Neuroprotective Role And Mechanism Of Neuregulinl In Traumatic Brain Injury

Traumatic brain injury (TBI) leads to longterm neurological deficits as well as cognitivedysfunction. Primary injury encompass the mechanical damage that occurs at the time oftrauma, and this initial traumatic insult contributes to the secondary injury evolves delayedbiochemical, metabolic and cellular changes. Over the past two decades, various molecularmechanisms of neuronal death after TBI have been dissected by many groups. Thesemechanisms often involve complex combinations of necrosis, apoptosis, necroptosis andautophagy. Defining these pathways offer the hope that effective therapeutic targets can beidentified. However, it is now increasingly recognized that in addition to neuronal death,responses in glial and vascular compartments should also play a key role in the progression ofsecondary injury after TBI.In the neurovascular unit (NVU), all the components interact to each other, builing ananatomical and funcational intergrity. Neuron in the central nervous system is finely regulatedby the surrounding microenvironment, and among this microenvironment, the most importantone is blood brain barrier (BBB). BBB is composed of endothelial cells line the entirevascularture of the brain, together with astrocytes and periytes foot processes.Indeed, when TBI patients are in the intensive care unit, major clinical challenges areoften centered on cerebrovascular instability perhaps related to glial swelling, BBB leakageand disruptions in flow-metabolism regulation. Taken together with emerging data in a widerange of experimental models, it is likely that developing therapies for brain trauma shouldrequire a careful rescue of the entire neurovascular unit, comprising interactions between allcell types in the damaged brain.The NRG1gene is a large and complex gene transcribing more than twenty differenttransmembrane proteins with a single membrane-spanning domain, and generating a largenumber of isoforms in tissue-and cell type–specific patterns. NRG1can be classified as type I,II, or III based on the type of amino-terminal sequence, and further characterized as type α or β according to the characteristics of the epidermal growth factor (EGF)-like domain. Recently,NRG1signaling in brain microvascular endothelial cells has been studied as well and hasbeen shown to play active roles in the function of brain microvascular endothelial cells. Ourprevious data show that co-incubation with NRG1reduces endothelial permeability duringIL-1β induced endothelial injury, and decreases acute BBB permeability in a mouse model ofbrain trauma. Our current experiments further confirm that NRG1ameliorates IL-1β inducedendothelial hyper-permeability, and investigate the pathways through which NRG1influencesendothelial permeability.To further understand the role of NRG1in the recovery phase of the TBI, and themechanisms through which NRG1plays protective function on endotheial cell, this study isdivided into two parts:Part1Neuroprotective role and mechanism of Neuregulin1in recovery phase ofadolescent traumatic brain injury in miceObjective: To investigate the neuroprotective role and the underying mechanism ofexogenous NRG1in recovery phase of adolescent traumatic brain injury. Methods:①Toestablish the cortical contusion injury model in adolescent mice, and randomly separate themice into NRG1treatment group and vehicle treatment group，implant an Alzet pump insubcutaneous to give the infusion of the drug for2weeks.②To evaluate sensorymotorfunction and cognitive founction during the recovery phase of TBI.③By histological andimmunological methods, to compare the pathophysiological changes in brain tissue includingthe contusion volume, neuronal death and changes in white matter. Results：①In therecovery phase of TBI, NRG1treatment group showed improved sensorymotor functiontested by NSS score, and Wire-grip score compared to the vehicle treatment group (p<0.05).②NRG1treatment group showed better learning and memory ability tested by Y Mazecompared to the vehicle treatment group (p<0.05).③NRG1treatment group failed todecrease the contusion volume compared to the vehicle treatment group.④In the ipsilateraland contralateral of the inury site, the number of total neuron showed no difference betweenNRG1and vehicle treatment. However, in the CA3region of the contralateral hippocampus, the number of PV positive interneuron is significantly higher in the NRG1treatment groupversous vehicle treatment group (p<0.05).⑤The NRG1treatment group showed longer andmore entact axon and more preserved myelin structure compared to the vehicle treatmentgroup.Conclusion：①NRG1is beneficial in sensorymotor and cognitive function recovery inrecovery phase of TBI.②NRG1could increase the number of inhibitory interneuron, protectthe white matter intergrity.Part2Protective role and mechanism of Neuregulin1in brain microvescularendothelial cell functionObjective：To study the protective role and mechanism of NRG1in brain microvascularendothelial function, we use IL-1β to injure the endothelial cell, and in the meantimeadminister the NRG1to study its effect. Methods:①when the primary human microvascularendothelial cell or human microvascular endothelial cell line reached confluency in the plate,treat the cells with IL-1β together with or without NRG1for corresponding time period.②Toevaluate the role of NRG1on cell toxicity and valibility by LDH and MTT.③To evaluatethe role of NRG1on endothelial permeability by TEER and Dextran extravasation；and toelucidate the mechanism, comparing the expression of tight junction and adhesive junctionprotein, cell cytoskeleton proteins by Western Blot and immunocytochemistry staining.④Toevaluate the role of NRG1on adhesive activity to the endothelial cells under inflammation byneuprophil adhesion assay; and further, to elucidate the mechanism through comparing theadhesive protein expression by westen blot. Results：①NRG1did not show significantbenefit in cell toxicity and viability.②NRG1decreased IL-1β induced increase in dextranextravasation, and preserved TEER; deactivated RhoA activity, inhibited the phosphorylationof MLC and stabilized the cytoskeleton (p<0.05).③NRG1ameliorated the IL-1β inducedincrease in neuprohil adhesion on endothelial cell; diminished the expression of adhesiveprotein ICAM-1，VCAM-1and E-selectin exression (p<0.05). Conclusion:①NRG1isprotective against IL-1β induced endotheial hyperpermeabiltiy by deactivating RhoA,inhibing MLC phosphorylation and stabilizing cytoskeleton.②NRG1decreases IL-1β induced neutrophil adhesion on endothelial cell by reducing the expression of adhesiveprotein.