Effects Of Metabolic Substrates And Oxidative Stress On ATP Production In Brain Tissues During And After Ischemic/hypoxic Injuries

Ischemia and hypoxic cerebrovascular disease is one of the most serious diseases of human health.In ischemia,the decreased ATP production can not adequately support vital cellular functions,which leads to a series of downstream damages and cell death.Ischemia and hypoxia can cause irreversible neural damage,but the exact mechanism remains unclear.Defining the core factors of the injuries is important for reducing injury and increasing recovery.Purposes In this study,we used brain tissues and cultured astrocytes under OGD and other conditions to simulate hypoxia and ischemia in vivo.Brain slices and optic nerve of C57 mice were used as gray matter and white matter,respectively.The effects of oxygen,metabolic substrates and microenvironment on ATP production were investigated in vitro to reveal the effects of metabolic substrates and oxidative stress on ATP production during cerebral ischemia,hypoxia and reperfusion.Methods A model of ischemia,hypoxia and reperfusion was established using the optic nerves and hippocampus slices.ATP contents in brain tissues under various treatments were measured.The effect of O2 on ATP levels was investigated by changing the oxygen levels with and without normal glucose supply.Phamacological intervention(mercaptosuccinic acid)was used to investigate the effect of endogenous oxidative stress on ATP production in brain tissues during and after hypoxia and ischemia.Astrocytes were cultured and stimulated by H2O2 with quantitative analysis of ATP to investigate the effect of oxidative stress on astrocyte ATP production.Results In this study,ATP contents in brain tissues were measured to represent the ATP production,we found that:(1)The best treatment window for brain tissues in vitro was 40~240min after preparation,and the age of mice most suitable for brain slice experiments were 1-3 months old.(2)The changes of ATP production in the white matter(optic nerve)after ischemia,anoxia and reperfusion were consistent with those of the gray matter(hippocampus slice).During ischemia and hypoxia,ATP production decreased significantly,to about 1%~6% of basal control levels.Once reperfused with oxygen and glucose,ATP production partially recovered,to approximately 15%~25% of the control value.(3)Oxygen and glucose deprivation of the brain tissue both reduced ATP production.with oxygen deprivation being more effective than glucose deprivation,indicating that oxygen supply is more important to energy production than glucose.(4)With the addition of SDT under ischemia and hypoxia,the ability of the brain tissues to produce ATP was further reduced,and the recovery in reperfusion was also affected.(5)In ischemia and hypoxia,addition of glutamate increased the production of ATP in brain tissues and cultured cells..(6)Glutathione peroxidase inhibitors decreased the ability of ATP production in brain tissues.Conclusions Glucose and oxygen deprivation plays an important role in brain damage under hypoxia,ischemia and oxidative stress.The damage from oxygen deprivation is greater than that of glucose deprivation.Glutamate,an important excitatory neurotransmitter in the brain,can be oxidatively metabolized in the three carboxylic acid cycle to synthesize ATP.The glutamate content in tissues and cells may play an important role in ameliorating ischemia and hypoxia injury.In the absence of glucose at least in white matter,glutamate functioning as metabolism substrate may overweight its role in excitatory neural injury.Oxidative stress is the main cause of reperfusion injury,and reducing oxidative stress during reperfusion is important to the treatment of hypoxic-ischemic brain damage.