Intermittent hypoxia conditioning protects mitochondrial cytochrome c oxidase of rat cerebellum from ethanol withdrawal stress

Abstract

Intermittent hypoxia (IH) conditioning minimizes neurocognitive impairment and stabilizes brain mitochondrial integrity during ethanol withdrawal (EW) in rats, but the mitoprotective mechanism is unclear. We investigated whether IH conditioning protects a key mitochondrial enzyme, cytochrome c oxidase (COX), from EW stress by inhibiting mitochondrially directed apoptotic pathways involving cytochrome c, Bax, or phosphor-P38 (pP38). Male rats completed two cycles of a 4-wk ethanol diet (6.5%) and 3 wk of EW. An IH program consisting of 5-10 bouts of 5-8 min of mild hypoxia (9.5-10% inspired O(2)) and 4 min of reoxygenation for 20 consecutive days began 3 days before the first EW period. For some animals, vitamin E replaced IH conditioning to test the contributions of antioxidant mechanisms to IH's mitoprotection. During the second EW, cerebellar-related motor function was evaluated by measuring latency of fall from a rotating rod (Rotarod test). After the second EW, COX activity in cerebellar mitochondria was measured by spectrophotometry, and COX, cytochrome c, Bax, and pP38 content were analyzed by immunoblot. Mitochondrial protein oxidation was detected by measuring carbonyl contents and by immunochemistry. Earlier IH conditioning prevented motor impairment, COX inactivation, depletion of COX subunit 4, protein carbonylation, and P38 phosphorylation during EW. IH did not prevent cytochrome c depletion during EW, and Bax content was unaffected by EW ± IH. Vitamin E treatment recapitulated IH protection of COX, and P38 inhibition attenuated protein oxidation during EW. Thus IH protects COX and improves cerebellar function during EW by limiting P38-dependent oxidative damage.

Fig. 1.

Ethanol intoxication-withdrawal program. Young adult male rats completed two cycles of consuming control dextrin or 6.5% ethanol diet for 4 wk, followed by 3 wk of ethanol withdrawal (EW). Intermittant hypoxia (IH) conditioning was applied during the last 3 days of the first ethanol diet and the first 17 days of subsequent EW. Rats were tested for overt signs of acute EW 24 h after the termination of the second ethanol diet, and recovered for 6 days before undergoing 5 days of Rotarod testing. Rats were killed at 3 wk of the second EW period, and cerebelli were harvested for biochemical analyses.

Fig. 2.

Behavioral signs of EW. Rats were evaluated for overt signs of EW 24 h after termination of the second ethanol diet. Behavioral signs of acute EW were attenuated by antecedent IH conditioning. Values are means ± SE for 7–10 rats/group. *Significant difference vs. respective dextrin group (P < 0.005). †Significant difference vs. EW (P = 0.006).

Fig. 3.

Motoric capacity. Motor function was evaluated by Rotarod testing on days 9–11 of the second EW or the corresponding postdextrin period. Rats were tested three times (three trials) per day with a 10-min interval between trials. EW rats fell from a rotating rod more quickly than dextrin controls (*P = 0.047). IH conditioning during the first EW period prevented motoric impairment during the second EW period (†P = 0.016 vs. EW). Values are means ± SE for 7–10 rats/group.

Fig. 4.

Cytochrome c oxidase content. COX activity (left) and COX subunit IV protein content (right) were measured by spectrophotometry and immunoblot, respectively, in cerebelli obtained at 3 wk of the second EW period. Loading control for immunoblots was β-actin. Values are means ± SE for 4–6 rats/group. *Significant difference vs. dextrin control (P < 0.005). †Significant difference vs. EW (P < 0.01).

Fig. 5.

Cytochrome c content. Mitochondria were isolated from cerebelli harvested at 3 wk of the second EW period for immunoblot measurements of cytochrome c content. Values are means ± SE for 6 rats/group. Voltage-dependent anion channel1 (VDAC1) was used as a positive loading control. *Significant difference vs. dextrin (P < 0.005).

Fig. 6.

Mitochondrial protein carbonylation. Mitochondria were isolated at 3 wk of the second EW period. DNPH derivatization was carried out to measure protein carbonyl contents. Values are means ± SE for 7–10 rats/group. *Significant difference vs. dextrin (P < 0.001). †Significant difference vs. EW (P = 0.02).

Fig. 7.

Vitamin E and COX activity. Vitamin E (275 mg·kg⁻¹·day⁻¹) was administered by gavage the last 3 days of the first ethanol diet and the first 17 days of the first EW period. COX was extracted from cerebelli taken from rats killed at 3 wk of the second EW period, and its activity was measured by a colorimetric assay. Values are means ± SE of 7–10 rats/group. Significant difference vs. dextrin: *P = 0.026; **P < 0.001. †Significant difference vs. EW (P = 0.002).

Fig. 8.

P38 phosphorylation. Phosphorylated P38 (pP38) was extracted from cerebelli taken from rats killed at 3 wk of the second EW period and detected by immunoblot. Values (means ± SE, 3/group) are expressed as percentages of the mean value of the dextrin control group. Loading control was β-actin. *Significant difference vs. dextrin (P < 0.01).

Fig. 9.

P38 and protein carbonyls in HT22 cells. HT22 cells were subjected to two cycles of 24 h of ethanol (50 or 100 mM) exposure + 4 h of ethanol washout. P38 inhibitor SB203580 (200 nM) or vehicle was added at the beginning of the first ethanol washout period; 4 h later, mitochondria were isolated to measure protein carbonyl content. Values are means ± SE of 4 experiments/group. *Significant difference vs. 0 mM ethanol (P < 0.05). †Significant difference vs. vehicle (P < 0.001).