The Neuroprotective Effects And Mechanism Study On Combination Therapy Of Mild Hypothermia And Phenothiazines In Rat Ischemic Stroke

Background and Purposes: Therapeutic Hypothermia(TH)has long been considered a promising neuroprotective agent of ischemic stroke,but the treatment’s relative complications along with the uncertain depth and duration of therapy significantly narrow its clinical scope.The Stroke Roundtable Conference have put forward that combination TH with other neurprotective agents might reduce its adverse reactions and enhance its neurological effects.In the present study,we combined TH with phenothiazine neuroleptics as additive neuroprotectants,with the aim of detecting whether our combination therapy could enhance the efficacy of TH,under the condition that not prolonging its duration and depth.These include chlorpromazine and promethazine,which have been shown to confer hibernation-like effects on the central nervous system(CNS).To elucidate the molecular mechanisms of our therapy’s outcomes,we also investigated its effects on the Phosphatidylinositol 3 kinase/Protein kinase B(PI3K/Akt)apoptotic pathway and several of its regulators.Methods: Sprague-Dawley rats were subjected to right middle cerebral artery occlusion(MCAO)for 2 h,and prepared the MCAO model.We used the sham group as an control group.MCAO rats were then given: no treatment(S),mild TH alone(S&H),phenothiazines alone(S&D),both TH and phenothiazines(S&HD),or combination therapy with a p-Akt inhibitor(S&HD&LY).Extent of brain injury was determined by infarct volume,neurological deficit,and apoptotic cell death 24 h after reperfusion.Expressions of p-Akt,cleaved Caspase-3,pro-apoptotic(AIF,Bax)and anti-apoptotic proteins(Bcl-2,Bcl-x L)were evaluated at 6 and 24 h after reperfusion.Results: The combination of mild hypothermia with pharmacological agents achieved reduction in MCAO rat temperatures in the rectum,cortex and striatum significantly(P<0.01)faster than hypothermia alone.Brain infarct volume and neurological deficits were significantly(P<0.01)reduced using our combination therapy as compared with monotherapies which conferred no specific protection.The combination of TH and pharmacological treatment significantly(P<0.05)increased expression of p-Akt and anti-apoptotic proteins(Bcl-2 and Bcl-x L),while reducing expression of pro-apoptotic proteins(AIF and Bax)and cleaved Caspase-3.Finally,the treatment’s neuroprotective effects were blocked by p-Akt inhibition.Conclusion: By combining mild hypothermia with phenothiazines,we remarkably enhanced the neuroprotective effects of TH.This study also sheds light on the possible mechanism for these neuroprotective effects which at least involve the PI3K/Akt signaling and apoptotic pathway.Thus,our combination therapy provides a promising approach to enhance the clinical outcomes of ischemic stroke using therapeutic hypothermia.