Effects And Mechanisms Of Oxiracetam On Hypoxic-ischemic Brain Damage In Neonatal Mice

BackgroundHypoxic-ischemic encephalopathy(HIE)is a hypoxic-ischemic(HI)injury caused by perinatal asphyxia,which is a key cause of neonatal death and permanent neurological dysfunction.Studies have shown that 1.5-4 out of an average of 1,000 live births have the disease,of which 15-25%of the sick will die in their newborns,while 20-30%of survivors will suffer(hypoxic-ischemic brain injury damage,HIBD)and accompanied by permanent neurological dysfunction disorders,such as epilepsy,intellectual disability and cerebral palsy etc..It brings severe physical and mental pressure to the sick children,and also causes heavy economic burden to the families of sick children.At present,clinical treatment mainly includes hyperbaric oxygen,sub-low temperature,dilatation of cerebrovascular and anti-free radicals,etc.,to delay or inhibit the damage and destruction of neurons caused by HIBD.However,the best treatment time window for these treatments is short,and secondary injury of ischemia-reperfusion is prone to occur after cerebral blood flow recanalization,so the therapeutic effect is not ideal.Therefore,it is urgent to comprehensive and deeply understand the pathogenesis of HIBD and find new treatment strategies.After HIBD,secondary injury causes the further development and deterioration of the disease,especially the overactivation of inflammation response,which leads to the gradual enlargement of the infarcts in the acute phase of HIBD,In the ischemic penumbra surrounding the infarct core,direct injury of HIBD is not sufficient to cause complete tissue necrosis.The histological changes were mainly shown as the apoptosis of neurons and the activation of microglia cells in penumbra.In addition,with the continuation of secondary injury,the penumbra tissue size develops gradually,and there is a significant risk of deterioration and the merger of infarcted cores.It has been one of the greatest challenges in neurology to avoid the deterioration of ischemic penumbra and restore the integrity of the tissue.As the"protagonist" of the inflammatory response,microglia play a "double-edged sword"effect in HIBD.It can promote tissue regeneration and recovery in the early stages of inflammation response.However,in the middle and later stages of the inflammation response,due to the over-activation of microglia caused by ischemia and hypoxia,and secretes a large number of pro-inflammatory factors such as IL-1 beta,TNF-alpha,IL-6,etc.These inflammatory factors will further cause the apoptosis of neurons,and further aggravate the primary injury.In HIBD,microglia not only play the role of inflammation,but also exhibit autophagy,which is closely related to the inflammatory response.Studies have shown that moderate autophagy has a positive effect on the repair of damaged cells,but autophagy reaches a certain threshold,cells will die.Therefore,how to retain the beneficial role of microglia in inflammation,and inhibit its overactivation,as well as how to establish a beneficial method to intervene the activation level of autophagy,is the key to the success of HIBD clinical treatment strategy.Oxiracetam(ORC)can selectively act on the hippocampus and cerebral cortex,can improve symptoms such as chronic brain functional defects and cerebral hypoxia,and restore damaged neurological functions.It is a neurotrophic drug.ORC can cross the blood-brain barrier,activate glycolysis during brain ischemia,improve the utilization efficiency of sugar and energy metabolism of brain tissues,and promote the recovery of nerve function of cerebral cortex.Some clinical practices have shown that ORC has achieved excellent effects in areas such as Alzheimer's disease,cerebral infarction and stroke.It is mainly applied in brain injury,neurological dysfunction,memory and intelligence insufficiency.Moreover,ORC has no central excitability and no direct vascular activity,which indicates that its side effects are small and safe.In recent years,it has been reported that ORC can be used in the clinical treatment of HIE,to inhibit the secretion lever of inflammatory factors,inhibit the apoptosis of nerve cells,reduce cerebral infarction,alleviate neurological function defects,and restore cognitive functions.However,the pathologic and molecular mechanisms of its treatment of HIBD are still unclear.In particular,what role ORC plays in inflammatory response and autophagy secondary to brain injury and how it affects the interaction between activated microglia and apoptotic neurons have not been reported.In addition,whether ORC can alleviate or reverse ischemic penumbra injury remains needs further study.ObjectiveThe HIBD model was established by Rice-Vannucci method to explore the effects and mechanism of ORC in nerve damage after HIBD in newborn mice.Methods1.Establishment of modelRice-Vannucci method was used to establish HIBD model to simulate HIE of human newborn:Take the Kunming mouse on the 7th day after birth to build the HIBD model.The mice were anesthetized with 2%isoflurane by inhalation,and the sterilized skin was fixed.After the skin was cut in the middle of the neck,the right common carotid artery was removed,and the wound was ligated with 4-0 suture line,and the skin was sterilized again.After the operation,the mice had a rest for 1h.The mice were placed in a constant temperature closed anoxic chamber at 37?,and a mixture of 8%oxygen and 92%nitrogen were injected,which was anoxic for 90min.The histopathological changes that develop under these conditions are similar to those in human newborns.If there is significant edema in the right brain three days after HIBD,the model is successfully established.The mice in the sham group were anesthetized and exposed to the right common carotid artery without ligation or hypoxia.2.The experimental group and the intervention methods7d newborn Kunming mice were randomly divided into 4 groups:control group,physiological saline(Sham),model group and physiological saline(HIBD),model group+low-dose ORC protection group(HIBD+1mg/kg ORC),model group+high-dose ORC protection group(HIBD+10mg/kg ORC),after the operation 6h was given the first drug treatment given 6 h after the surgery,then respectively in 24 h,48 h,72 h after HIBD abdominal delivery at a time.3.Detection methodBrain tissues of newborn mice were harvested 72 hours after HIBD,and the effects of ORC on autophagy,apoptosis and inflammatory factors in the damaged side of HIBD(right)cortex were detected by Reverse transcription-polymerase chain reaction(RT-PCR),Luminex,Immunofluorescence and Western blot.The effect of ORC on autophagosomes after HIBD was detected by Transmission electron microscope(TEM).The motor coordination and nerve reflexes ability were measured by righting reflex,negative geotaxis reflex and cliff avoidance reflex.Results1.Brain tissue injury1.1.Changes in water content of brain tissue3 days after HIBD,compared with the Sham group,the right hemisphere of mice in HIBD group showed significant edema,and the water content increased significantly;After ORC treatment,the degree of edema and the water content of brain tissue were significantly reduced.Moreover,the efficacy of ORC is dose-dependent.1.2.TTC stainingCompared with the Sham group,the HIBD group showed significant cerebral infarction area,while the ORC treatment group significantly reduced the area of cerebral infarction.1.3.Nissl stainingBrain tissue on the right side of the Sham group was clear and complete.Compared with Sham group,HIBD group suffered severe loss and obvious damage on the right side.and the right brain tissue in the ORC treatment group was significantly improved compared to the HIBD group.1.4.MRICompared with Sham group,the T2 image signal of infarction in HIBD group was higher,while T2 image signal of ORC group was lower.2.Behavioral test2.1.Righting reflexCompared with the Sham group,the time of landing on all fours in the HIBD group after turning over was prolonged.The time spent on all fours in the ORC group was significantly lower than that in the HIBD group.2.2.Negative geotaxis reflexCompared with Sham group,the time for mice to turn 180 degrees and start climbing was prolonged in HIBD group.The reflection time of ORC group was significantly shorter than that of HIBD group.2.3.Cliff avoidance reflexCompared with Sham group,the cliff avoidance time is longer stayed away from the table for 1cm.Compared with the HIBD group,the cliff avoidance time of the ORC treatment group was significantly reduced3.Apoptosis indicators3.1.TUNEL stainingThe number of TUNEL positive cells in the HIBD group was significantly higher than that in the Sham group.Compared with the HIBD group,the number of TUNEL positive cells in the ORC group was significantly reduced.3.2.Western blot resultsCompared with Sham group,the ratio of the Cleaved caspase-3/beta-actin in the HIBD group was significantly increased.Compared with the HIBD group,the ratio of Cleaved caspase-3/beta-actin in the ORC group was significantly reduced.3.3.Cleaved caspase-3/NeuN immunofluorescence stainingA significant increase in the number of Cleaved caspase-3/NeuN-positive cells in the HIBD group compared to the Sham group Compared with the HIBD group,the ORC group significantly reduced the number of Cleaved caspase-3/NeuN positive cells.4.Microglial cell indicators4.1.Western blot detection of microglia proliferationThe expression level of Iba1 in the HIBD group was significantly higher than that in the Sham group.After treatment with ORC,the expression level of Iba1 was decreased.4.2.Immunofluorescence staining with Cleaved caspase-3/Iba1The number of Cleaved caspase-3/Iba1 positive cells in the HIBD group was significantly higher than that in the Sham group.Compared with the HIBD group,the number of Cleaved caspase-3/Iba1 positive cells in the ORC group was significantly reduced,the ischemic penumbra was also significantly reduced,and the phagocytosis of microglia was significantly enhanced.4.3.Luminex detection of inflammatory mediatorsCompared with Sham group,the secretion of pro-inflammatory factors IL-1 beta,TNF-alpha,and IL-6 were significantly increased in HIBD group.The dose-dependent levels of proinflammatory factors induced by HIBD were reversed in the ORC group.The anti-inflammatory factors IL-4 and IL-10 showed an increasing trend after HIBD,and their secretion levels were enhanced after treatment with ORC.5.Autophagy indicators5.1.RT-PCRThe level of mRNA expression of LC3-B,Atg5 and Beclin-1 were increased in HIBD group compared with Sham group.The mRNA expression level was further increased after treatment with ORC.5.2.Beclin-1/Iba1 immunofluorescence stainingCompared with Sham group,the number of Beclin-1 positive cells in HIBD group significantly increased,and the number of Iba1 positive cells significantly increased.Compared with the HIBD group,the number of beclin-1 positive cells were further increased in the ORC group and the number of Iba1 positive cells was significantly decreased.LC3-B/Iba1 immunofluorescence staining showed the same results as Beclin-1/Iba1 immunofluorescence staining.5.3.Electron microscopy examination of autophagosomesCompared with Sham group,the number of autophagosomes increased.The ORC group further increased the number of autophagosomes.6.In vitro test6.1.CCK-8 experimentalCompared with the negative control group(NC),the proliferation activity of microglia BV-2 increased after OGD treatment,and the proliferation activity of HT-22 neurons decreased.After the addition of ORC,the activity of BV-2 cells was decreased in a dose-dependent manner and the value-added activity of HT-22 cells was increased.The proliferation activity of HT-22 cells was further increased after ORC was added and treated with supernatant of BV-2 cells.6.2.In vitro LuminexAfter OGD treatment,the inflammatory factors IL-1 beta,TNF-alpha,IL-6,IL-4,IL-10 have all increased.After ORC treatment,OGD induced BV-2 cell secretion of pro-inflammatory cytokines such as IL-1 beta,TNF-alpha and IL-6 significantly reduced,and anti-inflammatory cytokines such as IL-4,IL-10 were further increased,which was consistent with in vivo experiments.Conclusions:1.In the acute phase of HIBD,ORC can reduce cerebral edema,reduce the area of cerebral infarction and reduce brain damage.2.ORC effectively improves the behavioral defects in mice after HIBD.3.ORC can inhibit the apoptosis of neurons induced by HIBD.4.ORC can inhibit the proliferation of microglia,regulate the secretion characteristics of microglia,and translate the neurotoxic effect of microglia into neuroprotective effect,increase the phagocytosis of microglia,and reduce the ischemic penumbra.5.ORC can enhance the autophagy of cells.6.ORC can better protect neurons by regulating microglia.