Protective effect of α-tocopherol on brain cell membrane function during cerebral cortical hypoxia in newborn piglets

BRAIN RESEARCH ELSEVIER

Brain Research 653 (1994)45 50

Research report

Protective effect of a-tocopherol on brain cell membrane function during cerebral cortical hypoxia in newborn piglets Son Moon Shin c, Bharti Razdan a.,, Om P. Mishra ~', Lois Johnson

b

M a r i a D e l i v o r i a - P a p a d o p o u l o s ~' Departments of ~ Physiology attd Pediatrics and b Pennsyh'ania Hospital, Section of Newborn Pediatrics, Unit'ersity of Pennsyh'ania School of Medicine, Philadelphia, PA, USA c Department of Pediatrics, Yeungnarn Unit,ersity College of Medicine, Taegu, South Korea

Accepted 26 April 1994

Abstract

Protective effect of a-tocopherol on the structure and function of brain cell membranes was investigated by measuring Na+,K+-ATPase activity and products of lipid peroxidation (fluorescent compounds) in brain cell membranes obtained from newborn piglets. Four groups of anesthetized, ventilated piglets were studied: five hypoxic piglets and five normoxic piglets were pretreated with free a-tocopherol (20 mg/kg/dose i.m.), five additional hypoxic piglets received i.m. placebo and five normoxic piglets served as control. Placebo and a-tocopherol were given 48 and 3 h prior to onset of hypoxia. Hypoxic hypoxia was induced and cerebral hypoxia was documented as a decrease in the ratio of phosphocreatine to inorganic phosphate (PCr/P~) using 3~p NMR spectroscopy. PCr/P i decreased from baseline of 2.62 + 0.54 to 1.05 + 0.27 in a-tocopherol-pretreated and from 2.44 + 0.48 to 1.14_+ 0.30 in the placebo-pretreated group during hypoxia. Na+,K+-ATPase activity was unchanged in both normoxic and hypoxic a-tocopherol-pretreated groups. However, in placebo-pretreated hypoxic group, Na+,K+-ATPase activity decreased as compared with control (44.9 _+9.7 vs. 61.8 + 5.7 /xmol Pi/mg protein/h, P < 0.005). The level of fluorescent compounds increased in placebo-pretreated but not in a-tocopherol-pretreated group as compared with control. During hypoxia, serum a-tocopherol levels were higher in a-tocopherol-pretreated groups as compared with placebo-pretreated hypoxic group. The present data indicates that a-tocopherol protects brain cell membranes in newborn piglets from lipid peroxidative damage during tissue hypoxia probably by being incorporated in cell membrane and also as circulating antioxidant. Key words: Brain; Hypoxia; o~-Tocopherol; Na+,K+-ATPase; Lipid peroxidation

I. Introduction

The specific cellular changes which follow cerebral hypoxia may include adenosine 5'-triphosphate deficiency, alterations in intracellular calcium ion concentration, release of excitotoxie amino acid neurotransmitters and oxygen-free radical generation [9]. Fetal and neonatal brain is particularly susceptible to oxidative injury because of its high concentration of polyunsaturated fatty acids [27,33]. Our previous studies in fetal guinea pigs and newborn piglets have demon-

* Corresponding author. Address: Division of Neonatology,Hospi-tal of University of Pennsylvania, 2nd Floor Maloney Building, 3400 Spruce Street, Philadelphia, PA 19104, USA. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 00(16-8903(94)00519-I

strated increased lipid peroxidation and decreased Na+,K+-ATPase activity in the brain cell membranes following hypoxia [6,27,28]. Further, our in vitro studies have shown that peroxidation of cerebral cortical membrane lipids is associated with a decrease in Na+,K ÷ATPase activity [29]. Recent studies have demonstrated a possible causal relationship between decreased Na÷,K+-ATPase activity and neuronal necrosis following hypoxic-ischemic brain injury [21,31]. Oxygen-free radical-mediated membrane lipid peroxidation and resultant decrease in Na+,K+-ATPase activity has been proposed to be as one of the mechanisms of h y p o x i c - i s c h e m i c brain injury [6,13,26,36]. aTocopherol terminates the propagation step in peroxidation of the polyunsaturated fatty acids of the membrane phospholipids [39]. Several studies have sug-

46

S.M. Shin et al. / Brain Research 653 (1994) 45-50

gested a protective effect of anti-oxidants and radical scavengers in cerebral hypoxia [1,37,42,43]. The present study was performed to test the hypothesis that a-tocopherol, a non-enzymatic antioxidant, has a protective effect on brain cell membrane during cerebral hypoxia by reducing oxygen-free radical-mediated lipid peroxidation and inhibition of Na+,K +ATPase activity.

2. Experimental 2.1. Animal preparation Yorkshire piglets obtained from Willow Glenn Farm, Strasburg, PA, were anesthetized with 4% halothane in 0 2 and anesthesia maintained with 0.8% halothane. After local anesthesia with 1% lidocaine, tracheostomy was performed and polyethylene catheters were placed in the aorta through femoral artery to monitor arterial blood pressure and to collect blood samples for blood gas analysis and m e a s u r e m e n t s of vitamin E serum levels and inferior vena cava through femoral vein for drug administration. T h e scalp over the vertex of the skull was removed by electrocauterization and the cranium exposed for placement of a N M R coil in the hypoxic group of animals. After surgical preparation, the animals were paralysed with tubocurarine (2 m g / k g ) , received fentanyl ( 2 5 - 3 0 / x g / k g ) and were placed on a volume ventilator. Anesthesia was then maintained with nitrous oxide, 79%, and oxygen, 21%. T h e animals were allowed to recover over 60 min. Each animal received 2 5 - 3 0 / ~ g / k g fentanyl every 45-60 min throughout the experiment. Body temperature was maintained at 38.5°C with a warming blanket. Arterial blood pressure, arterial blood gases and hematocrit were monitored throughout the study. Ventilator settings were adjusted to maintain normal blood gas values (PaCO2 35-45 m m H g , PaO2 > 80 mmHg). Blood gases were m e a s u r e d using a Corning 178 blood gas analyser (CIBA; C o m i n g Diagnostics Corporation, Medfield, MA).

2.2. Experimental protocols 20 newborn piglets u n d e r 1 week of age were divided into four groups. Five hypoxic and five normoxic piglets were pretreated with vitamin E. Five additional hypoxic piglets received i.m. placebo (vehicle for vitamin E) and five normoxic piglets served as controls. 20 m g / k g / d o s e of vitamin E or equal volume of placebo were given 48 and 3 h prior to the study. Blood samples for a-tocopherol m e a s u r e m e n t were drawn prior to vitamin E administrations and at 1, 2 and 3 h after the second dose and at the end of the study. In the two normoxic groups (five control and five vitamin E-pretreated), normoxemia (PaO2 of 100-120 m m H g ) was maintained until the conclusion of the experiment, 60 min. At that time, the brain was removed, frozen in liquid nitrogen and stored at - 7 0 ° C until analysis of N a + , K + - A T P a s e activity and products of lipid peroxidation could be performed. 10 piglets (five vitamin E and five placebo-pretreated) were studied u n d e r hypoxic conditions using 31p N M R spectroscopy to continuously monitor cerebral oxygenation. T h e ratio of phosphocreatine (PCr) to inorganic phosphate (Pi) served as an index of level of oxidative phosphorylation. Following baseline P C r / P i measurements, FiO 2 was lowered to attain a 50% or greater decrease in P C r / P i ratio reflecting a diminished cerebral energy state. The achieved level of hypoxia was maintained until the conclusion of the experiment, 60 min. T h e brain was then removed and processed as described above.

2.3. [31p]NMRspectroscopy Phosphorus nuclear magnetic resonance spectroscopy non-invasively measures the relative concentrations of intracellular phosphate metabolites, including phosphocreatine, adenosine triphosphate, inorganic phosphate, and various mono- and diphosphoesters [5]. The P C r / P i ratio is related to the cellular phosphate potential and can be used as a marker of tissue oxygenation. The P C r / P i ratio was calculated for each spectrum. Since baseline P C r / P i values vary from one animal to another, the change in P C r / P i during hypoxia was assessed by comparing it to the corresponding baseline value for the same animal and calculating the percent decrease compared with baseline. In addition, through M i c h a e l i s - M e n t e n kinetics, intracellular p H (pH i) can be quantified by measuring the chemical shift between PCr and Pi peaks. Cerebral magnetic resonance spectroscopy was performed in vivo, using a 12-in horizontal bore, 2.7 Tesla superconducting magnet. A single turn, double-tuned circular radio frequency surface coil with a diameter of 3 cm was centered over the exposed vertex of the skull. Intracellular phosphate metabolites were measured from the area of cerebral cortex under the coil.

2.4. Brain cell membrane preparation Brain cell m e m b r a n e s were prepared according to the method described by Harik et al. [16]. Briefly, frozen cerebral cortex was homogenized in 10 m M Tris-HC1 buffer (pH 7.4) containing 0.32 M sucrose and 0.5 m M N a - E D T A . The homogenate was centrifuged at 1000× g for 10 min. The supernatant was centrifuged at 40,000× g for 60 min. The resultant pellet was rehomogenized in 10 m M Tris-HCl buffer (pH 7.4) containing 0.5 m M E D T A and centrifuged again at 40,000× g for 60 min. The final pellet was resuspended in 10 m M Tris-HC1/0.5 m M N a - E D T A buffer (pH 7.4). Protein content was determined according to the method of Lowry et al. [22].

2.5. Determination of Na +,K ÷-ATPase activity N a + , K ÷ - A T P a s e activity was determined as previously described [27]. A T P hydrolysis was carried out in 1 ml reaction mixture containing 100 m M NaCI, 20 m M KC1, 3 m M MgCI2, 3 m M Na2-ATP, 50 m M Tris-HCl buffer (pH 7.4) and 100 /~g m e m b r a n e protein. The reaction was carried out at 37°C in the presence and in the absence of 1 m M ouabain for 5 min, a period during which A T P hydrolysis is linear. KCI was not added to the tubes containing ouabain. The reaction was stopped by adding 12.5% trichloroacetic acid. The samples were placed on ice, centrifuged at 2000× g for 15 min and the supernatant was analysed for liberated inorganic phosphate by the method of Fiske et al. [11]. Ouabain-sensitive activity was referred to as Na+,K+-ATPase activity and expressed as ~ m o l P i / m g protein/h.

2.6. Determination of lipid peroxidation products Brain lipid peroxidation was determined by measuring the levels of conjugated dienes and fluorescent compounds. Lipids were extracted by the method of Folch-Pi et al. [12]. Brain tissue was homogenized in a c h l o r o f o r m - m e t h a n o l (2 : 1) mixture containing 0.5 m M E D T A and allowed to oscillate for 1 h under nitrogen at 30°C in a shaking incubator. Samples were filtered through glass fiber filters and mixed with 0.2 vol. of 0.88% NaCI. Following centrifugation, the lower phase was collected and dried under a steam of nitrogen. The samples were then redissolved in chloroform and dried u n d e r nitrogen. This process was repeated three times. Finally, the samples were dissolve in spectrophotometric grade n-heptane to provide 1 mg lipid/ml heptane solution and absorbance spectra were taken

S.M. Shin et al. / B r a i n Research 653 (1994) 45-50 between 200 and 300 nm. Difference spectra were obtained using computer software purchased from SLM Aminco. Conjugated dienes were calculated from difference spectrum using a molar extinction coefficient of 20,000 M 1.cm t [4]. The amount of conjugated dienes was expressed in terms of ~ m o l / m g lipid. Fluorescent compounds were determined spectrophotofluorometrically at excitation/ emission wavelengths of 360/435 nm according to the method of Dillard and Tappel [7] and expressed as fluorescent intensity, relative to a quinine sulfate standard, at 360/435, in /~g quinine s u l f a t e / r a g lipid. Serum vitamin E levels were measured by high performance liquid chromatography after the method of Bieri [2].

47

Table 2 [31P]NMR spectroscopy data during cerebral cortical hypoxia for hypoxia + vitamin E and hypoxia + placebo (n = 5, each group) groups of piglets Group Vitamin E Baseline Hypoxia Placebo Baseline Hypoxia

PCr/P i

pH~i I

2.62 _+0.54 1.05 +0.27 *

7.22 _+1~.07 6.96_+0.10 *

2.44 ± 0.48 1.14 _+0.30 *

7.23 + 0.05 6.96 ± (I. 13 *

* P < 0.005. Mean + S.D. 2. Z Statistical analysis Biochemical data from control and experimental groups were compared using Kruskall-Wallis test and, if the result of Kruskall-Wallis test revealed statistical significance, pairs of data sets were compared using Wilcoxon rank sum test.

PCr/P~ to 1.05 _+ 0.27 and 1.14 _+ 0.30 ( P < 0.005) with a concomitant significant decrease in intracellular pH from a baseline of 7.22 _+ 0.07 to 6.96 +_ 0.10 and from 7.23 _+ 0.05 to 6.96 _+ 0.13, during hypoxia, in vitamin E- and placebo-pretreated groups.

3. Results

3.2. Biochemical data

The physiological data for the control and experimental groups of animals are shown in Table 1. Baseline pH, P a C O 2 , P a O 2 and hematocrit were comparable in all four groups of animals. In vitamin E- and placebo-pretreated hypoxic groups, following a reduction in FiO 2, arterial pO:~ and pH decreased significantly from the baseline values, without a change in & C O 2, suggesting the presence of metabolic acidosis. Hematocrit remained unchanged throughout the study period.

The Na+,K+-ATPase activity in the brain cell membranes obtained from the control, vitamin E-pretreated normoxic, vitamin E-pretreated hypoxic and placebopretreated hypoxic group is shown in Fig. 1. The brain cell membrane Na+,K+-ATPase activity decreased significantly from 61.8 _+ 5.7 in control group to 44.9 _+ 9.7 p, mol P J m g p r o t e i n / h in placebo-pretreated hypoxic

80 ~* p<0.O05

3.1. N M R data

Cerebral cortical 31p N M R spectroscopy data during cerebral hypoxia from 10 hypoxic piglets is summarized in Table 2. The baseline PCr/Pi, as stabilized over a period of 60 min, was 2.62 _+ 0.54 and 2.44 _+ 0.48 in vitamin E- and placebo-pretreated groups, respectively. During hypoxia, there was a significant decline in

o "

:::::: ::::: :i:~i :::::: ::::::

60

E a.

D

_e O

E- i v

40 i'ii

Table 1 Physiological data for control, n o r m o x i a + v i t a m i n E, hypoxia+ vitamin E and hypoxia + placebo (n = 5, each group) groups of newborn piglets Group Control Normoxia + Vitamin E Vitamin E Baseline Hypoxia Placebo Baseline Hypoxia

pH a

PaCO2

PaO2

(mmHg)

(mmHg)

Hct (%)

7.44_+0.03 "7.40_+0.03

41+ 4 46_+ 3

107_+11 112_+14

30+1 30+2

7.44_+0.10 7.31_+0.09"

38+ 6 39_+ 6

154_+82 25_+ 7 * *

31_+1 31_+1

7.48+0.06 7.38_+0.02**

40_+10 43± 8

* P < 0.05: ** P < 0.005. Mean + S.D.

135_+ 7 21_+ 1 " *

35+4 35_+4

ii:: ¢)

O ¢B

20 i:ii: :xill :x:N :i:x

+g Control

Normoxia+

Hyp~xla

Hy%oxla

VIt. E

VIt. E

Placebo

Fig. 1. Na+,K+-ATPase activity in brain cell m e m b r a n e s prepared from cerebral cortex of control, n o r m o x i a + v i t a m i n E, hypoxia+ vitamin E and hypoxia+placebo (n = 5, each group) groups of newborn piglets. Each bar, mean _+S.D.

S.M. Shin et al. / Brain Research 653 (1994) 45-50

48

Table 3 Level of fluorescent compounds in cerebral cortex obtained from control, normoxia+vitamin E, hypoxia+vitamin E and hypoxia+ placebo (n = 5, each group) groups of newborn piglets Group

Fluorescent compounds (/zg quinine sulfate/g brain)

Control Normoxia + vitamin E Hypoxia + vitamin E Hypoxia + placebo

1.21 ± 1.35 + 1.13 + 1.72 ±

0.11 0.38 0.27 0.06 *

• P < 0.005. Mean ± S.D.

group (P < 0.005). In contrast, Na+,K+-ATPase activity in the vitamin E-pretreated hypoxic group was comparable to Na+,K+-ATPase activity in control and vitamin E-pretreated normoxic groups (66.0 + 7.0 vs. 61.8 + 5.7, 63.0 + 7.7/xmol P J m g protein/h, respectively). The comparison of level of fluorescent compounds in the brains obtained the four groups is shown in Table 3. The level of fluorescent compounds increased significantly from 1.21 + 0.11 in control to 1.72 + 0.06 /zg quinine sulfate/g brain in placebo-pretreated hypoxic group (P < 0.005), whereas the level of fluorescent compounds in the vitamin E-pretreated hypoxic group was comparable to the control and vitamin Epretreated normoxic groups (1.13 + 0.27 vs. 1.21 + 0.11, 1.35 + 0.38 /zg quinine sulfate/g brain, respectively) The level of conjugated dienes were similar in all four groups (zero reference value in control group). In vitamin E-pretreated normoxic and hypoxic groups, serum levels of vitamin E at 48 h after first dose and 3 h after second dose of vitamin E were higher than baseline vitamin E level in the two groups (1.73 + 0.50 vs. 0.56 + 0.16 baseline in normoxic group and 1.90 + 0.37 vs. 0.58 + 0.23 mg/dl baseline in the hypoxic group). These levels were maintained during the entire study period in the vitamin E-pretreated group of animals. The baseline vitamin E level in the placebo-pretreated group was comparable to the baseline vitamin E level in the two vitamin-pretreated groups (Table 4). 4. Discussion

Oxygen-free radical-mediated lipid peroxidation has been proposed as one of the major mechanism of cell

membrane damage and inactivation of Na+,K+-ATPase during cerebral hypoxia [17,20,27,35]. The present study investigated the protective effect of vitamin E on the structure and function of brain cell membrane during cerebral cortical hypoxia in newborn piglets by measuring brain cell membrane Na+,K+-ATPase activity and levels of products of lipid peroxidation in the brain. During hypoxia, the brain cell membrane Na+,K +ATPase activity in the placebo-pretreated hypoxic group decreased by 27% where as there was no change in the Na+,K+-ATPase activity in the vitamin E-pretreated hypoxic group as compared with the control and vitamin E-pretreated normoxic group. Also, there was a significant increase in the level of fluorescent compounds in the placebo-pretreated hypoxic group, while in the vitamin E-pretreated normoxic and hypoxic groups the level of fluorescent compounds was unaltered as compared with control. While it is known that decreased energy metabolism during hypoxia leads to cellular membrane injury, these results suggest that vitamin E has a protective effect on the structure and function of the brain cell membrane during hypoxia. Vitamin E is believed to have a wide range of physiological functions in man and animals, particularly as a free radical scavenger and natural antioxidant, protecting cell membranes against lipid peroxidation [8,10,15,23,34]. Pretreatment with orally administered (50 IU for 14 days) vitamin E to pregnant rats prevented lipid peroxidation in the fetal brain following ischemic-reperfusion injury induced by clamping and releasing of uterotubal vessels [19]. Vitamin E administration (20 mg i.v.) also suppressed the rise in lipid peroxides in the brains of adult hypertensive rats following ischemic-reperfusion injury [30]. Neuronal density in the CA 1 sector of the hippocampus following ischemic-reperfusion injury was preserved in vitamin E-preteated gerrbils (50 or 100 mg i.v.) [18]. a-Tocopherol-mediated protection of membranes against lipid peroxidation is dependent on the incorporation of a-tocopherol into membranes, and the extent of this protection is related to the quantity of atocopherol present in membranes [36]. a-Tocopherol in the membrane can break the free radical initiated and propagated chain of lipid peroxidation by reacting with hydroxyl radicals [38,39]. Among subcellular frac-

Table 4 Serum a-tocopherol level in normoxia + vitamin E, hypoxia + vitamin E (n = 5, each group) and hypoxia + placebo (n = 1) groups of newborn piglets Group Time (h)

0

45

46

47

48

Hypoxia + vitamin E Normoxia + vitamin E Hypoxia + placebo

0.58 ___0.23 0.56 + 0.16 0.30

0.67 + 0.17 1.00 + 0.27 0.20

1.50 + 0.20 1.40 + 0.35 -

2.00 + 0.40 1.75 ± 0.49 0.30

1.90 + 0.37 * 1.73 ± 0.50 * 0.25

Vitamin E 20 m g / k g dose. * P < 0.005. Mean ± S.D.

S.M. Shin et al. /Brain Research 653 (1994) 45-50

tions, vitamin E appears to be concentrated in the microsomal and mitochondrial fractions. These two fractions also have the highest levels of polyunsaturated fatty acids in their phospholipid fraction, especially in the inner mitochondrial membrane adjacent to electron transfer complexes associated with high oxygen radical activity. The number of a-tocopherol molecules relative to PUFA is particularly higher in the immediate vicinity of oxygen radical generation [3]. It has been postulated that a-tocopherol in microsomal membrane reacts with hydroxyl radicals preventing peroxidation of polyunsaturated fatty acids in the membrane [25] and that the vitamin also takes part in redox reactions in mitochondria and microsomes [40]. The protective effect of vitamin E in our study could have been either due to the chain-breaking action of vitamin E incorporated in the brain cell membrane or due to vitamin E as a circulating antioxidant. The serum level of vitamin E in the vitamin E-pretreated hypoxic group was higher than in the placebo-pretreated hypoxic group. Vitamin E administered 3 h prior to hypoxia in our study increased serum level of the vitamin within the physiologic range from the deficient levels of vitamin E in the newborn piglets. Although brain tissue levels of vitamin E were not measured in this study, administration of vitamin E 48 h prior to onset of hypoxia could have provided sufficient time for vitamin E to be incorporated into brain cell membrane. Prior studies on vitamin E kinetics have shown that following administration of high dose of vitamin E, liver and plasma levels of vitamin E increased more rapidly as compared with the level in brain [24]. Studies using radioactive a-tocopherol injected i.v. have demonstrated a rapid uptake of vitamin E into the brain within hours after administration even though the level of vitamin E in the brain was lower as compared with the level in the liver [14,40,41]. In an effort to increase the circulating vitamin E level and to allow the vitamin to be incorporated in the brain cell membranes, the vitamin was administered twice at different time intervals, prior to onset of hypoxia. In summary, vitamin E pretreatment resulted in protection of brain cell membrane structure and function as indicated by preservation of Na+,K+-ATPase activity and no increase in the level of products of lipid peroxidation in the vitamin E-pretreated hypoxic group. In contrast, in placebo-pretreated hypoxic group there was a decrease in the activity of Na+,K+-ATPase and an increase in the level of fluorescent compounds. In conclusion, the present study demonstrates that vitamin E has a protective effect on the structure and function of brain cell membrane, probably due to its antioxidant activity.

49

Acknowledgement The authors would like to thank D.M. Zurlo for her assistance in preparing the manuscript. This work was supported in part by NIH Grants HD-20337 and 5T32-HL07027 and presented in part at the 1992 Annual Meeting of the Society for Pediatric Research in Baltimore, MD.

References [1] Acosta, D., Kass, I.S. and Cottrell, J.E., Effect of o~-tocopherol and free radicals on anoxic damage in the rat hippocampal slice, Exp. Neurol., 97 (1987) 607-614. [2] Bieri, J.G., Tolliver, T.J. and Catignani, G.L., Simultaneous determination of a-tocopherol and retinol in plasma or red blood cells by high pressure liquid chromatography, Am. J. Clin. Nutr., 32 (1979) 2143-2149. [3] Buttriss, J.L. and Diplock, A.T., The relationship between atocopherol and phospholipid fatty acids in rat liver subcellular membrane fractions, Biochim. Biophys. Acta, 962 (1988) 81-90. [4] Chan, H.W.S. and Levett, A., Auto-oxidation of methyl linoleate. Separation and analysis of isomer mixture of methyl linoleate hydroperoxide and methylhydroxy linoleates, Lipids, 12 (1977) 99-106. [5] Delivoria-Papadopoulos, M. and Chance, B., In E.M. Scarpelli (Ed.), Pulmonary Physiology: Fetus, Newborn, Child and Adolescent, Lea and Berger, Philadelphia, PA, 1989, pp. 365-370. [6] DiGiacomo, J.E., Pan~, C.R., Gwiazdowski, S., Mishra, O.P. and Delivoria-Papadopoulos, M., Effect of graded hypoxia on brain cell membrane injury in newborn piglets, Biol. Neonate, 61 (1992) 25-32. [7] Dillard, C.J. and Tappel, A.L., Fluorescent damage products of lipid peroxidation, Methods Enzymol., 105 (1984) 337-34l. [8] Dormandy, Y.L., Free-radical oxidation and anti-oxidants, Lancet, i (1978) 647-750. [9] Espinoza, M.I. and Parer, J.T., Mechanism of asphyxial brain damage, and possible pharmacological interventions, in the fetus, Am. J. Obstet. Gynecol., 164 (1991) 1582-1591. [10] Farrell, P.M., Vitamin E deficiency in premature infants, J. Pediatr., 95 (1979) 869-872. [11] Fiske, C.H. and Subbarow, Y., The colorimetric determination of phosphorus, J. Biol. Chem., 66 (1925) 375-400. [12] Folch-Pi, J., Lees, M.J. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissue, J. Biol. Chem., 226 (1957) 497-509. [13] Girotti, A.W., Mechanism of lipid peroxidation, J. Free Radic. Biol. Med., 1 (1985) 87-95. [14] Goss-Sampson, M.A. and Muller, D.P.R., Studies on the neurobiology of vitamin E (a-tocopherol) and some other antioxidant systems in the rat, Neuropathol. Appl. Neurobiol., 13 (1987) 289-296. [15] Haider, S.S. and EI-Fakhri, M., Action of a-tocopherol on vanadium-stimulated lipid peroxidation in rat brain, NeurotoxL cology, 12 (1991) 79-85. [16] Harik, S.I., Douli, G.H. and Dick, A.P.K., Specific ouabain binding to brain microvessels and choroid plexus, Z Cereb. Blood FLow Metab., 5 (1985) 156-160. [17] Hexum, T.D. and Fried, R., Effect of superoxide radicals on Na+,K + transport adenosine triphosphate and protection by superoxide dismutase, Neurochem. Res., 4 (1979) 73-92.

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S.M. Shin et al. / Brain Research 653 (1994) 45-50

[18] Hideaki, H., Hiroyuki, K. and Kyuya, K., Protective effect of a-tocopherol on ischemic neuronal damage in the gerbil hippocampus, Brain Res., 510 (1990) 335-338. [19] Hozumi, I., Toshihiro, A. and Kenji, F., Protective effect of vitamin E on fetal distress induced by ischemia of the uteroplacental system in pregnant rats, Free Radic. Biol. Med., 8 (1990) 393-400. [20] Imaizumi, S., Kayama, T. and Suzuki, J., Chemiluminescence in hypoxic brain - the first report. Correlation between energy metabolism and free radical reaction, Stroke, 15 (1984) 10611065. [21] Lees, G.J., Lehmann, A., Sandberg, M. and Hamberger, A., The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor in the rat hippocampus, Neurosci. Lett., 120 (1990) 159-162. [22] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [23] Lucy, J.A., Functional and structural aspects of biological membranes: a suggested structural role for vitamin E in the control of membranes permeability and stability, Ann. N.Y. Acad. Sci., 203 (1972) 4-11. [24] Machlin, L.J. and Gabriel, E., Kinetics of tissue a-tocopherol uptake and depletion following administration of high levels of vitamin E, Ann. N.Y. Acad. Sci., (1982) 48-60. [25] McCay, P.B. and King, M.M., In L.J. Machlin (Ed.), Vitamin E A Comprehensive Treatise, Marcel Dekker, New York, NY, 1980, pp. 289-317. [26] McCord, J.M., Oxygen derived free radicals in postischemic tissue injury, NEJM, 312 (1985) 159-163. [27] Mishra, O.P. and Delivoria-Papadopoulos, M., Na÷,K+-ATPase in developing fetal guinea pig brain and their effect of maternal hypoxia, Neurochem. Res., 13 (1988) 765-770. [28] Mishra, O.P. and Delivoria-Papadopoulos, M., Lipid peroxidation in developing fetal guinea pig during normoxia and hypoxia, Dev. Brain Res., 45 (1989) 129-135. [29] Mishra, O.P., Delivoria-Papadopoulos, M., Cahillane, G. and Wagerle, L.C., Lipid peroxidation as the mechanism of modification of affinity of the Na+,K+-ATPase active sites for ATP, K+,Na ÷, and strophanthidin in vitro, Neurochem. Res., 14 (1989) 845-851. [30] Mitsuo, Y., Takeshi, S., Toru, U., Takashi, S., Kazuo, Y. and Takashi, K., A possible role of lipid peroxidation in cellular damages caused by cerebral ischemia and the protective effect of a-tocopherol administration, Stroke, 14 (1983) 977-982.

[31] Nagafuji, T., Koide, T. and Takato, M., Neurochemical correlates of selective neuronal loss following cerebral ischemia: role of decreased Na÷,K+-ATPase activity, Brain Res., 571 (1992) 265-271. [32] Nakamura, T. and Masugi, F., Transfer of a-tocopherol from plasma to erythrocytes in vitamin E-deficient rats, Int. J. ~tarn. Nutr. Res., 49 (1979) 364-369. [33] Ogihara, T., Kitagawa, M., Miki, M., Tamai, H., Yasuda, H., Okamoto, R. and Mino, M., Susceptibility of neonatal lipoproteins to oxidative stress, Pediatr Res., 29 (1991) 39-45. [34] Phelps, D.L., Vitamin E: where do we stand? Pediatrics, 63 (1979) 933-935. [35] Schaefer, A., Komlos, M. and Seregi, A., Lipid peroxidation as the cause of the ascorbic acid induced decrease of adenosine triphosphatase activities of rat brain microsomes and its inhibition by biogenic amines and psychotropic drugs, Biochem. PharmacoL, 24 (1975) 1781-1786. [36] Siejo, B.K., Cell damage in the brain: a speculative synthesis, J. Cereb. Blood Flow Metab., 1 (1981) 155-185. [37] Suzuki, J., Fujimoto, S., Mizoi, K. and Oba, M., The protective effect of combined administration of anti-oxidants and perfluorochemicals on cerebral ischemia, Stroke, 15 (1984) 672-679. [38] Takenaka, Y., Miki, M., Yasuda, H. and Mino, M., The effect of a-tocopherol an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites, Arch. Biochem. Biophys., 285 (1991) 344-350. [39] Tappel, A.L., Protection against free radical lipid peroxidation reactions, Adv. Exp. Med., 97 (1978) 111-131. [40] Vatassery, G.T., Angerhofer, C.K., Knox, C.A. and Deshmukh, D.S., Concentrations of vitamin E in various neuroanatomic regions and subcellular fractions, and the uptake of vitamin E by specific areas of rat brain, Biochim. Biophys. Acta, 792 (1984) 118-122. [41] Vatassery, G.T., Angerhofer, C.K. and Knox, C.A., Effect of age on vitamin E concentrations in various regions of the brain and a few selected peripheral tissues of the rat, and on the uptake of radioactive vitamin E by various regions of rat brain, J. Neurochem., 43 (1984) 409-412. [42] Yasuda, H., Shimada, O., Nakajima, A. and Asano, T., Cerebral protective effect and radical scavenging action, J. Neurochern., 37 (1981) 934-938. [43] Yoshida, S., Busto, R., Watson, B.D., Santiso, M. and Ginsberg, M.D., Postischemic cerebral lipid peroxidation in vitro: modification by dietary vitamin E, J. Neurochem., 44 (1985) 1593-1601.