| Objective Over the years,with the continuous improvement of the clinical first aid level,the survival rate of critical patients with suffocation,shock,CPR,myocardial infarction has been gradually improved,and these diseases will lead to the ischemia injury and reperfusion injury after ischemia,affecting the recovery of the brain.How to reduce the incidence of neuron death and mitochondrial injury after brain resuscitation,the influence of subhypothermia on cardiopulmonary nerve apoptosis and cardiopulmonary resuscitation,CPR by cold-induced RNA-binding protein（cold-inducible RNA-binding protein,CIRP）,hopes to provide more reliable experimental basis for future clinical applications.To explore the effects of mild hypothermia on neuronal apoptosis and mitochondria in cardiopulmonary resuscitation（CPR）rats through cold-inducible RNA-binding protein（CIRP）-mediated anti-apoptotic signal transduction pathway.Methods（1）Grouping method.First,45 rats were divided into sham operation group,normal temperature group and mild hypothermia group（15 rats in each group）.The CPR model is simulated by simulating cardiac arrest after asphyxia.In the sham operation group,the bilateral carotid arteries were separated and fully exposed,and craniotomy was adopted to expose the second avascular area of the basilar artery.After modeling,the esophageal temperature and rectal temperature of the rats in the normal temperature group were maintained at（37.0±0.5）℃,the operation group and the normal temperature group were both placed at room temperature,and the rats in the mild hypothermia group were maintained at（37.0±0.5）℃ after modeling.33.0±1.0)℃.After6 hours,tail vein blood samples and brain tissues were collected.Detect and compare the levels of inflammation,brain tissue damage,neuronal apoptosis,mitochondrial morphology,mitochondrial damage marker protein and CIRP expression in each group.Second,in addition,subdivide the mild hypothermia group into 1h to 24 h group,4h to24 h group and 8h to 24 h group,which will affect the results of different starting times of lower temperature.Third,μ-Calpain affects the survival of neurons.Choose male Long Evans adult rats,weighing between 370 g and 420 g.（2）Test results of the lost cell analyzer.First,place the fixed or fresh tissue on the 120 eye stainless steel mesh,use the ophthalmic scissors to cut the tissue,and then use the ophthalmic tweezers to gently knead the tissue block,rub simultaneously with normal saline cleaning,until the tissue is rubbed.Use a 300 mesh net to filter the suspension in the flat dish,remove the cell mass,and collect the cell suspension.Secondly,cell DNA staining,the internal parameter standard is: select 1×1050.1ML single cell suspension,add 10% concentration of samin-red dyeing,and sample synchronous dyeing,add 1ml of propymidine iodide,save for half an hour in the refrigerator with a temperature of 4℃,and then use 500 eyes copper mesh filtration.Then,the immunofluorescence marks the cells.1×1060.1ML monocell suspension was selected,and 0.1ML rat anti-human monoclonal antibody was added,incubated at room temperature for 30 minutes,then 10 MLPPS was added for washing,the upper cleaning fluid was abandoned and tested after 500 eyes of copper mesh filtration.Finally,through immunofluorescence data analysis,the EPi CS-XL2 type flow cytometry produced by American companies was introduced.Results First,（1）the results of the effect of mild hypothermia treatment on the survival rate of rats in different groups.3 in the sham operation group survived,with a survival rate of 20.0%;5 in the normal temperature group survived,accounting for33.3%;in the mild hypothermia group 8 survived,accounting for 53.3%.It can be found that the overall survival rate of rats in the mild hypothermia group is significantly higher than that of the other two groups.The survival rate at the initial time of 1h was 80.0%;the survival rate at the initial time of 4h was 60.0%,and the survival rate at the initial time of 8h was 20.0%.Among them,the survival rate at the starting time of 8h was lower than that of the normal temperature group.There are obvious differences in the neurological good rate of the three groups of rats.There was no significant difference in survival rate among the rats whose starting time was 1h,4h and 8h.（2）Three groups of imaging features.In the sham operation group,neurons were distributed evenly and the structure of the nucleus was clear.The staining of nuclei in the normal temperature group became darker and interstitial edema appeared.Neuronal damage and edema in the mild hypothermia group were lighter than those in the normal temperature group.（3）There were significant differences in the levels of inflammatory factors,mitochondrial damage and apoptosis marker protein expression in brain tissue,and CIRP m RNA and protein expression in the three groups of rats（P<0.05）.The degree of brain damage,apoptosis index,IL-1β,IL-6,mitochondrial damage,Cyt C,Caspase-9,and Caspase-3protein expression in the normal temperature group were significantly higher than those in the sham operation group（P<0.05）.In the mild hypothermia group,the degree of brain damage,apoptosis index（16.17±1.82 vs 38.29±3.79）,IL-1β [（39.71±5.09）pg/ml vs（74.24±8.25）pg/ml],IL-6 [（52.47）±7.30)pg/ml vs（88.36±10.12）pg/ml],Cyt C（0.32±0.03 vs 0.87±0.08）,Caspase-9（0.51±0.05 vs 0.92±0.09）,Caspase-3（0.46±0.05 vs1.05±0.10）was significantly lower than the normal temperature group（P<0.05）,and the expression levels of CIRP m RNA（1.58±0.14 vs 0.30±0.04）and CIRP protein（0.87±0.08 vs 0.12±0.01）were significantly higher than the normal temperature group（P<0.05）.（4）The SOD activity value of the sham operation group was the highest,which was（67.21±10.06）U/mg prot,followed by the normal temperature group,which was（55.34±1.06）U/mg prot,and the mild hypothermia group was（50.72±1.66）.)U/mg prot;the normal temperature group has the highest tissue water content,which is（86.31±2.15）%,followed by the mild hypothermia group（81.42±2.09）%,and the sham operation group is（78.11±1.02）%;the MDA content is the highest It is the sham operation group,（4.18±0.11）U/mg prot;followed by the normal temperature group,（0.89±0.31）U/mg prot,and finally the mild hypothermia group,（0.43±0.05）U/mg prot.The differences in SOD value,tissue water content and MDA content of the three groups of patients were significant and statistically significant（P<0.05）.Second,capn1 sh RNA played a certain role in preventing calpain cleavage,allowing neurons to be effectively protected.In the CA1 area after capn1 treatment,more neurons are in normal conditions,while CA1 after capn2 treatment The number of normal neurons in the area is significantly less.Conclusion Mild hypothermia may relieve mitochondrial damage by regulating the expression of CIRP,inhibit brain neuron apoptosis,and protect brain tissue damage after CPR. |
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