Melatonin Attenuate White Matter Injury Via Reducing Oligodendrocyte Apoptosis After Subarachnoid Hemorrhage In Mice

ObjectiveSubarachnoid hemorrhage(SAH)is a common disease in clinic caused by rupture of aneurysm with high morbidity and mortality.Recent theory holds that early brain injury(EBI)is the most promising target for treatment of SAH.EBI include neuronal death,surrounding apoptosis,microcirculation dysfunction,blood brain barrier(BBB)disruption,edema and other pathophysiological mechanisms in the early stage of subarachnoid hemorrhage.In human brain,approximately 50% tissue is white matter(WM)which contain neuronal axon and oligodendrocyte,but acute WM injury has been scarcely studied in experimental SAH.Currently,several clinical and experimental studies indicate WM may be involved in neurological outcome after SAH.Therefore,to alleviate white matter injury after SAH aim to improve neurological deficits,it is critical to develop novelty therapies.Preceding investigation indicated inflammation and apoptosis appeared early and paly critical role in neurological deficits after subarachnoid hemorrhage(2).NOD-like receptor family pyrin domain-containing 3(NLRP3)has been considered as key factor in both bacterial and aseptic inflammation during pathophysiological processes,by activated downstream caspase-1/Il-1?(25).Recently,melatonin(MLT)was found to protect and prevent neurological deterioration by reducing inflammation in preclinical experiment.MLT was amphipathic and easily to pass through blood-brain barrier(BBB),we wonder if MLT could affect the WM injury and attenuate early brain injury.Herein,we investigate the MLT in neurological impairment and pathophysiological changes of WM in mouse model of experimental SAH.We hypothesized that MLT may attenuate WM injury via preventing NLRP3 associated microglia activation and oligodendrocyte apoptosis,and sought to find more rigorous and reliable data to support this postulation.Materials and MethodsMale C57 BL / 6J mice weighing 22 G to 30 g were randomly divided into 6 groups:sham operation,SAH,SAH + vehicle,SAH + mlt50 mg / kg,SAH + mlt150 mg / kg and SAH + mlt300 / kg.Among the existing experimental methods of SAH model in mice,the intravascular puncture model is currently recognized to be the closest to the clinical symptoms of subarachnoid hemorrhage in patients.1% Pentobarbital Sodium(its concentration is 50 mg / kg)was injected intraperitoneally to anesthetize mice,and the SAH model was made by intravascular puncture.In the sham operation group,the thread bolt was inserted but the blood vessel was not punctured,and the rest was the same as the operation group.At 15 minutes after SAH,MLT was injected intraperitoneally at concentrations of 50 mg / kg,150 mg / kg and 300 mg / kg,respectively.The neurological scores were using the protocol of Modified Garcia score and Beam balance score at 24,48 and 72 hours post SAH,which have been previously described.Two blinded observers were employed for evaluating the mean of neurologic score.24 hours after SAH,the mice were killed to make brain sections,and the brain tissues were stained with Luxol fast blue staining(LFB staining)to observe the pathological changes of white matter after SAH.Mice were sacrificed at 24 hours and 72 hours after SAH,the brain specimens(n=6)were carefully remove from the skull and quickly cut into bilateral hemispheres,cerebellum and brain stem.Four separate parts were weighed on aluminum foil for wet weight.After drying at 57 ? for 48 hours,they were weighed with dry weight.The brain water content was measured by dry wet method to evaluate the degree of brain edema after SAH.Evans blue leakage evaluation BBB,gelatinase spectrum detection MMP9 activity;making brain tissue frozen section,make immunofluorescence staining and WB,observe the degradation of myelin,amyloid precursor protein(AMP The accumulation of protein,APP),apoptosis of oligodendrocytes and the expression of NLRP3,caspase-1,IL-1-?,Bcl-2 and Bim.ResultsWe revealed 50mg/kg MLT could improve neurological score,alleviate brain edema and reduce WM injury,at the same time,it can alleviate the permeability change of BBB.In addition,loss of myelin basic protein(MBP)and accumulation of amyloid precursor protein(APP),expressions in ipsilateral/left hemisphere were found after subarachnoid hemorrhage,and were reversed by MLT treatment.NLRP3 signal was activated in microglia and protein expression of caspase-1 and IL-1 ? increased.NLRP3 signal activation were found in microglia and apoptosis of oligodendrocytes were observed post SAH.MLT reducing oligodendrocytes apoptosis by regulating Bim and Bcl-2.ConclusionResults of the present study demonstrated that MLT attenuated EBI after SAH via NLRP3-induced apoptosis of oligodendrocytes.MLT exhibited the ability to cross the BBB in the central nervous system.therefore,it may be potentially beneficial to individuals who experience SAH.