Effects Of Mild Hypothermia Combined With Xenon On Pyroptosis Of HIBD Newborn Rat Neurons

Objective:Neonatal hypoxic-ischemic brain damage（HIBD）is associated with activation of inflammasome and cell pyroptosis in nerve cells.Mild hypothermia combined with xenon therapy may have a better neuroprotective effect.In this study,the HIBD newborn rat model was established,and the expression of NLRP-3 inflammasome and cell pyroptosis related indexes were detected.To explore the correlation between cell pyroptosis and HIBD.To explore whether mild hypothermia combined with xenon can reduce the degree of brain injury and improve the prognosis of nervous system by regulating the pyroptosis pathway of nerve cells.Methods:The 7-day-old SD newborn rats were randomly divided into 5 groups:Sham operation group（Sham group,n=27）、HIBD model group（HIBD group,n=27）、Xenon intervention group（Xe group,n=27）、mild hypothermia intervention group（HT group,n=27）、Mild hypothermia combined xenon intervention group（HT+Xe group,n=27）.The HIBD newborn rat model was established according to the modified Rice method.Sham group:after anesthesia,the left common carotid artery of newborn rats were separated without other intervention;HIBD group:after anesthesia,the left common carotid artery of newborn rats were separated and ligated,and were treated in anoxic box containing 8%nitrogen and oxygen mixture（8%O₂,92%N₂）for 1.5 h.Xe group:After preparing HIBD model,the HIBD model was treated in an airtight box containing xenon（50%Xe,30%O₂,20%N₂）for 3h;HT group:after the preparation of HIBD model,the rats were treated with mild hypothermia pad to maintain anal temperature of 32℃for 3h;HT+Xe group:after the preparation of HIBD model,the rats were treated with mild hypothermia pad to maintain anal temperature of 32℃for 3h in an airtight box containing xenon（50%Xe,30%O₂,20%N₂）.Monitoring indexes:72h after surgery,the left brain tissues of newborn rats in each group were used to perform HE staining,TTC staining and electron microscopy to observe the pathological changes of brain tissue cells;Immunohistochemistry（IHC）staining was used to detect the expression of NLRP-3、Caspase-1 mean optical density in the left hippocampus.Pyroptosis associated protein and m RNA（NLRP-3、ASC、GSDMD、Caspase-1、IL-1βand IL-18）in the left hippocampus were detect by Western blot and RT-PCR;The levels of inflammatory cytokines（IL-1β、IL-18）in brain tissue were detected by ELISA;3 days after surgery,the rats underwent neurobehavioral evaluation and righting reflex experiment;28 days after surgery,the capture rejection experiment and 45°slope experiment were performed.Results:1.HE staining:Compared with Sham group,the number of cell layers in the left hippocampal area of rats in HIBD group was significantly reduced,with irregular cell arrangement,disordered structure,small nucleus,nucleolar migration,and partial nuclear pyknosis;Compared with HIBD group,the number of left hippocampal cell layers in HT group and Xe group was significantly increased,with more orderly arrangement,improved cell morphology and clear nucleoli;Compared with HIBD group,the number of left hippocampal cell layers in HT+Xe group was significantly increased and arranged regularly,with roughly normal cell morphology and clear nucleoli without obvious migration;Compared with HT group and Xe group,the left hippocampal cell structure in HT+Xe group was normal,with more cell layers,more regular cell arrangement,round and clear nuclei.2.TTC staining:Compared with Sham group,HIBD group had a higher proportion of infarction area in the left cerebral hemisphere and the difference was significant（P<0.05）;Compared with HIBD group,the proportion of infarction area in left cerebral hemisphere of newborn rats in HT group and Xe group was relatively reduced（P<0.05）;The proportion of infarction area in left cerebral hemisphere of newborn rats in HT+Xe group was decreased while compared with HIBD group（P<0.05）;The proportion of infarction area in left cerebral hemisphere of newborn rats in HT+Xe group decreased while compared with HT group and Xe group（P<0.05）.3.Immunohistochemical staining:Compared with Sham group,the average optical density of NLRP-3、Caspase-1 in left hippocampus of HIBD group was higher（P<0.05）;The average optical density of NLRP-3、Caspase-1 in HT group and Xe group was slightly lower while compared with HIBD group（P>0.05）;Compared with HIBD group,the average optical density of NLRP-3、Caspase-1 in HT+Xe group was significantly lower（P<0.05）;The average optical density of NLRP-3、Caspase-1 decreased significantly in HT+Xe group while compared with HT group and Xe group（P<0.05）.4.The results of electron microscopy showed that the overall tissue structure of Sham group was basically normal,with round and clear nucleus,normal organelles and clear cell membrane.In HIBD group,the overall structure of the tissue was abnormal and obvious vesicular corpuscles were observed at the rupture and collapse of cell membrane,which were pyrosomes.The Xe group had abnormal overall tissue structure,nuclear chromatin fracture,nuclear membrane structure destruction and mitochondrial swelling.The HT group had abnormal overall structure,chromatin concentration and side convergence,mitochondrial swelling and crest fracture.The overall structure of the HT+Xe group was slightly abnormal,with round nuclei,nucleolar chromatin edge clustering,roughly normal organelles and clear cell membrane.5.Western blot and RT-PCR showed that the protein and m RNA expression levels of NLRP-3、ASC、GSDMD、Caspase-1、IL-1βand IL-18 in HIBD group were higher than those in Sham group（P<0.05）;Compared with HIBD group,the protein and m RNA expression levels of the above related molecules were lower in HT and Xe group（P<0.05）;The protein and m RNA expression levels of the above related molecules in HT+Xe group decreased significantly while compared with HIBD group（P<0.05）;Compared with HT group and Xe group,the protein and m RNA expression levels of the above related molecules decreased in HT+Xe group（P<0.05）.6.ELISA results showed that the expression of inflammatory cytokines IL-18 and IL-1βincreased significantly in HIBD group compared with Sham group（P<0.05）;Compared with HIBD group,the expression of above inflammatory factors decreased in HT and Xe group（P<0.05）;The expression of above inflammatory factors were significantly decreased in HT+Xe group while compared with HIBD group（P<0.05）;Compared with HT group and Xe group,the expression of above inflammatory factors were slightly lower in HT+Xe group（P<0.05）.7.Behavioral experiment results show:The neurobehavioral assessment,righting reflex experiment was performed 3 days after surgery and the capture rejection experiment,45°slope experiment was performed 28 days after surgery.The results showed that compared with the Sham group,the activity,sensitivity and alertness of the HIBD group were worse and the reflex time increased significantly（P<0.05）;Compared with HIBD group,HT group and Xe group had slightly higher activity,sensitivity,alertness and reduced reflex time（P>0.05）;HT+Xe group had higher activity,sensitivity,alertness and reduced reflex time significantly（P<0.05）;Compared with HT group and Xe group,HT+Xe group had higher activity,sensitivity,alertness and reduced reflex time（P<0.05）.Conclusion:1.NLRP-3 inflammasome and its mediated pyroptosis are involved in the pathophysiological process of HIBD in newborn rats.2.Mild hypothermia combined with xenon therapy can reduce neuronal injury and cerebral infarction area and play a neuroprotective role by inhibiting the pyroptosis mediated by inflammasome in HIBD newborn rat neurons.