Effects Of Translocator Protein Ligand Ro5-4864 On Inflammatory Response In Early Brain Injury After Subarachnoid Hemorrhage

Subarachnoid hemorrhage(SAH),a type of cerebral hemorrhagic stroke,has a high mortality and disability rate,which greatly threatens human health.Although the prognosis of SAH has improved with the continuous advancement of surgical interventions,there are still no effective therapeutic interventions.In addition to the primary damage caused by hemorrhage,injury mechanism includes secondary damage such as autophagy,inflammation,edema and blood-brain barrier destruction.In recent years,emerging evidence suggests that therapeutic agents targeting early brain injury(EBI)which caused by SAH within 72 hours,is neuroprotective under standardized basic research conditions.Multiple mechanisms,including inflammatory response are key contributors to neuronal damage after SAH.Inflammatory reactions occur rapidly after SAH,mainly involving microglia and astrocyte activation,peripheral inflammatory cell infiltration,and a large number of inflammatory factors.Since the involved nerve cells and cytokines regulate each other and play their respective roles,the study of neuroinflammation after SAH can help to understand the development of diseases after SAH and to guide the development of treatment plans.Previous studies have shown that TSPO can regulate the activation of inflammatory cells,which in turn affects the progression of inflammation.Translocator protein(TSPO)is a subunit of the previous peripheral benzodiazepine receptor and is widely distributed in body tissues.The subcellular structure of TSPO is mainly located on the outer membrane of mitochondria,and a small part is located on the perinuclear and cell surface,which plays an important role in various physiological processes of cells.It has been previously discovered that when brain tissue is exposed to a range of pathological environments,TSPO expression levels will be significantly up-regulated,and this phenomenon has been confirmed in a variety of disease models.So TSPO has been much used as a marker of inflammatory response in central nervous system diseases,combined with molecular imaging techniques.At the same time,TSPO and its ligands have shown great potential in the treatment of neurological diseases.However,there is a lack of related studies on TSPO expression and ligands after SAH.In addition,TSPO is involved in a variety of physiological processes,the most important of which is the transport of cholesterol substances,as a rate-limiting step in the process,can significantly regulate the synthesis of neurosteroids.Previous studies have demonstrated that TSPO,when combined with specific ligands,promotes steroidogenesis,relieves neuropathic pain,and reduces neuroinflammatory responses,and is therefore considered a potential therapeutic target.Ro5-4864(7-chloro-5-4-Chlo rophenyl-1,3-dihydro-1-methyl-2-H-1,4-benzod-iazepin-2)is a derivative of benzodiazepines.It has been confirmed in a variety of neurotoxic pathology experiments that neuroprotection can be exerted by regulating the synthesis of neurosteroid hormones.However,whether there is neuroprotective effect and possible mechanism of Ro5-4864 after SAH has not been reported.In this experiment,an experimental SAH animal model was established in mice.The first part will explore the expression of TSPO during EBI after SAH.The second part will initially study the effect of Ro5-4864 on inflammatory response in early brain injury after SAH to provide an experimental study for clinical treatment of SAH.I.Preliminary study on the early expression of TSPO after SAHObjective: To evaluate the temporal and spatial characteristics of TSPO expression in early brain tissue after SAH and its relationship with EBI.Methods: Forty-four C57BL/6J mice were randomly divided into sham group and SAH group.The SAH model was constructed by intravascular puncture.The neurological function of the mice was evaluated by modified Garcia score. The brain was detected by western blot after EBI.Tissue TSPO expression,PET-CT and immunofluorescence were used to evaluate TSPO spatial expression.Results: Compared with the sham group,the neurological function of the SAH group was significantly reduced.Western blot results showed that TSPO in brain tissue began to increase gradually 6 hours after SAH,and peaked at 48hours(P<0.05),and 72 hours was still higher than sham group.PET-CT showed that the expression of TSPO in the brain was higher than that in the sham group after SAH(P<0.05).The expression of TSPO in the right brain of SAH group was slightly higher than that on the left side(P>0.05).Immunofluorescence showed increased expression of TSPO in the parietal cortex,hippocampus and basal cortex of the hemorrhagic side.Pearson correlation test results showed that the neurological function score was negatively correlated with TSPO expression level(r=-0.6156,P<0.01).Conclusion: The expression of TSPO is widely increased after SAH and is time-dependent,which may be related to the occurrence and development of early brain injury.II.Effect of Ro5-4864 on inflammatory response in early brain injury after subarachnoid hemorrhage.Objective: To explore the effect of Ro5-4864 on the inflammatory response in early brain injury by observing the neurological deficit of mice after constructing subarachnoid hemorrhage modelsMethods: Twenty-seven C57BL/6J mice were randomly divided into sham operation group(sham group),subarachnoid hemorrhage group(SAH+DMSO group)and subarachnoid hemorrhage drug treatment group(SAH+Ro5-4864group).SAH+Ro5-4864 completed the first dose 30 minutes after model establishment,and was intraperitoneally injected with Ro5-4864(5 mg/kg,1%DMSO saline),re-administered 24 hours after modeling.And mice of sham group and SAH+DMSO group were given same volume 1% DMSO saline as a control.The body weight and neurological function scores were recorded before and after model establishment.The water content of brain tissue was measured by dry and wet weight method.The expression of TNF-? and IL-1?were determined by quantitative PCR and ELISA.Results: SAH can cause weight loss in mice,causing neuroinflammatory reactions and motor neurological dysfunction.Ro5-4864 alleviated the loss of body weight after SAH,reduced the water content of brain tissue and decreased the expression of TNF-?,which promoted the recovery of nerve function to a certain extent,but the effect on the expression of IL-1? was not obvious.Conclusion: TSPO ligand Ro5-4864 has a certain neuroprotective function,which can alleviate the weight loss and the increase of inflammatory factors in brain tissue after SAH in mice,and improve the neurological deficit.This could provide an experimental study for the comprehensive treatment of SAH.