Protective Effects And Mechanisms Of Dexmedetomidine On Cerebral Injury Induced By Ischemia-reperfusion

Acute cerebral injury will be induced by transient ischemia and hypoperfusion of brain,while during the reperfusion after ischemia,a series of complex mechanisms are initiated to cause apoptosis and necrosis of neurons,then,injury of brain will be further aggravated,which were called cerebral ischemia-reperfusion injury(IRI).Cerebral IRI is a common complication during perioperation,especially of neurosurgy or cardiovascular surgery and in the rescue process of critical patients with shock or sundden cardiac arrest,which is usually associated with increase of healthcare cost and great mental anguish by the delayed mental or motor dysfunction.Therefore,it is of important clinical significance to avoid or reduce cerebral IRI during perioperation.In recent years,a large number of studies have shown that apoptosis induced by inflammatory response in the central nervous system is an important mechanism of IRI.Phosphatidyl inositol 3 kinase/protein kinase B(PI3K/Akt)pathway is an important signal tansduction pathway related to inflammation and apoptosis.The activation of PI3K will generated PIP3,which as a second messenger of its downstream substrate will activate the phosphation of Akt.Phosphation-Akt wiIl inhibit neuronal apoptosis by inducting the generation of nerve growth factor and neurotrophic factors.PI3K/Akt pathway also can regulate the expression of its downstream signaling molecules such as NF-kB、Bax and Bcl-2,to inhibit the occurrence of inflammation or apoptosis and promote cells survival.Dexmedetomidine(Dex)is an new anesthesia auxiliary medicine commonly applied in clinical.Some recent experiments demonstrated that Dex probably provide kidney and heart protection by decreasing the apoptosis of renal tubular epithelial cells and cardiac myocytes during ischemia reperfusion.It also can reduce the release of pro-inflammatory factors to protect the organs from the injury of sepsis.All of these studies have shown that Dex has the effects of anti-inflammatory and anti-apoptosis.According to this,we hypothesized that Dex can provide brain protection and improve the outcome of neural prognosis in IRI.Therefore,the purpose of this research is to verify the hypothesis and further explore related mechanisms.The contents of this research include the following two parts:(1)To investigate if Dex can provide the brain protection via reducing inflammation and apoptosis at the level of living animal suffering from acute ischemia and reperfusion injury.(2)To investigate if PI3K/Akt and NF-κB pathways are involved in the neuroprotective effect of Dex at the level of cells in vitro.Part One:The protective effect of dexmedetomidine against acute cerebral ischemia-reperfusion injury of ratsObjective:Assess the neuroprotective effects of Dex against acute focal cerebral ischemia-reperfusion injury in rats.Methods:60 healty male Sprague-Dawley rats were randomly assigned into Sham group,Ischemia-reperfusion group(I/R),Ischemia-reperfusion with Dex treatment group(D+I/R).Focal brain I/R model was established by right middle cerebral artery occlusion(MCAO).Dex was given immediately after ischemia.Neural dysfunction and cerebral edema in rats were measured at 24h after reperfusion.Followed by HE staining for hippocampus histopathological assessment,TUNEL staining for evaluation of cell apoptosis in hippocampus,immuohistochemistry for expression of NF-κBp65 and Western blot for expressions of Bax,Bcl-2 and p-Akt,as well as ELISA for detection of inflammatory cytokine(IL-1β,IL-6,TNF-α)level in hippocampus.Results:1.1 Effect of Dex on neurological dysfunction and brain edema in I/R rats:The neurobehavioral scores,left-deflection percent,and degree of brain edema in I/R group were significantly higher than those in sham group.By contrast,neurological dysfunction of rats was alleviated in the D+I/R group,with a significant decrease in the Longa’s scales and the left-deflection percent,as well as the degree of brain edema compared with the I/R group(P<0.05).1.2 Effect of Dex on hippocampal morphology in I/R ratsBy HE staining,compared with sham group,I/R rats showed marked neuronal degeneration,with a triangular or irregular shape,concentrated cytoplasm and nucleus,and a damaged structure,especially in CA1 region of hippocampus.This pattern of injury was significantly alleviated in the D+I/R group.1.3 Effect of Dex on hippocampal apoptosis rates in I/R ratsTunnel positive cells(green fluorescent)were significantly increased in I/R rats.Compared with cerebral I/R group,there was a significant decrease of Tunnel positive cells in the rats hippocampus,and the difference has statistically significance(P<0.01).1.4 Effect of Dex on hippocampal IL-1β,IL-6,and TNF-α levels as well asmRNA expression in 1/R ratsFor rats subjected to cerebral I/R,the levels of IL-1β,IL-6 and TNF-a or mRNA expressions in hippocampus were all largely higher than those in Sham group(P<0.01),while in Dex treatment group,the levels of IL-1β,IL-6 and TNF-α or mRNA expressions were obviously reduced compared to I/R group(P<0.05).1.5 Effect of Dex on hippocampal Bax and Bcl-2 protein expression in I/R ratsIn I/R rats,the protein expression of Bax was significantly increased,while Bcl-2 was decreased respectively,compared with sham group(P<0.01).While the protein expression of Bax or Bcl-2 in the Dex treatment group were markedly downregulated or upregulated respectively(P<0.05).1.6 Effect of Dex on expression of NF-kBp65 positive cells in I/R ratsThere was a significant increase in the number of NF-kBp65-positive cells in hippocampus of I/R rats compared with the sham group(P<0.01).By contrast,the number of NF-kBp65-positive cells was markedly decreased in the D+I/R group(P<0.01).1.7 Effect of Dex on hippocampal p-Akt protein expression in I/R ratsCompared with sham group,p-Akt expression level in I/R rats decreased obviously(P<0.01),while in Dex treatment group,the expression of p-Akt in hippocampus is significantly higher than I/R rats(P<0.01).Part Two:The study of PI3K/Akt and NF-κB pathway involved in the mechanism of dexmedetomidine against hippocampal neuron injury induced by OGD/RObjective:To investigate if the PI3K/Akt/NF-κB pathway mediated the protective effect of Dex against cerebral ischemia-reperfusion in vitro.Methods:The primary cultivated hippocampal neurons in vitro were obtained from hippocampal tissues of the newborn SD rats(within 24 hours of the birth).After cultured for 7 days,I/R neuron model was induced by oxygen-glucose deprivation(OGD)for 2h and then reperfusion(R)for 12h(OGD/R).During the start of OGD,neurons were divided into control group,OGD/R group,10μM Dex treatment(D10+OGD/R)group,20μM LY294002 and 10μM Dex co-treatment(LY+D10+OGD/R)group,and were given pretreatments with Dex and LY294002(PI3K inhibitor).The morphology of cultured neurons was observed by inverted phase contrast microscope.The cell viability was assessed by cell activity assay kit cell counting kit-8(CCK-8).Apoptosis rate of neurons in each group was detected by Annexin V FITC/PI double dye flow cytometry.The protein expressions of Bcl-2,Bax,NF-κBp65,IκBa,p-lKBa and total Akt as well as p-Akt were detected by Western Blot method.Results:2.1 The primary cultured hippocampal neurons were all grown in typical morphology at different stages of culture.The purity of hippocampal neurons which were cultured for 7 days was 91.8%by detection of anti-MAP2-FITC staining.2.2 The viability of the hippocampal neurons in different treantment group:The cell activity was significantly decreased,and the survival rate of neurons was 46.3 ± 8.2%in OGD/R group,which was significantly different from the control group(100%,P<0.01).With the treatment of 10μM Dex,the survival rate of neurons was increased to 79.8 ± 7.3%(P<0.01 vs OGD/R group).While the intervention of LY294002 inhibited the cell viability,the survival rate decreased to 41.8 ± 4.1%,(P<0.01 vs D10+OGD/R group).2.3 The apoptosis rate of cells in different treantment group:Compared with control group,the apoptosis rate of neurons in OGD/R group was significantly higher(P<0.01).Compared with OGD/R group,the apoptosis rate in Dex treatment OGD/R group obviously decreased(P<0.01).While in co-treatment group with LY294002 and Dex,the apoptosis rate of cells was apparently higher than D10+OGD/R group(P<0.01).2.4 The protein expression levels of Bcl-2 and Bax in different treantment group:In OGD/R group,the expression of Bax was higher and Bcl-2 as well as the ratio of Bcl-2/Bax was lower than those of control group(P<0.01).Compared with OGD/R group,the expression level of Bax in D10+OGD/R group was significantly decreased,the expression level of Bcl-2 and the ratio of Bcl-2/Bax were significantly increased(P<0.01).Compared with D10+OGD/R group,the expression level of Bax in LY+D10+OGD/R group increased(P<0.01),and the level of Bcl-2 as well as ratio of Bcl-2/Bax significantly decreased(P<0.01).2.5 The activation of NF-κB in different treantment group:Compared with control group,the expression of NF-κBp65 protein and the level of phosphorylation IkBa were significantly increased in OGD/R cells,while the level of IkBa was decreased(P<0.01).Compared with OGD/R group,the expression levels of NF-kBp65 and p-IκBa were significantly decreased in Dex intervention OGD/R group(P<0.01),while the level of IκBa was increased(P<0.01).While in LY+D10+OGD/R group,the expression level of NF-kBp65,IκBa and p-IkBa in the cells was not significantly different from D10+OGD/R group(P>0.05).2.6 The activation of PI3K/Akt pathway in different treantment group:There was no significant difference of total Akt expression in each group.Compared with normal group,the phosphorylation level of Akt protein in the OGD/R neurons was decreased(P<0.05).The expression level of p-Akt protein in Dex intervention OGD/R cells was significantly higher than that in OGD/R group(P<0.01).The effect of Dex activating Akt was completely inhibited by the PI3K inhibitor(LY294002),and the expression of p-Akt protein in LY+D10+OGD/R group was extremely low(P<0.01).Conclusions1.Dexmedetomidine has neuroprotective effects against cerebral ischemia reperfusion injury.2.The neuroprotection effects afforded by dexmedetomidine against cerebral ischemia reperfusion injury are due to anti-apoptosis and anti-inflammation.3.PI3K/Akt and NF-κB pathway were involved in the neuroprotective effects of dexmedetomidine against cerebral ischemia reperfusion injury.