The Roles Of Oxygen Free Radical And JAK-STAT In Neuroprotection Induced By Sevoflurane Preconditioning In Rats

Background:Acute cerebral infarction often leads to neurological dysfunction, which does harm to the quality of patients'lives, even threaten their survivals when it become severe. Compared with the unfeasibility of ischemia preconditioning,many measures of non-ischemia preconditioning show great practical values for studying. In the course of ischemia-reperfusion, oxygen free radical and Janus kinase-signal transducer and activator of transcription(JAK-STAT)pathway play important roles. Our previous researches have investigated some points as follow. The neuroprotection of hyperbaric oxygen and isoflurane relate to the release of oxygen free radical (OFR), which up-regulate endogenous antioxidation and reduce reactive oxygen species (ROS) and OFR in ischemic phase. Hyperbaric oxygen and electroacupuncture preconditioning attenuate phosphorylation level of STAT3 in ischemic cortex and hippocampol. Moreover, the reduction of ROS after ischemia-reperfusion cuts down the phosphorylation level of STAT3. Therefore, we presume that ROS and JAK-STAT pathway participate the courses of cerebral ischemia-reperfusion and non-ischemia preconditionings. However, the relationship of these two factors and the specific mechanism of them have not yet been understood. As a new kind of inhalational anesthetics, sevoflurane has been used widely, neuroprotection effect of which has been testified. This research aims at investigating the effects of free radical and JAK-STAT on focal cerebral ischemia-reperfusion injury and sevoflurane preconditioning, with oxygen free radical scavenger DMTU and JAK2 inhibitor AG490 being used on middle cerebral artery occlusion (MCAO) model in rats.Part 1Effects of combined application of DMTU and AG490 on focal cerebral ischemia-reperfusion injury in ratsObjective1. To study neuroprotective effects of combined application of DMTU and AG490 on focal cerebral ischemia-reperfusion injury in rats.2. To investigate the dose-dependent relationship of neuroprotection induced by application of AG490 and DMTU.Method1. Neuroprotective effects of combined application of DMTU and AG490 on focal cerebral ischemia-reperfusion injury in rats.60 male SD rats were randomly divided into control (CON), dimethyl sulfoxide (DMSO), AG490, normal saline (NS), dimethylthiourea (DMTU) and combination (A+D) groups (n=10 each). In all rats, MCAO was induced by an occlusion of right internal carotid artery with a nylon monofilament. The neurological behavior scores (NBS) were assessed at 24h, 48h and 72h after reperfusion respectively, and the animals were then decapitated to determine the brain infarct volume.2. Dose-dependent relationship of neuroprotection induced by application of AG490 and DMTU.40 male SD rats were randomly divided into control group and three experimental groups administered with various doses of DMTU and AG490. NBS were assessed at 24h, 48h and 72h after reperfusion respectively, and the animals were then decapitated to determine the brain infarct volume.Result1. Neuroprotective effects of combined application of DMTU and AG490 on focal cerebral ischemia-reperfusion injury in ratsThe neurological behavior scores in A+D group, DMTU group and AG490 group were higher than those in control group at 24h, 48h and 72h after reperfusion, and their brain infarct volumes were obviously smaller than CON group as well. The brain infarct volumes in A+D group were obviously smaller in comparison with AG490 and DMTU alone.2. Dose-dependent relationship of neuroprotection induced by application of AG490 and DMTU.The values of NBS were higher and the brain infarct volumes were smaller in high dose and medium dose combination groups than those in low dose combination and control groups respectively. In addition, brain infarct volumes in high dose group were smaller than medium dose group, but there was no statistical difference between low dose and control groups. Part 2 The roles of ROS and JAK-STAT in sevoflurane preconditioningObjective1. To study dose-dependent relationship of sevoflurane preconditioning.2. To study the role of ROS in sevoflurane preconditioning.3. To study the role of JAK-STAT in sevoflurane preconditioning.Method1. Dose-dependent relationship of sevoflurane preconditioning.40 male SD rats were randomly divided into control group, 1% group, 2% group and 4% group. Rats in control group were administered with 100% O2, 1h/d, 5d consecutively. Rats in 1% group, 2% group and 4% group are administered with 1%, 2%and 4% sevoflurane respectively, 1h/d, 5d consecutively. MCAO was given at 24h after the last preconditioning. NBS were assessed at 24h, 48h and 72h after reperfusion respectively, and the animals were then decapitated to determine the brain infarct volume.2. The role of ROS in sevoflurane preconditioning.40 male SD rats were randomly divided into 4 groups: O2+NS group, SEV+NS group, O2+DMTU group, and SEV+DMTU group. DMTU or NS was administered to different groups before each preconditioning. After consecutive 5 days of preconditioning, rats were given MACO. NBS were assessed at 24h, 48h and 72h after reperfusion respectively, and the animals were then decapitated to determine the brain infarct volume. 3. The role of JAK-STAT in sevoflurane preconditioning.40 male SD rats were randomly divided into 4 groups: O2+DMSO group, SEV+DMSO group, O2+AG490 group and SEV+AG490 group. After consecutive 5 days of preconditioning, rats were given MACO. AG490 or DMSO was administered by lateral cerebral ventricle infusion 20min before MCAO. NBS were assessed at 24h, 48h and 72h after reperfusion respectively, and the animals were then decapitated to determine the brain infarct volume.Result1. Dose-dependent relationship of sevoflurane preconditioning.The values of NBS were higher in 2% and 4% sevoflurane group than those in 1% and control groups. NBS in 4% group was also higher than that in 2% group, but no statistical difference was found between 1% group and control group. After 72h reperfusion, brain infarct volumes in 2% group and 4% group were smaller than those in 1% and control groups. Brain infarct volumes in 4% were also smaller than that in 2%. There was no significant difference between 1% group and control group.2. The role of ROS in sevoflurane preconditioning.NBS of SEV+NS group were higher than those in O2+NS group, O2+DMTU group, and SEV+DMTU group, there were no statistical difference among the latter three groups. The brain infarct volumes in SEV+NS group were smaller than those in O2+NS group, O2+DMTU group, and SEV+DMTU group, there were no statistical difference among the latter three groups either.3. The role of JAK-STAT in sevoflurane preconditioning.NBS of SEV+DMSO group,O2+AG490 group and SEV+AG490 group were higher than those in O2+DMSO group, no statistical difference was found in former three groups. Brain infarct volumes of SEV+DMSO group,O2+AG490 group and SEV+AG490 group were smaller than those in O2+DMSO, there was no statistical difference among former three groups either.Conclusion1. Both ROS and JAK-STAT pathway play important roles in pathophysiological course of cerebral ischemia-reperfusion injury, DMTU and AG490 produce significant synergistic neuroprotective effects.2. Sevoflurane can induce dose-dependent neuroprotection effects.3. Neuroprotection induced by sevoflurane is related to release of ROS.4. Neuroprotection induced by sevoflurane may be related to attenuation of JAK-STAT activation level.