Influence And Mechanism Of Diazoxide On Brain Injury Induced By Deep Hypothermia Low Flow In Young Rats

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.