Arctic ground squirrel resist peroxynitrite-mediated cell death in response to oxygen glucose deprivation

Abstract

Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O₂^•-) to form peroxynitrite (ONOO^-), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400µm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, O₂^.- donors with and without OGD; pretreatment with inhibitors of NO, O₂^.- and ONOO^- followed by OGD. Perfusates collected every 15min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/O₂^.- alone is not sufficient to cause ischemic-like cell death, but with OGD enhances cell death more in rat than in AGS. A NOS inhibitor, SOD mimetic and ONOO^- inhibitor attenuates OGD injury in rat but has no effect in AGS. Rats also show a higher level of 3NT and 4HNE with OGD than AGS suggesting the greater level of injury in rat is via formation of ONOO^-.

Keywords: 3-nitrotyrosine; Hippocampal brain slices; Ischemia-reperfusion; Lactate dehydrogenase release; Nitric oxide; Oxygen glucose deprivation; Peroxynitrite; Superoxide.

Figure 1. AGS resist nitric oxide mediated LDH release

(a, d) Slices from rats and AGS were subjected to 30 min of aCSF, OGD, and OGD + NOSI. The time course of LDH release is shown. ^*p< 0.05 (aCSF vs. OGD), ^#p<0.05 (OGD vs. OGD + NOSI). (b, e) Total LDH (the sum of LDH released from the start of insult to the end of the experiment) is shown in a and b. ^*p< 0.05 vs. aCSF, ⁺p<0.05 vs. NO (10 μM), ^#p<0.05 vs. OGD + NOSI. (c, f) Total LDH release from additional experiments in which NO donor was included with OGD. *p< 0.05 vs. OGD. Grey bar indicates insult period.

Figure 2. AGS show better tolerance to O₂^•− mediated cell death

(a, c) Acute hippocampal slices from both rat and AGS were subjected to different concentrations of O₂^•− donor (HX-XO: 0, 15, 30, 45 mU/ml) and LDH release over a course of time was monitored. *p< 0.05 (0mU/ml vs. 45mU/ml), ⁺p<0.05 (0mU/ml vs. 30mU/ml). (b, d) Time course of LDH release was monitored in rat and AGS slices exposed to different concentrations of O₂^•−donor (HX-XO: 0, 15, 30, 45 mU/ml) along with OGD. ^*p< 0.05 (OGD+0mU/ml vs. OGD+45mU/ml), ⁺p<0.05 (OGD+0mU/ml vs. OGD+30mU/ml). (e, f) Acute hippocampal slices from rat and AGS pretreated with SOD mimetic (Tempol) followed by OGD. *p<0.05 (OGD vs OGD+Tempol (3mM)), ⁺p<0.05 (OGD vs OGD+Tempol (0.03mM)) (g) shows total 4-HNE–Protein adduct formation in rat and AGS slices exposed to OGD *p< 0.05 vs. aCSF.

Figure 3

AGS tolerate peroxynitrite mediated OGD injury. (a, b) rat and AGS slices were preincubated with FetMpyp (peroxynitrite decomposition catalyst) and LDH release was monitored. *p< 0.05 vs. OGD. (c, d) 3-NT was measured in rat and AGS slices subjected to NO donor along with OGD. *p< 0.05 vs. OGD, +p< 0.05 vs. aCSF. (e, f) 3-NT was measured in rat and AGS slices when subjected to O₂^•− donor along with OGD. *p< 0.05 vs. OGD.