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Neuroscience Letters, 128 (1991) 169-172 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100350U NSL 07867
Global elevation of brain superoxide dismutase activity following forebrain ischemia in rat Garnette Sutherland 1'2 Ranjan Bose 1, D e o n L o u w 1'2 and Carl Pinsky 1 Departments of 1Pharmacology and Therapeutics and 2Surgery ( Neurosurgery), The University of Manitoba, Winnipeg, Man. (Canada) (Received 18 January 1991; Revised version received 3 April 1991; Accepted 8 April 1991)
Key words: Lipid peroxidation; Superoxide dismutase; Forebrain ischemia; Astrocyte activation; Free radical Regional superoxide dismutase (SOD) activity and lipid peroxidation (as reflected by thiobarbituric acid-reactive substances (TBARS)) have been measured at increasing post-is.chemic time intervals following a (I0 min) forebrain ischemic insult in rat. All brain regions showed significant progressive increases in SOD activity with increasing post-ischemic time intervals. Lipid peroxidation also was significantly increased in frontal and parietal/ occipital regions at I and 24 h post-ischemia, although this was several orders of magnitude less than the increase in SOD activity. By 7 days postischemia lipid peroxidation had returned to control values for all regions. These data are consistent with the hypothesis that cerebral ischemia is accompanied by glial activation with an associated increase in SOD activity. Global increases in SOD activity may protect the brain from free radicals, thereby preventing large increases in lipid peroxidation.
Forebrain ischemia of short duration damages neurons in discrete regions of the CNS [10, 16]. Neuronal injury progressively worsens during the reperfusion period following a transient ischemic insult, a process termed ischemic maturation [10, 16]. Several processes occurring both during and following an ischemic insult determine the extent of neuronal injury [14]. Calcium fluxes possibly play a central role [14]. In addition, postischemic increases in tissue lactate [7, 9] indicate abnormal mitochondrial function which could result in generation of toxic free radicals due to the univalent reduction of oxygen [4]. Post-ischemic lipid peroxidation, a reflection of free radical chain reactions, has been observed in rats subjected to 30 min of global ischemia [19, 20] and in rats given an embolic stroke to one hemisphere [6]. Other investigators did not find significant post-ischemic changes in those brain phospholipids or fatty acids that would reflect free radical chain reactions [12]. The latter experiments were conducted on large multi-regional or whole brain samples and therefore significant regional changes could have been masked, yielding false negative results. Alternatively, in those studies, significant post-ischemic Correspondence: G. Sutherland, Departments of Pharmacology and Surgery (Neurosurgery), 61 Emily Street, Winnipeg, Man., Canada, R3A 1R9.
free radical generation did not occur or the brain was capable of mobilizing intrinsic cellular defense mechanisms that protected against free radical-related membrane injury. In view of this controversial evidence for impaired oxidative metabolism during and following ischemia [1 l, 14], we have measured regional lipid peroxidation and superoxide dismutase (SOD) activity following transient forebrain ischemia in rat. Twenty male Sprague-Dawley rats weighing 400-500 g were used. Five control rats were sham operated and then decapitated. The hippocampus, frontal lobes, parietal/occipital lobes and cerebellum were rapidly dissected on ice and stored in liquid nitrogen. As previously described [9, 18], transient (10 min) forebrain ischemia (bilateral carotid occlusion plus controlled hypotension (mean 50 mmHg)) was induced in the remaining 15 rats which were maintained normothermic (37.5°C), up to 1 h following ischemia, using a thermostatically controlled water blanket while mogitoring head temperature with a tympanic membrane:probe. A catheter was inserted into the tail artery for blood pressure monitoring and access to blood samples. We have shown that this model together with its anesthetic management (pentobarbital (20 mg/kg), chloral hydrate (150 mg/kg) and atropine (0.5 mg/kg)) produces neuronal damage confined to selectively vulnerable brain regions [18]. At increasing
170 post-ischemic time intervals (1 h, 24h, 7 days), animals in groups of 5 were decapitated and their brains dissected and stored as described above. SOD activity was measured in the brain regions by the method suggested by Minami and Yoshikawa [8]. Brain samples were homogenized in ice-cold normal saline (50 mg/ml), centrifuged (16,000 g for 60 min, 4°C) and kept on ice. The reaction mixture contained 0.5 ml of TRIScacodylate buffer (pH 8.0) containing 2.5 mM diethylene triamine pentaacetic acid, 0.2 ml of nitro blue tetrazolium (0.98 mM), 0.1 ml of Triton X-100 (16%), 0.2 ml of tissue supernatant, and 0.1 ml of pyrogallol (1.0 raM). Absorbance was measured at 540 nm, and the rate of reaction was estimated from the readings at 3 and 4 min after adding pyrogallol. Pure Cu-Zn SOD from bovine erythrocytes (Sigma) was used as a reference standard. Results are expressed as SOD units/g tissue. Brain samples were subjected to heat treatment by immersing the homogenate tubes in a boiling water bath for 10 minutes. These were then centrifuged at 16,000 g for 60 min and the supernatant assayed for residual SOD-like activity. To another batch of brain samples 0.2 ml of KCN (1 mM) was added during the pyrogallol assay to inhibit Cu-Zn SOD. SOD activity was also measured in untreated brain samples. Each of the above determinations was made in duplicate aliquots from six brain samples. Lipid peroxidation was measured by estimating thiobarbituric acid-reactive substances (TBARS) in the brain region samples [1]. The result were expressed as MDA equivalents based on a standard curve drawn for MDA bisdimethyl acetal (Aldrich). Intergroup comparisons were analyzed by ANOVA followed by Duncan's multiple comparison test.
The mean blood pressure was elevated immediately after the ischemic insult, increasing from 116.7+3.6 mmHg to 133.7+3.5 mmHg (t-test; P < 0 . 0 1 ) returning to pre-ischemic values by 15 20 min post-ischemia. The described method of provoking the ischemic insult did not produce a significant acidosis. At 2 min following reperfusion, blood gas analysis gave pH = 7.30+0.01; PaCO2 = 35.4_ 1.7 torr; HCO3- = 18.0___0.80 mM; and a negative base excess of - 6.6 + 0.7 mM (control values: pH = 7.37-+ 0.02; P a C O 2 = 34.0_ i.4 torr; HCO3= 1 9 . 8 + 4 . 4 mM; negative base excess = - 4 . 2 _ + 0 . 9 raM. Post-ischemic hematocrit (44.7_+ 1.2) was unchanged from the preischemic value (45.0 _+ I. 1). The effects of the ischemic insult on regional SOD activity are presented in Fig. 1. The hippocampus had the lowest inherent SOD activity (120_+ 70 units/g), being significantly less than either the frontal (410 _+60 units/g) or parietal/occipital (400 _+60 units/g) regions (P < 0.05). All regions showed a progressive increase in SOD activity with increasing postischemic time intervals. By 1 h post-ischemia, SOD activity was significantly elevated in the hippocampus ( P < 0.01) and at this time regional differences were no longer apparent. The further increase in SOD activity for all brain regions by 7 days was significantly higher than either the control or at other postischemic time intervals (P
Neuroscience Letters, 128 (1991) 169-172 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100350U NSL 07867
Global elevation of brain superoxide dismutase activity following forebrain ischemia in rat Garnette Sutherland 1'2 Ranjan Bose 1, D e o n L o u w 1'2 and Carl Pinsky 1 Departments of 1Pharmacology and Therapeutics and 2Surgery ( Neurosurgery), The University of Manitoba, Winnipeg, Man. (Canada) (Received 18 January 1991; Revised version received 3 April 1991; Accepted 8 April 1991)
Key words: Lipid peroxidation; Superoxide dismutase; Forebrain ischemia; Astrocyte activation; Free radical Regional superoxide dismutase (SOD) activity and lipid peroxidation (as reflected by thiobarbituric acid-reactive substances (TBARS)) have been measured at increasing post-is.chemic time intervals following a (I0 min) forebrain ischemic insult in rat. All brain regions showed significant progressive increases in SOD activity with increasing post-ischemic time intervals. Lipid peroxidation also was significantly increased in frontal and parietal/ occipital regions at I and 24 h post-ischemia, although this was several orders of magnitude less than the increase in SOD activity. By 7 days postischemia lipid peroxidation had returned to control values for all regions. These data are consistent with the hypothesis that cerebral ischemia is accompanied by glial activation with an associated increase in SOD activity. Global increases in SOD activity may protect the brain from free radicals, thereby preventing large increases in lipid peroxidation.
Forebrain ischemia of short duration damages neurons in discrete regions of the CNS [10, 16]. Neuronal injury progressively worsens during the reperfusion period following a transient ischemic insult, a process termed ischemic maturation [10, 16]. Several processes occurring both during and following an ischemic insult determine the extent of neuronal injury [14]. Calcium fluxes possibly play a central role [14]. In addition, postischemic increases in tissue lactate [7, 9] indicate abnormal mitochondrial function which could result in generation of toxic free radicals due to the univalent reduction of oxygen [4]. Post-ischemic lipid peroxidation, a reflection of free radical chain reactions, has been observed in rats subjected to 30 min of global ischemia [19, 20] and in rats given an embolic stroke to one hemisphere [6]. Other investigators did not find significant post-ischemic changes in those brain phospholipids or fatty acids that would reflect free radical chain reactions [12]. The latter experiments were conducted on large multi-regional or whole brain samples and therefore significant regional changes could have been masked, yielding false negative results. Alternatively, in those studies, significant post-ischemic Correspondence: G. Sutherland, Departments of Pharmacology and Surgery (Neurosurgery), 61 Emily Street, Winnipeg, Man., Canada, R3A 1R9.
free radical generation did not occur or the brain was capable of mobilizing intrinsic cellular defense mechanisms that protected against free radical-related membrane injury. In view of this controversial evidence for impaired oxidative metabolism during and following ischemia [1 l, 14], we have measured regional lipid peroxidation and superoxide dismutase (SOD) activity following transient forebrain ischemia in rat. Twenty male Sprague-Dawley rats weighing 400-500 g were used. Five control rats were sham operated and then decapitated. The hippocampus, frontal lobes, parietal/occipital lobes and cerebellum were rapidly dissected on ice and stored in liquid nitrogen. As previously described [9, 18], transient (10 min) forebrain ischemia (bilateral carotid occlusion plus controlled hypotension (mean 50 mmHg)) was induced in the remaining 15 rats which were maintained normothermic (37.5°C), up to 1 h following ischemia, using a thermostatically controlled water blanket while mogitoring head temperature with a tympanic membrane:probe. A catheter was inserted into the tail artery for blood pressure monitoring and access to blood samples. We have shown that this model together with its anesthetic management (pentobarbital (20 mg/kg), chloral hydrate (150 mg/kg) and atropine (0.5 mg/kg)) produces neuronal damage confined to selectively vulnerable brain regions [18]. At increasing
170 post-ischemic time intervals (1 h, 24h, 7 days), animals in groups of 5 were decapitated and their brains dissected and stored as described above. SOD activity was measured in the brain regions by the method suggested by Minami and Yoshikawa [8]. Brain samples were homogenized in ice-cold normal saline (50 mg/ml), centrifuged (16,000 g for 60 min, 4°C) and kept on ice. The reaction mixture contained 0.5 ml of TRIScacodylate buffer (pH 8.0) containing 2.5 mM diethylene triamine pentaacetic acid, 0.2 ml of nitro blue tetrazolium (0.98 mM), 0.1 ml of Triton X-100 (16%), 0.2 ml of tissue supernatant, and 0.1 ml of pyrogallol (1.0 raM). Absorbance was measured at 540 nm, and the rate of reaction was estimated from the readings at 3 and 4 min after adding pyrogallol. Pure Cu-Zn SOD from bovine erythrocytes (Sigma) was used as a reference standard. Results are expressed as SOD units/g tissue. Brain samples were subjected to heat treatment by immersing the homogenate tubes in a boiling water bath for 10 minutes. These were then centrifuged at 16,000 g for 60 min and the supernatant assayed for residual SOD-like activity. To another batch of brain samples 0.2 ml of KCN (1 mM) was added during the pyrogallol assay to inhibit Cu-Zn SOD. SOD activity was also measured in untreated brain samples. Each of the above determinations was made in duplicate aliquots from six brain samples. Lipid peroxidation was measured by estimating thiobarbituric acid-reactive substances (TBARS) in the brain region samples [1]. The result were expressed as MDA equivalents based on a standard curve drawn for MDA bisdimethyl acetal (Aldrich). Intergroup comparisons were analyzed by ANOVA followed by Duncan's multiple comparison test.
The mean blood pressure was elevated immediately after the ischemic insult, increasing from 116.7+3.6 mmHg to 133.7+3.5 mmHg (t-test; P < 0 . 0 1 ) returning to pre-ischemic values by 15 20 min post-ischemia. The described method of provoking the ischemic insult did not produce a significant acidosis. At 2 min following reperfusion, blood gas analysis gave pH = 7.30+0.01; PaCO2 = 35.4_ 1.7 torr; HCO3- = 18.0___0.80 mM; and a negative base excess of - 6.6 + 0.7 mM (control values: pH = 7.37-+ 0.02; P a C O 2 = 34.0_ i.4 torr; HCO3= 1 9 . 8 + 4 . 4 mM; negative base excess = - 4 . 2 _ + 0 . 9 raM. Post-ischemic hematocrit (44.7_+ 1.2) was unchanged from the preischemic value (45.0 _+ I. 1). The effects of the ischemic insult on regional SOD activity are presented in Fig. 1. The hippocampus had the lowest inherent SOD activity (120_+ 70 units/g), being significantly less than either the frontal (410 _+60 units/g) or parietal/occipital (400 _+60 units/g) regions (P < 0.05). All regions showed a progressive increase in SOD activity with increasing postischemic time intervals. By 1 h post-ischemia, SOD activity was significantly elevated in the hippocampus ( P < 0.01) and at this time regional differences were no longer apparent. The further increase in SOD activity for all brain regions by 7 days was significantly higher than either the control or at other postischemic time intervals (P
