Neuroprotective Effects Of Levosimendan On Sepsis-Associated Encephalopathy In Mice

Research Background and Objective:Sepsis-associated encephalopathy（SAE）is defined as diffuse impairment of brain function caused by sepsis.There is no relevant evidence to prove that it is caused by central nervous system infection.It is usually challenging to treat,and the mortality rate is relatively high,seriously affecting patients’safety.At present,there still needs to be the clinical treatment for SAEs.Levosimendan（Lev）is a new Ca²⁺sensitizer and K_ATPchannel opener,mainly used in the research and treatment of heart failure and cardiomyopathy.Studies have shown that levosimendan can improve a variety of nervous system diseases through anti-inflammatory,anti-oxidative stress,and vasodilation mechanisms.However,there are few studies on treating SAE with levosimendan at home and abroad.In this study,the Neuron-specific Enolase in the hippocampus of mice was detected by ELISA Neuron-specific Enolase（NSE）,S100β,glutathione peroxidase（GPx）,and thiobarbituric acid reactive substances（TBARS）were measured in mice.And it compared the number of neurons in pathological sections to explore the neuroprotective effect of levosimendan on SAE mice and provide new ideas and methods for treating SAE in clinics in the future.Research Method:Sixty healthy male C57BL/6 SPF mice were divided into four groups.The first group（Sham group）was given normal saline after cecal ligation and puncture.The second group was the same as the first group.Levosimendan was given after the operation as Sham+Lev group.In the third group,cecal ligation and puncture（CLP）established the sepsis mouse model.The appearance,autonomous consciousness,autonomous activity,response to stimulation,eye secretion,respiratory frequency,and respiratory quality of mice were evaluated by the general observation score of mice.The neurobehavioral score of mice determined the SAE model,and the SAE model was determined by a score≤6.After selecting the SAE model,normal saline was given as the SAE group.The fourth group was established as the same as the third group and was treated with levosimendan as the SAE+Lev group.Mice that did not meet the criteria for the SAE model were not included in the SAE group,and the mouse sepsis model was re-established to supplement.Some studies have shown that the sepsis model group showed inflammatory changes that gradually aggravated after 6 hours.The neurobehavioral scores of CLP mice began to decrease 3 hours after the operation,and the decrease was most significant at 24 hours.Therefore,the mice’s general conditions and neurobehavioral changes were observed again at 6 hours,12 hours,and 24 hours after operation in this experiment.The mice were killed at 6 hours,12 hours,and 24 hours after the operation.The brain tissues were taken and made into pathological sections by hematoxylin-eosin staining（HE）.The morphological and structural changes of neurons in the hippocampus of mice were observed.The number of neurons in pathological cells of each group under 200 times the visual field was counted by Image J software.The ELISA method detected the concentrations of NSE,S100β,GPx,and TBARS in the hippocampus of mice,and the data were analyzed by SPSS 26.0 statistical software.One-way ANOVA was used for multiple groups.The LSD test was used for comparison between groups.If the variance was uniform,Dunnett T 3 test was used.P<0.05 indicated that the difference was statistically significant.Results:1.Observation score of the general condition of mice:At 6 hours,12 hours,and24 hours the observation score of the general condition of mice in the SAE group was compared with that in the Sham group（P<0.05）,and the difference was statistically significant.At 6 hours,12 hours,and 24 hours,the general observation scores of mice in the SAE+Lev group were compared with those in the SAE group（P<0.05）,and the difference was statistically significant.2.Neurobehavioral changes in mice:At 6 hours,12 hours,and 24 hours,the neurobehavioral score of the SAE group was significantly lower than that of the Sham group and less than 6 points（P<0.05）,indicating that the mice in the SAE group were successfully modeled.At 6 hours,12 hours,and 24 hours,the neurobehavioral score of the SAE+Lev group was significantly higher than that of the SAE group（P<0.05）.3.Detection of brain tissue related factor level:（1）NSE concentration level:At 6 hours,12 hours,and 24 hours,the concentration of NSE in the SAE group was significantly higher than that in the Sham group（P<0.05）.At 6hours,12 hours,and 24 hours,the concentration of NSE in the SAE+Lev group was significantly lower than that in the SAE group（P<0.05）.（2）S100βconcentration level:S100βconcentration in the SAE group was significantly higher than that in the Sham group at 6 hours,12 hours,and 24 hours（P<0.05）,and S100βconcentration in the SAE+Lev group was significantly lower than that in the SAE group at 6 hours,12 hours and 24 hours（P<0.05）.（3）GPx concentration level:At 6 hours,12 hours,and 24 hours,the GPx concentration in the SAE group was significantly higher than that in the Sham group（P<0.05）,and the GPx concentration in the hippocampus of the SAE+Lev group was considerably lower than that in SAE group at 6 hours and 24h hours(P₆=0.000,P₂₄=0.003,P<0.05).There was no significant difference in the concentration of GPx between the SAE+Lev group and the SAE group at 12 hours(P₁₂=0.079,P>0.05).（4）TBARS concentration level:At 6 hours,12 hours,and 24 hours,there was no significant change in TBARS concentration between the SAE and the Sham groups（P>0.05）.At 6 hours,12 hours,and 24 hours,there was no significant difference in TBARS concentration between the SAE+Lev and SAE groups（P>0.05）.4.Neuropathological changes of mouse brain tissue:At 6 hours,12 hours,and 24 hours,,compared with the Sham group,the hippocampal neurons in the SAE group were more pyknotic,the boundary between the nucleus and cytoplasm was more blurred,and the deep staining of neurons was more prominent.Compared with the SAE group at each time point,only some neurons in the SAE+Lev group were slightly swollen,the neuronal pyknosis and deep staining were relieved obviously,and the cell structure was still evident.5.Number of neurons in pathological sections of each group under 200X visual field:At 6 hours,12 hours,and 24 hours,the SAE group was significantly lower than the Sham group（P<0.05）,and the difference was statistically significant.At 6 hours,12 hours,and24 hours,there were relatively more mice in the SAE+Lev group than in the SAE group,P<0.05,and the difference was statistically significant.Conclusion:Levosimendan can alleviate the neuronal apoptosis caused by SAE by inhibiting the release of NSE and S100βin SAE,lessen the damage to nervous system function in SAE mice to a certain extent,and produce a neuroprotective effect on SAE mice.Still,the correlation with anti-oxidative stress is not apparent.