The Role Of NMN In Hemorrhagic Stroke And The Hemorrhagic Transformation After Ischemic Stroke

BackgroundStroke is an acute cerebrovascular diseases caused by blood circulation disorders,which can be divided into ischemic stroke and hemorrhagic stroke,due to different circulatory disorders reasons.Ischemic stroke is also known as cerebral ischemic,accounting for about 80% of all strokes.Ischemic stroke is a regional cerebral blood disorder caused by a variety of reasons,leading to hypoxic-ischemic brain tissue lesions and necrosis,and a range of neurological disease damage.In the development of ischemic stroke,there is a state of both ischemic stroke and hemorrhagic stroke feature that cerebral ischemia hemorrhagic transformation(HT),in patients with cerebral ischemia,hemorrhagic transformation is associated with increased mortality.Thrombolytic therapy in acute phase of ischemic stroke is the only treatment approved by the US FDA.The most commonly used thrombolytic agents are tissue-type plasminogen activator(t PA),which can effectively improve the survival and prognosis of patients.But the use of thrombolytic treatments is limited,because it may increase the incidence of hemorrhagic transformation.Therefore,research on developing new drugs joint use with t PA to reduce the incidence of hemorrhagic transformation,extending its use of time windows have great potential for clinical application.Intracerebral Hemorrhage is a deadly type of strokes,which have a higher morbidity and mortality,accounting for 10~15% of all strokes.It is reported that mortality is about 40% in one month after the onset of cerebral hemorrhage.The improving of medical care over the past two decades does not reduce the mortality.Reason for the high rate of recurrence and mortality of cerebral hemorrhage due to lacking of effective treatment option,the existing clinical treatments is limited to primary care,such as control of intracranial pressure,treatment of hematoma and maintain hemodynamic stability.Therefore,the study of effective treatment is imperative.Nicotinamide phosphoribosyltransferase,referred Nampt,is the rate-limiting enzyme of NAD synthesis in mammalian cells that controls intracellular NAD concentration.Our previous study on cerebral ischemia in mice found that,Nampt can regulate SIRT1 deacetylation capacity by controlling the NAD synthesis,regulating AMPK upstream kinase LKB1 acetylation and intracellular redistribution,final regulation of AMPK active,exerting neuroprotective effect.In addition,we found that:Nicotinamide mononucleotide(NMN),enzyme product of Nampt,can play a neuroprotective effect in a rat MCAO model induced by suture method by intraventricular administration,and improve animal neurological function,including reducing cerebral infarction volume,neurological deficit and cell death in penumbra.In mouse MCAO reperfusion induced by suture method,NMN intraperitoneal injection once a day,continuous administration for more than 1 week,can significantly reduce stroke mortality,promote nerve regeneration and functional recovery.We further validate the role of NMN in ischemic stroke by different animal models and administration methods closer to clinical.We successfully constructed the MCAO hemorrhagic transformation model and intracerebral hemorrhage model,and explored the role of NMN in both models.Methods1.The methods of study in MCAO model and MCAO hemorrhagictransformation model(1)Cut off the middle cerebral artery by electric shock,to build electric coagulation MCAO model;(2)Blocked the middle cerebral artery by suture,to build suture MCAO model;(3)TTC staining and serial sectioning method were used to calculate the infarct volume;(4)t PA was administrated 5 hours after pMCAO to induce MCAO hemorrhagic transformation model;(5)QuantiChromTMhemoglobin assay kit was used to detect brain tissue hemoglobin.2.The methods of study in intracerebral hemorrhage model(1)Cerebral hemorrhage model was induced by stereotaxicion of 0.05 U collagenase IV into the striatum.(2)Beam walking experiments was used to evaluate mouse neurological function;(3)Serial sectioning method was used to calculate the volume of hematoma;(4)Real-time PCR technology was used to detect mRNA levels of inflammatory cytokines;(5)Immunohistochemical staining was used to detect the expression of inflammatory cytokines,chemokines and NOX1,and the activation of inflammatory cell;(6)TUNEL staining was used to detect cell death;(7)The activity of MDA,H2O2,CuZn/Mn-SOD and ABTSwere detected to evaluate Level of oxidative stress.Results1.NMN can reduce the cerebral infarct volume of rat electric coagulation MCAO model by intravenous administration.2.NMN can reduce the cerebral infarct volume of mouse suture MCAO model by pre-dosed or acute administration.3.Toxicity tests of NMN show that it has no significant acute toxicity.4.We successfully constructed the MCAO hemorrhage transformation model.5.NMN can alleviatespontaneous hemorrhagic transformation and t PA-induced hemorrhagic transformation after ischemic stroke.6.We successfully constructed the collagenase-induced ICH model(cICH).7.NMN can improve neurological function of cICH model mouse by intravenous administration in acute phase.8.NMN can relieve brain edema of cICH model mouse by intravenous administration in acute phase.9.NMN can reduce the expression of inflammatory cytokines(TNF-a,IL-6)and chemokines(Icam-1)in hematoma and surrounding tissue by intravenous administration in acute phase.10.NMN can reduce the activation of microglia and the infiltration of neutrophil in hematoma and surrounding tissue by intravenous administration in acute phase.11.NMN can reduce cell death in hematoma and surrounding tissue by intravenous administration in acute phase.12.NMN can reduce the activity of lipid oxidation and hydrogen peroxide and the expression of NOX1 in hematoma and surrounding tissue by intravenous administration in acute phase.13.NMN can promote the recovery of neurological function and weight of cICH model mouse by long-term administration.In summary,this study successfully constructed hemorrhagic transformation of cerebral infarction and cerebral hemorrhage model,and found the beneficial effects of NMN in model of ischemic stroke,hemorrhagic transformation of cerebral infarction and intracerebral hemorrhage.