Rearch On The Mechanism Of Vagus Nerve Stimulation Affect Post-Traumatic Cognitive Impairment In Controlled Cortical Impact Model

Objective:Traumatic brain injury（TBI）is an injury caused by external violence to the head,and post-traumatic cognitive impairment（PTCI）is one of its major complications and sequelae,manifesting as impairment of various cognitive functions such as memory and orientation after TBI.It has been found that transplantation of exogenous neural stem cells in the brain injury area of TBI model rats contributes to the recovery of their neurological functions.As a matter of fact,endogenous neural stem cells are naturally present in the adult CNS,mainly in the dentate gyrus（DG）and subventricular zone（SVZ）of the hippocampus.Studies have shown that adult hippocampal dentate gyrus neurogenesis can affect hippocampal correlated cognitive function.Vagus nerve stimulation（VNS）,a neuromodulation technique,has been widely used in the treatment of epilepsy and depression,however,its mechanism of action on cognitive impairment after TBI is still unclear.In the present experiment,we established a TBI model and acute and chronic VNS models to study the effects of VNS on cognitive dysfunction in TBI model mice,in order to observe the changes in neurogenesis in DG,and to explore the potential mechanisms of VNS.Methods:1.Based on Nestin-GFP^Tg/+transgenic mice,acute VNS model and chronic VNS model were constructed,on which controlled cortical impact（CCI）model mice were used as the disease model and VNS intervention treatment was given to construct CCI-VNS mice model.2.The experimental animals were divided into your groups:Naive group（n=6）,CCI only group（n=15）,CCI+c VNS group（n=8）,and CCI+c VNS-Sham group（n=6）,and behavioral experiments were conducted to explore the effects of VNS on cognitive dysfunction after TBI.3.The changes in hippocampal neurogenesis after VNS and CCI-VNS were studied using immunofluorescence staining techniques,and the possible signaling pathways affecting neurogenesis were explored by Western Blot experiments to investigate the role of VNS on adult neurogenesis after TBI.4.We investigated the changes in hippocampal neuronal loss and astrogliosis phenomenon after CCI and CCI-VNS by immunohistochemical staining technique and immunofluorescence staining technique to explore the possible therapeutic mechanism of VNS on TBI.Results:1.We successfully constructed an acute VNS mice model,a chronic VNS mice model,and a CCI-VNS mice model.2.The results of the MWM water maze experiment showed that in the positioning navigation experiment:CCI only group,CCI+c VNS group,and CCI+c VNS-Sham group showed a decreasing trend overall.The trend of change in the total distance of movement of each group was similar to the trend of change in the latency,and the difference between groups was not statistically significant（P>0.05）.The results of swimming speed showed that the difference in swimming speed among the 4 groups was not statistically significant（P>0.05）.The results of the positioning navigation experiment showed that there was no significant difference in the positioning navigation ability of the mice in the 4 groups（P>0.05）.The results of the spatial exploration experiment showed that CCI+c VNS group mice entered the escape platform more often than CCI+c VNS-Sham group,and the difference was statistically significant（P<0.05）.The retention time of the CCI+c VNS group platform was longer than the remaining three groups among the four groups,but this difference was not statistically significant（P>0.05）.The results of escape platform movement time,effective area entry number,effective area movement time,and effective area movement distance were consistent with the trend of escape platform entry number.3.Immunofluorescence measurements showed that the expression of the c-Fos protein was significantly upregulated in the granule cell layer of the hippocampal dentate gyrus after acute vagal stimulation（P<0.05）,and the difference in the number of Nestin-positive cells in the subgranular layer of the dentate gyrus was not significant（P>0.05）.The expression of Nestin-positive cells in the subgranular layer of the dentate gyrus of the hippocampus was upregulated by chronic vagus nerve stimulation for 7 days,and the difference was statistically significant（P<0.05）.The number of Nestin-positive cells in the subgranular layer of the dentate gyrus of CCI mice was significantly increased by further extending the stimulation days to 14 days,and the difference was statistically significant（P<0.01）.The number of Nestin-positive cells in the subgranular layer of the dentate gyrus of CCI model mice was reduced compared with that of Naive mice,but the difference was not statistically significant（P>0.05）.c-VNS intervention in CCI mice,In CCI+c VNS group mice,the number of Nestin-positive cells in the dentate gyrus was significantly increased（P<0.05）,and the number of Nestin-positive cells in CCI+c VNS-Sham group mice was not significantly different from that in CCI only group mice（P>0.05）.the number of DCX-positive cells was expressed similarly to Nestin.4.Western Blot results showed that hippocampal BDNF protein expression was elevated in CCI+c VNS group,and the difference in hippocampal BDNF protein expression between the remaining three groups was not statistically significant（P>0.05）;hippocampalβ-catenin 3expression was slightly higher in CCI+c VNS group than in Naive group and CCI only group,but the difference between the three groups was not statistically significant（P>0.05）.The expression of hippocampalβ-catenin 3 was significantly decreased in CCI+c VNS-Sham group compared with CCI+c VNS group.5.Nissl staining showed that the neurons in the CA1 and CA3 regions of the hippocampus of Naive group mice were full in shape and normal in morphology,and the overall cell layer was densely and neatly arranged.Compared with Naive group,CCI only group mice hippocampal CA1 and CA3 regions had a large number of neurons lost and showed vacuolation,some neurons had crumpled cell bodies,and the overall cell layer structure was disordered.In contrast,the overall cell arrangement in the CA1 and CA3 regions of CCI+c VNS group mice hippocampus was disorganized,but the cell morphology and structure were less damaged,and fewer neurons showed nuclear consolidation and vacuolation-like changes.CCI+c VNS-Sham group mice have a severe neuronal loss.6.The immunofluorescence staining results showed that compared with Naive group mice,CCI-only group mice had significantly more GFAP-positive cells in the hippocampal hilus region,CA3,and CA1 regions（P<0.05）,and the astrocyte morphology was significantly more swollen,pseudopod,and protruding.In contrast,the number of GFAP-positive cells in CA3 and CA1 regions of CCI+c VNS group mice treated with c-VNS was lower than that of CCI-only group,and the number of GFAP-positive cells and swollen astrocytes in CA1 and CA3 regions of CCI+c VNS-Sham group was significantly more than that of CCI+c VNS group mice（P<0.05）.The trend of GFAP positive cell number expression in the portal region was similar,but the difference was not statistically significant（P>0.05）.Conclusions:1.After acute VNS,c-FOS expression was elevated in the DG region of the hippocampus,and hippocampal neurons were activated;a-VNS had no significant effect on the proliferation of neural precursor cells,while after c-VNS,neural precursor cell proliferation was increased.2.CCI model mice with impaired cognitive function represented by spatial memory showed significant improvement in spatial memory after treatment with chronic VNS3.CCI model mice,given chronic VNS intervention,had elevated Nestin-positive cell expression in the DG region of the hippocampus,indicating increased proliferation of neural precursor cells.VNS was able to promote hippocampal dentate gyrus neurogenesis after CCI.4.In CCI model mice,hippocampal BDNF expression was upregulated after chronic VNS intervention,suggesting that chronic VNS has a neuroprotective effect after CCI,possibly by upregulating BDNF to promote neurogenesis in the dentate gyrus.5.A large number of neural pyramidal cells were lost in the CA1 and CA3 regions of the hippocampus after CCI in mice,but given chronic VNS treatment,the loss of hippocampal neurons would be reduced.6.Reactive astrocytes in the hippocampal hilus,CA1,and CA3 regions were increased after CCI in mice.Then given chronic VNS treatment,the Astrocytosis in the hippocampal CA1and CA3 regions would be reduced.