大鼠脑出血模型中神经细胞凋亡与自由基水平的相关性

目的 研究脑出血后血肿周围组织不同自由基水平对于细胞凋亡的影响.方法 成年SD大鼠随机分为4组:假手术组、模型组、1mg/kg依达拉奉组、3 mg/kg依达拉奉组,各组又根据造模后处死动物的不同时间(6 h,12 h,24 h,48 h,72 h,7 d,14 d)分为七个亚组,假手术组中每个亚组1只大鼠,其余三组中每个亚组6只大鼠.左尾壳核立体定向注入自体血80 μL,制作大鼠脑出血动物模型.分光光度计法检测血肿周围丙二醛(mflonaldehyde,MDA)及羟自由基含量,末端转移酶标记技术(terminal deoxynucleotidyltransfemse-mediated dUTP-biotin nick end labeling,TLINEL)检测血肿组织周围细胞凋亡数,并分析血肿周围组织自由基水平和凋亡相关性.结果 (1)模型组羟自由基及MDA含量较假手术组明显增加,四组间进行统计学分析,具有显著性差异.(2)模型组和两种剂量的依达拉奉组均于6 h即可观察到TUNEL阳性细胞,24 h明显增加,72 h时达到高峰,7 d时明显减少,14 d时于血肿周边仍可见少量阳性细胞.(3)凋亡细胞数与脑组织产生自由基能力(r=0.2003)及MDA含量(r=0.6563)具有相关性.结论 脑出血后细胞凋亡数和自由基水平变化趋势相同,二者具有相关性,提示自由基可能参与诱导出血后神经元及胶质细胞凋亡.

Correlation of free radical level and apoptosis after intracerebral hemorrhage in rats

Objective

To investigate the correlation of perihematomal free radical level and neuronal apoptosis following the intracerebral hemorrhage (ICH).

Methods

Animals were randomly divided into 4 groups: sham operation group, model group, 1 mg/kg edaravone group, and 3 mg/kg edaravone group. Each group was then divided into seven subgroups, in which the rats were correspondingly killed at 6 h, 12 h, 24 h, 48 h, 72 h, 7 d or 14 d (n = 1 in each subgroup of the sham group, and n = 6 in each subgroup of the other 3 groups). By Horseley-Clarke technique, autoblood (80 μL) were administered into the left caudate putamen of SD rats in a double administration-withdrawal way. Rats in the sham group were needled in but not administered with autoblood. The ICH model was then evaluated by Bederson’s scale. Around the hematoma, the levels of malonaldehyde (MDA) and hydroxyl radical were tested by spectrophotometer, and the process of apoptosis was tested by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) method.

Results

(1) ICH significantly increased the levels of MDA and hydroxyl radicals. Significant differences in MDA and hydroxyl radical contents were observed among the four groups. (2) In the sham group, a small number of TUNEL-positive cells were found. In the other three groups, the TUNEL-positive cells were observed at 6 h, increased significantly at 24 h, and reached peak level at 3 d, then fell profoundly at 7 d, but remained detectable at 14 d. (3) The positive correlation existed between apoptosis and free radical level (r = 0.2003), and existed between apoptosis and MDA content (r = 0.6563) in the brain.

Conclusion

Post-hemorrhagic apoptosis was related to the production of free radicals, indicating that the elevated free radicals following the ICH could induce neuron and glial cell apoptosis.