Study Of Neck Cooling Attenuates Brain Injury And Neuron Apoptosis In Cardiopulmonary Resuscitation Rabbits Through PI3K/Akt/GSK-3β Signaling Pathways

Cardiac arrest (cardiac arrest, CA)-induced global cerebral ischemia and reperfusioninjury is a major cause of lower patient survival after cardiac arrest recovery. Because ofthe tolerance of hypoxic-ischemic of brain tissue is low, and the unique response toischemia and reperfusion, and brain protection after cardiopulmonary resuscitation hasbeen the problem of the present study. CA/CPR cause brain damage, includingformation of free radicals, calcium overload, complex mechanisms such as enzyme-linkedreaction pathway activation and signal transduction death, the final outcome is a gradualneuronal cell apoptosis and necrosis. Therapeutic hypothermia as resuscitation has beenproven to increase the recovery success rates and significantly improved survival andneurological outcomes by a number of clinical studies, so it has been recommended by anumber of professional associations. The timing of the implementation of therapeutichypothermia and duration and range of herapeutic hypothermia there is no uniformstandard. It is now generally believed that after cardiac arrest cardiopulmonaryresuscitation should be an effective therapeutic hypothermia as soon as possible. However,the existing cooling methods more or less some deficiencies (poor effect, difficult tocontrol, complex, expensive, and there are potential side effects), and molecularmechanisms of brain protection remains unclear. We invented neck cooling device basedon our previous research about anatomical characteristics of rabbit neck, and previousanimal studies confirmed: it can effectively seize the window of cardiopulmonaryresuscitation and early treatment of brain injury, to rapidly reduce temperature of brainearly. To further study the protective effect of early CPR by neck cooling，This studyintends to adopt a rabbit model of cardiac arrest and a self-developed neck cooling device,quickly reduce the temperature of the neck while recovering.Therefore, the blood into thebrain tissue is cooled before the return of spontaneous circulation (ROSC) and target of thelow recovery of brain tissue reperfusion and quickly reducionof brain temperature isachieved. Compared with conventional resuscitation hypothermia, this method is observedto improve the successful recovery rate and survival rate and reduce nervous system damage. PCR, Westen blot, TUNEL are applied to explore the mechanism ofneuroprotective effect of PI3K/Akt/GSK-3β signaling pathway in mild hypothermia, thelocation of effective targets, the relationship between the regulation ofPI3K/Akt/GSK-3βactivation pathway and mitochondrial membrane stability which isclosely related to apoptosis, and expression of caspase, cytc and other apoptosis-relatedgenes. The molecular mechanisms of brain hypothermia protective effect is furtherelucidated, and experimental and theoretical basis for clinical treatment of mildhypothermia is provided.Part Ⅰ The establishment of rabbit model of cardiac arrest and resuscitationObjective To establish an effective rabbit model of cardiopulmonary cerebral resuscitation(CPCR) induced by right ventricular fibrillation.Methods10New Zealand white rabbits were induced cadiac arrest by cardiac fibrillation,which caused by alternating current in right ventricular catheter electrode. CPR began in4min after circulation stop, and serum NSE levels and neurological deficit score (NDS)were measured at different time points after ROSC. Vital signs, restoration of spontaneouscirculation time, the success rate of recovery,48h survival rate were recorded.Results In this experiment, all of10rabbits are successfully induced to CA. After theeffective electrical stimulation, femoral artery blood pressure occurs rapidly inrabbits showed that the modeling cycle stops. All of10rabbits were expressedas ventricular fibrillation (VF) after the electrical stimulation. Attempt recovery timewas326.22±154.78s,6of10rabbits were successful resuscitated, and48h survival ratewas50%. NDS scores of10rabbits before operation were zero, and NDS scores of10rabbits six hours after ROSC were the lowest. Neurological function gradually recoveredin12hours. AfterROSC,6hNSE has been maintained at a high level after level rise. Eachtime point were significantly different compared to baselineConclusions This model operation is not complicated, causing high success rate offibrillation, ROSC recovery rate and48h survival rate.It is similar with thetypical clinical CA/CPR process, and the results are stable and reliable, providingan ideal animal model of cardiopulmonary resuscitation Part Ⅱ The protective effects of neck rapid induced hypothermia on brain ofcardiopulmonary resuscitation rabbitObjective To compare the success rate of recovery, survival and nervous system functionamong intra-arrest therapeutic hypothermia (IATH)group, normothermia theat （NT）groupand post-arrest therapeutic hypothermia (PATH)group.Methods24healthy male New Zealand white rabbits, which was induced cadiac arrest by4min fibrillation, were randomly divided into three groups with8rabbits for eachgroup.(1) Normothermia theat group (NT): general resuscitation afterfibrillation induced cadiac arrest without cooling intervention.(2) intra-arrest therapeutichypothermia group (IATH): neck rapid cooling was started at the same time with CPR,the target brain temperature was34℃, and the target brain temperature was maintained for4hours after ROSC.(3) Post-arrest therapeutic hypothermia group (PATH): neck rapidcooling was started in1hour after the start of CPR, the target brain temperature was34℃,and the target brain temperature was maintained for4hours after ROSC. Changesof recovery success rate, brain temperature and rectal temperature within four hours,hemodynamics, respiratory function and24hours NDS score were observed.Results There are seven rabbits in IATH group and four in NT group and five in PATHgroup achieving successful resuscitation;induced ventricular fibrillation in4minuteslater, there was no significant differencebetween the groups rectal temperature,and braintemperature; After four minutes of cardiopulmonary resuscitation, the brain temperature ofIATH group, NT group, PATH group were37.4±0.7℃,38.2±0.3℃,38.1±0.5℃,IATH group was statistically significant than the other two group(p<0.05); rectaltemperature of each group was no significant difference. In CPR within four minutes,diastolic of IATH group from5.2mmHg rose to42mmHg, whilethe NT group andand PATH group increased onlyfrom5.7mmHg to32mmHg,and5.4mmHg increased to31mmHg (P <0.05). Systolic blood pressure during recoverygroups did not differsignificantly. IATH group of48hour survival rate was significantly higher than the NTthe NT group, the difference was statistically significant compared to the two groups (P <0.05).24hours after resuscitation NDSscore of each group are poor, no significantdifference between the groups, but IATH group was better than the NT group.Conclusion Cerebral temperature can be reduced, and the coronary perfusionpressure during recovery, the success rate of cardiopulmonary resuscitation and thesurvival rate in rabbits can be improved when neck rapid cooling was started at the sametime with CPR.Part Ⅲ The mechanism of PI3K/AKt/GSK3β signaling pathway on thebrain protective effect of neck cooling during resuscitationObjective To research on regulative changes of PI3K/Akt/GSK3β signaling pathway inneck cooling and explore the relationship between the brain protective effect of neckcooling and the phosphorylation of PI3K/Akt and GSK3β.Methods Rabbits were randomly divided into five groups, and models of cadiac arrestinduced by4min VF were applied. According to the different time points of4,9,24hoursafter resuscitation,each group was divided into three subgroups:(1) sham group (group1): the catheter was placed in the conventional operation way, CA was not induced, therabbits were killed and specimens were taken at30minutes after operation.(2)normothermia theat group(NT group): general recovered and the rabbits were killed andspecimens were taken at4,9,24-hour after operation.(3) intra-arrest therapeutichypothermia group (PATH group): neck rapid cooling was started at the same time withCPR, the target brain temperature was34℃, and the target brain temperature wasmaintained for4hours after ROSC. rabbits were killed and specimens were taken at4,9,24-hour after operation.(4) recovery cooling+LY294002(PATH+LY294002group):LY294002was injected intraventricularly at20minutes before resuscitation. neck rapidcooling was started at the same time with CPR, the target brain temperature was34℃, andthe target brain temperature was maintained for4hours after ROSC. rabbits were killedand specimens were taken at4,9,24-hour after operation.(5) post-arrest therapeutichypothermia group (PATH group): neck rapid cooling was started in1hour after the startof CPR, the target brain temperature was34℃, and the target brain temperature was maintained for4hours after ROSC.. Rabbits were killed and specimens were taken at4,9,24-hour after operation. Animals were killed by overdose anesthetic drug. If theresuscitation fails or the rabbits dead before the corresponding point, the experimental dataof the animal is excluded, and supplement animal experiments were done to ensure thateach group of five animals. Western blotting was used to detect the expression of Aktp-Akt GSK-3β p-GSK-3β (ser9) protein, and other methods as TUNEL were used toobserve apoptosis changes of tissues in each group.Results Compared with Sham group, Akt (Thr-308) phosphorylation (P-AKT) andP-GSK-3β levels in the brain cytoplasm of normothermia theat group in4,9,24hours afterCPR was significantly reduced, and showed a gradual reduction trend (p <O.05); theP-AKT and P-GSK-3β levels in the brain cytoplasm of intra-arrest therapeutic hypothermiagroup in4,9,24hours after CPR resuscitation were significantly enhanced compared withthose of normothermia the group (p <O.05); the protein expression levels of post-arresttherapeutic hypothermia group in one hour after resuscitation were significantly enhancedcompared with those of normothermia theat group (p <O.05), but weaker than those ofintra-arrest therapeutic hypothermia group. Intraventricularly injection of LY294002withdrawed the effect of hypothermia, indicating that LY294002inhibited thephosphorylation of Akt. Apoptosis cells were significantly reduced in intra-arresttherapeutic hypothermia group and ormothermia theat group compared with post-arresttherapeutic hypothermia group and LY294002groupConclusions Neck cooling can reduce apoptosis in rabbit brain cells after recovery, and theprotective effect of cooling immediate resuscitation best. LY294002specifically block thePI3K/Akt pathway, and the protective effect of cooling on the brain can be offset,indicatinghypothermia protects the neurological function via activation of PI3K/Akt pathway. Neckcooling protects the neurological function by activating PI3K/Akt/GSK-3β, promoting theAkt activation, and increasing the expression of P-GSK3β. Specific Akt inhibitorLY294002inhibits Akt phosphorylation of brain tissue recovery and further inhibit thephosphorylation of GSK-3β, thus offset neuroprotection effect of cooling Part IV Neck cooling by PI3K/Akt/GSK3β signaling pathway inhibition ofmitochondrial apoptotic pathwayObjective Explore the excitement of recovery after opening mPTP rabbit brainmitochondria, caspase-9, caspase-3cytc activation and release characteristics; and on thisbasis to further study whether the neck cooling via inhibition of mitochondrial apoptoticpathway PI3K/Akt/GSK3β pathways, thereby reducing brain injury..Methods Randomly divided into five groups, using4min VF models.(1) sham group,routine surgical procedure catheter, do not induce CA, in the specimens were sacrificedafter30minutes.(2) at room temperature resuscitation group (normothermia theat NTgroup)(3) recovering the cooling group (intra-arrest therapeutic hypothermia IATH group)at the same time start CPR neck rapid cooling, the target brain temperature is34℃, aftermaintaining the target brain temperature4hours to ROSC (4) recovery cooling+LY294002(LY294002group)(5) recovery1hour cooling group (post-arrest therapeutichypothermia PATH group) one hour after the start of the neck rapid cooling incardiopulmonary resuscitation, the target brain temperature is34℃, more than in thesame group3.. LY294002dissolved in DMSO was diluted to10μM, in animal stereotaxicinstrument in ROSC20min ago before giving intraventricular injection, the rest of thegroup were given the solvent DMSO. Further examined by Western blot caspase-9,caspase-3expression and cytc, spectrophotometry mPTP openness rabbit brain cellsResults sham group of neurons in caspase-9, caspase-3expression level and cytcexpression is minimal. Compared with Sham group, NT significantly increased mPTPopening and caspase-9, caspase-3expression and cytc protein (P <O.05). Compared withNT group, neck cooling can reduce mPTP opening, reducing caspase-9, caspase-3and cytcprotein (P <O.05). Similarly, P13K inhibitor LY294002inhibitory effects can be offset bythe neck to cool apoptosis. Cytoplasm of nerve cells seen under the electron microscopesham group mitochondriaand endoplasmic reticulum, theorganellemophologyintact myelin dense structure. NT group most nerve nuclear chromatin condensation, was ahollow core surrounded bymyelin irregular, partial mitochondrial swelling,mitochondrial damage body crest, there are many vacuoles in the cytoplasm. IATH group and PATHultrastructural damage thanthe NT group wassignificantly reduced, while LY294002gropthere are aggravatingultrastructural damage.Conclusions CPR neuronal mitochondria in brain tissue mPTP opening and caspase-9,caspase-3activation, cytc release; neck cooling signaling pathway by inhibiting theactivation PI3K/Akt/GSK3β mPTP opening and mitochondrial caspase-9, caspase-3isactivation, inhibition of mitochondrial apoptosis pathway, reducing ischemia andreperfusion cause death of neurons, which may be one of the mechanisms of hypothermiato reduce brain damage following cardiopulmonary resuscitation.