Abstract
Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with α-phenyl-N-tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.
Keywords
Cerebral ischemia/reperfusion is accompanied by enhanced production of free radicals (Siesjö, 1981) and mitochondrial dysfunction (Sims and Pulsinelli, 1987; Almeida et al., 1995). Oxidative damage to proteins and CA1 hippocampal cell death due to global cerebral ischemia are counteracted by the spin-trapping compound α-phenyl-N-tert-butyl-nitrone (PBN) in gerbils (Phillis and Clough-Helfman, 1990), whereas the effect—if any—is minimal in rats made ischemic by carotid occlusion and hypotension (Pahlmark and Siesjö, 1996).
Focal ischemia has a different pathophysiology, being long-lasting and with a strong inflammatory/immunologic component (Siesjö and Siesjö, 1996). Focal ischemia induced by occlusion of the middle cerebral artery affects mainly the lateral caudoputamen and the overlaying neocortex (Memezawa et al., 1992b). In these areas, the part that suffers the most marked reduction in cerebral blood flow, the “focus”, becomes infarcted unless recirculation is instituted promptly, but the surrounding tissue in which the flow is less severely reduced (the “penumbra”) can be salvaged potentially by pharmacologic treatment (Siesjö, 1992a,b) or recirculation (Memezawa et al., 1992a).
In focal areas, energy metabolism is severely impaired, and in penumbral areas, it is partially compromised; in both areas, an initial recovery of the bioenergetic state is observed with recirculation, but later a secondary deterioration of the cellular bioenergetic state is observed (Folbergrová et al., 1992, 1995). The PBN ameliorates brain damage (Zhao et al., 1994) and recovery of the bioenergetic state (Folbergrová et al., 1995) in both the focus and, to a lesser extent, in the penumbra. We have shown that in both penumbral and focal tissue homogenates, glutamate/malate-dependent mitochondrial respiration showed partial recovery after 1 hour of recirculation and a secondary deterioration after 2 to 4 hours; the latter was prevented significantly by PBN given after 1 hour of recirculation (Kuroda et al., 1996a,b).
To investigate the underlying cause of the observed decrease in respiratory rates and the mechanism of action of PBN, we measured the activity of the complexes of the respiratory chain and other mitochondrial enzymes under similar experimental conditions in the same areas, The results demonstrated that the decrease in respiratory rates is not accompanied by a decreased activity of respiratory chain complexes.
MATERIALS AND METHODS
Male Wistar rats (Møllegaard, Copenhagen, Denmark), 300 to 340 g, were used for transient middle cerebral artery occlusion (MCAO), as described previously (Memezawa et ah, 1992b). Animals were anesthetized with halothane/N2O:O2; the origin of the middle cerebral artery was occluded by a filament carrying a distal cylinder of silicon rubber, which was withdrawn after 2 hours to allow recirculation. Core temperature was maintained at 37°C. Physiologic parameters and the neurologic status (Bederson et al., 1986) of each rat were evaluated regularly; only rats that consistently circled toward the paretic side (grade 3) during the MCAO were included in this study.
Seven groups of animals (n = 6–7 each) were studied. Control animals were sham-operated and killed after 2 hours. All other animals were subjected to 2-hour MCAO, and killed immediately (MCAO) or at 1, 2, or 4 hours of recirculation. In two groups, PBN (100 mg/kg) or vehicle (saline) were administered intraperitoneally 1 hour after ischemia; recirculation was allowed for 1 or 3 hour hours.
The brains were removed rapidly and a 4-mm slice in the coronal plane was cut. The areas denoted “focus” and “penumbra” in previous bioenergetic and respiratory measurements (Folbergrová et al., 1995; Kuroda et al., 1996a), as well as a sample of contralateral neocortex, were dissected, homogenized in 10 volumes of ice-cold isolation medium (0.32 mol/L sucrose, 1 mmol/L potassium-edetic acid, 10 mmol/L Trishydrochloride, pH 7.4) using a glass–glass homogenizer, and stored at −70°C for no longer than 2 months. The small amount of tissue sampled did not allow further subcellular fractionation.
Samples were frozen/thawed three times before the assays. The specific activities of mitochondrial complex I (Ragan et al. 1987), complexes II and III (King, 1967), complex IV (Wharton and Tzagoloff, 1967), complex V (Soper and Pedersen, 1979), glutamate dehydrogenase (Leong and Clark, 1984), and citrate synthase (Shepherd and Garland, 1969) were measured spectrophotometrically at 30°C. Protein concentration also was measured (Lowry et al., 1951).
One-way analysis of variance and the Tukey test were used to compare the values in the three areas (contralateral cortex, penumbra and focus) within each experimental group.
RESULTS
Figures 1 and 2 show the activities of the enzymes in the seven experimental groups. A high variability in the activities was found in the cortex contralateral to the occlusion; this reflects individual differences between animals and the variability associated with the homogenates as opposed to purified fractions. Therefore, we have reported the data normalized to the value in the contralateral cortex of each group.
Only the activity of complex IV in the animals submitted to MCAO without reperfusion was lower in the focus (−27.0%; P < 0.05), and in the penumbra (−16.7%; P = not significant). This inhibition was reversible because it was not found in the reperfused groups, although a tendency toward decrease can be observed at 4 hours (−19.26%; P = not significant).
Enzyme activities of the complexes I (a), II-III (b), IV (c) in contralateral cortex, penumbra, and focus. Activities are expressed relative to the contralateral cortex in each group, ± SD (n = 6-7). The activity in the contralateral cortex, expressed in nmol min−1 mg protein−1 (k min−1 mg protein−1 for complex IV) is reported on the first bar of each group. *P = 0.019. □ Contralateral ctx, penumbra, ▪ focus.
Enzyme activites of complex V of the mitochondrial respiratory chain (a), glutamate dehydrogenase (b) and citrate synthase (c) in contralateral cortex, penumbra, and focus. Activities are expressed relative to the contralateral cortex in each group, ± SD (n = 6-7). The activity in the contralateral cortex, expressed in nmol min−1 mg protein−1 is reported on the first bar of each group. □ Contralateral ctx, penumbra, ▪ focus.
Treatment with PBN did not modify any of the enzyme activities compared with the corresponding recirculation time group.
DISCUSSION
The decrease in the activity of complex IV confirms findings obtained with other models of rat cerebral ischemia: perinatal forebrain ischemia (Nelson and Silverstein, 1994), carotid occlusion with hypotension (Dimlich et al., 1990), and postdecapitative ischemia (Canevari, 1996, unpublished observations).
The decrease in complex IV activity immediately after ischemia is unlikely to be responsible for the decrease in state 3 respiration observed in the previous experiments (Kuroda et al., 1996a). It has been shown that complex IV in nonsynaptic and synaptic mitochondria can be inhibited by cyanide by approximately 60% of the activity before it affects the respiration rate or the production of adenosine triphosphate (Davey and Clark, 1996; Davey, 1996, personal communication).
Nevertheless, preliminary experiments have shown a much higher activity of complex IV in all areas in ordinary control animals, compared with sham-operated controls (data not shown). This probably is due to the anesthesia: halothane is known to accumulate in brain membranes and modify their fluidity (Sastry et al., 1991) and to affect a number of membrane proteins and lipids (Franks et al., 1995), including decreasing complex IV activity in cardiac tissue (Caughey et al., 1993). It is possible that in our samples, the activity of complex IV was decreased by anesthesia in all areas of the brain, but not to a sufficient extent to affect oxygen consumption, whereas a further decrease induced by ischemia brought the level of activity below the threshold for an effect on respiration.
The secondary decrease in respiration observed after 4 hours of reperfusion and the protective effect of PBN cannot be explained by the data obtained in the present investigation, and they probably are mediated by a process other than the mitochondrial complexes activities. We believe that it is unlikely that the adenine translocator or the phosphate carrier are involved, because respiration in presence of an uncoupler was affected in the same way as state 3 respiration (Kuroda et al., 1996a). Pyruvate dehydrogenase activity, which has been shown to be impaired by cerebral ischemia and reperfusion (Zaidan and Sims, 1993), has not been considered because the change in respiration was observed using glutamate and malate as NADH-producing substrates. Possible targets are the dicarboxylate carrier (Groen et al., 1982) or the cyclosporin A-sensitive permeability transition pore on the mitochondrial inner membrane (Zoratti and Szabò, 1995): limitation of the access of substrates or calcium to mitochondrial dehydrogenases could influence the respiratory rate. Alternatively, modifications of the mitochondrial membranes properties could hinder the flow of reducing equivalents between the components of the respiratory chain.
Thus, the mitochondrial dysfunction observed in previous work (Kuroda et al., 1996a) does not appear to be mediated by a permanent, structural damage to the enzymes studied such as to cause a reduction in the specific activity, measured in standard conditions in vitro.