Recirculation following 2 h of focal
ischemia due to transient middle cerebral artery (MCA) occlusion has previously been found to be accompanied by an initial, partial recovery of the cellular bioenergetic state and of mitochondrial respiratory functions, with secondary deterioration during the first 2-4 h of reflow. Both the
free radical spin trap
alpha-phenyl-N-tert-butyl nitrone (PBN) and the
immunosuppressant drug FK506 ameliorate the damage incurred by the 2-h period of focal
ischemia, even when given 1-3 h after the start of the recirculation. The primary objective of this study was to find out if
FK506, like PBN, prevents the secondary deterioration of mitochondrial function, as this can be studied in vitro. Since this proved to be the case, we addressed the question of whether the secondary
mitochondrial dysfunction and bioenergetic failure were related to a secondary compromise of microcirculation and cellular
oxygen delivery. Six groups of male Wistar rats were studied for measurement of mitochondrial respiratory activity (total, n = 36). One group was used as control (n = 6). In the other groups of animals, MCA occlusion of 2 h duration was induced by an intraluminal filament technique, Neocortical focal and perifocal ("penumbra") tissues were sampled after 2 h of
ischemia (n = 6) and after 1 h (n = 6), 2 h (n = 6 with vehicle), and 4 h (n = 6 with vehicle; n = 6 with
FK506) of recirculation. The vehicle or 1.0 mg.kg-1 of
FK506 was injected intravenously after 1 h of recirculation. Homogenates were prepared, and stimulated (+
ADP), nonstimulated (-
ADP), and uncoupled respiratory rates were measured polarographically. The uncoupling agent used was
carbonyl cyanide m-chlorophenylhydrazone. Local CBF and tissue
oxygen tension were evaluated by
laser-Doppler flowmetry and PO2
microelectrodes, respectively, throughout the whole periods of 2 h of
ischemia and 4 h of recirculation, using a remote MCA occlusion technique. After 2 h of
ischemia, the penumbra showed a moderate decrease and the focus a marked decrease in
ADP-stimulated and uncoupled respiratory rates, with a marked fall in the respiratory control ratio, defined as
ADP-stimulated divided by nonstimulated respiration. Recirculation (1 h) brought about partial recovery, but continued reflow (2 and 4 h) was associated with a secondary deterioration of respiratory functions. The secondary deterioration was prevented by
FK506. The results thus confirm previous findings showing that secondary
mitochondrial dysfunction occurs following transient focal
cerebral ischemia and demonstrate that
FK506, like PBN, improves the in vitro performance of mitochondria in focal and penumbral areas. Following MCA occlusion, local CBF in a penumbral area and tissue PO2 in a focal area decreased to about 30 and 5% of control, respectively. However, recirculation brought about rapid recovery of blood flow and
oxygen delivery. During the whole 4-h period of recirculation, local CBF and tissue PO2 were maintained close to 100% and at about 160% of the preischemic level, respectively. The results make it highly unlikely that the secondary bioenergetic failure during recirculation is due to a compromised microcirculation. It follows that
oxygen delivery is not rate-limiting for recovery events. Very likely,
FK506 (and PBN) acts at the cellular level to improve mitochondrial energy functions.