The Role Of Mitochondria In Neonatal Mouse Cerebral Hypoxia-ischemia Brain Injury

IntroductionHypoxic ischemic encephalopathy(HIE)is the most important cause of morbidity and mortality of newborns in all over the world although antenatal and neonatal cares have been improved in recent years.Neonatal HIE is not a rare condition and is seen approximately in 2-3/1000 live births.Moreover,in the developing countries,its frequency has risen up to 26/1000 live births.About15-20%of these patients usually dies in neonatal intensive care units(NICU).Among the remaining alive newborns,cerebral palsy(10-20%),visual and auditory problems(about 40%),as well as motor and behavioral damages,such as epilepsy,global developmental delay,and autism are diagnosed.However,these treatments are not successful in all cases,and hypothermia is not suitable for very preterm infants,thus there is a pressing need for a better understanding of the mechanisms of brain injury and for conducting translational studies on how to reduce cell death,increase cell survival,and promote brain regeneration and repair after perinatal brain injury.In the pathogenesis of hypoxia-ischemia,time of injury and timing of treatment play important roles.In the primary phase,a partial healing process starts in the first 60 min after the acute insult of the injury.In the latent phase of the injury(in between first to sixth hours),oxidative metabolism,inflammation,and continuation of the activated apoptotic cascades take place.Six to 48 h after an HI injury,depletion of phosphate reserves,and release of excitatory neurotransmitters and free radicals occur in the second energy failure phase.In the tertiary phase months after the acute ischemia,late cell death,remodeling of the injured brain,and astrogliosis occur.Mitochondria are pluripotent organelles with multiple cellular functions,including the regulation of physiological metabolism,stress responses,and cell death,and they play an important role in the process of brain injury after insult.Mitochondria are key regulators of apoptotic cell death,and following an insult,mitochondria are permeabilized and cell death-related proteins are released from the mitochondria into the cytosol,including cytochrome c(Cyt c)and apoptosis-inducing factor(AIF),which,respectively,triggers caspase-dependent and caspase-independent apoptotic cell death.These processes are more prominent in the immature brain than in the adult brain,and interventions targeting mitochondria have shown promising results against immature brain injury.Autophagy-which is activated by many forms of stress-is an important physiological mechanism for degrading long-lived cytosolic protein complexes and aggregates and is the only known pathway for degrading organelles.Autophagy recycles amino acids and fatty acids to produce energy and removes damaged organelles.Thus,basal autophagy plays an essential role in cell survival.However inappropriate activation of autophagy can be directly involved in mediating cell death or can trigger the execution of apoptotic or necrotic cell death.Overall,the purpose of this study is characterizing the autophagy/mitochondrial in the development of immature brain injury in different ways through the clinical and animals studies.And find a useful therapeutic strategy to improve neurological outcomes in perinatal asphyxia-induced brain injury.Part ?:Haploinsufficiency in the mitochondrial protein CHCHD4 reduces brain injury in a mouse model of neonatal hypoxia-ischemiaBackgroundMitochondria contribute to neonatal hypoxic-ischemic brain injury by releasing potentially toxic proteins into the cytosol.CHCHD4 is a mitochondrial intermembrane space protein that plays a major role in the import of intermembrane proteins and physically interacts with apoptosis-inducing factor(AIF).The purpose of this study was to investigate the impact of CHCHD4 haploinsufficiency on mitochondrial function and brain injury after cerebral hypoxia-ischemia(HI)in neonatal mice.MethodsCHCHD4+/-and wild-type littermate mouse pups were subjected to unilateral cerebral HI on postnatal day 9.CHCHD4 haploinsufficiency reduced insult-related AIF and superoxide dismutase 2 releases from the mitochondria and reduced neuronal cell death.ResultsThe total brain injury volume was reduced by 21.5%at 3 days and by 31.3%at 4 weeks after HI in CHCHD4+/-mice.However,CHCHD4 haploinsufficiency had no influence on mitochondrial biogenesis,fusion,or fission;neural stem cell proliferation;or neural progenitor cell differentiation.There were no significant changes in the expression or distribution of p53 protein or p53 pathway-related genes under physiological conditions or after HI.ConclusionsThese results suggest that CHCHD4 haploinsufficiency afforded persistent neuroprotection related to reduce the release of mitochondrial intermembrane space proteins.The CHCHD4-dependent import pathway might thus be a potential therapeutic target for preventing or treating neonatal brain injury.Part ?:Dichloroacetate treatment improves mitochondrial metabolism and reduces brain injury in neonatal miceBackgroundThe purpose of this study was to evaluate the effect of dichloroacetate(DCA)treatment for brain injury in neonatal mice after hypoxia ischemia(HI)and the possible molecular mechanisms behind this effect.MethodsPostnatal day 9 male mouse pups were subjected to unilateral HI,DCA was injected intraperitoneally immediately after HI,and an additional two doses were administered at 24 h intervals.The pups were sacrificed 72 h after HI.ResultsBrain injury,as indicated by infarction volume,was reduced by 54.2%from 10.8± 1.9 mm3 in the vehicle-only control group to 5.0 ± 1.0 mm3 in the DCA-treated group at 72 h after HI(p = 0.008).DCA treatment also significantly reduced subcortical white matter injury as indicated by myelin basic protein staining(p =0.018).Apoptotic cell death in the cortex,as indicated by counting the cells that were positive for apoptosis-inducing factor(p = 0.018)and active caspase-3(p = 0.021),was significantly reduced after DCA treatment.The pyruvate dehydrogenase activity and the amount of acetyl-CoA in mitochondria was significantly higher after DCA treatment and HI(p = 0.039,p = 0.024).ConclusionsIn conclusion,DCA treatment reduced neonatal mouse brain injury after HI,and this appears to be related to the elevated activation of pyruvate dehydrogenase and subsequent increase in mitochondrial metabolism as well as reduced apoptotic cell death.Part ?:Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injuryBackgroundPerinatal asphyxia induces neuronal cell death and brain injury,and is often associated with irreversible neurological deficits in children.There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia(HI).MethodsPostnatal day 9(P9)Atg7 KO and WT mice were subjected to unilateral HI,and used to determine the effects of genetically inhibited autophagy on neuronal cell death and brain injury.Moreover,in order to determine whether autophagy could also be involved in human HIE brain damage,we investigated markers of autophagy in autopsied brain tissues of human term newborns who died from severe asphyxia with HIE focusing on the basal ganglia,a part of the brain highly susceptible to HI injury.ResultsNeuronal deletion of Atg7 prevented HI-induced autophagy;resulted in 42%decrease of tissue loss compared to wild-type mice after the insult,and reduced cell death in multiple brain regions,including apoptosis,as shown by decreased caspase-dependent and-independent cell death.Moreover,we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains,as indicated by the numbers of MAP1LC3B/LC3B(microtubule-associated protein 1 light chain 3)-,LAMP1(lysosomal-associated membrane protein 1)-,and CTSD(cathepsin D)-positive cells.ConclusionsThese findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.