Ischemic injury to rat forebrain mitochondria and cellular calcium homeostasis

Abstract

The three-vessel occlusion model of Kameyama et al. (Kameyama, M., Suzuki, J., Shirane, R. and Ogawa, A. (1985) Stroke 16, 489–493) was adapted with modifications to induce complete reversible rat forebrain ischemia. A fast and simple procedure for the isolation and purification of rat brain mitochondria, which provides high yield, is described. Mitochondria isolated from ischemic brain (12–30 min ischemia) exhibited decreases in State 3 respiratory rates of approx. 70% with NAD-linked respiratory substrates. Less effect was observed with succinate and rotenone. The State 4 respiratory activity remained near control levels except at 15 min of ischemia (25% increase) with NAD-linked substrates. Similarly, with succinate and rotenone, an approx. 30% increase in State 4 activity was observed at 20 min of ischemia. Consequently, the respiratory control indices (RCIs) were decreased. Both the respiratory rates and RCIs could be restored to near control levels upon the addition of EGTA(EDTA) or ruthenium red to the assay mixture. Analysis employing fura-2 as a Ca 2+ probe, indicated a great decrease in the first order rate constant for Ca 2+ uptake of ischemic mitochondria and a significant increase in Ca 2+ located externally to the inner mitochondrial membrane. These data suggest that ischemia disrupts cellular Ca 2+ homeostasis with an increase in the cytosolic Ca 2+ concentration which results in excessive association of Ca 2+ on the mitochondrial membrane and an inhibition of the respiratory chain-linked oxidative phosphorylation and Ca 2+-transport activity of forebrain mitochondria. These deficits are proportional to the duration of ischemia.