The good, the bad and the ugly of nitric oxide, superoxide and peroxynitrite signaling during oxygen‐glucose deprivation in rat and arctic ground squirrel

Abstract

Cerebral ischemia‐reperfusion (I/R) injury initiates a complex cascade of events, several of which involve the generation of nitic oxide (NO) and oxygen (O2•−) free radicals. NO and O2•− rapidly combine to form peroxynitrite (ONOO−) a potent oxidant and nitrating agent. Previous studies have revealed that the arctic ground squirrel (AGS; Urocitellus parryii) is a natural model of high tolerance to I/R injury, however, the mechanisms that contribute to the ability to tolerate this pathological scenario is still elusive. Here, we tested the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrosative stress associated with oxygen glucose deprivation (OGD).Hippocampal slices (400 micron) from rat and AGS were subjected to oxygen glucose deprivation (OGD) to mimic I/R in vivo using a novel microperfusion technique. Injury consisted of exposing the brain slices to: 1) NO and O2•− donors with and without OGD; and 2) pretreatment of slices with inhibitors of NO, O2•− and ONOO− followed by OGD. In control slices, treatment was administered by switching to standard aCSF. Perfusates were collected every 15 min and analyzed for LDH release, an indicator of cell death; slices were analyzed for nitrosative and oxidative damage using the markers 3‐nitrotyrosine (3NT) and 4‐hydroxynonenal (4HNE).Results show the effect of NO, O2•− and ONOO− mediated injury in ischemic tolerant AGS and ischemic susceptible rat. 1) Effect of NO mediated Injury: An NO donor alone is not sufficient to cause cell death, but with OGD enhances cell death more in rat than in AGS; an inhibitor of nNOS attenuates OGD‐induced injury in rat but has no effect in AGS. 2) Effect of O2•− mediated injury: A O2•− donor alone causes slight injury in rat and even less injury in AGS. Combining the O2•− donor with OGD aggravates the injury in rat; a SOD mimetic attenuates OGD injury in rat but has no effect in AGS. 3) Effect of ONOO− mediated injury: A ONOO− inhibitor attenuates OGD injury in rat but has no effect in AGS. Rat also shows higher levels of 3NT and 4HNE after OGD insult than AGS. These data demonstrate that in rat both NO and O2•− are required for injury and point to ONOO− as the damaging agent. This same mechanism of OGD‐induced cell death is lacking in AGS supporting the hypothesis that resistance to oxidative and nitrosative stress contributes to tolerance to cerebral I/R injury in AGS.Support or Funding InformationThis work was supported by the US Army Medical research and Material Command, No 0517800; the National Institute of Neurological Disorder and Stroke, Nos. NS041069‐06 and R15NS070779; Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395.