| Objective Establish a model of Delayed encephalopathy after acute carbon monoxide poisoning（DEACMP）in rats,and intervene the model with Lycium Barbarum Polysaccharides（LBP）to detect its inducible nitric oxide synthase(Inducible nitric oxide Synthase（iNOS）,neuronal nitric oxide synthase（nNOS）expression and its correlation with neuronal degeneration and necrosis,to explore the role of iNOS and nNOS protein in the pathogenesis of DEACMP and LBP to DEACMP Preliminary study of neuroprotective effects.Method 1.Preparation of DEACMP rat model:A modified intraperitoneal injection of CO method was used to establish a DEACMP rat model（the first injection of 99.99%CO pure gas120mL·kg-1,followed by three consecutive injections,each time according to 3,interval 4h）.Adult male SD rats were selected.After Morris water maze training,the rats that could not find the platform were removed after 60s.108 qualified SD rats were selected and randomly divided into DEACMP group,air group,and normal group.Gas,intraperitoneal injection of air,and abdominal cavity were not intervened,36 in each group.According to the 6 time selection points before and after modeling（before modeling,modeling 1d,modeling 7d,modeling 14d,modeling 21d,modeling 28d）,the DEACMP group,the air group,and the normal group are divided into 6 sub-groups.Group,6 in each subgroup.The three groups were monitored for arterial COHb concentration from 2 hours to 24 hours after the first exposure.Morris water maze and HE staining were used to observe degeneration and necrotic neurons in the hippocampal CA3 area to further validate the model.2.Expression of iNOS and nNOS proteins in the hippocampus and their correlation with neuronal degeneration and necrosis in the hippocampal CA3 region:72 male SD rats were selected and randomly divided into DEACMP group and normal group,with 36 in each group.According to different time periods before and after modeling,the two groups are further divided into 6subgroups before modeling,1d,7d,14d,21d,and 28d.On the segment,the neuron degeneration and necrosis of hippocampal CA3 area was observed under HE staining light microscope,and the expressions of iNOS and nNOS protein in hippocampus were detected by immunohistochemistry and Western blot.correlation analysis of the correlation between iNOS and nNOS protein expression and neuronal degeneration and necrosis.3.A preliminary study of the neuroprotective effect of LBP on DEACMP rats:adult male SD rats were selected and a modified intraperitoneal CO method was used to establish a DEACMP rat model.Take 99%wolfberry polysaccharide powder 500mg+10ml sterile physiological saline,and prepare a solution with a concentration of 50mg/ml（for future use）.On the 14th day of the modeling,Morris water maze was used to screen 48 DEACMP rat models.They were randomly divided into normal group[n=12,no intervention],DEACMP group[n=12,sterile saline],and high dose LBP group[n=12,Lycium barbarum polysaccharide solution 400mg·（kg·d）-1],LBP medium dose group[n=12,Lycium barbarum polysaccharide solution 200mg·（kg·d）-1],LBP low dose group[n=12,Lycium barbarum Polysaccharide solution 100 mg·（kg·d）-1].Starting from 14 days of DEACMP modeling,according to different groups,non-intervention,intraperitoneal injection of normal saline,and intraperitoneal injection of different doses of lycium barbarum polysaccharide solution were given once a day for 14 days in a continuous intraperitoneal injection,and 28 days after modeling,HE staining was performed.The neuron degeneration and necrosis in the hippocampal CA3 area was observed under a light microscope,and the hippocampal iNOS protein expression was detected by Western blot.The correlation between hippocampal iNOS protein expression and neuron degeneration and necrosis was analyzed by correlation.Results 1.DEACMP rat model preparation:（1）Morris water maze:before modeling,1d modeling,7d modeling,the average incubation period and number of crossing platforms of DEACMP group were not statistically significant compared with normal group and air group（P All>0.05）,and the difference was statistically significant in the 14d model,21d model and 28d model（all P<0.05）.There was no significant difference between the normal group and the air group at 6 time points before and after modeling（all P>0.05）.（2）HE staining to observe neuronal degeneration and necrosis in hippocampal CA3 area:the degeneration and necrosis count of DEACMP group compared with normal group and air group before and after modeling,1d and 7d,the difference was not statistically significant（P<0.05）,and the difference was statistically significant between the 14d model building,21d model building and 28d model building（all P>0.05）.There was no statistical difference between the normal group and the air group at 6 time points before and after modeling Academic significance（all P>0.05）.（3）correlation analysis:the correlation coefficient between the average latency of the DEACMP group and the neuron count of degeneration and necrosis in hippocampal CA3 area is r=0.915,which is correlated（P<0.05）2.The expression of iNOS and nNOS protein in hippocampus and its correlation with neuronal degeneration and necrosis in hippocampal CA3 area:（1）Immunohistochemical detection of iNOS and nNOS protein expression in hippocampal CA3 area:iNOS and nNOS positive staining is mainly expressed in the cell membrane,and the color is brown Deeply dyed particles.There was no statistically significant difference between the two groups of iNOS protein before modeling and on the 1st day of modeling（all P>0.05）,while on the 7th,14d,21d,and 28d models,the difference was statistically significant（P<0.05）;There was no statistically significant difference between the two groups of nNOS protein before and 28 days after modeling（all P>0.05）,and compared between 1d,7d,14d and 21d,The difference is statistically significant（P<0.05）.（2）Western blot method was used to detect the expression of iNOS and nNOS proteins in the hippocampus:the iNOS protein in the DEACMP group began to be expressed on the 7th day of modeling,and showed an increasing trend from 7d to 28d.Before modeling,the difference between the model 1d and the normal group was not statistically significant（all P>0.05）,and the difference was statistically significant between the model 7d,model 14d,model 21d,and model 28d（P were all<0.05）;DENOMP group nNOS protein began to be expressed on the 1st day of modeling,from the 1st day of modeling to the 21st day of modeling,there was an increasing trend.There was no statistically significant difference between the normal model and the 28th day before modeling and28 days after modeling.>0.05),and the difference was statistically significant in modeling 1d,modeling 7d,modeling 14d,and modeling 21d（all P<0.05）.（3）correlation analysis:The correlation coefficient between the expression level of iNOS protein and the count of degeneration and necrosis of neurons in hippocampal CA3 area is r=0.661,which is correlated（P<0.05）.The correlation coefficient between the expression level of nNOS protein and the count of degeneration and necrosis of neurons in hippocampal CA3 area was r=0.281,and there was no correlation（P>0.05）.3.Preliminary study of the neuroprotective effect of LBP on DEACMP rats:（1）The effect of LBP on neuronal degeneration and necrosis in the CA3 area of the DEACMP hippocampus:Compared with the LBP low dose group,the degeneration and necrosis count of the DEACMP group was not statistically significant（P>0.05）,compared with LBP medium-dose group and LBP high-dose group,the difference was statistically significant（all P<0.05）.（2）The effect of LBP on the expression of iNOS protein in DEACMP hippocampus:the expression of iNOS protein in DEACMP group was not statistically different from that of LBP low-dose group（P>0.05）,compared with LBP medium-dose group and LBP high-dose group Significance（all P<0.05）.（3）correlation analysis:The correlation coefficient r=0.868 of hippocampal iNOS protein expression and neuronal degeneration and necrosis count after LBP intervention was correlated（P<0.05）.Conclusion 1.Continuous intraperitoneal injection of poisoning is a practical method to establish a DEACMP rat model,which can establish an ideal model to study the mechanism of DEACMP.Morris water maze is a behavioral method that can objectively evaluate the cognitive ability of rats and can be used as a reliable method to judge whether DEACMP has occurred.2.iNOS continues to be highly expressed in the DEACMP hippocampus,which is positively correlated with the degeneration and necrosis count of neurons in the CA3 area of the hippocampus,indicating that iNOS plays an important role in the pathogenesis of DEACMP;while nNOS does not continue to be highly expressed in the DEACMP hippocampus,which is consistent with the hippocampus CA3 There was no correlation between degeneration and necrosis of neurons in the region.3.LBP can reduce the expression of iNOS in the hippocampus of the 28th day in the DEACMP group and reduce the count of neuronal deformation and necrosis in the hippocampal CA3 area.The neuroprotection of LBP may be through the NO/NOS system. |
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