Abstract:

Objective

To investigate the neuroprotective effect of drug-induced hypothermia in acute phase of ischemic stroke， as well as to elucidate the underlying mechanism involving DRP-1-mediated mitochondrial function.

Methods

A total of 45 adult male Sprague-Dawley rats were randomly assigned to three groups： sham operation （Sham）， middle cerebral artery occlusion （MCAO）， and MCAO with chlorpromazine and promethazine treatment （C+P） （n=15 per group）.Rats underwent 2 h of MCAO followed by 24 h or 48 h reperfusion.C+P was administered at the onset of reperfusion at a dose of 8 mg/kg，with one-third of the original dose injected 2 hours later to potentiate the drug's effects.Body temperature was monitored at pre-MCAO， initial drug， and 5 min， 10 min， 20 min， 30 min， 1 h， 2 h， 3 h， 6 h， 12 h， 24 h after administration.Infarct volumes and neurological deficits were assessed using 2，3，5-triphenyltetrazolium chloride （TTC） staining and scoring systems at 48 hours.TUNEL staining was used to detected the apoptotic cell death at 24 h.The lactate dehydrogenase （LDH）， cell death， ATP， reactive oxygen species （ROS）concentration， and mitochondrial respiratory chain complex （COX Ⅰ-Ⅳ） were evaluated by ELISA at 24 h.The mRNA and protein expressions of DRP-1 and Fis-1 were quantified by qPCR and western blotting at 24 h.LSD-t test or Dunnett's T3 method were used for inter-group comparisons.

Results

C+P administration（8 mg/kg） significantly reduced body temperature within 5 minutes post-MCAO in rats， reaching the lowest temperature ［（33.5±0.3） ℃］ after 2 hours， and maintaining a significantly reduced temperature for up to 12 hours before returning to normal.Compared to the MCAO group， C+P significantly reduced infarct volumes ［（29.73±2.32）% vs （48.46±0.48）%］ and neurological deficits［5 score： （2.0±0.1） vs （4.0±0.1）；12 score： （5.0±0.3） vs （8.0±0.2）； （5 score： t=2.917， P=0.008； 12 score： t=2.475， P=0.029）］.C+P also significantly mitigated mitochondrial dysfunction （LDH， ATP， ROS and COX I-IV） and cell death apoptosis（P＜0.05）.mRNA and protein expression of DRP-1 and Fis-1 were both significantly reduced by C+P（P＜0.05）.

Conclusion

Our findings suggest that C+P-induced hypothermia exerts neuroprotective effects after ischemic stroke by inhibiting DRP-1 phosphorylation and Fis-1 expression， and reducing mitochondrial dysfunction and apoptosis.

Key words: Stroke, Hypothermia, Mitochondrial fission, Ischemia-reperfusion injury, Apoptosis