Primary Research Of TSPO And Its Ligand Etifoxine After Intraventricular Hemorrhage

BackgroundIntracerebral hemorrhage(ICH),is subtype of stroke with high incidence,ranking second in all kind of stroke.There are approximately two million new cases of ICH a year in all the world.Intracerebral hemorrhage onset acute and progress fast with quite relatively disabling and fatality rate,and it is indeed a great threat to human health.However,there is no proven effective medical therapeutic measures for ICH exists.The mechanisms of ICH-related brain injury include primary injury which hematoma directly cause and consequent secondary injury.Recent studies indicate that neuro-inflmmation is a significant part during the progress of injury after ICH.Inflammatory response occurs rapidly once intracerebral hemorrhage happen,and the mechanisms involve resident microglia activation and infiltration of peripheral leukocyte,companied by the production of various inflammatory mediator and cytokines.The relationship between them is complicated.Thus,gain a better understand of inflammation and find out safe and effective therapeutic measures are really important.TSPO,namely translocator protein 18 k Da,used to be called peripheral benzodiazepine receptor.TSPO distribute widely throughout the body.Under the normal condition of brain,TSPO are mainly located in gila cell with low expression.When the brain is insulted by ischemia,hypoxia,mechanical injury and neurotoxicity,with activation of gila,TSPO is up-regulated sensitively and shortly.Therefore,the expression of TSPO has been regarded as a marker of neuro-inflammation.For this reason,imaging expression of TSPO ligand labeled by radioactive element with PET has been developed as a potentially clinically applicable tool for in vivo imaging of the patterns of neuroinflammation.TSPO PET imaging holds promise both for increasing understanding of the role of neuroinflammation in brain diseases and as a tool for assessing the efficacy of novel therapies.Up to now,there is no report about the application of TSPO ligand in ICH.In addition,TSPO owns a lot of physiological functions and most important is transport cholesterol.As rate-limit step,TSPO regulate neurosteroids synthesis.Endogenous and exogenous ligands have been showed the effect of promotion of neurosteroids synthesis and alleviate the neuroinflammation.Etifoxine,one of the TSPO ligands,is anxiolytic drug.In the animal model of multiple sclerosis,Etifoxine exert the neuroprotection by restrain the immune cell,reduce the cytokine levels,promote the oligodendroglial regeneration and improve the neurological deficits.The exact mechanism is still unclear yet.Our study established the experimental intracerebral hemorrhage model in mice.The first part focus on the primary study in the expression of TSPO aft er ICH.The second part is to investigate the potential neuroprotective effect of Etifoxine after ICH.Part 1 Primary research of the expression of TSPO after ICHObjective By establishing the experimental intracerebral hemorrhage model in mice,to investigate the expression of TSPO in peri-hematoma tissue and its location.Methods Kunming mouse were randomized into sham group,15 ?l-hematoma group,25?l-hematoma group.MRI scan and neurological deficit score are tested in 1d after ICH.Collect the brain tissue of the three groups mentioned above and 25 ?l-hematoma group's in 12 h,1d,3d,5d after ICH to test the expression of TSPO by Western Blot.Collect the brain slice of sham group and 25 ?l-hematoma group's in 12 h,1d,3d,5d after ICH,to test the expression of TSPO on microglia and astroglia by immunofluorescence.Results1.MRI shows we successfully establish the experimental intracerebral hemorrhage model in mice.25 ?l-hematoma group scores more than 15?l-hematoma group in neurological deficit score test in 1d after ICH.2.Western Blot indicates that,both groups express much more than sham group while 25?l-hematoma group expression more than 15 ?l-hematoma group.There is no evident increase expression of 25 ?l-hematoma group in 12 h after ICH,but it increase gradually in 1d,3d to 5d after ICH.3.Immunofluorescence shows that the expression of TSPO is obvious after ICH and it mainly located on the activated microglia and astroglia.ConclusionTSPO shows up-regulated expression after ICH and the expression level maybe is related with time after ICH and hematoma volume.TSPO mainly located on the activated microglia and astroglia suggest that is may involve in the neuroinflammation after ICH.Part 2 Primary research about neuroprotection of TSPO ligand Etifoxine after ICHObjective By establishing the experimental intracerebral hemorrhage model in mice,to investigate potential neuroprotection of Etifoxine after ICH.Methods1.Kunming mouse were randomized into sham group,ICH group,ICH+EFX group.ICH+EFX group get first intraperitoneal injection of Etifoxine(50mg/kg)within 6h after ICH,and then they get daily injection till 5d.Sham group and ICH group will get equal volume solvent as control.2.Weigh and record before ICH each group.Weigh and record before daily injection each group.3.At 1d,3d,5d after ICH,the neurological deficit score were measured in each group.4.At 5d after ICH,the brain tissue water contend and blood–brain barrier permeability were measured in each group.5.Test TNF-? and IL-1? level in serum in each group by ELISA at 5d.Conclusion1.The mouse ICH model cause mice weigh loss,acute edema,blood–brain barrier permeability damage,cytokine level increase and neurological deficits.2.Etifoxine relieve the weight loss and brain edema of mice after ICH,reduce the level of IL-1? in serum,and improve the neurodeficit scores while it is not evident in protection of blood–brain barrier permeability.ConclusionTSPO ligand Etifoxine maybe a neruoprotection after ICH.Etifoxine can alleviate weight loss,brain edema,cytokine level and neurological deficit score.It may be one choice of future comprehensive therapy in intracerebral hemorrhage.