基于大鼠臂丛残根模型应用醋酸格拉替雷保护脊髓前角运动神经元的实验研究  

Experimental study on the protection of spinal motoneurons by using glatiramer acetate based on a rat model of nerve stumps

作　　者：李亮 黄家俊 顾立强[3] LI Liang;HUANG Jiajun;GU Liqiang(Department of Spine Surgery,Nanxishan Hospital of Guangxi Zhuang Autonomous Region,Guilin,Guangxi 541002,China;不详)

机构地区：[1]广西壮族自治区南溪山医院脊柱外科,广西桂林541002 [2]广西壮族自治区南溪山医院运动系统疾病研究与精准治疗实验室,广西桂林541002 [3]中山大学附属第一医院显微创伤手外科,广东广州510080

出　　处：《中国临床研究》2024年第12期1921-1927,共7页Chinese Journal of Clinical Research

基　　金：广西科技计划项目(桂科AD20238020);广西壮族自治区卫生健康委员会科研课题(Z20190041);广西医疗卫生重点培育学科建设项目(桂卫科教发〔2022〕4号);广西壮族自治区南溪山医院院级科研课题(NY2019006)。

摘　　要：目的明确臂丛神经损伤(BPI)后胶质细胞活化情况,探讨醋酸格拉替雷(GA)能否通过抑制胶质细胞的活化,提高残根脊髓前角运动神经元存活率。方法将76只SD大鼠经手术建立大鼠BPI后残根模型,并随机分为GA组(n=38)和对照组(n=38)。术后,GA组即每日给予GA皮下注射,对照组每日给予等体积生理盐水皮下注射。在术后/给药的第1、3、7、14、28天这5个时点进行下述各指标检测。采集大鼠脑脊液,酶联免疫吸附法检测脑脊液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及脑源性神经营养因子(BDNF)水平。取C5残根及脊髓;脊髓进行Nissl染色观察组织病理学变化,并进行星形胶质细胞活化标记物[胶质纤维酸性蛋白(GFAP)]和小胶质细胞活化标记物[离子钙接头蛋白分子1(Iba-1)]免疫组化染色及western blot检测,同时对突触小泡蛋白(SYP)也行western blot检测。残根用于苏木精伊红(HE)染色、轴突生长标记物[生长相关蛋白43(GAP-43)]及施旺细胞标记物S100的免疫组化染色,并进行甲苯胺蓝染色以观察和计算再生神经有髓神经纤维密度。结果所有大鼠均成功建立BPI残根模型。与对照组术后/给药后各时点比较,GA组脑脊液中TNF-α、IL-6降低,BDNF增高(P<0.05);GA组损伤侧脊髓前角运动神经元存活百分比增高(P<0.05);GA组脊髓中GFAP和Iba-1表达降低,SYP表达增高(P<0.01);GA组残根单位面积的GAP43和S100阳性率除第一天外均增高(P<0.05);GA组残根有髓神经纤维密度均增高(P<0.05)。此外,残根HE染色示,对照组神经结构混乱,GA组有序。结论BPI后胶质细胞活化,大鼠臂丛残根功能状态及脊髓前角运动神经元数量降低;经GA保护的脊髓前角运动神经元与其残根功能状态得以改善与维持;GA能抑制小胶质细胞和星形胶质细胞活化。Objective To determine the activation of glial cells after brachial plexus injury(BPI),and to explore whether glatiramer acetate(GA)can improve the survival rate of spinal motoneurons of nerve stumps by inhibiting the activation of glial cells.Methods A nerve stumps model of 76 SD rats after BPI was established by operation,and the rats were randomly divided into GA group(n=38)and control group(n=38).After operation,GA group was given subcutaneous injection of GA,and control group was given subcutaneous injection of normal saline of equal volume.The following indexes were detected at 5 time points on the 1st,3rd,7th,14th,and 28th day after operation and administration.The cerebrospinal fluid(CSF)of rats was collected and the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and brain-derived neurotrophic factor(BDNF)in CSF were measured by ELISA.The C5 stump and spinal cord were removed.The spinal cord was stained with Nissl to observe the histopathological changes.Immunohistochemistry and western blot were used to detect the astrocyte activation marker[glial fibrillary acidic protein(GFAP)]and microglia activation marker[ionic calcium adaptor protein-1(Iba-1)]in spinal cord,and western blot was used to detect the synaptophysin(SYP)in spinal cord also.HE staining and immunohistochemical staining were used for detecting axonal growth marker[growth-associated protein 43(GAP-43)]and Schwann cell marker S100 in nerve stump,and toluidine blue staining was used to observe myelinated nerve fiber density of regenerated nerve.Results The nerve stump model of all rats after BPI was successfully established.Compared with the control group at each time point after surgery and administration,TNF-αand IL-6 levels decreased and BDNF increased in the GA group(P<0.05);the survival percentage of motorneurons in the injured anterior horn increased in GA group(P<0.05);the expressions of GFAP and Iba-1 decreased and the expression of SYP increased in the GA group(P<0.01);the positive rates of GAP43 and S100 immunohistoch

关 键 词：醋酸格拉替雷 脊髓前角运动神经元 臂丛神经损伤 胶质细胞活化 突触小泡蛋白 

分 类 号：R651[医药卫生—外科学] R-33[医药卫生—临床医学]