大鼠实验性脊髓损伤后胶质瘢痕分布规律研究

目的观察慢性脊髓损伤胶质瘢痕形成的时间与空间分布特征。方法应用Allen′s法建立大鼠脊髓打击伤模型,通过神经功能评分、体感及运动诱发电位、组织病理学及免疫荧光双标记方法确定胶质瘢痕形成时间及分布。结果神经功能评分、诱发电位、组织病理学显示伤后4周进入相对稳定状态,伤后4周损伤区形成空洞,胶质瘢痕位于空洞壁。结论脊髓损伤后4周进入慢性期,研究伤后脊髓空洞及胶质瘢痕形态,为判断切除胶质瘢痕的界限提供形态学依据。     

移植人脐带间充质干细胞修复大鼠脊髓损伤

背景：已知人脐带间充质干细胞对脊髓损伤存在着潜在的治疗价值,然而,当前对移植人脐带间充质干细胞治疗脊髓损伤及机制方面研究很少。 目的：观察人脐带间充质干细胞对脊髓损伤大鼠的治疗效果。 方法：40只Wistar大鼠建立脊髓损伤模型,38只造模成功后随机摸球法分为3组：空白对照组：只接受单纯损伤,不做任何移植;DMEM移植组：损伤后1周予以5 μL DMEM局部移植;细胞移植组：损伤后1周予以5 μL准备好的人脐带间充质干细胞局部移植(细胞数1×106)。移植后对实验动物通过BBB评分、体感诱发电位与运动诱发电位观察后肢功能恢复情况。分别于损伤后2,4,6,8,10周随机于细胞移植组抽取大鼠2只,免疫组织化学染色观察人脐带间充质干细胞存活、迁移、分化,通过胶质纤维酸性蛋白阳性细胞染色比较各组损伤局部胶质瘢痕形成面积。 结果与结论：BBB评分损伤后4周细胞移植组高于其他两组(P < 0.05),损伤后12周细胞移植组与其他两组相比SEP、MEP潜伏期缩短、波幅值增高(P < 0.05)。免疫组织化学染色示人脐带间充质干细胞可向神经元、星形胶质细胞和少突胶质细胞分化,分化的少突胶质细胞并包绕轴突形成髓鞘。细胞移植组损伤局部胶质瘢痕面积均小于其他两组(P < 0.05),空白对照组、DMEM移植组间差异无显著性(P > 0.05)。提示未经体外诱导的人脐带间充质干细胞可于损伤大鼠脊髓体内向神经元、星形胶质细胞、少突胶质细胞分化,减小胶质瘢痕,并促进脊髓损伤大鼠神经功能的恢复。     

再程序化脂肪干细胞移植治疗大鼠脊髓损伤的机制

目的制备再程序化脂肪干细胞(ADSCs),并在体研究再程序化ADSCs移植入大鼠脊髓损伤模型后促进损伤脊髓神经功能恢复的作用和机制。方法体外培养、纯化和鉴定大鼠ADSCs,并利用慢病毒包装神经元生成素2(Ngn2)基因转染ADSCs制备再程序化干细胞。体内实验将48只雌性SD大鼠随机分成3组:SCI对照(A)组、单纯ADSCs移植(B)组和Ngn2-ADSCs移植(C)组。采用BBB评分评价大鼠运动功能,并通过HE染色、免疫组化和免疫荧光等方法检测脊髓组织学改变和相关蛋白的表达水平,进而观察实验动物脊髓功能恢复情况。结果 Ngn2-ADSCs移植组在运动功能评分、胶质瘢痕的形成、脊髓损伤后病理变化和分泌神经营养因子BDNF和VEGF蛋白含量明显优于其他组。结论 Ngn2-ADSCs移植后能有效地存活,并分化为神经细胞,抑制胶质瘢痕形成,减小脊髓损伤空洞,增加BDNF和VEGF表达,最终促进SCI大鼠的运动功能恢复,较单纯应用ADSCs能更好地促进SCI修复。     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

拉莫三嗪对小鼠脊髓背侧半横断损伤的保护作用

目的探讨拉莫三嗪在小鼠脊髓损伤修复中的作用。方法雌性C57BL/6小鼠80只采用脊髓背侧半横断损伤模型,随机分为模型组:单纯脊髓损伤;治疗组:脊髓损伤后每天给予腹腔注射拉莫三嗪,按照25 mg/kg的计量,连续7 d;对照组:脊髓损伤后每天给予腹腔注射0.9%生理盐水,按照25 mg/kg的计量,连续7 d。术后分别在1 d、7 d、14 d对各组小鼠行BBB评分,并分别在1 d、7 d、14 d在各组随机抽取6只小鼠,进行免疫荧光染色观察胶质细胞变化,并在术后第7天各组取6只小鼠组织行ELISA检测,观察炎症因子表达情况。结果损伤后7、14 d治疗组小鼠BBB评分明显高于模型组和对照组,差异有统计学意义(P0.05);脊髓损伤后矢状切面损伤灶下游的GFAp+(星形胶质细胞的标记)细胞的免疫荧光面积百分比,发现术后7 d和14 d治疗组(9.87±0.96,4.79±1.02)的明显低于模型组(12.34±1.72,7.46±1.28)和对照组(11.89±1.70,7.53±1.70),差异均有统计学意义(P0.05)。ELISA检测术后7 d小鼠T9~T11的IL-1、IL-10、TNF-α表达情况,IL-1、IL-10的表达治疗组明显低于模型组和对照组,差异有统计学意义(P0.05)。结论拉莫三嗪可以改善脊髓损伤小鼠的运动功能,通过前期减少炎症因子的分泌降低胶质细胞的活化来实现的。     

人羊膜间充质干细胞移植对大鼠脊髓损伤神经功能恢复的影响

目的 观察人羊膜间充质干细胞( hAD - MSCs)移植对大鼠脊髓损伤神经功能恢复的影响.方法 建立大鼠脊髓全横断损伤模型,脊髓横断后立即以明胶海绵吸附10μl hAD- MSCs(约2×105个)或等量PBS液植入脊髓两断端之间.术后每周应用BBB评分评价大鼠后肢运动功能;采用免疫荧光染色观察hAD - MSCs在脊髓内的存活、分化情况;免疫组织化学染色观察受损脊髓远端组织NF - 200表达.结果 hAD - MSCs移植组神经功能明显恢复,BBB评分逐周增加,与对照组比较差异有统计学意义(P＜0.05).hAD - MSCs植入后2周在宿主脊髓中存在MAB1281染色阳性细胞,但不表达MAP -2和GFAP.hAD - MSCs移植后大鼠受损脊髓远端神经组织NF - 200表达明显强于对照组.结论 hAD - MSCs移植可促进大鼠脊髓损伤后的神经功能恢复,其机制可能与hAD - MSCs促进受损脊髓远端组织表达NF - 200有关.     

大鼠脊髓冲击伤模型制作方法的改良探索

目的:探索一种制作稳定性强、重复性好的脊髓损伤模型的简易方法。方法:应用简易重物坠落打击装置建立大鼠脊髓中度损伤组(5g×8cm)、重度损伤组(5g×16cm)模型和假手术组,每组大鼠均n=10。通过运动功能评分法(BBB)评分、苏木精-伊红染色观察脊髓损伤后两组大鼠功能及病理的变化特点,以评价该制作方法的可靠性。结果:BBB评分显示两组大鼠后肢运动功能均有不同程度的恢复,但中度损伤大鼠的恢复明显优于重度损伤大鼠(P<0.05)。组织病理学观察显示脊髓损伤后脊髓结构紊乱,有胶质瘢痕及空洞形成,重度损伤组脊髓空洞面积明显大于中度组(P<0.05)。结论:该方法制作的大鼠脊髓模型能将不同打击力度造成的损伤区分开,并且模型的行为学与病理学结果相吻合,说明此模型具有良好的稳定性、重复性和一致性,适合脊髓损伤实验研究的应用。     

miRNA-9修饰骨髓间充质干细胞治疗大鼠脊髓损伤的实验研究

目的 通过观察miRNA-9在骨髓间充质干细胞(MSCs)分化为神经元过程中的作用,探讨基因修饰在脊髓损伤治疗中的作用.方法 分离培养大鼠MSCs并构建miRNA-9-1慢病毒载体.成功建立84只大鼠急性脊髓损伤模型,并按照随机数字表法分为对照组、MSCs组及miRNA组,每组28只.脊髓损伤后1周,对MSCs组大鼠进行MSCs移植,对miRNA组大鼠移植miRNA-9-1慢病毒载体感染的MSCs,对照组大鼠仅在损伤部位注射等量的生理盐水.选择不同时间点对大鼠后肢进行Basso Beattie Bresnahan(BBB)评分,并行神经丝蛋白200(NF-200)和胶质纤维酸性蛋白(GFAP)免疫组织化学染色,对各组阳性表达面积百分比进行比较.结果 细胞移植4周后,各组大鼠BBB评分差异有统计学意义,其中miRNA组评分较MSCs组及对照组明显提高,差异有统计学意义(P＜0.05).免疫组织化学染色示miRNA组的NF-200阳性面积较MSCs组及对照组明显增大,GFAP阳性面积较MSCs组及对照组明显减小,各组间的差异具有统计学意义(P＜0.05).结论 miRNA-9在MSCs横向分化为神经元中起重要调控作用,并通过促进轴突再生,减少脊髓损伤部位反应性胶质细胞的数量等机制促进脊髓损伤后的功能修复.     

骨髓间充质干细胞不同移植途径治疗大鼠脊髓损伤的比较

背景：在适当的生长环境下，中枢神经系统内的一些受损的神经元轴突有少许再生，并能与靶细胞形成功能性的突触联系。 目的：比较局部注射和尾静脉注射途径移植骨髓间充质干细胞对大鼠脊髓损伤神经功能恢复的作用。 设计、时间及地点：细胞组织学对照观察，于2007-03/2008-04在承德医学院完成。 材料：健康成年雄性SD大鼠40只，由解放军军事医学科学院动物中心（北京）提供。 方法：取4只大鼠，采用密度梯度离心法和贴壁法体外分离培养骨髓间充质干细胞，传至第2代于临用前24 h行BrdU标记。余36只大鼠均建立T12脊髓损伤模型，1周后随机分为3组，局部注射组于损伤部位上下位点注射1×106个骨髓间充质干细胞至损伤大鼠体内；尾静脉注射组通过尾静脉移植等量骨髓间充质干细胞至损伤大鼠体内；模型对照组不行细胞移植。 主要观察指标：神经功能缺损BBB评分，苏木精-伊红染色病理学检测，细胞分化免疫组化染色结果。 结果：细胞移植后2，4，6周，模型对照组神经功能缺损BBB评分均显著低于局部注射组、尾静脉注射组（F=721.373，F=1 114.450，F=1 004.099，P均 < 0.01）；局部注射组神经功能缺损BBB评分均显著高于尾静脉注射组（t=55.261，t=71.385，t=78.135，P均 < 0.01）。苏木精-伊红染色结果显示，模型对照组损伤脊髓组织有较多空腔，横断处形成大量空泡；局部注射组无明显空腔，空泡小而少，间质水肿较轻。移植后4，6周，部分植入的骨髓间充质干细胞呈微管相关蛋白2及胶质纤维酸性蛋白双阳性表达。 结论：局部注射和尾静脉注射两种途径移植的骨髓间充质干细胞均可在脊髓损伤处存活、分化并改善神经功能，且局部注射的效果优于尾静脉注射。     

咯利普兰对大鼠脊髓损伤后保护作用及其机制

目的探讨咯利普兰对大鼠脊髓损伤(SCI)后的保护作用及其可能机制。方法将84只SD大鼠随机分为三组:假手术组(n=4)、损伤组(n=40)和治疗组(n=40)。大鼠SCI模型采用纽约大学脊髓重物坠落伤模型。治疗组伤后即刻腹腔内注射咯利普兰,剂量为0.5 mg(/kg.d),2次/d,连续3d。损伤前后对损伤组和治疗组大鼠进行开放场地试验(BBB)评估大鼠脊髓功能。免疫组化染色分析三组大鼠损伤前后脊髓兴奋性氨基酸转运蛋白4(EAAT4)表达情况。结果损伤组和治疗组伤后即刻大鼠BBB评分均为0,处于完全瘫痪状态;然后BBB评分逐渐增高;伤后42、56和64d,治疗组BBB评分明显高于损伤组(P<0.01)。假手术组脊髓组织形态正常,EAAT4表达较少;脊髓损伤1周后,损伤中央出现较大空洞,EAAT4表达明显升高;而治疗组,组织空洞缩小,EAAT4表达较损伤组明显增加。结论咯利普兰有助于大鼠脊髓损伤后功能恢复,其机制可能与增加EAAT4表达有关。     

丹酚酸B促进大鼠急性脊髓损伤修复及量效关系探讨

目的 研究腹腔注射丹酚酸B(Sal B)对大鼠急性脊髓损伤(SCI)模型的神经保护作用和促功能恢复作用,探讨Sal B在急性SCI治疗的应用价值,并探讨其量效关系. 方法参照Allen法制作SD大鼠T9脊髓节段急性损伤模型,腹腔注射Sal B或PBS液,按照注射液的不同分为4组:Sal B高剂量组(20 mg/kg组),Sal B中剂量组(10 mg/kg组),Sal B低剂量组(2 mg/kg组)和对照组(注射PBS液),每组12只.用比色法检测髓过氧化物酶活性;用免疫组织化学染色法检测损伤脊髓节段MMP-1、c-Fos抗体表达情况,用干湿重法评价的水肿程度,并采用后肢功能评分(BBB)评分评价10 d内大鼠的运动功能恢复情况. 结果损伤后4 h Sal B治疗组髓过氧化物酶活性下降,损伤后1 dHE染色切片显示SalB组治疗后局部组织损伤减轻,炎性细胞浸润数量减少,损伤后1d免疫组化染色结果显示,Sal B治疗组比对照组MMP-1表达减少,c-Fos表达下调;Sal B治疗组水肿程度轻于对照组,从SCI后第7天起,SalB组高剂量组(20 mg/kg组)和对照组之间的BBB评分有显著性差异(P<0.05).各指标改善情况与Sal B剂量呈正相关性. 结论 Sal B可减轻大鼠SCI后的组织损伤,下调损伤相关因子MMP-1和c-Fos的表达,降低损伤局部髓过氧化物酶活性,减轻组织水肿,并能促进损伤大鼠的功能恢复.     

丹酚酸B促进大鼠急性脊髓损伤修复及量效关系探讨

目的 研究腹腔注射丹酚酸B(Sal B)对大鼠急性脊髓损伤(SCI)模型的神经保护作用和促功能恢复作用,探讨Sal B在急性SCI治疗的应用价值,并探讨其量效关系. 方法参照Allen法制作SD大鼠T9脊髓节段急性损伤模型,腹腔注射Sal B或PBS液,按照注射液的不同分为4组:Sal B高剂量组(20 mg/kg组),Sal B中剂量组(10 mg/kg组),Sal B低剂量组(2 mg/kg组)和对照组(注射PBS液),每组12只.用比色法检测髓过氧化物酶活性;用免疫组织化学染色法检测损伤脊髓节段MMP-1、c-Fos抗体表达情况,用干湿重法评价的水肿程度,并采用后肢功能评分(BBB)评分评价10 d内大鼠的运动功能恢复情况. 结果损伤后4 h Sal B治疗组髓过氧化物酶活性下降,损伤后1 dHE染色切片显示SalB组治疗后局部组织损伤减轻,炎性细胞浸润数量减少,损伤后1d免疫组化染色结果显示,Sal B治疗组比对照组MMP-1表达减少,c-Fos表达下调;Sal B治疗组水肿程度轻于对照组,从SCI后第7天起,SalB组高剂量组(20 mg/kg组)和对照组之间的BBB评分有显著性差异(P<0.05).各指标改善情况与Sal B剂量呈正相关性. 结论 Sal B可减轻大鼠SCI后的组织损伤,下调损伤相关因子MMP-1和c-Fos的表达,降低损伤局部髓过氧化物酶活性,减轻组织水肿,并能促进损伤大鼠的功能恢复.     

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。     

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND: Transplantation of human umbilical cord blood-derived mesenchymal stem cells (MSCs) has been shown to benefit spinal cord injury (SCI) repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE: To observe changes in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and interleukin-8 (IL-8) expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS: Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS: A total of 62 Wister rats were randomly assigned to control (n = 18), model (n = 22, SCI + PBS), and transplantation (n = 22, SCI + MSCs) groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine (BrdU)-labeled MSCs (24 hours before injection) were intravascularly transplanted. MAIN OUTCOME MEASURES: The rats were evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation.RESULTS: A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group (P< 0.05), which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks (P < 0.05), but IL-8 levels remained unchanged (P > 0.05).CONCLUSION: MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.     

Upregulated protein O-GlcNAcylation promoted functional and structural recovery of the contused spinal cord injury in rats by Thiamet-G treatment

ABSTRACT

Objectives-Elevated protein O-GlcNAcylation could benefit cell survival and promote organ functional recovery. Thiamet-G (O-GlcNAcase inhibitor) could upregulate protein O-GlcNAcylation level to improve dyskinesia in models of neurodegenerative diseases without any obvious detrimental side-effects. Therefore, we conducted this study to investigate the effects of protein O-GlcNAcylation upregulation by Thiamet-G on the spinal cord injury (SCI) in rats. Methods-We randomly assigned 74 rats to three groups: sham-operated group (Sham) with no lesion (n = 22), injured control group (SCI+SS) with saline solution (n = 26), and Thiamet-G treated group (SCI+Thiamet-G) (n = 26). We assessed Locomotor behavior using the Basso, Beattice, and Bresnahan (BBB) scale and evaluated histopathological alterations by morphometry and histochemistry. We also assessed potential inflammatory effects by microglia/macrophages immunohistochemistry, and potential apoptosis effects by caspase-3 western blot. Results-Thiamet-G treatment improved hindlimb motor functional recovery by inducing elevated protein O-GlcNAcylation, and mitigated the severity, reduced the lesion size and promoted the structural recovery of the injured spinal cord. Thiamet-G treatment also inhibited microglia/macrophages infiltration at the injury sites and the caspase-3 mediated apoptosis pathway. Discussion-We conclude that Thiamet-G induced elevated protein O-GlcNAcylation to ameliorate acute SCI, which could provide a potential novel therapeutic approach for SCI treatment.     

PKH67示踪骨髓间充质干细胞迁移至损伤脊髓的实验研究

目的 探讨SCI后体外移植PKH67标记的BMSCs迁移至脊髓损伤处并进行增值和分化的动员情况。方法 用梯度离心法分离和培养出SD 大鼠第3代BMSCs,用绿色荧光染料PKH67标记; 采用钳夹法制备脊髓损伤(SCI)模型,分为实验组(n=15)、对照组(n=16)、假手术组(n=16); SCI术后对脊髓损伤组织进行HE染色,实验组和假手术组于术后尾静脉移植含有1×10⁷个BMSCs的0.5 mL生理盐水,对照组注射等量生理盐水; 分别于术后1、7、14、21 d观察大鼠后肢运动功能恢复情况,并做BBB分; 术后21 d后取脊髓组织,行免疫荧光染色,观察BMSCs的迁移,增值和分化情况。结果(1)镜下可见损伤脊髓形成的空洞、坏死及炎性细胞的增多;(2)共聚焦荧光显微镜观察显示术后21 d实验组脊髓损伤部位可见移植的BMSCs, 部分BMSCs呈GFAP和Nestin阳性表达; 假手术组无 PKH67标记的BMSCs; 实验组GFAP和Nestin阳性细胞数较对照组和假手术组明显增加(P<0.05),对照组较假手术组增加不明显(P>0.05);(3)实验组和对照组BBB评分均有增加,但实验组BBB评分显著高于对照组(P<0.05)。结论 PKH67示踪的BMSCs可迁移至损伤脊髓部位,进行增值并分化为神经元样细胞,促进损伤脊髓的神经功能恢复。     

Combined transplantation of bone marrow mesenchymal stem cells and pedicled greater omentum promotes locomotor function and regeneration of axons after spinal cord injury in rats*★

BACKGROUND： According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells （BMSCs） into neural cells. OBJECTIVE： Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETFING： A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007. MATERIALS： Fifteen male inbred Wistar rats, weighing （200±20） g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS： Cell culture： We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention： The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury （SCI） only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the Tl0 level for 30 seconds to produce the SCI model. 5 μ L PBS containing 10^5 BMSCs was injected in     

Temporal kinetics of macrophage polarization in the injured rat spinal cord

Local activated macrophages derived from infiltrating monocytes play an important role in the damage and repair process of spinal cord injury (SCI). The present study investigates the dynamic change of classically activated proinflammatory (M1) and alternatively activated anti‐inflammatory (M2) cells in a rat model with contusive SCI by flow cytometry (FCM) and immunohistochemistry. The macrophage subsets were immunophenotyped by using antibodies against cluster of differentiation (CD)?68, C‐C chemokine receptor type 7 (CCR7), CD163, and arginase 1 (Arg1). The CD68⁺CD163^– and CD68⁺CCR7⁺ cells were determined to be M1 subsets, whereas the CD68⁺CD163⁺ and CD68⁺Arg1⁺ cell subpopulations represented M2 cells. The subsets of macrophages in the injured spinal cord at 1, 3, 5, 7, 14, and 28 days postinjury (dpi) were examined. In the sham‐opened spinal cord, few M1 or M2 cells were found. After SCI, the phenotypes of both M1 and M2 cells were rapidly induced. However, M1 cells were detected and maintained at a high level for up to 28 dpi (the longest time evaluated in this study). In contrast, M2 cells were transiently detected at high levels before 7 dpi and returned to preinjury levels at 14 dpi. These results indicate that M1 cell response is rapidly induced and sustained, whereas M2 induction is transient after SCI in rat. Increasing the fraction of M2 cells and prolonging their residence time in the injured local microenvironment is a promising strategy for the repair of SCI. © 2015 Wiley Periodicals, Inc.     

Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion

Neuronal cell death and the failure of axonal regeneration cause a permanent functional deficit following spinal cord injury (SCI). Administration of recombinant glial cell line-derived neurotrophic factor (GDNF) has previously been reported to rescue neurons following severe SCI, resulting in improved hindlimb locomotion in rats. In this study, thus, GDNF gene therapy using an adenoviral vector (rAd-GDNF) was examined in rats following SCI induced by dropping the NYU weight-drop impactor from a height of 25 mm onto spinal segment T9-T10. To evaluate the efficacy of intraspinal injection of recombinant adenovirus into the injured spinal cord, we observed green fluorescent protein (GFP) gene transfer in the contused spinal cord. GFP was effectively expressed in the injured spinal cord, and the most prominently transduced cells were astrocytes. The expression of GDNF was detected only in rats receiving rAd-GDNF, not the controls, and remained detectable around the injured site for at least 8 days. Open-field locomotion analysis revealed that rats receiving rAd-GDNF exhibited improved locomotor function and hindlimb weight support compared to the control groups. Immunohistochemical examination for the neuronal marker, calcitonin gene-related peptide (CGRP), showed an increase in CGRP+ neuronal fibers in the injured spinal cord in rats receiving rAd-GDNF treatment. Collectively, the results suggest that adenoviral gene transfer of GDNF can preserve neuronal fibers and promote hindlimb locomotor recovery from spinal cord contusion. This research should provide information for developing a clinical strategy for GDNF gene therapy.     

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.