The Role Of NKCC1and Its Correlation With Homer1a After Traumatic Brain Injury In Mouse

Traumatic brain injury (TBI) is one of the main reasons to cause disability amongpeople.The brain tissue go through primary brain injury and secondary brain injury aftertraumatic brain injury. Secondary brain injury plays very important role in traumaticbrain injury. Recent studies have proved that lipid oxidation medium release, cellapoptosis and free radical production are involved in the pathological mechanisms ofsecondary brain damage after TBI. A lot of key molecules and interactions are related tothe mechanisms of TBI.NKCC1, a kind of water channel in cell membrane regulate cell volume and playsvery important role in maintaining homeostasis.Recent studies have shown that NKCC1regulates a lot of cellular and molecular functions in brain diseases. However, the role ofNKCC1in traumatic brain injury remains unclear. Homer1a is an immediate early gene(IEG) and plays vital role in traumatic brain injury Alzheimer’s and Parkinson’sdisease and so on. Moreover, the latest studies shown that Homer1a plays a protectiverole in traumatic brain injury. Therefore, this study was to investigate the role of NKCC1in traumatic brain injury and the correlation with Homer1a.Part IMechanical injured model of mice cortical culturesObjective:In order to mimic the in vitro traumatic brain injury, we have established anreliable and effective mechanical injury model of mice cortical cultures.Methords:Themechanical injury model was achieved by using a steriled21-gauge plastic needle to drawparallel scratches on the cultured neurons.After that, morphological changes, lactatedehydrogenase (LDH) release and TUNEL assay was used to elucidate the mechanicalinjury effects.Results: The neuronal nucleus was large and clear with elliptical or roundoutline.The nucleolus and membrane of nucleus could be easily found by the usage ofmicroscope.The LDH level was notablely increased after mechanical injury and theTUNEL-positive cells was increased at24hours after mechanical injury.Conclusion:This mechanical injury model of cortical neurons in mice is simple andeffective for thetraumatic brain injury study in vitro.Part2: Closed head injury model of mouce induced by a weight-drop deviceObjective:In order to futher study thepathophysiological changes and the mechanisms oftraumatic brain injury in mouce,we establish a simple but reliable traumatic brain injurymodel.Methords:We exemine the changes in brain water content of mice after the closedhead injury. TUNEL assay and the HE staining was used to clarify the braindamge.Results:The normal mouse brain tissue of HE staining showed that the cellstructure in cortical and hippocampal is clear and tightly packed. Neuronal cell bodies arecomplete and in normal morphology. But there are many degenerated glial and neuronscells at the edge of the damage area and the number of neurons is reduced after aftertraumatic brain injury. Some nucleoli of neurons are swelling, tightly stained. Somenuclear condensation and fragmentation could be found in damage area. A lot ofTUNEL-postive cells can be seen in the damage area surrounding cortical tissue aftertraumatic brain injury. Brain water content was notablely increased at12h after traumatic brain injury.Conclusion: This closed head injury model of mouse induced by aweight-drop device is simple but effective model to mimic traumatic brain injury.Part3:NKCC1and Homer1a expression after traumatic brain injury in vitro and invivo.Objective:To investigate the expression of NKCC1and Homer1a after traumatic braininjury in vivo and in vitro.Methords: We studied the changes of NKCC1and Homer1aprotein using western blotting and immunohistochemistry in traumatic brain injurymodel.we investigated the mRNA of NKCC1by using polymerase chain reaction.Results:NKCC1was significantly increased6h after after traumatic brain injury andpeaked at12h.The expression of Homer1a expression was significantly increased6hafter traumatic brain injury and peaked at12h. Double immunofluorescence stainingshowed that NKCC1and Homer1a are co-expressed in neurons12h after brain damage.Conclusion:The protein level of NKCC1and Homer1a were increased aftertraumaticbrain injury, suggesting that NKCC1and Homer1a may play vital roles in traumatic braininjury.Part4: Effects of NKCC1on Homer1a and ERK expression after traumatic braininjury in vivo and in vitro.Objective:In this experiment, we studied the effect of NKCC1on Homer1a andERKexpression after traumatic brain injury in vivo and in vitro.Methods: Westernblotanalysis of the expression of Homer1a and p-ERK in mice with Bumetanideinhibitor ofNKCC1or transfected with si-RNA of NKCC1after traumatic brain injury in vivo and invitro. We exemine the changes in brain water content of mice after the treatment ofBumetanide.Results:Comparing with the empty vector, inhibite of NKCC1cansignificantly increase the expression of Homer1a but reduce the expression of p-ERKafter traumatic brain injury in vivo and in vitro. Using the inbitor or si-RNA of NKCC1,we found that inhibit the expression of NKCC1and reduce the brainedema.Conclusion:Theses results suggest that NKCC1may play a harmful role intraumatic brain injury.