二氮嗪对幼龄大鼠深低温低流量脑损伤的影响及机制研究
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摘要
【目的】
通过建立幼龄大鼠深低温低流量(DHLF)脑损伤模型,研究二氮嗪通过线粒体凋亡通路的脑保护机制。
【方法】
将198只3周龄SD大鼠随机分为三组:假手术组、模型组和二氮嗪组。模型组和二氮嗪组在大鼠肛温降至(21±0.5)℃时阻断两侧颈总动脉120 min后再开放。假手术组除不阻断两侧颈总动脉外,其余操作相同。利用激光多普勒血流监测仪连续监测假手术组、模型组和二氮嗪组(每组6只)大鼠局部脑血流量。然后,每组60只大鼠再随机分成5个亚组:再灌注后1h,6h,24h,72h和7d(n=12)。二氮嗪组于缺血前30min腹腔内注射二氮嗪5 mg/kg(溶解于0.25 ml二甲基亚砜(DMSO)),模型组和假手术组则予腹腔内注射等体积的DMSO。TUNEL法检测各组脑组织细胞凋亡,RT-PCR检测各时间点细胞色素C和Caspase-3的mRNA表达的变化,免疫组织化学检测各时间点细胞色素C和Caspase-3的蛋白表达的变化。
【结果】
大鼠局部脑血流量在模型组和二氮嗪组均显著下降。模型组和二氮嗪组在DHLF 0～5 min内分别降至正常水平的(15.73±3.47)%和(14.74±3.60)%,且在DHLF期间无明显改变。模型组中TUNEL-阳性细胞在再灌注后6h,24h,72h和7d均显著增高(P＜0.01),细胞色素C和Caspase-3的mRNA表达在再灌注后6h,24h和72h亦明显增高(P＜0.05 or P＜0.01),同时细胞色素C和Caspase-3的蛋白表达分别在再灌注后6 h～72 h和24 h～72 h增高(P＜0.05 orP＜0.01)。二氮嗪组TUNEL阳性细胞显著下降,并且明显抑制了细胞色素C的释放和Caspase-3的活化(P＜0.05 or P＜0.01)。
【结论】
本实验结果表明幼龄大鼠深低温低流量模型可近似于临床心脏手术深低温低流量过程。二氮嗪在深低温低流量脑缺血损伤中通过阻止细胞色素C的释放和抑制Caspase-3的活化发挥抗细胞凋亡的保护作用,为临床防治深低温低流量术后神经系统损伤提供新的治疗思路,具有重要的临床研究应用价值。
Objective:
The purpose of this study was to determine the effects of diazoxide on apoptosis and the relative mechanisms in a model of brain injury induced by deep hypothermia low flow (DHLF).
Methods:
198 three-week-old Sprague-Dawley rats were randomly and equitably divided into sham-operated group, placebo-treated group and diazoxide-treated group respectively. Placebo-treated and diazoxide-treated rats were experienced bilateral common carotid arteries (CCAs) occlusion for 120 min during deep hypothermia at (21±0.5)℃. Sham-operated rats were only without occlusion of the CCAs. Specific examination of the regional cerebral blood flow (rCBF) was measured in the three groups (n=6 for each group) continuously during the operation by laser Doppler flowmetry (LDF). Then 60 rats for each group was redistributed into five subgroups: 1 hour (1 h), 6 hours (6 h), 24 hours (24 h), 72 hours (72 h) and 7 days (7 d) after reperfusion. Diazoxide (5 mg/kg) or the same volume of DMSO was administered intraperitoneally 30 min before brain ischemia. In situ end-labeling of nuclear DNA fragmentation (TUNEL) was employed to determine the level of apoptosis. The mRNA expressions of cytochrome c and caspase-3 were determined by RT-PCR, and the protein expressions of cytochrome c and caspase-3 were determined by immunohistochemistry.
Results:
rCBF was significantly decreased in both of placebo-treated and diazoxide-treated group. rCBF showed flow reduction to (15.73±3.47)% (placebo-treated group) and (14.74±3.60)% (diazoxide-treated group) of baseline value just after ischemia in the time interval 0-5 min, and had no obviously changes in all the time intervals during the operation. In the placebo-treated group, the increased the percentage of TUNEL-positive staining cells were significantly observed at 6 h, 24 h, 72 h and 7 d after reperfusion (P<0.01 vs sham). The mRNA expressions of cytochrome c and caspase-3 were also increased at 6 h, 24 h and 72 h after reperfusion (P<0.05 or P<0.01 vs sham), while the protein expressions of cytochrome c and caspase-3 were increased from 6 h or 24 h to 72 h after reperfusion, respectively (P<0.05 or P<0.01 vs sham). Diazoxide preconditioning markedly decreased the percentage of TUNEL-positive staining cells, and inhibited the release of cytochrome c and activation of caspase-3 (P<0.05 or P<0.01 vs placebo).
Conclusion:
These results suggest that this animal model is approximately to clinic cardiac operation DHLF procedure, and the protective effects of diazoxide preconditioning in our model correlated with the reduction of DNA fragmentation, the prevention of mitochondrial cytochrome c release and inhibition of caspase-3 activation. These findings demonstrate that diazoxide may become a therapeutic drug for the brain injury during DHLF cardiopulmonary bypass.
通过建立幼龄大鼠深低温低流量(DHLF)脑损伤模型,研究二氮嗪通过线粒体凋亡通路的脑保护机制。
【方法】
将198只3周龄SD大鼠随机分为三组:假手术组、模型组和二氮嗪组。模型组和二氮嗪组在大鼠肛温降至(21±0.5)℃时阻断两侧颈总动脉120 min后再开放。假手术组除不阻断两侧颈总动脉外,其余操作相同。利用激光多普勒血流监测仪连续监测假手术组、模型组和二氮嗪组(每组6只)大鼠局部脑血流量。然后,每组60只大鼠再随机分成5个亚组:再灌注后1h,6h,24h,72h和7d(n=12)。二氮嗪组于缺血前30min腹腔内注射二氮嗪5 mg/kg(溶解于0.25 ml二甲基亚砜(DMSO)),模型组和假手术组则予腹腔内注射等体积的DMSO。TUNEL法检测各组脑组织细胞凋亡,RT-PCR检测各时间点细胞色素C和Caspase-3的mRNA表达的变化,免疫组织化学检测各时间点细胞色素C和Caspase-3的蛋白表达的变化。
【结果】
大鼠局部脑血流量在模型组和二氮嗪组均显著下降。模型组和二氮嗪组在DHLF 0～5 min内分别降至正常水平的(15.73±3.47)%和(14.74±3.60)%,且在DHLF期间无明显改变。模型组中TUNEL-阳性细胞在再灌注后6h,24h,72h和7d均显著增高(P＜0.01),细胞色素C和Caspase-3的mRNA表达在再灌注后6h,24h和72h亦明显增高(P＜0.05 or P＜0.01),同时细胞色素C和Caspase-3的蛋白表达分别在再灌注后6 h～72 h和24 h～72 h增高(P＜0.05 orP＜0.01)。二氮嗪组TUNEL阳性细胞显著下降,并且明显抑制了细胞色素C的释放和Caspase-3的活化(P＜0.05 or P＜0.01)。
【结论】
本实验结果表明幼龄大鼠深低温低流量模型可近似于临床心脏手术深低温低流量过程。二氮嗪在深低温低流量脑缺血损伤中通过阻止细胞色素C的释放和抑制Caspase-3的活化发挥抗细胞凋亡的保护作用,为临床防治深低温低流量术后神经系统损伤提供新的治疗思路,具有重要的临床研究应用价值。
Objective:
The purpose of this study was to determine the effects of diazoxide on apoptosis and the relative mechanisms in a model of brain injury induced by deep hypothermia low flow (DHLF).
Methods:
198 three-week-old Sprague-Dawley rats were randomly and equitably divided into sham-operated group, placebo-treated group and diazoxide-treated group respectively. Placebo-treated and diazoxide-treated rats were experienced bilateral common carotid arteries (CCAs) occlusion for 120 min during deep hypothermia at (21±0.5)℃. Sham-operated rats were only without occlusion of the CCAs. Specific examination of the regional cerebral blood flow (rCBF) was measured in the three groups (n=6 for each group) continuously during the operation by laser Doppler flowmetry (LDF). Then 60 rats for each group was redistributed into five subgroups: 1 hour (1 h), 6 hours (6 h), 24 hours (24 h), 72 hours (72 h) and 7 days (7 d) after reperfusion. Diazoxide (5 mg/kg) or the same volume of DMSO was administered intraperitoneally 30 min before brain ischemia. In situ end-labeling of nuclear DNA fragmentation (TUNEL) was employed to determine the level of apoptosis. The mRNA expressions of cytochrome c and caspase-3 were determined by RT-PCR, and the protein expressions of cytochrome c and caspase-3 were determined by immunohistochemistry.
Results:
rCBF was significantly decreased in both of placebo-treated and diazoxide-treated group. rCBF showed flow reduction to (15.73±3.47)% (placebo-treated group) and (14.74±3.60)% (diazoxide-treated group) of baseline value just after ischemia in the time interval 0-5 min, and had no obviously changes in all the time intervals during the operation. In the placebo-treated group, the increased the percentage of TUNEL-positive staining cells were significantly observed at 6 h, 24 h, 72 h and 7 d after reperfusion (P<0.01 vs sham). The mRNA expressions of cytochrome c and caspase-3 were also increased at 6 h, 24 h and 72 h after reperfusion (P<0.05 or P<0.01 vs sham), while the protein expressions of cytochrome c and caspase-3 were increased from 6 h or 24 h to 72 h after reperfusion, respectively (P<0.05 or P<0.01 vs sham). Diazoxide preconditioning markedly decreased the percentage of TUNEL-positive staining cells, and inhibited the release of cytochrome c and activation of caspase-3 (P<0.05 or P<0.01 vs placebo).
Conclusion:
These results suggest that this animal model is approximately to clinic cardiac operation DHLF procedure, and the protective effects of diazoxide preconditioning in our model correlated with the reduction of DNA fragmentation, the prevention of mitochondrial cytochrome c release and inhibition of caspase-3 activation. These findings demonstrate that diazoxide may become a therapeutic drug for the brain injury during DHLF cardiopulmonary bypass.
引文
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[2] Jonas RA. Hypothermia circulatory arrest and the pediatric brain[J]. J Cardio Thorac Vasc Anesth. 1996, 10: 66-74.
[3] Vannucci RC. Mechanisms of perinatal ischemic brain damage. In Janos RA, Newburger JW, Volpe JJ, eds. Brain injury and pediatric cardiac surgery. Newton: Butterworth-Heinemann. 1996, 201-204.
[4] Jonas RA. Brain injury and pediatric cardiac surgery. 1995, 201-204.
[5] Kristian T, Siesjo BK. Calcium-related damage in ischemia[J]. Life Science. 1996, 59:357-367.
[6] Karibe H, Chen SF, Zarow GJ, et al. Mild intra ischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats [J]. Brain Res. 1994, 649: 12-18.
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