| Objective:The purpose of this experiment is to establish a rat model of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP)by modified interval intraperitoneal injection method,to investigate the dynamic expression of nuclear factorκB(NF-κB)and nerve adhesion molecule(NCAM)and the mechanism of synaptic remodeling in delayed encephalopathy after acute carbon monoxide poisoning(DEACMP),and to evaluate the intervention effect of specific inhibitor of nuclear factorκB,pyrrolidone dithiocarbamates(PDTC).Methods:1.150 male SD rats with an average weight of 250-300 g were randomly divided into 3 groups,group K:air group(normal saline+air);Group C:carbon monoxide poisoning group(normal saline+CO);Group P:carbon monoxide+PDTC group(CO+PDTC);Each large group was divided into 5 subgroups according to the monitoring points on the 1st,3rd,7th,14th and 21st days after exposure,and 10 rats were placed in each subgroup.The divided rats were weighed and marked.The rats in group P were injected intraperitoneally with the prepared PDTC at a dose of 100mg/kg 30min before molding,and then were injected PDTC at the same time every day until they were killed.At the same time,the air control group and CO group were given intraperitoneal injection of physiological saline of corresponding volume every day.2.The acute CO poisoning rat model was established by modified interval intraperitoneal injection method.Blood was taken from the rat the femoral artery and COHb concentration was measured by blood gas analyzer.Morris water maze test was used to observe the behavioral performance of rats,to judge whether the model was successfully established,and to screen rats with delayed encephalopathy and mark them.3.Rat specimens were made at various time points after CO poisoning.The expressions of NF-κB and NCAM in hippocampus were detected by immunofluorescence,the morphological changes of cells in hippocampus were observed by HE staining,the changes of synaptic structure in hippocampus were observed by transmission electron microscope,and the expressions of NF-κB and NCAM in hippocampus were detected by Western Blot.SPSS23.0 was used to analyze the classified data,the theoretical distribution of the observed values of the samples is expressed by the mean standard deviation(~-x±S).Results:1.Behavioral Changes of Rats after Exposure:after intraperitoneal injection of CO,rats showed mild activity,dysphoria,shortness of breath,and some rats showed obvious mania and hyperextension of angular arch.When CO was added again,the rats showed limp limbs,cyanosis of oral mucosa and cherry red at the extremities,even coma and death.There was no obvious behavioral change in the air control group rats.2.Morris water maze test:Inter-group comparison:the average escape latency of group C was longer than that of group K and group P at the time points of the 7th,14th and 21st days(P<0.05).The average escape latency of rats in group C and group P was not significantly delayed at the three time points of pre-modeling,on the 3rd and on the 7th respectively,and the difference was not statistically significant(P>0.05),while the average escape latency at the two time points of the 14th and 21st days was not statistically significant(P>0.05).However,the average escape latency was significantly delayed on the 14th and 21st days compared with the days before,on the 3rd and on the 7th,respectively,with statistical significance(P<0.05).There was no significant difference in the average escape latency of rats in group K(P>0.05).In the space exploration experiment,the residence time in the platform target quadrant of group c was significantly reduced compared with group k and group p on the 14th and 21st days,and the difference was statistically significant(P<0.05).3.HE staining results:In the air group,the cells in hippocampal CA1 region were arranged in order and the cell structure was complete at all time points.In the CO poisoning group,neurons in hippocampus swelled,and nuclei showed shrinkage and fragmentation,and with the extension of time,the degree of necrosis increased,which was most significant on the 21st day.Compared with CO poisoning group,PDTC intervention group significantly reduced hippocampal tissue edema and neuronal cell necrosis at various time points.4.expression of NF-κB and NCAM:NF-κB and NCAM were detected by immunofluorescence.in the hippocampal tissue of air group,NF-κB and NCAM had no obvious change at each time point,and the difference was not statistically significant(P>0.05);The expression of NF-κB in hippocampus of group C was continuously increased,and the expression of NF-κB in group P gradually decreased after reaching the peak on the 3rd day.the difference between group C and groups K and P at various time points was statistically significant(P<0.05);The expression of NCAM in group C and group P decreased gradually after reaching the peak on the 3rd day,and the expression of NCAM in group P increased and had statistical difference compared with that in group C and group K(P<0.05).Western blot analysis was consistent with immuno-fluorescence results.5.The results of projection electron microscope:In the air group,the fibers were arranged in order and the synaptic structure was clear and complete at all time points.On the 3rd day after CO poisoning,nerve fibers began to appear disorder,loose,uneven in size,enlarged synaptic space and decreased synaptic vesicles.The damage reached its peak on the 14th and 21st days after CO poisoning,with floccules with low electron density in synapses,unclear internal organelles,discontinuous presynaptic membrane,uneven thickness,dissolution of synaptic vesicles and formation of vacuoles.On the14th day,compared with group C,synaptic vesicles and complete organelles were visible,and synaptic structure was relatively clear.Conclusion:1.Interval intraperitoneal injection method is an ideal method for making DEACMP model of rats.It is simple to operate,requires low equipment,has relatively high success rate,and can well simulate acute severe CO poisoning of human body.2.Morris water maze experiment is widely used in learning and memory evaluation as evidence of validity of hippocampal dependent spatial navigation and reference memory measurement.The changes of cognitive function in the experimental results,combined with behavioral and hippocampal histopathological results,determine the success of DEACMP model.3.The dynamic expression of NF-κB and NCAM and synaptic remodeling participate in the occurrence of delayed encephalopathy induced by carbon monoxide poisoning.PDTC can inhibit the expression of NF-κB in hippocampus,increase the expression of NCAM,and improve delayed neuronal necrosis caused by CO poisoning,thus protecting brain tissue. |
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