Hypothermia-induced neuroprotection is associated with reduced mitochondrial membrane permeability in a swine model of cardiac arrest

Abstract

Increasing evidence has shown that mild hypothermia is neuroprotective for comatose patients resuscitated from cardiac arrest, but the mechanism of this protection is not fully understood. The aim of this study was to determine whether prolonged whole-body mild hypothermia inhibits mitochondrial membrane permeability (MMP) in the cerebral cortex after return of spontaneous circulation (ROSC). Thirty-seven inbred Chinese Wuzhishan minipigs were successfully resuscitated after 8 minutes of untreated ventricular fibrillation (VF) and underwent recovery under normothermic (NT) or prolonged whole-body mild hypothermic (HT; 33°C) conditions for 24 or 72 hours. Cerebral samples from the frontal cortex were collected at 24 and 72 hours after ROSC. Mitochondria were isolated by differential centrifugation. At 24 hours, relative to NT, HT was associated with reductions in opening of the mitochondrial permeability transition pore, release of pro-apoptotic substances from mitochondria, caspase 3 cleavage, apoptosis, and neurologic deficit scores, as well as increases in mitochondrial membrane potential and mitochondrial respiration. Together, these findings suggest that mild hypothermia inhibits ischemia-induced increases in MMP, which may provide neuroprotection against cerebral injury after cardiac arrest.

Figure 1

Mitochondrial permeability transition at 24 and 72 hours after ROSC. Data represent the mean±s.d. of five to seven independent experiments. HT, mild hypothermia; NT, normothermia; SC, surgery control; ROSC, restoration of spontaneous circulation.

Figure 2

Effect of mild hypothermia on mitochondrial membrane potential in frontal cortex of pigs, assessed by the red/green JC-1 fluorescence in NT and HT as a percentage of SC. *P<0.05 compared with SC; ^#P<0.05 compared with NT24h. HT, mild hypothermia; NT, normothermia; SC, surgery control.

Figure 3

Parameters of mitochondrial respiration in frontal cortex of pigs, including state 3 respiration (A), state 4 respiration (B), respiratory control ratio (RCR; state 3/state 4 respiration) (C), and ADP/O ratio (D). Glutamate (5 mmol/L) and malate (5 mmol/L) were used as respiration substrates. Oxygen consumption is expressed in nmol O₂/min per mg protein (mean±s.d.). *P<0.05 compared with SC; ^#P<0.01 compared with NT24h. HT, mild hypothermia; NT, normothermia; SC, surgery control.

Figure 4

Expression of cytoplasmic cytochrome c, Smac, cleaved caspase 3, and nuclear AIF in frontal cortex of pigs. Western blotting was used to measure cytoplasmic cytochrome c, Smac, and cleaved caspase 3 (A), and nuclear AIF (B). Western blots of Porin (C) showed bands only within the mitochondrial fractions, confirming successful separation of mitochondria. The optical density (OD) of each band was measured using Image J software. Values of cytoplasmic cytochrome c (Cyto c) (D), Smac (E), and cleaved caspase 3 (F) were normalized to GAPDH. Nuclear AIF (G) was normalized to lamin B2. Data are expressed as the mean±s.d. Group sizes were SC, n=5; NT24h, n=6; HT24h, n=7; NT72h, n=5; and HT72h, n=7. *P<0.05 compared with SC; ^#P<0.01 compared with NT24h. AIF, apoptosis inducing factor; HT, mild hypothermia; NT, normothermia; SC, surgery control.

Figure 5

TUNEL (TUNEL, deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) analysis of apoptosis in frontal cortex of pigs in SC (A), NT (B), and HT (C) groups and apoptosis index (D). Magnification, × 200. *P<0.05 compared with SC; ^#P<0.01 compared with NT24h. HT, mild hypothermia; NT, normothermia; SC, surgery control.

Figure 6

Neurologic deficit scores. Scale range is from 0 (normal) to 400 (brain death). Values are given as median±interquartile range. *P<0.01 compared with NT24h; ^#P<0.01 compared with NT72h. HT, mild hypothermia group; NT, normothermia group; SC, surgery control.