Therapeutic time window and regulation of autophagy by mild hypothermia after intracerebral hemorrhage in rats

Although recent studies have shown that mild hypothermia has neuroprotective effects on intracerebral hemorrhage (ICH), the therapeutic time window of the therapy and the role of autophagy as a potential neuroprotective mechanism remain unclear. This study was aimed to investigate the appropriate time window of mild hypothermia and the regulation of autophagy during the treatment in a rat model of autologous blood-injected ICH injury. The rats were divided into Sham, normothermic (NT) and hypothermic (HT) groups. HT groups received mild hypothermia (33 °C-35 °C) for 48 h starting from 3 h (HT3), 6 h (HT6), and 12 h (HT12) respectively after ICH. The neurological function, brain edema, blood brain barrier (BBB) permeability and volume of tissue loss were tested. The expression of metrix metalloproteinase 9 (MMP-9) and tight junction (TJ) protein including Occludin and Claudin-5 around the hematoma were detected by Western blot. Moreover, autophagy after ICH was detected by the ratio of LC3B-II/I, and the expression of Beclin-1 and p62, while apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dURP nick end labelling (TUNEL) staining and expression of Bcl-2, Bim, cleaved Caspase-3. Compared with NT group, neurological deficit, brain edema and BBB permeability were attenuated in HT6 and HT12 groups, but not in HT3 group, while volume of tissue loss was reduced only in HT12 group. The expression of MMP-9 and the degradation of Occludin and Claudin-5 were suppressed only in HT6 and HT12 groups, especially in the latter one. Moreover, neuronal autophagy and apoptosis induced by ICH were downregulated in HT12 group. The results suggested that mild hypothermia initiated at 6 h or 12 h post-injury was neuroprotective in ICH model of rats, especially at 12 h post-injury, via suppression of autophagy upregulated by ICH.