The Role And Mechanism Of Mild Hypothermia Therapy On Early Brain Injury In Rats With Subarachnoid Hemorrhage

Objectives: Subarachnoid hemorrhage(SAH)is a prevalent yet severe subtype of stroke that accounts for approximately 5 % of all stroke cases.Intracranial aneurysms are the most common cause,accounting for approximately 85%,and have a high rate of disability and death.Mild hypothermia(MH)therapy has significant neuroprotective effects and is widely used in the treatment of patients with severe cranial injury and after resuscitation from cardiac arrest.However,there are few researches on its use in EBI after SAH.Therefore,this research aimed to clarify the role of MH therapy in EBI after SAH in rats and its potential mechanism.Methods:(1)Experiment 1: Verification of the neuroprotective effect of MH therapy in EBI after SAH.54 SD rats(63 of which underwent surgery and 9 were excluded due to death or SAH grading score < 8)were completely randomized into 3groups,including control group(Sham group),subarachnoid hemorrhage group(SAH group)and subarachnoid hemorrhage mild hypothermia group(SAH + MH group)with 18 rats in each group.The SAH rat model was constructed by using the intravascular puncture method.The rectal temperature(RT)was reduced to a predetermined temperature(33 ± 0.5°C)by ice packs in a short period of time(15 ～ 30min)in the MH group and maintained at 33°C ± 1°C for 4 h by ice packs and insulation blankets,followed by rewarming within 1.5 hours.The brain temperature(BT),RT and intracranial pressure(ICP)of each rat were monitored promptly after modeling.After 24 h of modeling,the neurological deficits in each group of rats were assessed using the modified Garcia score and the beam balance test.Then,we assessed the expression of inflammatory factors(IL-1β,IL-18 and TNF-α)in rats by enzymelinked immunosorbent assay(ELISA)(n = 6),and analyzed the extent of cell death in each group of 6 rats by terminal deoxynucleotidyl transferase d UTP nick end labeling method(TUNEL staining)and hematoxylin and eosin(HE)staining.Finally,the brain water content of the remaining 6 rats was assessed using the classical wet and dry weight method.(2)Experiment 2: To validate that MH therapy inhibits pyroptosis through AMPK-mediated signaling pathway,thereby attenuating EBI after SAH in rats.72 SD rats(84 of which underwent surgery and 12 were excluded due to death or SAH grading score < 8)were completely randomized into 4 groups,including control group(Sham + Vehicle group),subarachnoid hemorrhage group(SAH + Vehicle group),subarachnoid hemorrhage mild hypothermia group(SAH + MH + Vehicle group),and subarachnoid hemorrhage mild hypothermia + Compound C(SAH + MH+ Compound C group),with 18 rats in each group.Compound C,a AMPK inhibitor,was administered intravenously(i.v)30 minutes prior to SAH modeling to inhibit the AMPK pathway and reverse the neuroprotective effects of MH treatment.Compound C is soluble in saline solution,and Vehicle is the same amount of saline solution.The Garcia score and beam balance test were used to assess the neurological deficits in rats24 h after modeling.Brain edema was assessed by measuring the brain water content of each group of 6 rats via wet and dry weight method.6 rats in each group were used for TUNEL staining and immunofluorescence staining,and the remaining 6 were used for Western blot and ELISA.The degree of cell death in rats was analyzed by TUNEL staining.Western blot and immunofluorescence staining were used to compare the effects of different treatments on AMPK phosphorylation and expression of pyroptosisrelated proteins(NLRP3,ASC,Cleaved-caspase 1,GSDMD).The levels of inflammatory factors(IL-1β and IL-18)were measured by ELISA 24 h after SAH induction.Results:(1)Experiment 1: Compared with the Sham group,the SAH group caused significant changes in BT,RT and ICP.Not only that,but also inflammatory reaction,brain edema and morphological changes in brain histology occurred,which impaired neurological function after SAH in rats.In contrast,MH therapy reduced SAH-induced increases in BT,RT and ICP,and decreased the release of proinflammatory factors(IL-1β,IL-18 and TNF-α),reducing brain damage after SAH in rats.(2)Experiment 2: Compared with the Sham + Vehicle group,the SAH+ Vehicle group had elevated expression levels of pyroptosis-related proteins(NLRP3,ASC,Cleaved-caspase 1,GSDMD,IL-1β and IL-18)and more severe brain injury.furthermore,MH therapy alleviates EBI after SAH by inhibit the level of pyroptosisassociated protein expression via promoting AMPK phosphorylation.In contrast,the protective effects of MH treatment on AMPK phosphorylation levels,focal deathrelated protein expression levels,and brain injury after SAH were inhibited by intravenous administration of Compound C to rats.In contrast,the protective effects of MH therapy on AMPK phosphorylation levels,pyroptosis-related protein expression levels,and brain injury in SAH 24 h were inhibited after intravenous administration of Compound C to rats.Conclusions: This research demonstrated that MH treatment alleviated EBI after SAH in rat model by modulating AMPK/NLRP3 inflammasome-mediated pyroptosis and neuroinflammation.Meanwhile,MH treatment effectively suppressed the trend of elevated BT,RT and ICP after SAH.