Function Of Toll Like Receptor 2 On Proliferation And Differentiation Of Neural Stem Cells After Traumatic Brain Injury

BackgroundThere is high morbidity of traumatic brain injury(TBI)during wartime and usual time morbidity and high mortality and disability rate is the main characteristic of TBI.In the pathophysiological process of TBI,the non-infectious inflammatory process in brain is of great significance to the repair and outcome of nerve function after TBI.Adult mammalian neural stem cells(NSCs)can play an important role in the repair of central nervous system(CNS)damage and functional reconstruction through the process of proliferation,differentiation and migration.However,it is urgently needed in clinical practice that activation and regulation of NSCs by induction or stimulation,especially for the proliferation and differentiation caused by the inflammatory state after the change of the intracranial microenvironment homeostasis caused by trauma.Toll-like receptors(TLRs)usually play an important role in inflammatory activities caused by pathogen-derived or non-pathogen-derived factors,and also play an important role in tissue and cell damage recognition,repair initiation,and environmental induction effect.Studies have shown that expression of TLRs can be seen on the surface of NSCs in CNS,especially expression of TLR2,and changes in TLRs expression are of great significance to the change of NSCs,especially the change of NSCs proliferation and differentiation,regardless of whether neurons or glial cells.In addition,TLRs may also play an important role in CNS regeneration.Therefore,due to its "recognition" and "initiation" effects and special effects on regeneration,we speculated whether NSCs can recognize microenvironmental changes and trauma status through TLRs receptors,which were on the surface of NSCs,under the inflammatory environment in the brain after TBI and affect its own behavioral capabilities including proliferation,differentiation and migration.We realize that the TLRs receptors expressed on the surface of NSCs may have greater significance in the activation,regulation,control and CNS function repair of NSCs after TBI.But so far,the research on the function of TLRs on the surface of NSCs after craniocerebral injury,especially the study of the relationship between TLR2 and NSCs after trauma was rarely reported.NSCs generally refer to a family of cells that have great potential to differentiate into neurons and glial cells including oligodendrocytes,astrocytes,and microglia,and can selfrenew.Adult mammals generally have a large number of NSCs,but in normal time,the NSCs are generally dormant,with only a very small number of newly borned nerve cells.When the pathophysiological state of CNS occurs,NSCs may proliferate,then migrate to functional sites and enter into the CNS network through differentiation and integration to play a role in tissue and cell repair and functional repair.The nerve regeneration in the adult mammalian brain runs through the life cycle.The proliferation and differentiation of NSCs are of great significance to the repair and function maintenance of the brain during the whole process of nerve regeneration.The theory of nerve cells’ non-regeneration has been completely changed,and it provides important ideas in the exploration of ways and methods of the treatment of CNS-related diseases.Adult mammalian NSCs are abundantly present in the subventricular zone of the lateral ventricle(SVZ)and the subgranular zone of dentate gyrus(SGZ).Because of the important role of SGZ in learning and memory functions,it is our focus of research.However,how to activate and affect NSCs in adult mammals is still unclear,and the mechanism of injury-induced regeneration is not clear.In addition,the regeneration of nerve cells after CNS injury is still difficult to meet the needs of cell,tissue and functional repair.Therefore,measures to affect the behavioral changes of NSCs should be sought.At present,there is still a lack of interventions that act on the molecular targets of NSCs,especially in the disordered intracranial microenvironment after brain injury.TLRs usually have the following characteristics.First,they can recognize changes in the microenvironment and undergo autologous changes;second,TLR2,as an important member of the TLRs family,is closely related to CNS-related diseases;third,the dependent signal transduction pathways mediated by TLRs family members are different.And there are literatures showing that TLRs family members especially TLR2 can expressed on hematopoietic stem cells and mesenchymal stem cells.Since the inflammatory response after TBI plays a key role in the repair of CNS damage,it is of great significance for in-depth discussion of inflammation and immune diseases after trauma.The inflammatory response after TBI usually precedes the expression of pro-inflammatory factors including TNF-α and IL-1β,followed by the expression of adhesion factors including ICAM-1,and finally the expression of antiinflammatory factors.The effects of these inflammatory factors are bidirectional,which is to say that they can promote repair and aggravate damage.To regulate the inflammatory process after TBI,and to improve and to promote the repair function is our lines of inquiry.ObjectiveThrough the establishment of TBI model to simulate the traumatic environment and change the expression of TLR2,the use of Modified Neurological Severity Scores(m NSS),neurological behavior and emotional methods,western blotting,H&E staining,immunofluorescence staining technology,cell proliferation and differentiation labeling,real-time quantitative PCR and other methods,explain the changes in the expression level of TLR2 on the surface of NSCs in the inflammatory microenvironment after TBI,and the influence and the relationship with NSCs,and try to explain the relationship between TLR2 expression changes and regeneration after TBI,and provide data support and theoretical basis for further research on cells and tissue repair after TBI and clinical TBI treatment.Methods1.TBI model establishment: using PCI-3000(precision cortical impactor-3000)device,and establishing a stable rat medium-sized brain injury model based on the data such as dural contact time,dural downward movement distance and trauma speed when contacting with dura.2.m NSS and neurological behavior and emotional test: The changes in neurofunctional and behavioral levels between different groups after trauma were detected by rat m NSS,field test,elevated plus maze and Morris water maze experiments.3.Changes in proliferation cells and TLR2 after TBI: at different time points after TBI,three immunizations with Brd U,TLR2 and DAPI were used to explore the relationship between proliferation cells and TLR2 changes in the inflammatory microenvironment after TBI.4.Expression of key molecules in TLR2 signal transduction pathway: use C29 to inhibit TLR2 expression,and use Cu-T12-9 to promote TLR2 expression,explore MYD88 and NF-κB protein and m RNA expression changes.5.The relationship between proliferating cells and NSCs and the relationship between NSCs and TLR2 expression changes: After the establishment of the TBI model,nestin was used to label NSCs,and the proportion of NSCs in proliferating cells and the expression of newly-generated NSCs after TLR2 expression changes were explored.6.The relationship between NSCs proliferation and differentiation: use different markers to label early,middle,and late neurons and various types of glial cells to explore the differentiation of nerve cells and analyze the relationship with TLR2 expression.Results1.After the successful construction of the medium-sized TBI model,it was found that the scores of the TBI group was significantly higher than that of the sham group,and the m NSS after the increase of TLR2 expression was lower than that of the downregulated TLR2 expression group and the control group.2.In the field test,the total distance and central activity time of the TBI group were less than that of the sham group.After the up-regulated of TLR2 expression,the total distance and central activity time of rats were longer than those of the down-regulated group and the control group.3.In the elevated plus maze,the number and time of entering the open arm in the TBI group were less than those in the sham group.After the up-regulated of TLR2 expression,the above two indexes were higher than those in the down-regulated group and the control group.4.In the Morris water maze,the latent period of the sham group was significantly shorter than that of the TBI group.The latent period of the TLR2 up-regulation group was shorter than that of the down-regulation group and the control group.The platform was removed on the last day and explore the number of crossing times of the platform area.It was found that the number of crossing times in the TBI group was less than that of the sham group.The number of crossing times in up-regulated group increased compared with the down-regulated group and the control group.5.After stimulating the expression of TLR2,the protein and m RNA expression of MYD88 and NF-κB were significantly up-regulated.6.After up-regulating the expression of TLR2,the expression of TLR2 on the surface of proliferating cells was detected 1h-7d after TBI,and it was found that the most TLR2 expression appeared at 3-day post TBI.7.It could be found that NSCs are the main proliferating cells in the hippocampal dentate gyrus during exploring the expression of NSCs in proliferation cells.It could also be found that a large number of TLR2 expressions appeared on the surface of NSCs after stimulating TLR2 expression during exploring the expression of TLR2 on the surface of NSCs.8.The proliferation of NSCs in the dentate gyrus of the hippocampus was detected again 3 days after TBI,and a large number of newly-generated NSCs were expressed.3days-post TBI was selected,different CNS cells were labeled with DCX,MAP-2,Neu N,CNP,GFAP,and IBA1,and nerve cells appeared more after stimulation of TLR2 expression,and the expression level of the early immature neurons was higher than that of the middle and late mature neurons.Conclusion1.In this study,a TBI model of adult rats is successfully established,and behavioral level experiments such as neurofunctional scores,mine experiments,elevated cross experiments,and water maze experiments found that the rats’ exercise ability,learning and memory ability decreased,and and anxiety appeares through the m NSS,field test,elevated plus maze and Morris water maze test.2.The TLR2 agonist CU-T12-9 is used to stimulate the expression of TLR2,and the detection of protein and m RNA expression levels of the MYD88 and NF-κB further confirmes that TLR2 stimulation is effective.3.The TBI model is successfully established and the expression of endogenous NSCs is found to be increased after TLR2 up-regulation.By observing the expression of NSCs at different time points from 1h to 7d after trauma,it is found that the highest expression of NSCs was 3-days after the model establishment,which indicating that TLR2 up-regulation is effective for the proliferation of NSCs and the peak proliferation is at 3-day post trauma.4.After TLR2 up-regulation,neurons and various types of glial cells are labeled with different markers,and it is found that new neurons and various types of glial cells are produced.Among them,the new neurons are mainly early immature neurons.