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Lipid peroxidation in neonatal mouse brain subjected to two different types of hypoxia
Lipid Peroxidation in Neonatal Mouse Brain Subjected to Two Different Types of Hypoxia Koh Hasegawa, MD, Hiroshi Yoshioka, MD, Tadashi Sawada, MD and Hiroyasu Nishikawa, MD
To elucidate the role offree radicals in the pathogenesis of neonatal hypoxic encephalopathy, we determined the content of thiobarbituric acid reactants (TBARs), as an index of lipid peroxidation related with a free radical reaction, in the brains of newborn mice dUring hypoxia and recovery from hypoxia. Hypoxic stress was induced by 100% nitrogen gas breathing (N2 group) or 100% carbon dioxide gas breathing (C02 group). TBARs increased with 20 minutes of hypoxia and returned to the control level during the recovery period in both groups. The increase in TBARs in the C02 group was greater than that in the N2 group. These results may suggest that free radical reaction occurs during the hypoxic period and that CO2 hypoxia is more effective on free radical production in the newborn brain than N2 hypoxia. Key words: Lipid peroxidation, thiobarbituric acid reactants, free radical, neo1llJtai hypoxic encephalopathy. Hasegawa K, Yoshioka H, Sawada T, Nishikawa H. Lipid peroxidation in neonatal mouse brain subjected to two different types of hypoxia. Brain Dev 1991;13:101-3
Brain tissue is highly rich in polyunsaturated fatty acids, which are susceptible to oxidation [1]. The role of free radicals in ischemic brain damage has been investigated by many workers [2-6]. However, less is known about the production of free radicals in the hypoxic brain, especially in the brain of perinatal asphyxiated infants, which is one of the most important problems for neonatologists. In previous papers, we reported the influence of hypoxia induced by 100% nitrogen gas breathing (N2 group) or 100% carbon dioxide gas breathing (C~ group) on the neonatal mouse brain [7,8]. We found that cell proliferation in the cerebellum was suppressed in the C02 group, but not in the N2 group [7]. In addition, brain energy metabolism was investigated by 31 P-NMR spectroscopy in these two kinds of hypoxia [8]. The results of NMR studies suggest that the suppression of the cerebellar neuronal production in the CO 2 group might result from the remarkable tissue acidosis or transient energy
From the Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto (KH, HY, TS); and Department of Physiology, Meiji College of Oriental Medicine, Kyoto (HN). Received for publication: July 16, 1990. Accepted for pUblication: February 6, 1991. Correspondence address: Dr. Koh Hasegawa, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyou-ku, Kyoto, Japan.
failure. In this study, we determined the effects of two kinds of hypoxia on lipid peroxidation in the brains of neonatal mice and examined the role of free radicals in the pathogenesis of neonatal hypoxic encephalopathy.
MATERIALS AND METHODS Jc1-ICR strain male mice (one-day-old, body weight, 1.5-1.9 g) were used in this study. The survival rate was 100% up to 20 minutes in the cases of N z and C~ hypoxia at 25°C [7]. The mice were put into plastic chambers (20 x 14 x 6.5 cm) which were constantly flushed with 100% C02 gas or 100% N z gas for up to 20 minutes at the rate of 3-4 litres/min through water at 25° C [9]. The oxygen concentration in the chamber could be kept at 0% with this flow rate. Mter the hypoxic insult, mice which survived were kept in room air. Groups (n = 6) of mice were decapitated after 5, 10, 15 and 20 minutes of hypoxia and after 5, 10, 20 and 30 minutes of the recovery period. The whole cerebrums were isolated and frozen in liquid nitrogen. The frozen cerebral tissue, after the addition of nine volumes of ice-cold saline, was homogenized with a teflon homogenizer under a nitrogen atmosphere. The brains of mice not subjected to hypoxic insult were used as controls. Complete cerebral ischemia was produced by decapitation followed by keeping the heads in room air for 5, 10,
15 and 20 min. Then, the cerebral tissue was isolated and used for measurements, as described above. The lipid peroxide contents were estimated by means of the thiobarbituric acid (TBA) test described byOhkawa et al [10]. 0.2 m1 of a 10% (w/v) tissue homogenate was added to 0.2 m1 of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of a 20% acetic acid solution adjusted to pH 3.5 and 1.5 ml of a 0.8% aqueous solution of TBA. The mixture was made up to 4.0 ml with distilled water and then heated in boiling water for 60 minutes. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine (15: 1, v/v) were added, followed by vigorous shaking. After centrifugation at 4,000 rpm for 10 minutes, the organic layer was taken and its absorbance at 553 nm was measured by means of spectrofluorometry, using an excitation wave length of 515 nm. 1,1,3,3-tetraethoxypropane (TEP) was used as an external standard and the level of lipid peroxide was expressed as nmol of malondialdehyde (MDA). Chemicals: TBA was obtained from Sigma Chemical Co (St Louis, Mo, USA). SDS and TEP were obtained from Nakarai Chemicals (Kyoto, JAPAN). All chemicals were of the highest purity available. Statistics: Measurements were performed for six mice for each point. The significance of the differences between mean values was examined by means of Student's t test, P < 0.05 being considered to be significant.
ischemic brain are generated by the ischemically injured electron transport system of brain mitochondria. Kukreja et al [11] reported that free radicals, including the superoxide anion, were generated during arachidonic acid metabolism via cyclooxygenase and lipoxygenase. But the actual mechanism underlying the reaction has not been clarified. It is difficult to detect free radicals in a biological system directly in vivo,. because their lives are very short and their concentrations are very low [12]. The TBA method is one of the commonly used methods for measuring free radical reactions indirectly. A number of studies have shown that lipid peroxidation occurs in the early stage of recirculation following ischemia in many tissues, including heart, kidney, lung and brain [4, 13-15]. It is of interest that we observed an increase in TBARs not in the early stage of recovery but during the hypoxic period in this study. This may suggest that free radicals that initiate lipid peroxidation are produced during the hypoxic period. The lipid peroxidation is propagated by the remaining oxygen molecules in the brain, resulting in accumulation of TBARs. There was no change in TBARs in the complete ischemic group. This
5 o
C02 group
•
N2 group
RESULTS The changes in the TBA reactive substances (TBARs) concentration during hypoxia and recovery from hypoxia are illustrated in Fig 1. In the N2 group, the TBARs concentration increased significantly from the control value, 2.75 ± 0.26 nmol/ 100 mg wet wt (mean ± SD), to 3.57 ± 0.31 nmol/lOO mg wet wt at 20 minutes of hypoxia, and then rapidly recovered to the control level during the recovery phase. In the C02 group, the TBARs concentration increased significantly to 3.18 ± 0.25 nmol/ 100 mg wet wt and continued to increase to 4.20 ± 0.44 nmol/100 mg wet wt at 20 min of hypoxia. During the recovery period, it decreased to the control value, but the rate of decrease was slower than that for the N2 group. The TBARs value at 20 min of hypoxia was significantly higher in the C02 group than in the N2 group. In the complete ischemic group, the TBARs concentration of 0, 5, 10, 15 and 20 minutes of ischemia was 2.57 ± 0.28, 2.41 ± 0.20, 2.46 ± 0.21, 2.40 ± 0.25 and 2.47 ± 0.24 nmol/lOO mg wet wt respectively. There was no significant change in the TBARs level before and during ischemia (Fig 2). DISCUSSION Kogure et al [2] have speculated that free radicals in the
102 Brain & Development, Vol 13, No 2, 1991
VI
C
~
~
«
CD
2
1
I-
OLO'--5r--10r-~15~~W--~25--~30----~40----~~~~~8~O-TIME(min.l
Fig 1 Time course of the thiobarbituric acid reactants (TBARs)
level during hypoxia and recovery from hypoxia. Values are means ± SD and expressed as nmol/100 mg of wet weight (n = 6). * p < 0.05, ** p < 0.01, *** P < 0.001 vs controls, * p < 0.05, ** P < 0.01 vs the N, group.
Time course of the thiobarbituric acid reactants (TBARs) level during complete ischemia. There are no signifi· cant changes before and during complete ischemia (n = 6). Fig 2
OLO.,.----5,.---...,10c-....... 15----::"20:-
TIM E (min.)
shows that the supply of oxygen to the brain tissue had completely ceased. We previously reported a change in the acid-base balance of blood collected from the severed neck vessels of newborn mice during hypoxia and recovery from hypoxia [7]. DUring hypoxia, P02 decreased in both groups compared with the control values, and was maintained at 20-30 mmHg. Siesjo [16] reported that in arachidonic acid metabolism complete interruption of the oxygen supply would abolish cyclooxygenase activity, but that at oxygen levels exceeding about 20 J.Ltl101 (P0 2 about 12 mmHg), the synthesis ofprostaglandins and endoperoxides, which could be the origin of lipid peroxides, was determined by the arachidonic acid availability. On the other hand, Yoshida et al showed a striking increase in polyunsaturated fatty acids, including arachidonic acid, in the brains of gerbils during bilateral carotid occlusion [4] . These reports may support the possibility of the production of lipid perioxides during hypoxia in our experimental model. Our fmdings did not indicate an increase in lipid peroxidation during the recovery phase, which was mentioned in other reports [4,14]. Since all of the mice were survived spontaneously after the hypoxic insult in this study, we speculate that the degree of hypoxia in brain tissue was not as severe as in other ischemic models involving mechanical ventilation, and that reperfusion injury could not occur in our hypoxic model. The TBARs value was higher in C02 hypoxia than in N2 hypoxia. As described above, the P02 values in the two groups were the same. Furthermore, we studied the brain energy metabolism in newborn mice in vivo by means of 31 P-NMR spectroscopy during hypoxia and recovery from hypoxia [8]. Tissue acidosis of the brain, which was calculated from the chemical shift of inorganic phosphate, was more remarkable in the CO2 group. During hypoxia, the pH of the brain tissue decreased to 7.02 in the N2 group and 6.64 in the CO 2 group, respectively. Siesjo et al [17] reported that the TBARs level in brain tissue was greatly increased at pH 6.0-7.0, while the tissue a-tocopherol level decreased. They concluded that acidosis has the potential of triggering increased free radical formation. Therefore, the difference in the levels of TBARs in the two groups may be dependent upon the degree of acidosis in the hypoxic brain. During the recovery phase, the TBARs values returned to the control level more rapidly in the N2 group than in the C02 group. This may be due to the rapid recovery of respiration in the N2 group after hypoxia. We reported changes in the respiration and heart rates of mice during and following these two kinds of hypoxia [18]. The respiration of mice after CO2 hypoxia was irr~gular, while it became regular soon after N2 hypoxia. In the N2 group, the recovery of the heart rate to the control level occurred more quickly than in the C02 group. Thus, the hypoxic condition in the brain tissue disappeared more quickly in
the N2 group, being the result of washing-out of TBARs through blood recirculation. In conclusion, the present study demonstrated that neonatal hypoxia caused the production of free radicals in the brain, that appears to initiate lipid peroxidation. It is suggested that tissue acidosis may participate in free radical production. REFERENCES 1. Tappel AL. Lipid peroxidation damage to cell components. Fed Proc 1974; 32:1870-4. 2. Kogure K, Arai H, Abe K, Nakano M. Free radical damage of the brain following ischemia Brain Res 1985;63:237-59. 3. Yoshida S, Busto R, Watson BD, Santiso M, Ginsberg MD. Postischemic cerebral lipid peroxidation in vitro: modification by dietary vitamin E. J Neurochem 1985;44: 1593-601. 4. Yoshida S, Inoh S, Asano T, et al. Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain. J Neurosurg 1980;53:323-31. 5. Tominaga T, Imaizumi S, Yoshimoto T, Suzuki J, Fujita Y. Application of spin-trapping study to rat ischemic brain homogenate incubated with NADPH and Fe-EDT A. Brain Res 1987;402:370-2. 6. Imaizumi S, Tominaga T, Uenohara H, Yoshimoto T, Suzuki J, Fujita Y. Initiation and propagation of lipid peroxidation in cerebral infarction models. Neurol Res 1986;8:214-20. 7. Yoshioka H. Neonatal asphyxia and subsequent brain development in the mouse. In: Yabuuchi H, Watanabe K, Okada S, eds. Neonatal brain and behavior. Nagoya: The University of Nagoya Press, 1987 :27-34. 8. Yoshioka H, Fujiwara K, Ishimura K, et al. Brain energy metabolism in two kinds of total asphyxia: an in vivo phosphorus nuclear magnetic resonance spectroscopic study. Brain Dev (Tokyo) 1988; 10:88-91. 9. Yoshioka H, Ochi M, Morikawa Y, Kasubuchi Y, Kusunoki T. Changes in cell proliferation kinetics in the mouse cerebellum after total asphyxia. Pediatrics 1985; 76:965-9. 10. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. 11. Kukreja RC, Kontos HA, Hess ML, Ellis EF. PGH synthase and lipoxygenase generate superoxide in the presence of NADHorNADPH. GrcRes 1986;59:612-9. 12. Watson BD, Ginsberg MD. Mechanism of lipid peroxidation potentiated by ischemia in brain. In: Halliwell B, ed. Oxygen radicals and tissue injury, Proceedings of a Brook Lodge Symposium; Augusta, Michigan, USA, 1987:81-91. 13. Ganduel Y, Duvelleroy MA. Role of oxygen radicals in cardiac injury due to reoxygenation. J Mol Cell Cardiol 1984; 16:459-70. 14. Granger DN, Hollwarth ME, Parks DA. Ischemia reperfusion injury: role of oxygen derived free radicals. Acta Physiol Scand 1986;548(suppl):47-63. 15. McCord 1M. Oxygen-derived free radicals in postischemic tissue injury. N EnglJ Med 1985; 312:159-63. 16. Siesjo BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metabol 1981; 1: 155-85. 17. Siesjo BK, Bendek G, Koide T, Westerberg E, Weiloch T. Influence of acidosis on lipid peroxidation in brain tissue in vitro. J Cereb Blood Flow Metabol 1985; 5:253-8. 18. Fujiwara K. Effects of neonatal asphyxia on the central nervous system in mice. 1. Brain damages caused by two different types of total asphyxia (in Japanese). No To Hattatsu (Tokyo) 1985; 17:558-64.
Hasegawa et al: Lipid peroxidation in hypoxic brain
103
To elucidate the role offree radicals in the pathogenesis of neonatal hypoxic encephalopathy, we determined the content of thiobarbituric acid reactants (TBARs), as an index of lipid peroxidation related with a free radical reaction, in the brains of newborn mice dUring hypoxia and recovery from hypoxia. Hypoxic stress was induced by 100% nitrogen gas breathing (N2 group) or 100% carbon dioxide gas breathing (C02 group). TBARs increased with 20 minutes of hypoxia and returned to the control level during the recovery period in both groups. The increase in TBARs in the C02 group was greater than that in the N2 group. These results may suggest that free radical reaction occurs during the hypoxic period and that CO2 hypoxia is more effective on free radical production in the newborn brain than N2 hypoxia. Key words: Lipid peroxidation, thiobarbituric acid reactants, free radical, neo1llJtai hypoxic encephalopathy. Hasegawa K, Yoshioka H, Sawada T, Nishikawa H. Lipid peroxidation in neonatal mouse brain subjected to two different types of hypoxia. Brain Dev 1991;13:101-3
Brain tissue is highly rich in polyunsaturated fatty acids, which are susceptible to oxidation [1]. The role of free radicals in ischemic brain damage has been investigated by many workers [2-6]. However, less is known about the production of free radicals in the hypoxic brain, especially in the brain of perinatal asphyxiated infants, which is one of the most important problems for neonatologists. In previous papers, we reported the influence of hypoxia induced by 100% nitrogen gas breathing (N2 group) or 100% carbon dioxide gas breathing (C~ group) on the neonatal mouse brain [7,8]. We found that cell proliferation in the cerebellum was suppressed in the C02 group, but not in the N2 group [7]. In addition, brain energy metabolism was investigated by 31 P-NMR spectroscopy in these two kinds of hypoxia [8]. The results of NMR studies suggest that the suppression of the cerebellar neuronal production in the CO 2 group might result from the remarkable tissue acidosis or transient energy
From the Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto (KH, HY, TS); and Department of Physiology, Meiji College of Oriental Medicine, Kyoto (HN). Received for publication: July 16, 1990. Accepted for pUblication: February 6, 1991. Correspondence address: Dr. Koh Hasegawa, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyou-ku, Kyoto, Japan.
failure. In this study, we determined the effects of two kinds of hypoxia on lipid peroxidation in the brains of neonatal mice and examined the role of free radicals in the pathogenesis of neonatal hypoxic encephalopathy.
MATERIALS AND METHODS Jc1-ICR strain male mice (one-day-old, body weight, 1.5-1.9 g) were used in this study. The survival rate was 100% up to 20 minutes in the cases of N z and C~ hypoxia at 25°C [7]. The mice were put into plastic chambers (20 x 14 x 6.5 cm) which were constantly flushed with 100% C02 gas or 100% N z gas for up to 20 minutes at the rate of 3-4 litres/min through water at 25° C [9]. The oxygen concentration in the chamber could be kept at 0% with this flow rate. Mter the hypoxic insult, mice which survived were kept in room air. Groups (n = 6) of mice were decapitated after 5, 10, 15 and 20 minutes of hypoxia and after 5, 10, 20 and 30 minutes of the recovery period. The whole cerebrums were isolated and frozen in liquid nitrogen. The frozen cerebral tissue, after the addition of nine volumes of ice-cold saline, was homogenized with a teflon homogenizer under a nitrogen atmosphere. The brains of mice not subjected to hypoxic insult were used as controls. Complete cerebral ischemia was produced by decapitation followed by keeping the heads in room air for 5, 10,
15 and 20 min. Then, the cerebral tissue was isolated and used for measurements, as described above. The lipid peroxide contents were estimated by means of the thiobarbituric acid (TBA) test described byOhkawa et al [10]. 0.2 m1 of a 10% (w/v) tissue homogenate was added to 0.2 m1 of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of a 20% acetic acid solution adjusted to pH 3.5 and 1.5 ml of a 0.8% aqueous solution of TBA. The mixture was made up to 4.0 ml with distilled water and then heated in boiling water for 60 minutes. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n-butanol and pyridine (15: 1, v/v) were added, followed by vigorous shaking. After centrifugation at 4,000 rpm for 10 minutes, the organic layer was taken and its absorbance at 553 nm was measured by means of spectrofluorometry, using an excitation wave length of 515 nm. 1,1,3,3-tetraethoxypropane (TEP) was used as an external standard and the level of lipid peroxide was expressed as nmol of malondialdehyde (MDA). Chemicals: TBA was obtained from Sigma Chemical Co (St Louis, Mo, USA). SDS and TEP were obtained from Nakarai Chemicals (Kyoto, JAPAN). All chemicals were of the highest purity available. Statistics: Measurements were performed for six mice for each point. The significance of the differences between mean values was examined by means of Student's t test, P < 0.05 being considered to be significant.
ischemic brain are generated by the ischemically injured electron transport system of brain mitochondria. Kukreja et al [11] reported that free radicals, including the superoxide anion, were generated during arachidonic acid metabolism via cyclooxygenase and lipoxygenase. But the actual mechanism underlying the reaction has not been clarified. It is difficult to detect free radicals in a biological system directly in vivo,. because their lives are very short and their concentrations are very low [12]. The TBA method is one of the commonly used methods for measuring free radical reactions indirectly. A number of studies have shown that lipid peroxidation occurs in the early stage of recirculation following ischemia in many tissues, including heart, kidney, lung and brain [4, 13-15]. It is of interest that we observed an increase in TBARs not in the early stage of recovery but during the hypoxic period in this study. This may suggest that free radicals that initiate lipid peroxidation are produced during the hypoxic period. The lipid peroxidation is propagated by the remaining oxygen molecules in the brain, resulting in accumulation of TBARs. There was no change in TBARs in the complete ischemic group. This
5 o
C02 group
•
N2 group
RESULTS The changes in the TBA reactive substances (TBARs) concentration during hypoxia and recovery from hypoxia are illustrated in Fig 1. In the N2 group, the TBARs concentration increased significantly from the control value, 2.75 ± 0.26 nmol/ 100 mg wet wt (mean ± SD), to 3.57 ± 0.31 nmol/lOO mg wet wt at 20 minutes of hypoxia, and then rapidly recovered to the control level during the recovery phase. In the C02 group, the TBARs concentration increased significantly to 3.18 ± 0.25 nmol/ 100 mg wet wt and continued to increase to 4.20 ± 0.44 nmol/100 mg wet wt at 20 min of hypoxia. During the recovery period, it decreased to the control value, but the rate of decrease was slower than that for the N2 group. The TBARs value at 20 min of hypoxia was significantly higher in the C02 group than in the N2 group. In the complete ischemic group, the TBARs concentration of 0, 5, 10, 15 and 20 minutes of ischemia was 2.57 ± 0.28, 2.41 ± 0.20, 2.46 ± 0.21, 2.40 ± 0.25 and 2.47 ± 0.24 nmol/lOO mg wet wt respectively. There was no significant change in the TBARs level before and during ischemia (Fig 2). DISCUSSION Kogure et al [2] have speculated that free radicals in the
102 Brain & Development, Vol 13, No 2, 1991
VI
C
~
~
«
CD
2
1
I-
OLO'--5r--10r-~15~~W--~25--~30----~40----~~~~~8~O-TIME(min.l
Fig 1 Time course of the thiobarbituric acid reactants (TBARs)
level during hypoxia and recovery from hypoxia. Values are means ± SD and expressed as nmol/100 mg of wet weight (n = 6). * p < 0.05, ** p < 0.01, *** P < 0.001 vs controls, * p < 0.05, ** P < 0.01 vs the N, group.
Time course of the thiobarbituric acid reactants (TBARs) level during complete ischemia. There are no signifi· cant changes before and during complete ischemia (n = 6). Fig 2
OLO.,.----5,.---...,10c-....... 15----::"20:-
TIM E (min.)
shows that the supply of oxygen to the brain tissue had completely ceased. We previously reported a change in the acid-base balance of blood collected from the severed neck vessels of newborn mice during hypoxia and recovery from hypoxia [7]. DUring hypoxia, P02 decreased in both groups compared with the control values, and was maintained at 20-30 mmHg. Siesjo [16] reported that in arachidonic acid metabolism complete interruption of the oxygen supply would abolish cyclooxygenase activity, but that at oxygen levels exceeding about 20 J.Ltl101 (P0 2 about 12 mmHg), the synthesis ofprostaglandins and endoperoxides, which could be the origin of lipid peroxides, was determined by the arachidonic acid availability. On the other hand, Yoshida et al showed a striking increase in polyunsaturated fatty acids, including arachidonic acid, in the brains of gerbils during bilateral carotid occlusion [4] . These reports may support the possibility of the production of lipid perioxides during hypoxia in our experimental model. Our fmdings did not indicate an increase in lipid peroxidation during the recovery phase, which was mentioned in other reports [4,14]. Since all of the mice were survived spontaneously after the hypoxic insult in this study, we speculate that the degree of hypoxia in brain tissue was not as severe as in other ischemic models involving mechanical ventilation, and that reperfusion injury could not occur in our hypoxic model. The TBARs value was higher in C02 hypoxia than in N2 hypoxia. As described above, the P02 values in the two groups were the same. Furthermore, we studied the brain energy metabolism in newborn mice in vivo by means of 31 P-NMR spectroscopy during hypoxia and recovery from hypoxia [8]. Tissue acidosis of the brain, which was calculated from the chemical shift of inorganic phosphate, was more remarkable in the CO2 group. During hypoxia, the pH of the brain tissue decreased to 7.02 in the N2 group and 6.64 in the CO 2 group, respectively. Siesjo et al [17] reported that the TBARs level in brain tissue was greatly increased at pH 6.0-7.0, while the tissue a-tocopherol level decreased. They concluded that acidosis has the potential of triggering increased free radical formation. Therefore, the difference in the levels of TBARs in the two groups may be dependent upon the degree of acidosis in the hypoxic brain. During the recovery phase, the TBARs values returned to the control level more rapidly in the N2 group than in the C02 group. This may be due to the rapid recovery of respiration in the N2 group after hypoxia. We reported changes in the respiration and heart rates of mice during and following these two kinds of hypoxia [18]. The respiration of mice after CO2 hypoxia was irr~gular, while it became regular soon after N2 hypoxia. In the N2 group, the recovery of the heart rate to the control level occurred more quickly than in the C02 group. Thus, the hypoxic condition in the brain tissue disappeared more quickly in
the N2 group, being the result of washing-out of TBARs through blood recirculation. In conclusion, the present study demonstrated that neonatal hypoxia caused the production of free radicals in the brain, that appears to initiate lipid peroxidation. It is suggested that tissue acidosis may participate in free radical production. REFERENCES 1. Tappel AL. Lipid peroxidation damage to cell components. Fed Proc 1974; 32:1870-4. 2. Kogure K, Arai H, Abe K, Nakano M. Free radical damage of the brain following ischemia Brain Res 1985;63:237-59. 3. Yoshida S, Busto R, Watson BD, Santiso M, Ginsberg MD. Postischemic cerebral lipid peroxidation in vitro: modification by dietary vitamin E. J Neurochem 1985;44: 1593-601. 4. Yoshida S, Inoh S, Asano T, et al. Effect of transient ischemia on free fatty acids and phospholipids in the gerbil brain. J Neurosurg 1980;53:323-31. 5. Tominaga T, Imaizumi S, Yoshimoto T, Suzuki J, Fujita Y. Application of spin-trapping study to rat ischemic brain homogenate incubated with NADPH and Fe-EDT A. Brain Res 1987;402:370-2. 6. Imaizumi S, Tominaga T, Uenohara H, Yoshimoto T, Suzuki J, Fujita Y. Initiation and propagation of lipid peroxidation in cerebral infarction models. Neurol Res 1986;8:214-20. 7. Yoshioka H. Neonatal asphyxia and subsequent brain development in the mouse. In: Yabuuchi H, Watanabe K, Okada S, eds. Neonatal brain and behavior. Nagoya: The University of Nagoya Press, 1987 :27-34. 8. Yoshioka H, Fujiwara K, Ishimura K, et al. Brain energy metabolism in two kinds of total asphyxia: an in vivo phosphorus nuclear magnetic resonance spectroscopic study. Brain Dev (Tokyo) 1988; 10:88-91. 9. Yoshioka H, Ochi M, Morikawa Y, Kasubuchi Y, Kusunoki T. Changes in cell proliferation kinetics in the mouse cerebellum after total asphyxia. Pediatrics 1985; 76:965-9. 10. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. 11. Kukreja RC, Kontos HA, Hess ML, Ellis EF. PGH synthase and lipoxygenase generate superoxide in the presence of NADHorNADPH. GrcRes 1986;59:612-9. 12. Watson BD, Ginsberg MD. Mechanism of lipid peroxidation potentiated by ischemia in brain. In: Halliwell B, ed. Oxygen radicals and tissue injury, Proceedings of a Brook Lodge Symposium; Augusta, Michigan, USA, 1987:81-91. 13. Ganduel Y, Duvelleroy MA. Role of oxygen radicals in cardiac injury due to reoxygenation. J Mol Cell Cardiol 1984; 16:459-70. 14. Granger DN, Hollwarth ME, Parks DA. Ischemia reperfusion injury: role of oxygen derived free radicals. Acta Physiol Scand 1986;548(suppl):47-63. 15. McCord 1M. Oxygen-derived free radicals in postischemic tissue injury. N EnglJ Med 1985; 312:159-63. 16. Siesjo BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metabol 1981; 1: 155-85. 17. Siesjo BK, Bendek G, Koide T, Westerberg E, Weiloch T. Influence of acidosis on lipid peroxidation in brain tissue in vitro. J Cereb Blood Flow Metabol 1985; 5:253-8. 18. Fujiwara K. Effects of neonatal asphyxia on the central nervous system in mice. 1. Brain damages caused by two different types of total asphyxia (in Japanese). No To Hattatsu (Tokyo) 1985; 17:558-64.
Hasegawa et al: Lipid peroxidation in hypoxic brain
103