Long-term Neurologic Outcome And Mechanisms Of Sevoflurane Postconditioning On Neonatal Rats With Hypoxic–ischemic Brain Damage

【Objective】The present study was designed to investigate long-term neurologic outcome on the neonatal hypoxic-ischemic brain damage (HIBD) rats after sevoflurane post- conditioning and explore the possible mechanism about PI3K/Akt-mPTP signaling pathway.【Methods】A rat model with HIBD at age 7day (P7) was set up as Rice reported (Rats was induced through left common carotid artery legation,and then breathing 8%oxygen +92%nitrogen for two hours). All rats were randomly divided into ten groups (n=12 per group) including: Sham without hypoxic- ischemic,HI/Control without other interventions,HI+Sev (Sevoflurane post- conditioning) treated the same as HI/ Control and then put into the container for sevoflurane treatment(2.5%sevoflurane in 30%oxygen +70% nitrogen for 30min),and other groups treated the same as HI/ Control(HI+Atr and HI+CsA) or HI+Sev(HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Sev+Atr and HI+Sev+CsA) and separately given PI3K inhibitor LY294002 0.3mg/kg,eNOS inhibitor L-NAME 10mg/kg,GSK-3βinhibitor SB216763 0.2mg/kg,the mPT pore opener atractyloside (Atr) 10mg/kg and inhibitor cyclosporin A (CsA) 5mg/kg by injecting slowly into the lateral cerebral ventricle with microsyringe 5min before sevoflurane or 30% oxygen treatment. Sham,HI/Control and HI+Sev groups injected with Normal Saline. The rats , brain sensorimotor functional outcome was evaluated by suspension test (P21-P27) and the balancing test (P15-P29),long-term learning- memory capacity was evaluated by the novel object recognition test(P30-P35) and Moriss Water Maze test(P37-P42),The morphological and histological changes of the brain tissues,including brain weight,surviving neurons in hippocampal CAl and CA3 region,was observed under the electron microscope by Haematoxylin-eosin staining and by eyes-directly. Another 120 SD rats were treated as what was mentioned above,and expression of phosphorylate Akt,total Akt,phosphorylate eNOS,total eNOS,phosphorylate GSK-3βand total GSK-3βwere evaluated by Western-bloting,and mPT by spectrophotometry at age 8 day (P8).【Results】(1)Sensorimotor functional outcome:â‘ Suspension test : The time of suspension did not have clear difference between each group (F=0.430,P=0.916);â‘¡Balancing test:The score of the balancing test in the HI+Sev,HI+CsA and HI+Sev+CsA groups were much more than that in the HI/Control group(P<0.01),but the HI+Atr and HI+Sev+Atr groups had no clear difference compared with the HI/Control group(P>0.05). Compared with the HI+Sev group,the score in the HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups reduced significantly (P<0.01),but there were no notable changes in the HI+CsA and HI+Sev+ CsA groups (P>0.05).The novel object recognition test : About the discrimination index (DI) including DI0,DI3,DI24:the HI+Sev,HI+CsA and HI+Sev+CsA were significantly higher then the HI/Control group(P<0.01),but there were no clear difference between the HI+Atr,HI+Sev+Atr and HI/Control groups(P>0.05);Compared with the HI+Sev group,the three index of the HI+Sev+LY,HI+Sev+L-N,HI+Sev +SB,HI+Atr and HI+Sev+Atr groups reduced deeply (P<0.05),but there were no significant changes in the HI+CsA and HI+Sev+ CsA groups (P>0.05). Moriss Water Maze test :â‘ Place Navigation test : Compared with the HI/ Control group,the HI+Sev,HI+CsA and HI+Sev+ CsA groups reduced deeply in the mean escape latencie(sP<0.05),but the HI+Atr and HI+Sev+Atr groups had no clear difference(P>0.05);Compared with the HI+Sev group,the mean escape latencies of the HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups prolonged clearly (P<0.01),but the HI+CsA and HI+Sev+ CsA groups had no notable changes(P>0.05);About the moving routes,the HI+Sev,HI+CsA and HI+Sev+CsA groups probed mainly in straight strategy or tendency strategy(learning strategy),and there were no significant difference between these three groups ,but the HI/Control,HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups probed mainly in marginal strategy or random strategy(not learning strategy) .â‘¡Space Exploration test:About the learning and memory capacity including cross number,probe time,probe length,the HI+Sev,HI+CsA and HI+Sev+CsA were clearly higher then the HI/Control group(P<0.01),but there were no significant difference between the HI+Atr,HI+Sev+Atr and HI/Control groups(P>0.05);Compared with the HI+Sev group,the three factors above in the HI+Sev+ LY,HI+Sev+L-N,HI+Sev+ SB,HI+Atr and HI+Sev+Atr groups reduced deeply (P<0.01),but there were no significant changes in the HI+CsA and HI+Sev+ CsA groups (P>0.05);About probe speed,there were not clearly different between each group(F=0.504,P=0.869).The morphological and histological in brain tissues:â‘ The general brain injury:Compared with the HI/Control group witch was injury seriously,the HI+Sev,HI+CsA and HI+Sev + CsA groups were alleviated clearly(P=0.007;P=0.002;P= 0.002), but the HI+Atr and HI+Sev+Atr groups had no significant difference(P= 1.000;P=0.679);Compared with the HI+Sev group, the HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups were injury more seriously (P=0.015;P=0.041;P=0.022), but the HI+CsA and HI+Sev+ CsA groups had no significant difference(P=0.192;P=0.192).â‘¡Left and right brain weight:the HI+Sev,HI+CsA and HI+Sev+ CsA groups were higher then the HI/ Control group(P<0.01), but the HI/ Control group,HI+Atr and HI+Sev+Atr groups had no clear difference(P>0.05);Compared with the HI+Sev group,the weight of left and right brain in the HI+Sev+LY,HI+Sev+L-N,HI+Sev+ SB,HI+Atr and HI+Sev+Atr groups reduced clearly (P<0.05),but the HI+CsA and HI +Sev+ CsA groups had no significant changes(P>0.05).â‘¢The atrophy degree of brain:The HI+Sev,HI+CsA and HI+Sev+CsA groups were smaller than the HI/Control group(P<0.01),but the HI+Atr and HI+Sev+Atr groups had no clear difference compared with the HI/ Control group(P>0.05);Compared with the HI+Sev group, the atrophy degree of the HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups increased significantly (P<0.01), but there were no notable changes in the HI+CsA and HI+Sev+ CsA groups (P>0.05).â‘£The number of surviving neurons in left hippocampus CA1 and CA3 region:the HI+Sev,HI+CsA and HI+Sev+ CsA groups were more then the HI/ Control group(P<0.01),but the HI/ Control group,HI+Atr and HI+Sev+Atr groups had no significant difference(P>0.05); Compared with the HI+Sev group, the number of surviving neurons in CA1 and CA3 region of the HI+Sev+LY,HI+Sev+L-N,HI+Sev+ SB,HI+Atr and HI+Sev+Atr group reduced clearly (P<0.01),but the HI+CsA and HI +Sev+ CsA groups had no clear changes(P>0.05);The HI+Sev+L-N group was more then HI+ Sev+SB group significantly (CA1:P=0.037;CA3:P=0.042).(2)Western-bloting : Compared with the HI/ Control group Sevoflurane post- conditioning (HI +Sev) could activate PI3K route which up-regulated expression of phosphorylate Akt,eNOS and GSK-3β(P<0.05); However,Compared with the HI +Sev group , PI3K inhibiter LY294002,eNOS inhibitor L-NAME,GSK-3βinhibitor SB216763 could separately inhibit the expression of phosphorylate Akt,eNOS and GSK-3β(P<0.05) and completely block the PI3K-Akt-eNOS/ GSK-3βsignaling pathway in Sevoflurane post- conditioning (HI +Sev).The decrease of absorbance values of mitochondrial(ΔOD540 in 5min) : Compared with the HI/ Control group, the HI+Sev,HI+CsA and HI+Sev+ CsA groups reduced deeply inΔOD₅40(P<0.01)witch represented the degree of mitochondrial permeability transition, but the HI+Atr and HI+Sev+Atr groups had no clear difference(P>0.05);Compared with the HI+Sev group, theΔOD540 in the HI+Sev+LY,HI+Sev+L-N,HI+Sev+SB,HI+Atr and HI+Sev+Atr groups increased clearly (P<0.05),but the HI+CsA and HI+Sev+ CsA groups had no significant changes(P>0.05);The HI+Sev+L-N group was less then HI+ Sev+SB group significantly(P=0.024).【Conclusions】(1) Sevoflurane post-conditioning might result in a protective effect against HIBD-induced long-term neurologic outcome including brain morphological and histological deficits and spatial learning and memory disability;(2) Sevoflurane post-conditioning protected the brain of neonatal rats with HIBD probably through enhancing the activation of PI3K/Akt/eNOS and PI3K/Akt/GSK-3βsignaling pathway,and blockage of mPT may be involved in attenuation of hypoxic-ischemic injury.