亚低温抑制大鼠弥漫性脑损伤后细胞凋亡的研究

Study on mild hypothermia inhibition of apoptosis after diffuse cerebral injury in rats

目的 探讨大鼠不同程度弥漫性脑损伤后脑组织的凋亡变化过程及亚低温治疗对脑细胞凋亡的抑制作用。方法 采用大鼠Marmarou颅脑创伤装置制作弥漫性脑损伤模型,然后将128只Wistar大鼠分为未损伤组(对照组)、重度损伤组、轻度损伤组和亚低温治疗组。通过电子显微镜、组织切片原位末端标记DNA片段(TUNEL染色)、琼脂糖凝胶电泳(DNA Ladder法)等方法,观察和比较不同程度脑损伤后,大鼠脑皮层及海马区凋亡细胞的形态、特点和数量。结果 (1)损伤后24～48 h,皮层及海马区可见大量细胞皱缩、核碎裂、核不规则等细胞凋亡现象,48 h较24 h更为严重;亚低温治疗后24～48 h,电子显微镜观察皮层及海马区未见细胞皱缩、核碎裂等细胞凋亡现象。(2)TUNEL染色结果显示,随着损伤程度的加重凋亡明显加重,损伤后48 h达高峰,然后逐渐下降。轻度损伤组细胞凋亡主要限于海马CA2和CA3区;重度脑损伤组细胞凋亡涉及整个海马结构,同时还广泛累及额顶区皮质。损伤后第24、48、72 h,皮层及海马区的凋亡细胞数量较同期未治疗组明显减少。(3)重度损伤后48 h,海马和皮层区细胞琼脂糖电泳可见典型的DNA梯状带,其他时间未见梯状带。亚低温治疗组、轻度脑损伤组及未损伤组亦未见梯状带。结论 轻度弥漫性脑损伤后,脑细胞凋亡多发生于海马CA2和CA3?

Objective The apoptosis processes of cerebral tissue after various degree of diffuse cerebral injury in rat and the mild hypothermia therapy for inhibition of apoptosis were studied. Methods The diffuse injury model of Wistar rat was established with Marmarou craniocerebral injury device. A total of 128 Wistar rats were divided into control group (without injury) , severe injury group, mild injury group and mild hypothermia treatment group. The apoptosis cell morphological characteristics and amount of cerebral cortex and hippocampus in rat with various degrees of injury were observed and compared by using electronic microscope (EM) , orthotopic terminal labeled DNA fragment in tissue section(TUNEL staining) and agarose gel electrophoresis(DNA Ladder technique) . Results (1) A great deal of programmed cell death phenomena including of cell shrink, karyopy-knosis, irregular nucleus were found in cortex and hippocampus after injury in 24 - 48 h and more severe features were found in 48 h than that in 24 h. The above apoptosis phenomena were not found in 24 - 48 h after mild hypothermia treatment under the EM examination for cortex and hippocampus. (2) The result of TUNEL orthotopic labeling showed that obviously exacerbation of apoptosis was associated with the exacerbation of injury intensity. The peak was reached in 48 h after injury, then declined gradually. The apoptosis was limited in CA2 and CA3 area of hippocampus in mild injury group. But the apoptosis was involved in whole hippocampus and spreaded to frontal cortex in severe injury group. The apoptosis cell count in cortex and hippocampus was significantly less in therapeutic group than that in untreated group in 24, 48, 72 h after injury. (3)The typical ladder zone of DNA in agarose gel electrophoresis was found in hippocampus and cortex cells in 48 h after injury, but it was not found in other time and other groups eg. mild hypothermia treatment group, mild cerebral injury group and control group. Conclusion The apoptosis is usually found in CA2 and CA3 area of hippocampus after mild diffuse cerebral injury. The widespread apop-tosis in cortex and hippocampus is found after severe cerebral injury. The apoptosis is exacerbating as the injury exacerbating, and its peak is occurred at the 2 nd day after injury. The mild hypothermia treatment is effective for inhibiting the apoptosis in rat after diffuse cerebral injury.