The Role Of MGluR5in Traumatic Brain Injury And Its Relationship To Homer1a

As a leading cause of morbidity and disability, traumatic brain injury (TBI) resultsin a series of physiological and pathological changes, leading to progressive neuronaldegeneration and long-term functional deficits. Although the mechanisms leading toneuronal injury after TBI have not been fully understood, excessive release of glutamateand activation of its receptors are considered as one of the important factors. In thecentral nervous system (CNS), metabotropic glutamate receptor (mGluR), with limiteddistribution and side effects, has multiple regulatory activities, and is considered to beone of the ideal approaches for the treatment of neurological diseases, including TBI.mGluR regulates intracellular signaling transduction through combining with G protein,and plays an important role in neuronal excitability regulating, presynaptic inhibition andneurodegeneration. mGluRs can be divided into3groups: group I mGluRs includemGluR1and5; group II mGluRs include mGluR2and3; group III mGluRsincludemGluR4,6,7and8. Group I mGluRs are highly expressed in postsynapticdensity (PSD) and lead to phosphoinositide (PI) hydrolysis and intracellular calcium overload via activating phospholipase C (PLC). However, group II and III mGluRshave no close relationship with adenyl cyclase. Previous studies have demonstrated thatthe activation of group II and III mGluRs aggravates neuronal injury after TBI, while therole of group I mGluRs (especially mGluR5) in traumatic neuronal injury and thepotential mechanism are disputable. Homer1a protein, which belongs to the morerecently discovered postsynaptic scaffolding proteins Homer, is the product of animmediately early gene (IEG), and is shown to regulate intracellular signalingtransduction via binding to group I mGluRs. The aim of this study is to investigate therole of mGluR5in an in vitro TBI model in primary cultured crotical neurons, as well asthe potential mechanisms with focus on Homer1a protein.Part ⅠMechanical neuronal injury model in cultured cortical neuronsObjective: In order to mimic traumatic brain injury in vitro, we established aneffective and reliable mechanical neuronal injury model in cultured rat cortical neurons.Methods:Cortical neurons were cultured from Sprague-Dawley rats, and culture mediumwas changed every other day. Traumatic neuronal injury was induced by scratchingneurons with10μl pipettor at7days later. The lactate dehydrogenase (LDH) release intoculture medium was measured to dectect the neuronal injury. Results:After mechanicalinjury, LDH in culture medium was significantly increased and peaked at12and24hafter injury. Conclusion: Our in vitro TBI model could cause obvious neuronal injuryas determined by increased LDH release. This experimental model, easily being operatedand highly succussed, is an effective neuronal injury model, which provide a reliablemodel to investigate molecular mechanism.Part II Closed head injury model in miceObjective: To establish a reliable and stable closed brain injury model in mice,laying the foundation for further study of mechanism after injury. Methods:TBI wasinduced by a closed brain injury model in mice as mentioned by Flierl. Brain watercontent and TUNEL staining were used to measure neuronal injury after TBI. Results:Brain water content was obviously increased at12h after TBI, and lasted for more than24h. TUNEL staining confirmed that TBI caused apoptotic cell death at12h after injury in cortical neurons. Conclusion: Our in vivo model was a sinple and effective model ofTBI in mice as measured by increased brain water content and obvious apoptosis incortical neirons.Part Ⅲ Expression of mGluR5and Homerla afterTBIObjective: To detect the expression of mGluR5and Homer1a after TBI in vitro andin vivo. Methods: Wester blot was used to detect the expression levels of mGluR5protein and Homer1a protein after TBI. Results: The expression of mGluR5protein incultured cortical neurons started to increase at1h after TBI and peaked at12h. Theexpression of Homer1a in cultured cortical neurons increased at6h after TBI, and theincrease lasted for more than24h. The expression of mGluR5started to increase at1hafter TBI in vivo, peaked at12h and lasted for more than24h. The expression ofHomer1a started to increase at3h after TBI in vivo, peaked at12h and lasted for morethan48h. Conclusion: The expression levels of mGluR5and Homer1a proteins aresignificantly increased after TBI, and they probably play important roles in neuronalinjury after TBI.Part Ⅳ Protective effects of mGluR5agonists against TBIObjective: To investigate the neuroprotective effects of mGluR5specific agonistson TBI and the potential mechanism. Methods: LDH release and caspase-3activityassay were used to examine the neuroprotection of (R,S)-2-chloro-5–hydroxy–phenylgcine (CHPG) and3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide(CDPPB), two mGluR5specific agonists, after mechanical neuronal injury; Westernblot was used to detect the expression changes of extracellular signal-regulatedkinase(ERK),c-jun amino acid terminal kinase (JNK), and p38and elucidate thepotential mechanisms underlying neuroprotection of CHPG and CDPPB. Results:LDH release and caspase-3activity were increased after mechanical neuronal injury.Pretreatment with CHPG and CDPPB reduced the elevation of LDH release andcaspase-3activity in a dose-dependent manner. By western blot analysis, pretreatmentwith CHPG and CDPPB significantly increased the phosphorylation of ERK, but didnot affect the phosphorylation of JNK and p38. Conclusion: CHPG and CDPPB two mGluR5agonists, have protective effects on mechanical neuronal injurythroughregulating ERK pathway.Part Ⅴ Effects of mGluR5on Homer1a expression after TBI in vivo and vitroObjective: To investigate the effects of mGluR5agonist-dependent activity andagonist-independent activity on expression level of Homer1a protein after TBI. Methods:Cortical neurons were pretreated with the mGluR5inhibitors,2-methyl-6-phenyl ethynylpyrimidine (MPEP) and α-methyl-4-carboxyphenyl glycine (MCPG) for1h, and injuredby mechanical injury model. Mice were pretreated with MPEP or MCPG for1h andsuffered by TBI in vivo. The expression levels of Homer1a protein in vitro and in vivowere detected by Western blot. Results: Homer1a expression was decreased afterpreatment of two different dose MPEP, while MCPG was not effective. Conclusion:The expression of Homer1a after TBI is partly dependent on the agonist-independentactivation of mGluR5.