获得性免疫反应与海人藻酸引起的C57BL/6小鼠海马损伤的关系(英文)

Adaptive immune response is involved in kainic acid- induced hippocampal injury in C57BL/6 mice

背景:海人藻酸引起在啮齿类动物的海马损伤是用于研究人类神经退行性病变的优质模型。虽然许多研究已证实炎症分子和反应参与并且促进了疾病的进程,但是否获得性免疫反应,尤其是免疫活性细胞,如T细胞和B细胞是否参与了神经退行性病变尚不清楚。目的:观察B和T细胞亚型在海人藻酸引起的海马神经元退行性病变中的作用。设计:随机对照动物实验。单位:吉林大学第一医院耳鼻喉头颈科和神经内科,瑞典卡若林斯卡医学院Huddinge医院神经医学系老年科。材料:实验于2000-06/09在瑞典KarolinskaInstitute医学院神经医学系完成。选用20只的雄性C57BL/6小鼠(野生型),同样基因背景的雄性CD4(-/-),CD8(-/-),CD4/CD8(-/-)和Igh-6(-/-)基因敲除C57BL/6小鼠分别为17,19,15,14只,鼠龄5￣6周,体质量18￣20g。3只鼠龄和体质量相匹配的C57BL/6小鼠经鼻给水作为对照。试剂和仪器:海人藻酸购自Sigma,USA。双色流式细胞仪和CellQuest购自BectonDickinson,CA,USA。方法:①所有小鼠经麻醉后,按48mg/kg剂量将7.69g/L海人藻酸滴入85只小鼠双侧鼻孔中。3只对照小鼠鼻内滴入等量水。②观察小鼠临床症状,临床症状分级如下:0￣6分,0分:正常;6分:死亡。③给予海人藻酸4.0￣5.0h后,采用流式细胞术测定脾细胞表面标记的表达。④给药7d后,麻醉所有小鼠,取脑,固定,包埋。采用尼氏染色方法评估(海马)切片神经元形态,判断神经细胞和神经退行的严重程度。应用半定量系统,即病理评分判断神经细胞和神经退行性病变的严重程度:0￣6分,0分:正常;6分:重度神经元脱失(CA3区有>40%神经元脱失)。⑤多组间差异比较采用单因素方差分析,两组间差异比较采用t检验。主要观察指标:各组小鼠临床级别、海马神经病理学改变和脾细胞表面细胞分子表达。结果:小鼠85只均进入结果分析。①临床级别:所有CD4(-/-)小鼠表现出严重癫痫发作,其临床级别明显高于野生型小鼠(P<0.01)。CD4/CD8(-/-)小鼠临床级别明显低于野生型小鼠(P<0.01)。而CD8(-/-)andIgh-6(-/-)临床级别与野生型小鼠无明显差异。对照小鼠无临床症状。②海马神经病理变化:CD4/CD8(-/-)小鼠病理变化最轻(病理级别最低),而Igh-6(-/-)小鼠海马CA3区病理损害较CD8(-/-)小鼠和野生型小鼠更为严重。③脾细胞亚群变化:CD8(-/-)鼠和野生型鼠CD4+T细胞百分比明显升高(给药前后分别为8.4%,14.2%;18.2%,31.5%);Igh-6(-/-)小鼠CD8+T细胞升高(给药前后分别为2.1%和7.4%);CD4(-/-)鼠B细胞升高(给药前后分别为22.7%,32.8%)。结论:获得性免疫反应参与海人藻酸引起的小鼠海马神经元退行性病变,B细胞和T细胞亚型在海人藻酸引起的海马损伤中起到不同作用。

BACKGROUND： Kainic acid （KA）-induced hippocampal injury in rodents is a good model for studying human neurodegenerative diseases. Although many studies have evidenced that inflammatory molecules and responses participate in and accelerated the process of disease, it is still unclear whether adaptive immune response, especially immune competent cells, such as T and B cells, is involved in the pathogenesis of neurodegenerative diseases. OBJECTIVE： To observe the roles of B and T cell subsets in KA-induced hippocampal neurodegeneration DESIGN： Randomized controlled animal tda SETTING： Department of Otolaryngology and Head, and Department of Neurology, First Hospital, Jilin University; Division of Geriatrics, Department of Neurotec, Huddinge Hospital, Karolinska Institute. MATERIALS： This trial was conducted in the Department of Neurotec, Huddinge Hospital, Karolinska Institute during June to September 2000. Twenty male C57 BL/6 mice （wide-type）, and knockout mice CD4（-/-） （n =17）, CD8（-/-）（n =19）, CD4/CD8（-/-） （n =15） and lgh-6（-/-） （n =14） of C57BL/6 background were involved in this trial. They were aged 5 to 6 weeks,weighing 18 to 20 g. Three age-and body mass-matched C57BL/6 mice received water as controls. Reagent and instruments： KA （Sigma, USA）. Bicolor flow cytometer and CellQuest （Becton Dickinson, CA, USA）. METHODS ： ① Eighty-five anesthetized mice were slowly administrated with 7.69 g/L KA by micropipette which was connected to nose of mouse at the dose of 48 mg/kg. Three control C57BL/6 mice received the same amount of water intranasally. ②Clinical symptoms of mice were monitored. Seizures were graded using a 6-point scale, 0： normal; 6： death. ③After 4 to 5 hours of administration of KA, surface immunofluorescence staining of spleen cells was measured with flow cytometer. ④After 7 days of administration of KA, all the mice were anesthetized, and their brains were harvested, then fixed and embedded. For assessment of the severity and extent of hippocampal neurodegeneration by Nissl＇s staining, the sections were scored by a semiquantitative grading system with a 6-point scale： 0： normal; 6： severe loss of neurons （more than 40% neuron loss in area CA3）; ⑤One-factor analysis of variance was used for the comparison of difference among groups and students＇ ttest was used between two groups. MAIN OUTCOME MEASURES： Clinical grade, hippocampal neuropathological changes and the molecular expression of splenic monocytes of mice in each group. RESULTS： Eighty-five mice were involved in the result analysis. ① Clinical grade： All CD4 （-/-） mice displayed severe seizures, and their clinical symptoms were significantly severer than those of wild type mice （P 〈 0.01 ）. Clinical scores of CD4/CD8 （-/-） mice were significantly lower than those of wide-type mice （P 〈 0.01 ）. However, the responses of CD8 （-/-） and lgh-6 （-/-） mice did not differ notably from those of the wild-type mice. The clinical grade of control mice was the lowest. ②Hippocampal neuropathological changes： Neurodegeneration was the mildest in CD4/CD8 （-/-） mice and severest in lgh-6 （-/-） mice. ③ Spleen cell subsets changes： the number of splenic CD4＋T cells was significantly increased in CD8（-/-） mice and wide-type mice （before and after administration of KA： 8.4%,14.2%;18.2% ,31.5%）; CD8＋ T cells were up-regulated in lgh-6（-/-） mice （ before and after administration： 2.1% and 7.4%）; B cells rose numerically in CD4（-/-） （Before and after administration： 22.7% and 32.8%）. CONCLUSION： Aadaptive immune response is involved in the KA-induced hippocampal neurodegeneration in mice, and B and T cell subsets contribute differently to the pathogenesis.