Isoflurane Preconditioning Protects Rat Brains Against Focal Ischemia By Toll-like Receptor 4 Myeloid Differentiation Factor 88-dependent Signal Pathway

Ischemic cerebral diseases often leads to neurological dysfunction,Which does influence the quality of patients'life and even threaten their survivals. Cerebral infarction is a complex pathophysiological process and can be affected by multiple factors. It has long been recognized that injured tissue releases endogenous molecules, which induce an inflammatory immune response. Several of these endogenous components were identified as ligands for TLR4. TLR4 play an important role in Cerebral ischemia and induction of immune-inflammatory reaction. TLR4 is a key component of the innate immune system, functioning as pattern recognition receptor that recognises the lipopolysaccharide (LPS). Considering the role of TLR4-Myd88 dependent signaling pathway,we infer that Iso pretreatment may be associated with TLR4 signaling pathway in the damage of cerebral ischemia/reperfusion.Part 1 The dose-effect relationship of isoflurane preconditionObjective:To evaluation dose-effect relationship of isoflurane precondition.Methods: 40 male SD rats, weighing 250-300g, were randomly divided into four groups.Animals in control groups were Inhaled with 100% O2 1 hour every day ,for 5d.Rats in 1% iso groups,2% iso groups,3% iso groups received administered with 1%,2%,3% isoflurane respectively,1h/d,for 5d. A middle cerebral artery occlusion (MCAO) model was made after at 24h the last preconditioning. At 24h,48h and 72h after the reperfusion the neurological deficit scores were evaluated,and infarct size were measured at 72h after reperfusion.Results: The NDS in 2% and 3% Iso groups at24,48 and 72h after reperfusion were significantly higher than those in 1% Iso group and control group.and NDS in 2% was also higher than that in 3% Iso group.But there were no significant difference between the in control group and 1% Iso group.The infarct volume at 72h after reperfusion in 2% and 3% iso group were smaller than those in control group and 1% Iso group.Brain infarct volumes in 2% was also smaller than that in 3% Iso group. But in control group and 1% Iso group,there were no significant difference.Conclusion:Isoflurane can induced dose-dependent neuroprotective effects.Part 2 The expression of Toll-like receptor 4(TLR4) on glial cell and neuron about isoflurane preconditioning.Objective: To explore the expression of toll-like receptor 4(TLR4) on glial cell and neuron about isoflurane preconditioning.Methods: 48 Male SD rats weighing 250~300g were randomized into three groups. Rats in control group ,don't insert nylon thread into right Internal carotid artery, just strip vessel,(n = 16) , Mcao group,in which the focal cerebral ischemia was induced by nylon monofilament for 2h.(n = 16), animals in Iso group were administered with 2% isoflurane O2 1h/d,for 5d.(n = 16) , MCAO was induced after 24h under isoflurane preconditioning. Immunofluorescence staining were used to analyse the expression of TLR4 on glial cell(GFAP ,OX42)and nerve cell after 24 h following reperfusion.Results: At 24h after reperfusion,there was no TLR4 positive cell detected in sham group,But a large number of TLR4 positive cells were detected by immunohistochemistry in Mcao group.The TLR4 was mainly located in the GFAP positive astrocytes,and TLR4were minor located in NeuN positive nerve cell and OX-42 positive microglia.The number of immunoreative cells were obviously higher in Mcao group than those in Sham group and Iso group. But there were no significant difference between Sham group and Iso group.Conclusion: Isoflurane preconditioning protected the brain from damage may be involved in the mechanism through downregulation the expression of TLR4 on glial cell.Part 3 The protein expression of TLR4,Myd88 and NF-κB in isoflurane precondition.Objective: To study the protein expression of TLR4,Myd88 and NF-κB in isoflurane precondition.Methods:54 Male SD rats weighing 250~300g were randomly divided into three group: control group ,don't insert nylon thread into right Internal carotid artery, just strip vessel,(n = 16) , Mcao group,in which the focal cerebral ischemia was induced by nylon monofilament for 2h.(n = 16), animals in Iso group were administered with 2% isoflurane O2 1h/d,for 5d.(n = 16) , MCAO was induced after 24h under isoflurane preconditioning. Western blot were used to analyse the expression of TLR4, Myd88, and NF-κB protein level in ischemic brain tissue after 24h,48h and 72h.Results: .The TLR4 was first detected at 24h of reperfusion,peaked at 48h of reperfusion,and declined at 72h of reperfusion. the protein levels of TLR4 in Iso group was obviously lower than those in Mcao group. the change of Myd88 and NF-κB protein have the same trend with TLR4 in the cerebral cortex of the ischemic.Conclusion:1.Isoflurane preconditioning protected the brain from damage may be involved in the mechanism of throug downregulation of TLR4, Myd88, and NF-κB2.Iso preconditioning protected the brain from damage may be involved in the mechanism of throug downregulation of TLR4-Myd88.