再发性低血糖致幼鼠脑损伤的实验研究

目的探讨再发性低血糖幼鼠脑Caspase-3表达及神经元变性的动态变化。方法将48只15日龄C57BL／6野生型小鼠，分为正常对照组（n=8）及低血糖组（n=40），再分为低血糖后12h，24h．48h，72h组，每组10只）。低血糖组给予短效胰岛素腹腔注射3次，每次剂量为5U／kg。采用免疫组化方法观察小鼠脑内caspase-3表达的动态变化；FJB染色观察小鼠脑内神经元变性的时程及分布情况。结果再发性低血糖后12h幼鼠脑内caspase-3表达即增多，24h达高峰，至72h仍高于正常对照组；开始阳性染色主要位于细胞质。以后逐渐向细胞核转移，caspase-3表达最强的部位是海马齿状回，皮质次之，海马CA1区仅见少量caspase-3阳性细胞。再发性低血糖后12h，脑内即可见FJB阳性细胞，24h及48hFJB阳性细胞逐渐增多，至72h阳性细胞最多，染色最强；从分布上看，纹状体内FJB阳性细胞数最多（尤以枕部皮质与纹状体交界处为著），皮质次之，海马回仅见少量FJB阳性细胞。结论再发性低血糖可致幼鼠脑损伤，神经元凋亡与坏死并存，易损部位为海马齿状回、枕部皮质与纹状体交界处，以选择性神经元死亡为主。

Study of developing brain damage in young mouse following recurrent hypoglycemia

Objective] To explore the mechanisms involved in neuronal loss in developing brain subjected to hypoglycemia. Methods] Fourty-eight mice were randomly assigned into control group (n=8) and hypoglycemia group (n=40). The mice in hypoglycemia group were i.p. injected with insulin three times at the dose of 5 U/kg, and were killed at 12 h, 24 h, 48 h, and 72 h after the third injection. Caspase-3 immunohistochemistry and Fluoro-Jade B staining were used to determine the time course of caspase-3 expression and cellular localization of neuronal degeneration. Results] Immunohistochemistry indicated that caspase-3 positive cells in the brain following hypoglycemia increased at 12 h, peaked at 24~48 h, and decreased at 72 h. The widespread caspase-3 staining was seen in the dentate gyrus of the hippocampal, moderate in the cortex, and scattered in the hippocampal CA1 region. Fluoro-Jade B staining revealed that in striatal sections, FJB positive cells were seen at 12 h, and the intensity of staining increased progressively at 24 h and 48 h, peaked at 72 h after the third injection. With scattered positive neurons within the cortex, and less FJB positive cells were seen in the hippocampus. The most vulnerable region was the borderline between the occipital cortex and the striatum. Conclusion] We conclude that caspase-3 contributes to neuronal damage after hypoglycemia, and FJB is a useful marker of cellular degeneration following hypoglycemia.