Discrimination of degrees of mitochondrial damage: Progression of mitochondrial disruption with time of recovery. (A) Graded degrees of mitochondrial structural disruption: Discriminating criteria and representative images. The table illustrates the spectrum of mitochondrial morphologies as described. To be rated as intact, mitochondria were generally elongated and had intact outer membrane and clear abundant cristae. Mitochondria were rated as showing mild damage if they showed evidence of early swelling and some loss of cristae but with an intact outer membrane (blue arrows highlight sparse cristae), and were considered to be severely damaged if there was apparent disruption of the outer membrane (red arrows); virtually all mitochondria with outer membrane rupture showed no discernible cristae. Such severely damaged mitochondria also displayed a spectrum of damage, ranging from early membrane rupture (left) to substantial rupture with loss of mitochondria contents (middle) or complete absence of membrane (right). (B) Mitochondrial damage progresses with increasing recovery time. To assess possible progression of mitochondrial disruption over time after ischemia, we re-examined the same postischemic images (as in Fig. 3), only evaluating the mitochondria that both appeared damaged and contained Zn2+ precipitates. Each mitochondrion was rated as mildly damaged if it was judged to have an intact outer membrane (OM; blue) or severely damaged if its OM was disrupted (red). Note the substantial progression of mitochondrial disruption over time, with a far greater percentage of mitochondria showing OM disruption after 4-hour than after 1-hour recovery. Bars show percentage of injured mitochondria in each category (intact or disrupted OM), and represent mean ± SEM from 3 to 4 independent animals (≥55 mitochondria counted from ≥10 sections, each animal; ** Indicates p < 0.01 by 2-tailed Student t-test).