Correlation Between Rho/ROCK Pathway And Delayed Encephalopathy After Acute Carbon Monoxide Poisoning

BackgroundsThe main pathological change of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP)is extensive demyelination of the white matter of the brain.Previous studies have found that the abnormal expression of Rho/ROCK signaling pathway related factors may be related to central nervous system damage.When the adult central nervous system is stimulated,the activation of the Rho/ROCK signaling pathway causes the overexpression of downstream factors,which leads to neuronal damage and demyelination.At this stage,whether the abnormal function of the Rho/ROCK signal pathway is related to the pathological changes of DEACMP has not yet been verified.ObjectivesExplore the changes in the expression of Rho/ROCK signaling pathway related factors in DEACMP model rats,and whether the DEACMP model rats’ cognition,exercise,and white matter demyelination are improved after drug intervention in the Rho/ROCK signaling pathway,and understand Rho/ROCK signaling Correlation between pathways and the pathogenesis of DEACMP.Methods1.107 SPF male SD rats,with a weight standard of 240～260 g,were placed in individual ventilated cages(IVC)and reared adaptively for one week.Randomly grouped Supported by the National Science Foundation of China(81141071,81671319)Henan Provincial Key Labaratory of Biological Psychiatry(Henan International Joint Laboratory of Psychoneurology)into model and control(Control)group.CO gas was quickly injected into the abdominal cavity of the modeled rats at 100 m L/kg,and injected into the abdominal cavity every 4hours.The volume of the gas injected was halved from the second time,and the gas was injected 4 times in total.In the Control group,the same volume of air was injected in the same way.2.On the first day after modeling,the model rats were randomly divided into CO+Y-27632 group and CO group,5 mg/kg intraperitoneally injected ROCK inhibitor into CO+Y-27632 group,once a day at the same time point Continuous administration for one week;continuous intraperitoneal injection of 0.9% normal saline for one week is the CO group.In the Control group,0.9% normal saline was continuously injected intraperitoneally for one week.Complete the open field,runner fatigue test,Y-maze,and new object recognition behavioral tests at the first,second,and third week time nodes after modeling,to observe the cognitive and motor function of each group of rats change.Hematoxylin-eosin(HE),luxol-fast-blue(LFB)staining and immunofluorescence techniques were used to detect the pathological changes of rats in each group at each time node after modeling.Select areas sensitive to hypoxia,such as hippocampus,frontal lobe,and striatum,and use western blotting to detect various groups of nerve injury markers at different time nodes,such as neuron-specific enolase(NSE),glial Fibrillary acidic protein(glial fibrillary acidic protein,GFAP)and factors related to the repair of myelin damage,such as the calcium-dependent cell adhesion molecule family(N-Cadherin),Subfamily members of the small G protein superfamily Rho A,Rho-related protein kinase ROCK,myelin basic protein(myelin basic protein,MBP)molecular expression levels.3.The independent sample t test was used to statistically analyze the data between the two groups,and the nonparametric test was used for the data that did not conform to the normal distribution.P<0.05 indicates statistical significance.Results1.DEACMP model preparation(1)The behavioral results showed that compared with the Control group,the exploration time of entering the new arm in the CO group was significantly reduced in the first week,second week,and third week after modeling(P=0.03,P=0.03,P= 0.02),in the first week and second week after modeling,the discrimination index of exploring new objects decreased significantly(P=0.01,P=0.04),and the total distance of exercise was significant in the first week,second week,and third week after modeling Decrease(P=0.01,P=0.01,P=0.01),the average activity time of the runner track was significantly reduced in the first week,second week,and third week after modeling(P=0.01,P=0.03,P= 0.01).(2)The pathological results showed that: compared with the Control group,a large number of spotted and patchy necrotic neurons appeared in the hippocampal CA1 area within three weeks after modeling in the CO group,the hippocampal pyramidal cell layer became thinner,and the cells on both sides were sparse;the corpus callosum area The sheath is broken or lost.In CO group,the fluorescence intensity of GFAP protein in hippocampal CA1 area increased at different time nodes,and the fluorescence intensity of myelin MBP protein in corpus callosum area decreased at different time nodes.The above behavioral and pathological changes are basically consistent with the clinical features of DEACMP,it indicates that the DEACMP model is successfully prepared.2.The correlation between Rho/ROCK pathway and DEACMPProtein results showed that compared with the Control group,the hippocampus,frontal lobe,and striatum ROCK protein levels in the CO group increased significantly in the first week after modeling,and the hippocampus,striatum Rho A,and ROCK protein levels in the second week after modeling.Significant increase,the hippocampus,striatum Rho A,and ROCK protein levels increased significantly in the 3rd week after modeling(all P<0.05).In summary,the results from the protein level suggest that the Rho/ROCK pathway has a certain correlation with DEACMP.After drug intervention in DEACMP model rats,all detection indicators have improved3.Changes of various indexes after drug intervention in DEACMP model rats(1)The behavioral results showed that compared with the CO group,the exploration time of entering the novel arm in the first week and the second week after drug intervention in the CO+Y-27632 group was significantly increased(P=0.04,P=0.04),drug intervention After the first week and the second week and the third week,the discrimination index of exploring new objects increased significantly(P=0.01,P=0.01,P=0.04),and the total distance of exercise increased significantly in the second and third weeks after the drug intervention(P=0.04,P= 0.02),the average activity time of the wheel track in the first week,the second week,and the third week after the drug intervention increased significantly(P=0.01,P=0.04,P=0.01).In summary,the detection of various indicators suggests that ROCK inhibitors may be effective therapeutic drugs for DEACMP.(2)The pathological results showed that compared with the CO group,in the CO+Y-27632 group,the neuron nuclear membrane and nucleolus in the CA1 area of the hippocampus were more complete within three weeks after drug intervention,the demyelination of the corpus callosum was reduced,and the GFAP fluorescence in the CA1 area of the hippocampus.The intensity decreases,and the fluorescence intensity of MBP in the myelin sheath of the corpus callosum increases.(3)The protein results showed that compared with the CO group,the expression levels of GFAP and NSE proteins in the hippocampus and striatum of the CO+Y-27632 group decreased significantly,and the expression levels of NSE and ROCK proteins in the frontal lobe decreased significantly in the first week after drug intervention.The expression levels of Rho A and ROCK protein decreased significantly,the expression levels of MBP protein in the hippocampus and striatum increased significantly,and the expression levels of N-Cadherin protein in the hippocampus and frontal lobe increased significantly;the CO+Y-27632 group drug intervention in the second week of the hippocampus and frontal lobe GFAP,ROCK protein expression levels decreased significantly,hippocampus,striatum GFAP,Rho A,ROCK protein expression levels decreased significantly,hippocampus,striatum N-Cadherin,MBP protein expression levels increased significantly;CO+Y-27632 group drug intervention in the third week The expression levels of GFAP,NSE,and ROCK proteins in the hippocampus,frontal lobe,and striatum significantly decreased,and the expression levels of N-Cadherin and MBP proteins in the hippocampus,frontal lobe,and striatum increased significantly(all P<0.05).In summary,the results of various indicators suggest that ROCK inhibitors interfere with the Rho/ROCK pathway to improve the behavioral phenotype of DEACMP model rats and reduce neuropathological damage.Conclusion1.The pathogenesis of DEACMP is related to the activation of the Rho/ROCK pathway.2.ROCK inhibitors can reduce the degree of nerve damage in DEACMP model rats by intervening in the Rho/ROCK pathway.