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The importance of free hydroxyl radicals to hypoxia preconditioning
Brain Research 868 (2000) 147–149 www.elsevier.com / locate / bres
Short communication
The importance of free hydroxyl radicals to hypoxia preconditioning Christine Rauca*, Renate Zerbe, Hannelore Jantze, Manfred Krug Department of Pharmacology and Toxicology, Faculty of Medicine, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany Accepted 4 April 2000
Abstract Hypoxia preconditioning states that a sublethal hypoxia period will afford neuroprotection against a second harmful event. In our experiments, we carried out a procedure for the development of hypoxia preconditioning in adult male Wistar rats using hypoxic exposure (9% O 2 ; 91% N 2 ) for 1 h. The protection against pentylenetetrazol (PTZ)-induced seizures was studied. For this, rats were tested by a single injection of PTZ (55 mg / kg i.p.) on days 1–21 after hypoxia exposure. The hypoxia exposure significantly prevented the development of acute PTZ convulsion at different times after hypoxia. The present study was designed to determine the effect of N-t-butyl-a-phenylnitrone (PBN), an electron-trapping agent and free radical scavenger, on hypoxia preconditioning against PTZ seizures 7 days after hypoxia exposure. PBN abolished the protective action of hypoxia exposure. The generation of free hydroxyl radicals in the brains of animals exposed to hypoxia was determined in a second experiment. For this purpose, the rats were i.p. pretreated with 30 mg / kg PBN and NaCl, respectively, 20 min before the start of hypoxia exposure. Forty-five minutes later the rats were i.p. injected with 300 mg / kg sodium salicylate and once again exposed to hypoxia for 15 min. Immediately after that the animals were decapitated and the free hydroxyl radicals and the salicylate content were estimated in the whole brain without cerebellum. Hypoxia preconditioned animals pretreated with NaCl showed a significantly higher extent of free hydroxyl radicals in the brain compared with PBN-injected preconditioned animals and with naive and sham exposed controls. The results pointed out that the generation of free reactive oxygen species under hypoxic conditions in the brain is involved in the development of the hypoxic preconditioning phenomenon. 2000 Elsevier Science B.V. All rights reserved. Keywords: Hypoxia preconditioning; Pentylenetetrazol (PTZ); Seizure; Free hydroxyl radical; Salicylate trapping
Preconditioning caused by a prior moderate, normobaric hypoxia period protects the brain against acute pentylenetetrazol (PTZ)-induced seizures in mice [7] and rats [4]. Although over the last few years many reports have appeared involving the preconditioning phenomenon, the mechanisms which induce and mediate preconditioning are still unknown. An initial step in the cascade of intracellular events mediating protection would be the increased formation of free radicals during conditioning hypoxia. In our experiments, we examined the importance of the free radicals formed following hypoxic exposure to the induction of hypoxic preconditioning. For this, we tested the influence of the free radical scavenger N-t-butyla-phenylnitrone (PBN) on the development of preconditioning when PBN was injected before the hypoxic period. The generation of free hydroxyl radicals was measured indirectly by estimation of 2,3-dihydroxybenzoic acid *Corresponding author. Fax: 149-391-6715869. E-mail address: [email protected] (C. Rauca)
(DHBA) which was formed from salicylate in the presence of free hydroxyl radicals. In contrast to 2,5-DHBA which may also be formed enzymatically, the production of 2,3-DHBA appears to be dependent exclusively on the presence of hydroxyl radicals. Consequently, the content of 2,3-DHBA exclusively reflected the generation of free hydroxyl radicals in the brain of hypoxic rats during the last 15 min of the hypoxia period in comparison with sham exposed and naive controls. In our experiments 8-week-old male Wistar rats weighing 250–300 g were exposed to normobaric hypoxic conditions (9% O 2 ; 91% N 2 ) for 1 h. At different times after hypoxia the rats were analyzed with regard to susceptibility to an acute intraperitoneal injection of 55 mg / kg PTZ. In another experiment, the rats were pretreated with PBN (30 mg / kg i.p.) or NaCl 20 min before the start of hypoxia and tested 7 days later with regard to the induction of clonic–tonic seizures following an acute application of 55 mg / kg PTZ. After each PTZ injection the convulsive behaviour was observed for 20 min and the
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02388-X
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C. Rauca et al. / Brain Research 868 (2000) 147 – 149
resultant seizures were classified according to a modified RACINE scale [6]. Parallel to this study, we investigated the generation of free hydroxyl radicals in rat brains after pretreatment with PBN or NaCl during the hypoxic period. To this end, rats were injected with 30 mg / kg PBN 20 min before the beginning of hypoxia exposure. After a hypoxic period of 45 min the rats were taken out of the hypoxia chamber and intraperitoneally treated with 300 mg / kg sodium salicylate (SAL) and once again exposed to the hypoxic conditions for 15 min. Immediately after the end of hypoxia the animals were decapitated, the brains were dissected and frozen on dry ice. The tissue was stored at 2708C until analysis. Each frozen brain was ultrasonicated in 1 M perchloric acid. SAL and the stable adduct 2,3-DHBA which was formed from SAL under the influence of free hydroxyl radicals were extracted from homogenate and quantitatively analyzed using HPLC separation with electrochemical detection and a variable wavelength detector, respectively. The analyses were performed as described by Rauca et al. [8]. The results were expressed as the ratio of the adduct 2,3-DHBA to SAL concentration in the brain to normalize the 2,3-DHBA level to differing brain concentrations of SAL. Statistical differences were determined using the x 2 -test (behavioural studies) or the one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test with P,0.05 required for significance (biochemical studies). Fig. 1 demonstrates the time course of susceptibility to PTZ-induced seizures following hypoxic preconditioning and sham exposure in comparison to naive controls from
Fig. 1. The time course of susceptibility of rats to acute pentylenetetrazol (PTZ)-induced seizure (55 mg / kg PTZ, intraperitoneally applied) following hypoxic preconditioning and sham exposure in comparison to naive controls from days 1 to 21 after hypoxia expressed as percentage of rats showing convulsions of stage 5 during the 20-min period following PTZ injection. A significant protection (*P,0.05) against PTZ seizures could be established 1 and 7 days following hypoxia exposure. The statistical significance was determined by x 2 -test by comparison of preconditioned rats with naive controls. Fifteen animals were investigated per group.
days 1 to 21 after hypoxia exposure expressed as percentage of rats showing seizures of stages 5 during the 20-min period following PTZ injection. A significant protection against PTZ convulsions could be established 1 and 7 days following hypoxia exposure. In contrast to previous experiments with rats which were exposed to hypoxic conditions for 8 h, in the present study we reduced the duration of hypoxia to 1 h as described for mice to achieve protection against PTZ-induced seizures [7]. In our experiments, the protective action of preconditioning against PTZ seizure was blocked by pretreatment of rats with 30 mg / kg PBN 20 min before the beginning of hypoxia, when this was tested 7 days later (see Fig. 2). Fig. 3 shows the significant enhancement of the formation of free hydroxyl radicals in the rat brain during the last 15 min of the hypoxic period in comparison with sham exposed and naive controls. PBN at a dose of 30 mg / kg intraperitoneally applied 20 min before the start of hypoxia exposure inhibited the increased formation of these reactive oxygen species. Our results indicate that the increased formation of free hydroxyl radicals in the brain and their metabolic consequences, respectively, are involved in the induction of preconditioning against PTZ seizure. However, the present findings cannot explain which mechanisms are ultimately responsible for triggering the decrease in susceptibility to the chemical convulsant. It is known from the relevant literature that both endogenous scavenging enzymes such as oxygen radicals removing manganese-superoxide dismutase [5] and enzymes of oxidative phosphorylation may play an important role in the preconditioning [2]. This fact is supported by the findings that the inhibition of protein synthesis with cycloheximide prevented the protective effect of hypoxia on kainic acid-induced status epilepticus in rats [3]. In addition to this, an increased neuropeptide Y
Fig. 2. The significant protective action of hypoxic preconditioning against pentylenetetrazol (PTZ)-induced seizure (55 mg / kg PTZ, intraperitoneally applied) (*P,0.05), and their inhibition by pretreatment of rats with N-t-butyl-a-phenylnitrone (PBN) (30 mg / kg, intraperitoneally applied) 20 min before the beginning of hypoxia (8P,0.05), when this was tested 7 days later. The statistical significance was determined by x 2 -test. Twenty animals were investigated per group.
C. Rauca et al. / Brain Research 868 (2000) 147 – 149
149
References
Fig. 3. The formation of free hydroxyl radicals expressed as the ratio of the adduct 2,3-dihydroxybenzoic acid (DHBA) to salicylate (SAL) concentration in the brain (without cerebellum). Following hypoxia preconditioning the extent of 2,3-DHBA was significantly increased in comparison to sham exposed and naive controls (*P,0.05); the pretreatment with N-t-butyl-a-phenylnitrone (PBN) (30 mg / kg, intraperitoneally applied) significantly blocked the increased formation of 2,3-DHBA during the hypoxia period (8P.0.05). The statistical significance was determined by one-way-ANOVA with Tukey’s multiple comparison test. Eight animals were investigated per group.
immunoreactivity [10] and several other endogenous factors, such as phospholipids or free fatty acids, are discussed in the context of the mechanisms of preconditioning [1,9]. However, it can be excluded that the protective effect afforded to preconditioning animals is the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia [11]. Our results indicate that the increased generation of reactive oxygen species in the brain is involved in the development of protection against PTZ seizure following hypoxia preconditioning.
Acknowledgements This study was supported by a grant of the Bundes¨ Bildung und Forschung (FKZ:07 NBL ministerium fur 04 / 01 ZZ 95050).
[1] C. Duan, F. Yan, G. Lu, H. Liu, N. Yin, Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia, Biol. Signals Recept. 8 (1999) 261–266. [2] C. Duan, F. Yan, X. Song, G.W. Lu, Changes of superoxide dismutase, glutathione peroxidase and lipid peroxides in the brain of mice preconditioned by hypoxia, Biol. Signals Recept. 8 (1999) 256–260. [3] M.R. Emerson, S.R. Nelson, F.E. Samson, D.L. Pazdernik, Hypoxia preconditioning attenuates brain edema associated with kainic acidinduced status epilepticus in rats, Brain Res. 825 (1999) 189–193. [4] W. Pohle, C. Rauca, Hypoxia protects against the neurotoxicity of kainic acid, Brain Res. 644 (1994) 297–304. [5] W. Pohle, C. Rauca, Does a moderate hypoxia induce manganesesuperoxide dismutase or heat shock proteins to act as possible protective factors?, Naunyn-Schmiedeberg’s Arch. Pharmacol. 356 (1997) 746–749. [6] R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizures, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 281–285. ¨ [7] C. Rauca, H.-L. Ruthrich, Moderate hypoxia reduces pentylenetetrazol-induced seizures, Naunyn-Schmiedeberg’s Arch. Pharmacol. 351 (1995) 261–267. [8] C. Rauca, R. Zerbe, H. Jantze, Formation of free hydroxyl radicals after pentylenetetrazol-induced seizure and kindling, Brain Res. 847 (1999) 347–351. [9] M.W. Riepe, F. Esclaire, K. Kasischke, S. Schreiber, H. Nakase, O. Kempski, A.C. Ludolph, U. Dirnagl, J. Hugon, Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: ‘chemical preconditioning’, J. Cereb. Blood Flow Metab. 17 (1997) 257–264. [10] C. Schwarzer, G. Sperk, C. Rauca, W. Pohle, Neuropeptide Y and somatostatin immunoreactivity in the rat hippocampus after moderate hypoxia, Naunyn-Schmiedeberg’s Arch. Pharmacol. 354 (1996) 67–71. [11] R.C. Vannucci, J. Towfighi, S.J. Vannucci, Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates, J. Neurochem. 71 (1998) 1215–1220.
Short communication
The importance of free hydroxyl radicals to hypoxia preconditioning Christine Rauca*, Renate Zerbe, Hannelore Jantze, Manfred Krug Department of Pharmacology and Toxicology, Faculty of Medicine, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany Accepted 4 April 2000
Abstract Hypoxia preconditioning states that a sublethal hypoxia period will afford neuroprotection against a second harmful event. In our experiments, we carried out a procedure for the development of hypoxia preconditioning in adult male Wistar rats using hypoxic exposure (9% O 2 ; 91% N 2 ) for 1 h. The protection against pentylenetetrazol (PTZ)-induced seizures was studied. For this, rats were tested by a single injection of PTZ (55 mg / kg i.p.) on days 1–21 after hypoxia exposure. The hypoxia exposure significantly prevented the development of acute PTZ convulsion at different times after hypoxia. The present study was designed to determine the effect of N-t-butyl-a-phenylnitrone (PBN), an electron-trapping agent and free radical scavenger, on hypoxia preconditioning against PTZ seizures 7 days after hypoxia exposure. PBN abolished the protective action of hypoxia exposure. The generation of free hydroxyl radicals in the brains of animals exposed to hypoxia was determined in a second experiment. For this purpose, the rats were i.p. pretreated with 30 mg / kg PBN and NaCl, respectively, 20 min before the start of hypoxia exposure. Forty-five minutes later the rats were i.p. injected with 300 mg / kg sodium salicylate and once again exposed to hypoxia for 15 min. Immediately after that the animals were decapitated and the free hydroxyl radicals and the salicylate content were estimated in the whole brain without cerebellum. Hypoxia preconditioned animals pretreated with NaCl showed a significantly higher extent of free hydroxyl radicals in the brain compared with PBN-injected preconditioned animals and with naive and sham exposed controls. The results pointed out that the generation of free reactive oxygen species under hypoxic conditions in the brain is involved in the development of the hypoxic preconditioning phenomenon. 2000 Elsevier Science B.V. All rights reserved. Keywords: Hypoxia preconditioning; Pentylenetetrazol (PTZ); Seizure; Free hydroxyl radical; Salicylate trapping
Preconditioning caused by a prior moderate, normobaric hypoxia period protects the brain against acute pentylenetetrazol (PTZ)-induced seizures in mice [7] and rats [4]. Although over the last few years many reports have appeared involving the preconditioning phenomenon, the mechanisms which induce and mediate preconditioning are still unknown. An initial step in the cascade of intracellular events mediating protection would be the increased formation of free radicals during conditioning hypoxia. In our experiments, we examined the importance of the free radicals formed following hypoxic exposure to the induction of hypoxic preconditioning. For this, we tested the influence of the free radical scavenger N-t-butyla-phenylnitrone (PBN) on the development of preconditioning when PBN was injected before the hypoxic period. The generation of free hydroxyl radicals was measured indirectly by estimation of 2,3-dihydroxybenzoic acid *Corresponding author. Fax: 149-391-6715869. E-mail address: [email protected] (C. Rauca)
(DHBA) which was formed from salicylate in the presence of free hydroxyl radicals. In contrast to 2,5-DHBA which may also be formed enzymatically, the production of 2,3-DHBA appears to be dependent exclusively on the presence of hydroxyl radicals. Consequently, the content of 2,3-DHBA exclusively reflected the generation of free hydroxyl radicals in the brain of hypoxic rats during the last 15 min of the hypoxia period in comparison with sham exposed and naive controls. In our experiments 8-week-old male Wistar rats weighing 250–300 g were exposed to normobaric hypoxic conditions (9% O 2 ; 91% N 2 ) for 1 h. At different times after hypoxia the rats were analyzed with regard to susceptibility to an acute intraperitoneal injection of 55 mg / kg PTZ. In another experiment, the rats were pretreated with PBN (30 mg / kg i.p.) or NaCl 20 min before the start of hypoxia and tested 7 days later with regard to the induction of clonic–tonic seizures following an acute application of 55 mg / kg PTZ. After each PTZ injection the convulsive behaviour was observed for 20 min and the
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02388-X
148
C. Rauca et al. / Brain Research 868 (2000) 147 – 149
resultant seizures were classified according to a modified RACINE scale [6]. Parallel to this study, we investigated the generation of free hydroxyl radicals in rat brains after pretreatment with PBN or NaCl during the hypoxic period. To this end, rats were injected with 30 mg / kg PBN 20 min before the beginning of hypoxia exposure. After a hypoxic period of 45 min the rats were taken out of the hypoxia chamber and intraperitoneally treated with 300 mg / kg sodium salicylate (SAL) and once again exposed to the hypoxic conditions for 15 min. Immediately after the end of hypoxia the animals were decapitated, the brains were dissected and frozen on dry ice. The tissue was stored at 2708C until analysis. Each frozen brain was ultrasonicated in 1 M perchloric acid. SAL and the stable adduct 2,3-DHBA which was formed from SAL under the influence of free hydroxyl radicals were extracted from homogenate and quantitatively analyzed using HPLC separation with electrochemical detection and a variable wavelength detector, respectively. The analyses were performed as described by Rauca et al. [8]. The results were expressed as the ratio of the adduct 2,3-DHBA to SAL concentration in the brain to normalize the 2,3-DHBA level to differing brain concentrations of SAL. Statistical differences were determined using the x 2 -test (behavioural studies) or the one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test with P,0.05 required for significance (biochemical studies). Fig. 1 demonstrates the time course of susceptibility to PTZ-induced seizures following hypoxic preconditioning and sham exposure in comparison to naive controls from
Fig. 1. The time course of susceptibility of rats to acute pentylenetetrazol (PTZ)-induced seizure (55 mg / kg PTZ, intraperitoneally applied) following hypoxic preconditioning and sham exposure in comparison to naive controls from days 1 to 21 after hypoxia expressed as percentage of rats showing convulsions of stage 5 during the 20-min period following PTZ injection. A significant protection (*P,0.05) against PTZ seizures could be established 1 and 7 days following hypoxia exposure. The statistical significance was determined by x 2 -test by comparison of preconditioned rats with naive controls. Fifteen animals were investigated per group.
days 1 to 21 after hypoxia exposure expressed as percentage of rats showing seizures of stages 5 during the 20-min period following PTZ injection. A significant protection against PTZ convulsions could be established 1 and 7 days following hypoxia exposure. In contrast to previous experiments with rats which were exposed to hypoxic conditions for 8 h, in the present study we reduced the duration of hypoxia to 1 h as described for mice to achieve protection against PTZ-induced seizures [7]. In our experiments, the protective action of preconditioning against PTZ seizure was blocked by pretreatment of rats with 30 mg / kg PBN 20 min before the beginning of hypoxia, when this was tested 7 days later (see Fig. 2). Fig. 3 shows the significant enhancement of the formation of free hydroxyl radicals in the rat brain during the last 15 min of the hypoxic period in comparison with sham exposed and naive controls. PBN at a dose of 30 mg / kg intraperitoneally applied 20 min before the start of hypoxia exposure inhibited the increased formation of these reactive oxygen species. Our results indicate that the increased formation of free hydroxyl radicals in the brain and their metabolic consequences, respectively, are involved in the induction of preconditioning against PTZ seizure. However, the present findings cannot explain which mechanisms are ultimately responsible for triggering the decrease in susceptibility to the chemical convulsant. It is known from the relevant literature that both endogenous scavenging enzymes such as oxygen radicals removing manganese-superoxide dismutase [5] and enzymes of oxidative phosphorylation may play an important role in the preconditioning [2]. This fact is supported by the findings that the inhibition of protein synthesis with cycloheximide prevented the protective effect of hypoxia on kainic acid-induced status epilepticus in rats [3]. In addition to this, an increased neuropeptide Y
Fig. 2. The significant protective action of hypoxic preconditioning against pentylenetetrazol (PTZ)-induced seizure (55 mg / kg PTZ, intraperitoneally applied) (*P,0.05), and their inhibition by pretreatment of rats with N-t-butyl-a-phenylnitrone (PBN) (30 mg / kg, intraperitoneally applied) 20 min before the beginning of hypoxia (8P,0.05), when this was tested 7 days later. The statistical significance was determined by x 2 -test. Twenty animals were investigated per group.
C. Rauca et al. / Brain Research 868 (2000) 147 – 149
149
References
Fig. 3. The formation of free hydroxyl radicals expressed as the ratio of the adduct 2,3-dihydroxybenzoic acid (DHBA) to salicylate (SAL) concentration in the brain (without cerebellum). Following hypoxia preconditioning the extent of 2,3-DHBA was significantly increased in comparison to sham exposed and naive controls (*P,0.05); the pretreatment with N-t-butyl-a-phenylnitrone (PBN) (30 mg / kg, intraperitoneally applied) significantly blocked the increased formation of 2,3-DHBA during the hypoxia period (8P.0.05). The statistical significance was determined by one-way-ANOVA with Tukey’s multiple comparison test. Eight animals were investigated per group.
immunoreactivity [10] and several other endogenous factors, such as phospholipids or free fatty acids, are discussed in the context of the mechanisms of preconditioning [1,9]. However, it can be excluded that the protective effect afforded to preconditioning animals is the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia [11]. Our results indicate that the increased generation of reactive oxygen species in the brain is involved in the development of protection against PTZ seizure following hypoxia preconditioning.
Acknowledgements This study was supported by a grant of the Bundes¨ Bildung und Forschung (FKZ:07 NBL ministerium fur 04 / 01 ZZ 95050).
[1] C. Duan, F. Yan, G. Lu, H. Liu, N. Yin, Changes in phospholipids and free fatty acids in the brains of mice preconditioned by hypoxia, Biol. Signals Recept. 8 (1999) 261–266. [2] C. Duan, F. Yan, X. Song, G.W. Lu, Changes of superoxide dismutase, glutathione peroxidase and lipid peroxides in the brain of mice preconditioned by hypoxia, Biol. Signals Recept. 8 (1999) 256–260. [3] M.R. Emerson, S.R. Nelson, F.E. Samson, D.L. Pazdernik, Hypoxia preconditioning attenuates brain edema associated with kainic acidinduced status epilepticus in rats, Brain Res. 825 (1999) 189–193. [4] W. Pohle, C. Rauca, Hypoxia protects against the neurotoxicity of kainic acid, Brain Res. 644 (1994) 297–304. [5] W. Pohle, C. Rauca, Does a moderate hypoxia induce manganesesuperoxide dismutase or heat shock proteins to act as possible protective factors?, Naunyn-Schmiedeberg’s Arch. Pharmacol. 356 (1997) 746–749. [6] R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizures, Electroencephalogr. Clin. Neurophysiol. 32 (1972) 281–285. ¨ [7] C. Rauca, H.-L. Ruthrich, Moderate hypoxia reduces pentylenetetrazol-induced seizures, Naunyn-Schmiedeberg’s Arch. Pharmacol. 351 (1995) 261–267. [8] C. Rauca, R. Zerbe, H. Jantze, Formation of free hydroxyl radicals after pentylenetetrazol-induced seizure and kindling, Brain Res. 847 (1999) 347–351. [9] M.W. Riepe, F. Esclaire, K. Kasischke, S. Schreiber, H. Nakase, O. Kempski, A.C. Ludolph, U. Dirnagl, J. Hugon, Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: ‘chemical preconditioning’, J. Cereb. Blood Flow Metab. 17 (1997) 257–264. [10] C. Schwarzer, G. Sperk, C. Rauca, W. Pohle, Neuropeptide Y and somatostatin immunoreactivity in the rat hippocampus after moderate hypoxia, Naunyn-Schmiedeberg’s Arch. Pharmacol. 354 (1996) 67–71. [11] R.C. Vannucci, J. Towfighi, S.J. Vannucci, Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates, J. Neurochem. 71 (1998) 1215–1220.