission protein, showed the opposite kinetics. At 2–24 hours after acute spinal cord injury, malondialdehyde content, cytochrome c levels and caspase-3 expression were increased, but glutathione content, adenosine triphosphate content, Na+-K+-ATPase activity and mitochondrial membrane potential were gradually reduced. Furthermore, mitochondrial morphology altered during the acute stage of spinal cord injury. Fusion was important within the first 8 hours, but fission played a key role at 24 hours. Oxidative stress was inhibited, biological productivity was diminished, and mitochondrial membrane potential and permeability were reduced in the acute stage of injury. In summary, mitochondrial apoptosis is activated when the time of spinal cord injury is prolonged.     

Ependymal cell proliferation and apoptosis following acute spinal cord injury in the adult rat

BACKGROUND: Studies have reported that spinal cord injury can induce the reactive proliferation of ependymal cells and secondarily cause the apoptosis of nerve cells. However, there is no generally accepted theory on the apoptotic characteristics of ependymal cells in the injured spinal cord.OBJECTIVE: To observe the reactive proliferation and apoptosis of ependymal cells in adult rats following acute spinal cord injury.DESIGN, TIME AND SETTING: A randomized control study based on neuropathology was performed in the Third Military Medical University of Chinese PLA between 2005 and 2007.MATERIALS: Forty healthy, adult, Wistar rats were included in the present study.METHODS: Moderate spinal cord injury was established in twenty rats using Feeney's method, while the remaining 20 rats served as controls and were only treated with laminectomy. All rats were injected intraperitoneally with 1.25 mL of BrdU solution (10 mg BrdU/mL saline) 3 times at 4 hours intervals during the 12 hours prior to sacrifice.MAIN OUTCOME MEASURES: Ependymal cell proliferation and apoptosis in the rat spinal cord were determined by BrdU and nestin immunofluorescence double-labeling, as well as the TUNEL method, at 1, 3, 7, and 14 days after operation.RESULTS: In the moderate spinal cord injury rats, nestin expression was observed in the cytoplasm of ependymal cells. One day immediately following surgery, ependymal cells were BrdU-labeled. The number of BrdU-positive cells increased at 3 days, reached a peak at 7 days, and gradually reduced thereafter. The ependyma developed ti'om a constitutive monolayer cells to a multi-layer cell complex. Some BrdU/Nestin double-positive ependymal cells migrated out from the ependyma. TUNEL-positive cells were also detected in the ependyma in the central region, as well as ischemic regions of the injured spinal cord. In addition, TUNEL-positive cells were visible in the ependyma. No TUNEL-positive ependymal cells were observed in the normal spinal cord.CONCLUSION: Proliferating ependymal cells induced apoptosis in the central and surrounding region following spinal cord injury.     

移植分泌神经营养素3的人胚神经干细胞在脊髓损伤大鼠体内存活及对运动功能的影响

BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes.
OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3.
DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007.
MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls.
METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale.
MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats.
RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）.
CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.     

Protective effects of human umbilical cord mesenchymal stem cell vein transplantation against spinal cord ischemia/reperfusion injury in rats

BACKGROUND: The majority of studies addressing spinal cord ischemia/reperfusion injury (SCIRI) have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reports that human umbilical cord mesenchymal stem cell (hUCMSC) transplantation achieves good therapeutic effects, but the mechanisms underlying nerve protection remain poorly understood.OBJECTIVE: To observe survival of transplanted hUCMSCs in SCIRI rat models and the influence on motor function in the hind limbs, to determine interleukin-8 expression and cellular apoptosis in spinal cord tissues, and to verify the hypothesis that hUCMSC transplantation exhibits protective effects on SCIRI.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Laboratory of the Department of Orthopedics in the First Affiliated Hospital of Soochow University,China between January 2007 and December 2008.MATERIALS: hUCMSCs were harvested from umbilical cord blood of healthy pregnant women after parturition in the Obstetrical Department of the First Affiliated Hospital of Soochow University, China. Rabbit anti-human BrdU monoclonal antibody was provided by DAKO, USA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit and enzyme-linked immunosorbent assay (ELISA) Kit were purchased by Wuhan Boster, China. METHODS: A total of 72 healthy, Wistar, adult rats were randomly assigned to three groups: sham-surgery, model, and transplantation, with 24 rats in each group. SCIRI was induced in the model and transplantation groups via the abdominal aorta block method. The inf rarenal abdominal aorta was not blocked in the sham-surgery group. Prior to abdominal aorta occlusion, 0.2-0.3 mL bromodeoxyuridine (BrdU)-labeled hUCMSCs suspension (cell concentration 5 × 10~3/μL) was injected through the great saphenous vein of the hind limb, and an equal volume of physiological saline was administered to the model and sham-surgery groups.MAIN OUTCOME MEASURES: Pathological observation of rat spinal cord tissues was performed by hematoxylin-eosin staining at 6, 24, and 48 hours post-surgery. Immunohistochemistry was applied to determine hUCMSCs survival in the spinal cord. The amount of cellular apoptosis and interleukin-8 expression in spinal cord tissues was assayed utilizing the TUNEL and ELISA methods, respectively. Motor function in the hind limbs was evaluated according to Jacob's score. RESULTS: Numerous BrdU-positive cells were observed in spinal cord tissues from the transplantation group. The number of apoptotic cells and interleukin-8 levels significantly decreased in the transplantation group (P < 0.05), pathological injury was significantly ameliorated, and motor function scores significantly increased (P < 0.05) compared with the model group. CONCLUSION: Via vein transplantation, hUCMSCs were shown to reach and survive in the injury area. Results suggested that the transplanted hUCMSCs contributed to significantly improved pathological changes in the injured spinal cord, as well as motor function, following SCIRI. The protective mechanism correlated with inhibition of cellular apoptosis and reduced production of inflammatory mediators.     

The Effect of Methylprednisolone on Caspase-3 Activation after Rat Spinal Cord Transection

Purpose: Caspase-3 is known as a crucial effector for apoptotic cell death. Apoptosis has recently been recognized as an important cell death mechanism after spinal cord injury (SCI). This study attempts to define the effect of methylprednisolone (MP) on the activation of caspase-3 in the lesioned area following SCI. Methods: Forty-eight rats with a complete transection of the thoracic spinal cord received a placebo or MP (30 mg/kg, iv.) at 5 min, 2 and 4 h post-injury and were then sacrificed at 12, 24 h, 3 or 7 days thereafter. Results: Caspase-3 positive cells in the lesioned area were immunocytochemically observed in both cord stumps and decreased in number with increasing distance from the lesion site. More caspase-3 positive cells were present in the MP-treated group than the control group at all time points, but the differences were not statistically significant. Conclusion: These results suggest that the MP-induced decrease of tissue loss following SCI may not involve a reduction of apoptotic cell death.     

依达拉奉对大鼠急性脊髓损伤后神经细胞凋亡的影响

目的:观察依达拉奉对大鼠急性脊髓损伤后细胞凋亡的影响,探讨脊髓保护的作用机制。方法:120只SD雄性大鼠,Allen法建立脊髓损伤模型,随机分为依达拉奉组、假手术组和对照组（均n=40）。依达拉奉组给予依达拉奉10mg·kg^-1,每日2次腹腔注射,连续6d;对照组给予等量同次数的生理盐水腹腔灌注;假手术组仅行椎板切除,不损伤脊髓,不给药。在伤后第1、7、14、28天观察各组大鼠活动情况行BBB评分,取受伤脊髓节段检测抑制羟自由基能力、caspase-3蛋白表达、原位脱氧糖核苷酸末端转移酶介导的原位末端标记法（TUNEL法）标记凋亡细胞。结果:依达拉奉组抑制羟自由基能力明显高于假手术组和对照组（P<0.05）;依达拉奉组caspase-3与对照组比各时间点的表达明显降低（P<0.05）;依达拉奉组与对照组比各时间点凋亡细胞显著减少（P<0.05）。结论:依达拉奉能减少急性脊髓损伤部位的羟自由基,下调caspase-3表达,抑制脊髓神经细胞凋亡,对继发性脊髓损伤有保护作用。     

脊髓全横断大鼠模型的构建☆

背景：脊髓全横断模型在造模时常难以保证神经纤维的完全离断。 目的：构建大鼠脊髓全横断损伤模型。 方法：将大鼠随机分为模型组和假手术组。模型组构建脊髓T10节段全横断模型;假手术组动物仅打开椎管与硬脊膜而后缝合,但不损伤脊髓。建模后1,3,5,7 d分别进行BBB评分以评估后肢运动功能,检测其体感诱发电位和运动诱发电位来评估神经传导通路的完整性,并行形态学观察来评估脊髓肉眼观病理形态。 结果与结论：与假手术组相比,模型组大鼠在建模后1,3,5,7 d时,其BBB评分降低(P < 0.01),未检测出体感和运动诱发电位。形态学观察结果显示模型组大鼠脊髓完全横断,而假手术组脊髓形态完整。结果提示实验成功构建了大鼠脊髓全横断模型。     

MK-801在脊髓缺血中的神经保护作用

目的地佐环平(MK-801)对脊髓缺血中神经保护作用的研究。方法建立新西兰兔脊髓缺血模型,利用HE染色、原位末端转移酶标记技术(TUNEL)、免疫组化、逆转录反应系统(RT-PCR)等技术检测N-甲基-D-天门冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)、诱导型一氧化氮合酶(inducible Nitric Oxide Synthase,iNOS)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达水平,观察不同剂量MK-801在脊髓缺血中的神经保护作用。结果对照组脊髓结构完全消失,低剂量组结构较完整,高剂量组缺血损害程度最轻,假手术组脊髓结构正常。NMDAR、iNOS、Caspase-3等蛋白在神经元中有明确表达。凋亡指数、NMDAR、iNOS、Caspase-3 mRNA表达水平在对照组最高,低剂量组、高剂量组,假手术组则逐渐降低,差别具有统计学意义(P<0.05)。结论 MK-801能抑制神经细胞凋亡,对脊髓缺血具有神经保护作用。     

Differentiation of endogenous neural precursors following spinal cord injury in adult rats☆

BACKGROUND： Studies have shown that cell death can activate proliferation of endogenous neural stem cells and promote newly generated cells to migrate to a lesion site.
OBJECTIVE： To observe regeneration and differentiation of neural cells following spinal cord injury in adult rats and to quantitatively analyze the newly differentiated cells.
DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Institute of Orthopedics and Medical Experimental Center, Lanzhou University, between August 2005 and October 2007.
MATERIALS： Fifty adult, Wistar rats of both sexes; 5-bromodeoxyuridine （BrdU, Sigma, USA）; antibodies against neuron-specific enolase, glial fibrillary acidic protein, and myelin basic protein （Chemicon, USA）.
METHODS： Twenty-five rats were assigned to the spinal cord injury group and received a spinal cord contusion injury. Materials were obtained at day 1, 3, 7, 15, and 29 after injury, with 5 rats for each time point. Twenty-five rats were sham-treated by removing the lamina of the vertebral arch without performing a contusion.
MAIN OUTCOME MEASURES： The phenotype of BrdU-labeled cells, i.e., expression and distribution of surface markers for neurons （neuron-specific enolase）, astrocytes （glial fibrillary acidic protein）, and oligodendrocytes （myelin basic protein）, were identified with immunofluorescence double-labeling. Confocal microscopy was used to detect double-labeled cells by immunofluorescence. Quantitative analysis of newly generated cells was performed with stereological counting methods.
RESULTS： There was significant cell production and differentiation after adult rat spinal cord injury. The quantity of newly-generated BrdU-labeled cells in the spinal cord lesion was 75-fold greater than in the corresponding area of control animals. Endogenous neural precursor cells differentiated into astrocytes and oligodendrocytes, however spontaneous neuronal differentiation was not detected. Between 7 and 29 d after spinal cord injury, newl     

凝血酶大鼠脑内注射对细胞凋亡的影响及其机制的实验研究

目的探讨凝血酶(Thrombin,TM)脑内注射对周围组织Caspase-3蛋白表达和细胞凋亡的影响。方法TM、TM 组织蛋白酶G(Cathepsin G,CATG)、TM Caspase-3抑制剂(DEVD-fmk)脑内立体定向注射制作动物模型,应用Western-blot技术检测Caspase-3蛋白表达,TUNEL法检测细胞凋亡。结果TM脑内注射后6hCaspase-3蛋白开始增加,与对照组比较差异具有显著性(P<0.01),注射后24h达高峰(P<0.01),然后逐渐下降,96h时恢复至正常水平;TM脑内注射后24h凋亡细胞数目开始增加,与对照组比较差异具有显著性(P<0.05),48h达高峰(P<0.01),然后逐渐下降。TM DEVD-fmk脑内注射后凋亡细胞数目与对照组比较差异无显著性(P>0.05)。TM CATG脑内注射后Caspase-3蛋白表达和凋亡细胞数目与对照组比较差异均无显著性(P>0.05)。结论TM通过蛋白酶激活受体-1(protease activated receptor-1,PAR-1)激活Caspase-3,诱导细胞凋亡。     

Protective effects and anti-apoptotic role of nerve growth factor on spinal cord neurons in sciatic nerve-injured rats

Abstract

Objectives:

The purpose of this study is to demonstrate a dependence of spinal cord motoneurons on the communication with their targets, sciatic nerves, and investigate whether the effects of nerve growth factor (NGF) on the spinal cord neuron apoptosis and surviving through the regulation of nuclear factor-kappa B (NF-kappaB) in Schwann cells (SCs) in sciatic nerve injured rats.

Methods:

Ninety healthy adult Sprague–Dawley rats were divided randomly into normal control group, crushing group, and NGF-intervened group. When sciatic nerve crushed 1, 3, 7, 14, and 21 days, the expression of NF-kappaB in SCs and the apoptosis regulator Bcl-2 and Caspase-3 in spinal cord were examined by immunohistochemistry staining, Western blot analysis, and immunofluorescence double-labeling method, the motor neuron apoptosis were investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the surviving neurons were tested by toluidine blue (Nissl) staining, respectively. All the data were further analyzed with SPSS10·0 application software.

Results:

The level of the expression of NF-kappaB in crushing group enhanced at 1 day after crushing, reached peak at 3 days, and reduced at least until 21 days, which was markedly higher than that in the normal control group. The expression of NF-kappaB in NGF-intervened group showed the same changes, reached peak at 7 days, and reduced until 21 days. However, when compared with crushing group, the expression of NF-kappaB in NGF-intervened group was down-regulated significantly until 3 days after injury, and up-regulated obviously with time going on. The same trend was observed in the time course on motor neuron apoptosis in crushing group and NGF-intervened group after sciatic nerves injury, while the reversing change was found in the surviving neurons. Moreover, the kinetics of Bcl-2 expression in spinal cord was consistent with that of NF-kappaB, while reversing with that of Caspase-3.

Conclusion:

The findings revealed that NGF may play a pivotal role of anti-apoptosis in spinal cord neurons through retrograde transport of NF-kappaB in SCs following sciatic nerve injury in rats.