Normoxic resuscitation after cardiac arrest protects against hippocampal oxidative stress,metabolic failure,and neuronal death

Background

Animal models of global cerebral ischemia and reperfusion indicate that neurologic outcome is improved using normoxic compared to hyperoxic resuscitation; however the molecular basis for this improvement is unknown. This study tested the hypothesis that normoxic ventilation (21% O2) during the first hour after 10 min cardiac arrest in dogs protects against oxidative protein tyrosine nitration and loss of the critical metabolic enzyme complex pyruvate dehydrogenase (PDHC) in the hippocampus, an area selectively vulnerable to delayed neuronal cell death. The study also tested the hypothesis that normoxic resuscitation also improves oxidative cerebral energy metabolism early during reperfusion and protects against delayed neuronal cell death.

Methods

Chloralose-anesthetized adult beagles underwent 10 min ventricular fibrillation cardiac arrest, followed by electrical defibrillation and ventilation with either 21% or 100% O2. At 1 hr post-resuscitation, the ventilator was adjusted to maintain normal blood gas levels in both groups. Brains were perfusion-fixed at 2 hr reperfusion and used for semi-quantitative immunohistochemical measurements of nitrotyrosine and pyruvate dehydrogenase (E1 alpha subunit). In other animals, mitochondria were isolated from the hippocampi of unfixed brains. Pyruvate dehydrogenase activity was measured spectrophotometrically and pyruvate-dependent mitochondrial respiration measured polarographically. Other animals were provided with constant critical care for 24 hr, then perfusion fixed for quantitative neuropathology using stereologic analysis of hippocampal neuronal cell death based on morphological changes apparent with cresyl violet stained tissue.

Results

In hyperoxic ventilated dogs, E1 alpha immunostaining diminished by approximately 90% in CA1, CA3, and dentate gyrus compared to sham-operated dogs, while neuronal PDHC staining in normoxic animals was not significantly different from non-ischemic dogs. Protein nitration in hippocampal pyramidal neurons of hyperoxic animals was 2–3 times greater than either sham-operated or normoxic resuscitated animals at 2 hr reperfusion. In 2 hr hyperoxic animals, hippocampal mitochondrial PDHC enzyme activity was 35% less, and ADP-stimulated respiration was 50% less than non-ischemic controls, whereas these activities in normoxic animals were not significantly different from controls. Stereologic quantification of neuronal death at 24 hr reperfusion demonstrated a significant reduction in the percentage of dying neurons ± s.d. using normoxic compared to hyperoxic resuscitation (CA1: 32 ± 8% vs. 48 ± 2; CA3: 27 ± 6% vs. 49 ± 3%; p<0.05).

Conclusions

These results indicate that postischemic hyperoxic ventilation promotes oxidative stress that exacerbates prelethal loss of pyruvate dehydrogenase, respiratory dysfunction, and delayed hippocampal neuronal cell death. Moreover, these findings indicate the need for clinical trials comparing the effects of low and high ventilatory oxygen levels on neurologic outcome after cardiac arrest.