Mechanism Underlying Dexmedetomidine Function In Cerebral Ischemia Reperfusion Injury

BackgroundIschemic stroke is the most common type of stroke,which occurs in the neck or cerebral vascular obstruction and seriously threatens human health,especially in preoperative acute ischemic stroke(PAIS).Cognitive dysfunction caused by ischemia reperfusion injury has become a thorny problem for clinicians to solve,which seriously affects the prognosis of patients,especially aged patients,and also imposes a serious economic burden on patients’ families and even the whole society.Medical researchers have paid more and more attention to the patients who has postoperative cerebral ischemic dysfunction without risk of cerebral ischemic stroke during preoperative.The pathogenesis of ischemic stroke is very complex,involving a variety of pathophysiological mechanisms and a process of accumulation and offset of multiple factors.Oxidative stress is now considered to be associated with brain injury after ischemic stroke.In recent years,both clinical and basic studies have pointed out that endoplasmic reticulum stress may be involved in cerebral ischemia reperfusion injury,and its specific mechanism is still unclear.Dexmedetomidine,the alpha 2-adrenoceptor agonist,is now widely used in clinical anesthesia and ICU.Studies have shown that adrenergic 2 receptor(ADRA2)is involved in the process of oxidative stress in cells.It also has been found that dexmedetomidine can highly selectively activate ADRA2,reduce the release of inflammatory mediators and neuroendocrine hormones,inhibit the oxidative stress of neurons and reduce apoptosis in global cerebral ischemia-reperfusion injury in rats.Dexmedetomidine can also reduce morphological and functional changes caused by central nervous system ischemia and traumatic injury.Our previous experimental data also showed that dexmedetomidine preconditioning can increase the tolerance of PC 12 cells to hypoxia,however,the specific molecular mechanism remains to be explored.ObjectiveIn the current study,we aimed to investigate whether dexmedetomidine preconditioning has protective effects on neurons after ischemia and improves cognitive function,and explore the role of endoplasmic reticulum stress in this process.MethodsPart 1 In vivo study:SPF-grade health male Sprague-Dawley(SD)rats were recruited in this study.Global cerebral ischemia was induced by 20min occlusion of bilateral common carotid arteries combined with hypotension.The rats were randomly divided into 3 groups:Sham group(control group),I/R group(global cerebral ischemia reperfusion group)and DEX+I/R group(dexmedetomidine preconditioning group).Intraoperative ECG and EEG monitoring and blood gas analysis were performed.Neurological evaluation was performed using the Neurologic Deficit Scores(NDS)system at 6h,24h and 72h;HE staining and TUNEL kit were used to observe the pathology and neuronal apoptosis of hippocampal area;Western blot was used to detect the expression of Bax,Bcl-2,Cleaved caspase-3,MANF,STIM1,GRP78,CHOP and ATF-6 in the hippocampus;MANF was assayed by immunohistochemical and GRP78 and CHOP and ATF-6 was assayed by immunofluorescence.Part 2 In vivo study:SPF-grade aged health male SD rats were recruited in this study.Global cerebral ischemia was induced by 20min occlusion of bilateral common carotid arteries combined with hypotension.The rats were randomly divided into 3 groups:Sham group(control group),I/R group(global cerebral ischemia reperfusion group)and DEX+I/R group(dexmedetomidine preconditioning group).Intraoperative ECG and EEG monitoring and blood gas analysis were performed.The cognitive function of rats was determined by a step-down type passive avoidance test and Morris Water Maze test on the third day(72h)after reperfusion;HE staining and TUNEL kit were used to observe the pathology and neuronal apoptosis of hippocampal CA1 region;the levels of HMGB1,TNF-α and IL-1β in the hippocampus,serum S100β and NSE were detected by ELISA kit;TUNEL kit was used to detect choroid plexus microstructure changes,and Evans Blue assay was used to detect blood brain barrier permeability.Part 3 In vitro study:Norvegicus adrenal pheochromocytoma PC12 cells was routinely subculture and randomly divided into 5 groups:1)Normal group(NC group):PC 12 cells was incubated in prepared normal medium for 24h after synchronization;2)OGD/R group(Control group):after synchronization,PC 12 cells was incubated in glucose-free medium in an oxygen-free incubator,according to the method to establish the OGD/R model;3)DEX 4μg/mL+OGD/R group(DEX+4μg/mL):after synchronization,PC 12 cells was incubated in glucose-free medium in an oxygen-free incubator,and dexmedetomidine was added to make the final concentration 4μg/mL,then the OGD/R model was established;4)DEX 1μg/mL+OGD/R group(DEX+1μg/mL):after synchronization,PC 12 cells was incubated in glucose-free medium in an oxygen-free incubator,and dexmedetomidine was added to make the final concentration 1μg/mL,then the OGD/R model was established;5)DEX 0.25μg/mL+OGD/R group(DEX+0.25μg/mL):after synchronization,PC 12 cells was incubated in glucose-free medium in an oxygen-free incubator,and dexmedetomidine was added to make the final concentration 0.25μg/mL,then the OGD/R model was established.NC group cells was cultured under an atmosphere of 5%CO2 and 95%air at 37℃;Cells in the OGD/R group was cultured under an atmosphere of 1%O2,5%CO2,94%N2 for 24h,and then re-oxygenation 12h under an atmosphere of 5%C02 and 95%air at 37℃.Cell viability was detected by MTT assay kit;assay kits was used to detect the levels of LDH and MDA;DHE fluorescence probe was used to detect ROS production;Hoechst 33342 staining and flow cytometry were used to detect cell apoptosis;flur-4 Ca2+ probe was used to detect the concentration of Ca2+ in the cytoplasm;mRFP-GFP-LC3 adenovirus was used to detect the formation and degradation of autophagosomes detecting autophagy body formation and degradation;MANF,STIM1,GRP78,CHOP,ATF-6,Bax,Bcl-2 and Cleaved caspase-3 were detected by Western blot.ResultsPart 1 In vivo study:1.Compared with Sham group,the NDS of I/R group and DEX+I/R group were significantly increased at 6h,24h and 72h after global cerebral ischemia reperfusion;The histological damage of hippocampal and TUNEL positive cells were significantly increased;Bax/Bcl-2 ratio and the expression of Cleaved caspase-3 expression were significantly enhanced in I/R group and DEX+I/R group;MANF,STIM1,GRP78,CHOP and ATF-6 expression were significantly increased in I/R group and DEX+I/R group.2.Compared with I/R group,DEX+I/R group significantly reduced the NDS at 24h and 72h after global cerebral ischemia reperfusion.The histological damage of hippocampal and TUNEL positive cells were significantly reduced.Bax/Bcl-2 ratio and the expression of Cleaved caspase-3 were significantly reduced,and STIM1,GRP78,CHOP and ATF-6 expression were also significantly decreased,while MANF expression was significantly increased.Part 2 In vivo study:1.Compared with Sham group,the response time,response error times and the latency of I/R group were significantly increased,and the step-down latency was significantly shortened.The response time and response error times of DEX+I/R group were significantly increased,and the latency was significantly shortened;the escape latency of the water maze test was significantly prolonged from day 1 to day 5 in I/R group,and from day 1 to day 3 in DEX+I/R group,and the number of platform crossings in quadrant 1 was significantly shortened in both I/R group and DEX+I/R group;the damaged cone cells and TUNEL positive cells in hippocampal CA1 region of the I/R group were significantly increased,and also increased in DEX+I/R group;HMGB1,TNF-α,IL-1β and serum levels of S100β and NSE were significantly increased in I/R group,and HMGB1,TNF-α and IL-1β were significantly increased in DEX+I/R group,while serum levels of S100β and NSE were increased without significant statistical difference;the content of EB in brain tissue was significantly increased 72h after reperfusion in I/R group and DEX+I/R group;a large number of vacuoles appeared in the epithelial cells of the choroid plexus in I/R group,with irregular cyton and obscure border,scattered vacuoles appeared in the epithelial cells of the choroid plexus in DEX+I/R group,with slight changes in cyton and basically evident border.2.Compared with I/R group,the response time,response error times and the escape error times in DEX+I/R group were significantly reduced,and the latency was significantly increased;the escape latency of the water maze test was significantly prolonged from day 1 to day 5,and the number of platform crossings in quadrant 1 was significantly increased;the damaged cone cells and TUNEL positive cells in hippocampal CA1 region were significantly decreased;HMGB1,TNF-α,IL-1β and serum levels of S100βand NSE were significantly decreased;the content of EB in brain tissue was significantly decreased 72h after reperfusion in I/R group and DEX+I/R group;the damaged epithelial cells of the choroid plexus was also significantly decreased.Part 3 In vitro study:1.Compared with NC group,each group has lower cell viability of PC 12 cells OGD 24h,higher release of LDH,MDA and ROS,and higher ration of apoptotic cells;the expression of MANF,STIM1,CHOP,GRP78 and UPR related gene ATF-6 were significantly increased in Control group and DEX+1μg/mL+OGD/R group;the concentration of Ca2+ in the cytoplasm and the number of autophagosomes not degraded by autolysosome were also significantly increased.2.Compared with Control group,1 and 0.25μg/mL dexmedetomidine preconditioning can significantly increase the cell viability of PC12 cells OGD 24h,reduce the release of LDH,MDA and ROS,and the cell apoptosis;1μg/mL dexmedetomidine preconditioning significantly reduces the expression of Bax/Bcl-2 and Cleaved caspase-3,and also significantly inhibited the expression of STIM1,CHOP,GRP78 and UPR related gene ATF-6,while enhanced the expression of MANF;the concentration of Ca2+ in the cytoplasm and the number of autophagosomes not degraded by autolysosome were also significantly decreased;4μg/mL dexmedetomidine preconditioning significantly increased the expression of Bax/Bcl-2 and Cleaved caspase-3.ConclusionPart 1 In vivo study:1.Endoplasmic reticulum stress may be an important cause of hippocampal neuron apoptosis after global cerebral ischemia reperfusion in rats.2.Dexmedetomidine preconditioning can effectively inhibit endoplasmic reticulum stress and reduce the apoptosis of neurons after global cerebral ischemia reperfusion in rats.Part 2 In vivo study:1.The decrease and apoptosis of pyramidal cells in the hippocampal CA1 region may be caused by the destruction of BBB due to the change of the microstructure of choroid plexus in the lateral ventricle after global cerebral ischemia reperfusion in aged rats.2.The changes of cognitive function may be caused by apoptosis of neurons in the hippocampus CA1 region and infiltration of inflammatory cells after destruction of BBB after global cerebral ischemia reperfusion in aged rats.3.Dexmedetomidine preconditioning can significantly reduce the release of inflammatory cytokines and the change of the microstructure of choroid plexus in the lateral ventricle,thereby reducing the apoptosis of pyramidal cells in the hippocampus CA1 region and improving the cognitive dysfunction after global cerebral ischemia reperfusion in aged rats.Part 3 In vitro study:1.Oxidative stress,ER stress,and the down-regulation of autophagy level may be an important cause of cell damage and apoptosis after OGD/R in PC 12 cells.2.Dexmedetomidine preconditioning can reduce oxidative stress,inhibit the expression of UPR related gene ATF-6 and ERS related protein,decrease the overload concentration of Ca2+ in the cytoplasm to stabilize mitochondrial function,as well as regulate the level of autophagy,so as to reduce the cell damage and apoptosis of PC12 cells after OGD/R.