The Anti-neuroinflammation Effects And Mechanisms Of Methylene Blue In Early Brain Injury After Subarachnoid Hemorrhage

BackgroundSubarachnoid Hemorrhage,also called SAH,is a fatal form of stroke which is very common in the clinic.A ruptured aneurysm is responsible for about 85%of SAH.Patients who survive the acute phase of SAH usually suffer from chronic neurological deficits,which cause a heavy burden to the families and society.In a long time in the past,delayed cerebral vasospasm was regarded as the major cause of the poor outcome of SAH;however,as clinical trials using anti-vasospastic drugs failed to improve the outcomes of SAH patients,early brain injury(EBI)in the first 72 h after SAH has now became the research focus of mitigating the effect of SAH.The pathological mechanisms of EBI are complex,including the direct stimulation of the blood,sharp increase of intracranial pressure,decrease of cerebral perfusion,decrease of cerebral blood flow and so on.These phenomenons after SAH induce the activation of a complex cell signaling network downstream,and finally induce neurological deficits.The severity of EBI is directly related with the neurological outcome of clinical patients and animal models.It is critical to explore the mechanisms of EBI,especially neuroinflammation,and discover effective pharmacotherapies targeting the neuroinflammatory process in managing SAH.Methylene blue(MB)is an older drug which was used for treating methemoglobinemia and cyanide poisoning for several decades.It can readily penetrate the blood brain barrier(BBB)and concentrate in ’brain tissue after administration.So many researchers have used methylene blue to cure experimental central nervous system(CNS)injury,and found it’s neuroprotective role in neurodegenerative diseases,ischemic brain injury and traumatic brain injury.However,there is no study about whether MB could exerts a neuroprotective role after SAH.Recently,Chen et al.discovered the role of myocyte enhancer factor 2D(MEF2D)in the neuroprotective effect of MB in a glutamate-induced toxicity cell model and also found that MB potentiates Akt and downregulates the MEF2 inhibitor GSK-3β,which suggested that the Akt/GSK-3β signaling pathway could mediate the expression of MEF2D.Another recent study demonstrated that MEF2D could regulate anti-inflammatory cytokine IL-10 secretion in microglia to protect neuronal cells from neuroinflammation.Thus in this study,we use endovascular filament puncture model to create SAH rat models,explore the neuroprotective role of MB in EBI after SAH,and MB’s anti-neuroinflammation role through the Akt/GSK-3b/MEF2D pathway.We hope this study could provide new targets for SAH management.MethodsPart 1SD rats were randomly divided into four groups:the sham group,sham + MB group,SAH + vehicle group and SAH + MB group.The motality,neurological scores and SAH scores were recorded.Using the wet-dry weighting method to measure the water content of the left hemisphere tissue,as the representative of the extent of brain edema.Using Evans blue dye extravasation method to measure the damage of blood bra:in barrier(BBB).Real time PCR was used to measure the mRNA level of TNF-a and western blot was used to measure the protein level of TNF-α,IL-1β and IL-6.Part 2SD rats were randomly divided into seven groups:sham,SAH 3 h,SAH 6 h,SAH 12 h,SAH 24 h,SAH 48 h,and SAH 72 h.The motality was recorded.Western blot was used to measure the protein levels of MEF2D in each group of this part and the level of MEF2D in nuclear and cytoplasm in part 1’s samples.Real time PCR and western blot was used to measure the mRNA level of IL-10 and immunofluorescence staining was used to observe the localization of MEF2D and IL-10 in neuron,microglia and astrocyte.Part 3SD rats were randomly divided into four groups:SAH + vehicle group,SAH + MB group,SAH + MK2206 group and SAH + MB + MK2206 group.The motality,neurological scores and SAH scores were recorded.Western blot was used to measure the phosphorylation levels of Akt and GSK-3β in this part and part 1’s samples.immunofluorescence staining was used to observe the co-localization of Akt and MEF2D.The number of IL-10,Iba-1 and MPO-positive cells were counted.Real time PCR was used to measure the mRNA level of IL-10 and TNF-α,and western blot was used to measure the protein level of IL-10,TNF-α,IL-1β and IL-6.ResultsPart 124 h after SAH induction,the neurological scores was significantly decreased.The brain water content of the left hemisphere was significantly higher,and the Evans blue level of the left hemisphere was significantly increased in the SAH + vehicle group.The level of proinflammatory cytokines TNF-α,IL-1β and IL-6 were significantly increased.Administration of 1.5 mg/kg MB in total could improve the neurological scores significantly,decrease the brain water content and Evans blue dye extravasation of the left hemisphere after SAH.MB could also suppress the expression of proinflammatory cytokines TNF-α,IL-1β and IL-6.Part 2Western blot showed that the MEF2D levels were significantly decreased at 6 h and 12 h and reached a nadir at 24 h.After that,the MEF2D expression returned to baseline levels at 48 h and 72 h.Besides,SAH could significantly decrease nuclear MEF2D levels at 24 h,with a significant increase of it in cytosol.Immunofluorescence staining also showed the MEF2D was expressed in the cytosol of a few MEF2D-positive cells in the cortical area of SAH rats at 24 h,while these phenomena were rarely observed in the sham rats.MB administration could not only increase nuclear MEF2D levels but also cytosolic levels of MEF2D.Double immunostaining of MEF2D and IL-10 with different cell biomarkers in the SAH 24 h group suggested that MB was expressed in neurons while IL-10 was mainly expressed in neurons and microglia.The IL-10 level was increased 24 h after SAH,and MB could further augmented IL-10 mRNA and protein expression.Part 3Double immunofluorescent staining revealed co-localization of Akt and MEF2D.24 h after SAH,the level of pSer473-Akt and pSer9-GSK-3β were significantly decreased.MB could reverse these changes.When managed with Akt inhibitor MK2206,the neurological scores of the rats were significantly decreased,the phosphorylation of Akt and GSK-3β and the protein level of MEF2D in nuclear and cytosol were also significantly decreased.MB administration could significantly reduce the number of MPO-positive cells and Iba-1-positive cells.MK2206 reversed these changes.Conclusion1.MB can be a neuroprotective agent after experimental SAH through alleviating brain edema,suppressing blood brain barrier disruption and inhibiting neuroinflammation.2.MB can increase the level of MEF2D in nuclear,and promote the expression of IL-10,thus to play a neuroprotective role against neuroinflammation after SAH.3.These effects of MB may modulate by the Akt/GSK-3β pathway.