Ischemic Postconditioning And Hypothermia Attenuates The Whole Ischemic Brain Injury Possibly Through JAK2-STAT3and Wnt5a Signaling Pathways

Congenital heart disease has become the most common birth defectdisease in China. Surgery for congenital heart disease accounts for55%-60%of all heart surgeries in the Chinese mainland. Although infantcardiopulmonary bypass technology has gradually been improving,cardiopulmonary bypass is a controlled process involving ischemia andhypoxia, especially in aortic arch surgery in some infants. Such surgerywill inevitably induce deep hypothermia, circulatory arrest and globalcerebral ischemia. Therefore, the brains of these patients will be subjectto ischemia/reperfusion injury and subsequent potential damage.Ischemic brain damage of the whole brain mainly originates from adisorder in oxygen and energy metabolism caused by ischemia and hypoxia,which ultimately cause brain tissue damage. Under the state of globalcerebral ischemia, oxygen and energy supply to brain cells is reduced,while anaerobic metabolism is increased, accompanied by high-energyphosphate compound reserves being exhausted. These changes induce aseries of secondary pathological processes, such as activation ofexcitatory amino acid (EAA) cell toxicity; overload of intracellularcalcium (Ca²⁺) in nerve cells, excessive formation of oxygen free radicalswithin specific neurons, free radical chain reactions; production ofinflammatory cytokines, accumulation of free fatty acids and disorder ofthe internal environment, among others. If no intervention is undertaken,these processes eventually lead to impaired function of brain cells, whichthen disintegrate and die. In recent years, a large number of studies haveshown that organ blood flow recovery after ischemia can lead to furthertissue damage and dysfunction in some cases. This further damage occursafter blood flow perfusion is restored and is known asischemia/reperfusion injury. The mechanism of ischemia/reperfusioninjury is complex and is related to the triggering and release of endogenous substances, overload of intracellular and mitochondrial Ca2,an increase in oxygen free radicals in reperfusion, activation ofneutrophils, and many other factors. Among these factors, aninter-constitutional negative feedback network is formed, which leads tofurther damage in ischemic organs and tissues after blood flow isrestored.Numerous studies have attempted to find effective interventions ordrug treatments to alleviate organ and tissue ischemia/reperfusion injury.While some drugs or treatment measures are effective in experimentalanimals, few of them have been successfully applied in clinical use.In2003, Zhao et al. found that repeated transient myocardial ischemiareperfusion before myocardial ischemia reperfusion can protect themyocardium against subsequent reperfusion injury, which led to theconcept of ischemic post-conditioning. Ischemic post-conditioninginitiates its protection by stimulating inherent protection mechanisms.Studies have shown that the mechanism of ischemic post-conditioning mayinclude the triggering and release of protective endogenous substances,a reduction in the release of oxygen free radicals, as well as partialactivation of intracellular signaling pathways. Since post-conditioningcan be implemented after ischemia, it is highly controllable with a simpleclinical operation; therefore, its potential clinical application may bedirect and effective.Many experiments have confirmed the protective effect of hypothermiaon global cerebral ischemia. The cerebral protective effect ofhypothermia is not confined to the reduction of energy metabolism andoxygen consumption, but also includes inhibition of the release ofexcitatory amino acids, inhibition of excessive formation of oxygen freeradicals and inhibition of intracellular Ca²⁺overload, whichsignificantly increases the tolerance of brain tissue to ischemia. Clinical cardiac surgery during cardiopulmonary bypass has beensuccessfully applied with various degrees of hypothermia forintra-operative brain protection, especially deep hypothermiccirculatory arrest (DHCA), which is used in aortic arch surgery in someinfants.Janus kinase (JAK) signal transducer and activator of transcription(STAT) is a newly discovered intracellular signal transduction pathway.The JAK protein family has four members: JAK1, JAK2, JAK3and TYK2, whilethe STAT protein family includes seven cloned STAT members: STAT1, STAT2,STAT3, STAT4, STAT5a, STAT5b, and STAT6. Studies on the JAK-STAT pathwayhave mostly focused on tumor and liver diseases, the generation of nervecells, hematopoietic system diseases and heart disease. The JAK-STATsignal pathway is involved in ischemic brain injury. Studies have shownthat hypothermia can reduce the expression of JAK1and STAT1, and inhibitapoptosis induction of the JAK1-STAT1pathway after ischemia and hypoxia.This can further affect the transcription of STAT1downstream target genes,inhibiting the expression of the pro-apoptotic genes caspase-3and BAX,and increasing the expression of the anti-apoptotic factors Bc1-2andBcl-x1. According to the above mechanisms, hypothermia treatment willreduce brain injury caused by ischemia-reperfusion. Research has shownthat the JAK2-STAT3pathway is involved in the mediation of ischemicmyocardial protection.The Wnt gene encoding the Wnt proteins family is a secreting type ofglycoprotein, which combines with receptors located on the cell membranethrough a paracrine or autocrine mechanism. Wnt proteins activateintracellular signaling molecules at all levels, and regulate theexpression of target genes. The Wnt/Ca²⁺pathway is mainly activated bywnt5a and wntll, possibly through G-protein activation of phospholipaseC (PLC) and protein kinase C (PKC), accompanied by increased intracellular Ca²⁺concentrations and activated Ca²⁺sensitive signal components. Inrecent years, the role of this pathway in inflammation and apoptosis hascaused increasing concern.Taking into account the features of hypothermia and global cerebralischemia in infant aortic arch surgery and the findings from theabove-mentioned research, we designed this study to investigate whetherthe JAK2-STAT3pathway and the non-classical Wnt5a signaling pathway areinvolved in molecular mechanisms mediating brain protection ofhypothermia and ischemic post-conditioning, to further guide theirclinical application. This study was divided into the following sections:Part I: Investigation of the best ischemic treatment option for braininjury caused by global cerebral ischemia.Objective: To investigate the best ischemic treatment options forbrain injury caused by global cerebral ischemia.Methods: The four-vessel occlusion method was applied to adult maleSD rats by clamping bilateral common carotid arteries for20minutes toestablish a global cerebral ischemia model. Before releasing bilateralcommon carotid arteries for reperfusion, a variety of transient ischemicand post-conditioning procedures were applied, which were implemented byseven experimental groups: the ischemia-reperfusion group the15seconds×1time (15×1) group, the15seconds×3times (15×3) group, the30seconds×1time (30×1) group, the30seconds×3times (30×3) group, the60seconds×1time (60×1) group, and the control group.There were six SD rats in each group. After48hours, the neurologicalinjury score was determined and the rats were sacrificed. HE staining andTUNEL staining were performed in forebrain slices. The levels of bcl-2and pro-caspase-3expression in brain tissue were determined usingwestern blotting to observe the difference in brain tissue damage bydifferent ischemic post-conditioning conditions after global cerebral ischemic reperfusion.Results: Clear neuronal damage was observed in the frontal andparietal cortex and subcortical structures in rats with ischemiareperfusion, and the degree of injury was the most serious. Compared withthe ischemia-reperfusion group, five types of ischemic post-conditioningconditions reduced brain damage induced by cerebral ischemia andreperfusion, but to different extents; the15×3treatment group showedthe strongest effect of ischemic post-conditioning on reduced braindamage.Conclusion: Transient ischemic post-conditioning has aneuroprotective effect on rat brain cerebral ischemia, in which ischemicpost-conditioning of15seconds×3times may be the most effective.Part II: Ischemic post-conditioning and hypothermic reduction ofbrain injury caused by global cerebral ischemia reperfusion through theJAK2-STAT3and non-classical Wnt5a signaling pathways.Objectives:(1) To study expression of the JAK2-STAT3andnon-classical Wnt5a signaling pathways in the rat brain after cerebralischemia at different times.(2) To study the role of the JAK2-STAT3and non-classical Wnt5asignaling pathways in reducing brain injury caused by global cerebralischemia-reperfusion using ischemic post-conditioning and hypothermicconditions.Methods:(1) Adult male SD rats were used for the study to establisha global cerebral ischemia model at room temperature. Six rats wereassigned to each group and were subjected to cerebral ischemia andreperfusion after2,6,12,24,48and72hours. A control group was alsoincluded. The neurological injury score was determined, andimmunohistochemistry and western blotting were used to detect JAK2,p-JAK2, STAT3, p-STAT3, Wnt5a, and β-catenin expression. (2) Six SD rats in each group were used. Seven groups were used inthe experiment, which included the ischemia and reperfusion control group,the ischemic post-conditioning (15×3) group, the hypothermia group,the ischemic post-conditioning (15×3)+hypothermia group, the ischemicpost-conditioning (15×3)+hypothermia+AG490group, the ischemicpost-conditioning (15×3)+hypothermia+specific Ca²⁺inhibitor group,and the ischemic post-conditioning (15×3)+hypothermia+AG490+specific Ca²⁺inhibitor group. The above treatments were performed andperfusion was restored. Twenty-four hours after cerebral ischemia, theneurological injury score was determined and western blotting was usedto detect expression levels of JAK2, p-JAK2, STAT3, p-STAT3, and Wnt5ain brain tissue.Results: After cerebral ischemia and reperfusion in the rat, JAK2,p-JAK2, STAT3, p-STAT3, and Wnt5a expression in the cerebral cortexreached peak levels by24hours. Ischemic brain tissue morphology showednerve injury and necrosis. However, post-conditioning and hypothermicmeasures reduced this brain injury after ischemia and reperfusion. Thesetwo protective measures could be superimposed on each other. However,these two protective measures could also be inhibited by AG490or specificCa²⁺ inhibitors or a combination.Conclusion: the JAK2-STAT3and non-classical Wnt5a signalingpathways are activated early in cerebral ischemia and reperfusion, whichreach a peak in24hours. Ischemic post-conditioning and hypothermia canmediate the JAK2-STAT3and non-classical Wnt5a signaling pathways toreduce brain damage after global cerebral ischemia-reperfusion.Part III: Inflammatory response inhibition of the JAK2-STAT3andnon-classical Wnt5a signaling pathways by ischemic post-conditioning andhypothermia may be one of the mechanisms that reduce brain injury in globalcerebral ischemia and reperfusion in rats. Objectives:(1) To investigate global cerebral inflammation andinflammatory mediator expression levels at different times after braininjury due to cerebral ischemia and reperfusion.(2) To further investigate methods to inhibit the inflammatoryresponse through the JAK-STAT and non-classical Wnt5a signaling pathwaysto reduce brain injury in global cerebral ischemia-reperfusion.Methods:(1) Adult male SD rats were used in the study to establisha global cerebral ischemia model at room temperature. Six SD rats wereassigned to each group subjected to cerebral ischemia and reperfusionafter2,6,12,24,4and72hours. A control group was included. Thecontent of myeloperoxidase enzyme (MPO) was determined, andimmunohistochemistry and western blotting were used to detect expressionlevels of proinflammatory factors in ischemic brain tissue, includingTLR2, TLR4, nf-kb, p65and COX-2. The ELISA method was used to determineTNF-α levels in serum.(2) Six SD rats were assigned to each group in a total of eight groups,which included the ischemia and reperfusion group, the control group, theischemic post-conditioning (15×3) group, the hypothermia group, theischemic post-conditioning (15×3)+hypothermia group, the ischemicpost-conditioning (15×3)+hypothermia+AG490group, the ischemicpost-conditioning (15×3)+hypothermia+specific Ca²⁺inhibitor group,and the ischemic post-conditioning (15×3)+hypothermia+AG490+specific Ca²⁺inhibitor group. The above treatments were performedand perfusion was restored. Twenty-four hours after cerebral ischemia,we determined the expression of MPO and TNF-a. We used western blottingto detect the expression levels of proinflammatory factors in ischemicbrain tissue including COX-2, nf-kb and p65.Results: The content of the inflammatory marker MPO reached its peakby24hours after cerebral ischemia. MPO levels were maintained for48 hours, and gradually declined by72hours, but were still higher thannormal levels. Expression levels of TLR2, TLR4, COX-2and TNF-α reacheda peak by24hours after ischemia. However, post-conditioning andhypothermic measures reduced brain injury due to ischemia reperfusion.These two protective measures could be superimposed on each other. However,these two protective measures could also be inhibited by AG490or specificCa²⁺inhibitors or a combination.Conclusion: Rat brain injury by cerebral ischemia reperfusion caninduce inflammation and proinflammatory cytokine expression, which reacha peak by24hours after injury. In addition, ischemic post-conditioningand hypothermia inhibit the expression of inflammatory mediators andinflammatory responses through the JAK2-STAT3and non-classical Wnt5asignaling pathways, which may be one of the mechanisms that reduce braincerebral ischemic reperfusion injury.Part IV: Inhibition of neuronal apoptosis through the JAK2-STAT3andnon-classical Wnt5a signaling pathways may be one of the mechanisms whereischemic post-conditioning and hypothermia reduce brain injury caused byglobal cerebral ischemia-reperfusion.Objectives:(1) To investigate neuronal apoptosis levels at differenttime points after brain injury caused by global cerebralischemia-reperfusion.(2) To investigate the inhibition of neuronal apoptosis through theJAK2-STAT3and non-classical Wnt5a signaling pathways to reduce braininjury caused by global cerebral ischemia-reperfusion.Methods:(1) Adult male SD rats were used in the study to establisha global cerebral ischemia model at room temperature. Six SD rats wereassigned to each group subjected to cerebral ischemia and reperfusionafter2,6,12,24,48and72hours. A control group was also included.We used the Nissl staining method and western blotting to determine levels of bcl-2/BAX and caspase-3.(2) Six SD rats were assigned to each group in a total of eight groups,which included the ischemia and reperfusion control group, the controlgroup, the ischemic post-conditioning (15×3) group, the hypothermiagroup, the ischemic post-conditioning (15×3)+hypothermia group, theischemic post-conditioning (15×3)+hypothermia+AG490group, theischemic post-conditioning (15×3)+hypothermia+specific Ca²⁺inhibitor group, and the ischemic post-conditioning (15×3)+hypothermia+AG490+specific Ca²⁺inhibitor group. The above treatmentswere performed and perfusion was restored. Twenty-four hours aftercerebral ischemia, we performed HE and Nissl staining to detect the levelof neuronal apoptosis. Western blotting was used to examine the levelsof bcl-2/BAX and caspase-3.Results: Brain injury caused by cerebral ischemia and reperfusioninduced neuronal apoptosis. The level of neuronal apoptosis reached a peakby24hours, and was maintained from48to72hours. Neuronal apoptosisthen started to decrease after this time, but it was still higher thannormal levels. Ischemic post-conditioning and hypothermia decreased theexpression levels of BAX and caspase-3in brain injury caused by braincerebral ischemia-reperfusion. The expression of bcl-2was increased.These two protective effects could be superimposed on each other. However,these two protective measures could also be inhibited by AG490or specificCa²⁺inhibitors or a combination.Conclusion: Cerebral ischemia and reperfusion in the rat inducesneuronal apoptosis and activates BAX and caspase-3expression. Ischemicpost-conditioning and hypothermia can inhibit BAX and caspase-3activation and apoptosis of brain cells through the JAK2-STAT3andnon-classical Wnt5a signaling pathways, which may be one of the mechanismsto reduce brain injury in the rat after cerebral ischemic reperfusion.