Effect of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in newborn piglets

Brain Research 922 (2001) 276–281 www.elsevier.com / locate / bres

Research report

Effect of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in newborn piglets Won Soon Park a , Yun Sil Chang a , So Hee Chung a , Dae Won Seo b , Sung Hwa Hong c , a, Munhyang Lee * a

Department of Pediatrics, Samsung Medical Center, 50 Ilwon-Dong, Kangnam-Gu, Sungkyunkwan University School of Medicine, Seoul, South Korea b Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea c Department of Otorhinolaryngology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea Accepted 5 October 2001

Abstract The aim of this study was to evaluate the effects of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy metabolism in the developing brain. Thirty-seven newborn piglets were divided randomly into four groups: normothermic control (NC, n59); hypothermic control (HC, n57); normothermic bilirubin infusion (NB, n511); and hypothermic bilirubin infusion (HB, n510) groups. In bilirubin infusion groups (NB and HB), a loading dose of bilirubin (35 mg / kg) was given over 5 min, followed by a continuous infusion (25 mg / kg / h) for 4 h. The control groups (NC, HC) received a bilirubin-free buffer solution. Sulfadimethoxine was administered to animals in all experimental groups. Rectal temperature was maintained between 38.0 and 39.08C in normothermic groups, and between 34.0 and 35.08C in hypothermic groups for 4 h after the start of bilirubin infusion. The final blood and brain bilirubin concentrations in the bilirubin infusion groups (NB and HB) were not significantly different. Decreased cerebral cortical cell membrane Na 1 ,K 1 –ATPase activity and increased lipid peroxidation products observed in the NB group, indicative of bilirubin-induced brain damage, were significantly attenuated in the HB group. Hypothermia also significantly improved the bilirubin-induced reduction in brain ATP and phosphocreatine levels and increase in blood and brain lactate levels. In summary, hypothermia significantly attenuated the bilirubin-induced alterations in brain cell membrane function and energy metabolism in the newborn piglet. These findings suggest the possibility that hypothermia could be a good neuroprotective therapeutic modality in neonatal bilirubin encephalopathy.  2001 Elsevier Science B.V. All rights reserved. Keywords: Bilirubin; Hypothermia; Brain protection; Cerebral metabolism; Newborn piglet

1. Introduction Bilirubin encephalopathy (kernicterus) is a major complication of the toxic effect of bilirubin on the newborn brain [7,17,18,24]. Although the precise mechanisms of its toxic effect are not completely understood, bilirubin has been reported to act as a generalized cellular poison and affect a variety of cellular functions. Disruption of membrane function, compromise of energy metabolism, decreased membrane potential, alteration in enzymatic function, inhibition of protein and DNA synthesis, and ex-

*Corresponding author. Tel.: 182-2-3410-3522; fax: 182-2-34100043. E-mail address: [email protected] (M. Lee).

citotoxicity are some of the toxic mechanisms implicated [7,17,18,20,23–25,28–30,32,35]. The neuroprotective role of hypothermia during hypoxia–ischemia [9,34,36], meningitis [21,22] and traumatic brain injury [10,12] has been well known. Hypothermia has been demonstrated to have a beneficial effect at multiple points of brain injury progression. Hypothermia reduced nitric oxide generation, cytokine production and the release of excitotoxic amino acids [10,12,21,22,34]. A decrease in cerebral metabolism and preservation of energy stores as well as a decrease in cerebral edema has also been documented [9,22,36]. Therefore, these features make hypothermia an attractive therapeutic modality to prevent or treat bilirubin-induced brain injury. This study was done to determine whether hypothermia could ameliorate the bilirubin-induced brain injury in the

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )03186-9

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developing brain. We tested the hypothesis that hypothermia attenuates the bilirubin-induced alterations in brain cell membrane function and energy metabolism in the newborn piglet. Changes in brain cell membrane function and energy metabolism were determined by measuring Na 1 ,K 1 –ATPase activity, lipid peroxidation products (conjugated dienes), and concentrations of high-energy phosphate compounds in the cerebral cortex.

2. Materials and methods

2.1. Animal preparation The experimental protocols described herein were reviewed and approved by the Institutional Animal Care and Use Committee of the Samsung Biomedical Research Center, Seoul, Korea. This study also followed the institutional and National Institutes of Health guidelines for laboratory animal care. Newborn piglets less than 3 days old were anesthetized with sodium thiopental (5 mg / kg, intravenously), paralyzed with pancuronium (1 mg / kg, intravenously), tracheotomized, and artificially ventilated with a mechanical ventilator (Sechrist Infant Ventilator, IV-100B, Sechrist Industries Co., Anaheim, CA, USA). Femoral arteries and veins were cannulated for blood pressure monitoring, blood sampling, and for medication and fluid infusion, respectively. Electrocardiograph (ECG), oxygen saturation and blood pressure were continuously monitored using Hewlett–Packard neonatal monitoring system (Hewlett– Packard Model M1276A, Hewlett–Packard Co., MA, USA).

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solution without bilirubin was given. In all experimental groups, 200 mg / kg of sulfadimethoxine (Sigma) was given at 1 h after the start of bilirubin infusion to promote net transfer of free bilirubin into the brain [28]. In normothermic groups (NC and NB), the piglet was placed under the servo-controlled warmer (Airshields Inc., Hatboro, PA, USA) and rectal temperature was maintained between 38.0 and 39.08C throughout the experiment. In hypothermic groups (HC and HB), animals were cooled rapidly by applying ice water packs to the trunk just after the start of bilirubin infusion, and rectal temperature was kept between 34.0 and 35.08C during the experiment. Rectal and brain temperatures were equivalent in our previous pilot study of hypothermia in the newborn piglet. Glucose (5 mg / kg / min) and electrolyte solution was given at a rate of 4 ml / kg / h to each experimental group during the experiment. Continuous monitoring of ECG, systemic blood pressure and oxygen saturation was done during the experiment. Arterial blood gas analyses, concentrations of glucose and lactate, and bilirubin in the blood were measured at baseline, and every 1 h for 4 h after the start of bilirubin infusion. Arterial blood gases were measured on a blood gas analyzer (Ciba-Corning Diagnostics Corp., Medfield, MA, USA), concentrations of glucose and lactate were measured using an YSI model 2300 dual analyzer (Yellow Springs Instrument Co., Yellow Springs, OH, USA), and bilirubin level was measured using bilirubin tester (Wako Pure Chemical Industries Ltd., Osaka, Japan). At the end of the experiment the piglets were decapitated using a guillotine, the harvested brains were quickly frozen in liquid nitrogen, and stored at 2808C for further biochemical analyses.

2.3. Biochemical analyses of brain cortex 2.2. Experimental protocol After surgery and a stabilization period of 1 h, 37 newborn piglets were randomly divided into the following four experimental groups: nine in the normothermic control (NC); seven in the hypothermic control (HC); 11 in the normothermic bilirubin infusion (NB); and 10 in the hypothermic bilirubin infusion (HB) groups. In bilirubin infusion groups (NB and HB), a loading dose of bilirubin (35 mg / kg) was given over 5 min, followed by a continuous infusion at a rate of 25 mg / kg / h for 4 h [20]. Bilirubin (Sigma Chemical Co., St Louis, MO, USA) was dissolved in a buffer solution containing 18.5 vol% 0.1 N NaOH, 44.5 vol% human albumin (5%), and 37 vol% 0.055 M Na 2 HPO 4 , with the final concentration and pH adjusted to 3 mg / ml and 7.4, respectively [20]. The bilirubin solution was prepared fresh immediately prior to use in a darkened room, and was shielded from light with aluminum foil throughout the experiment. In control groups (NC and HC), a same volume of buffer

Methods of brain cell membrane preparation and determination of cerebral cortical cell membrane Na 1 ,K 1 – ATPase activity, levels of conjugated dienes, tissue glucose and lactate concentrations, ATP and phosphocreatine were described in detail previously [8,31]. Brain bilirubin levels were measured by a modified diazo technique after homogenization and acid chloroform extraction [4].

2.4. Statistical analysis The principal statistical tests used were one-way analysis of variance and Scheffe’s correction. To detect significant changes over time within each group, data were compared using repeated measures analysis of variance with Bonferroni correction. Statistical analysis described above was done using SAS software program version 6.12. A P-value of ,0.05 was considered significant. Data were given as mean6S.D.

W.S. Park et al. / Brain Research 922 (2001) 276 – 281

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Table 1 Physiological data at baseline and 4 h of experiment in each group of newborn piglets NC (n59)

HC (n57)

Heart rate ( /min) Baseline 187632 4h 193625

NB (n511)

HB (n510)

181630 189631

13968 a 165638 a

Mean arterial pressure (mmHg) Baseline 70615 68612 4h 7267 46613 a,b

72610 7468

7068 5565 a,b

Arterial base excess (mEq /l) Baseline 1.863.4 2.262.1 4h 1.262.4 23.264.0 a,b

2.361.8 1.961.7

1.861.5 22.062.1 a,b

198629 181645 b

Arterial pH Baseline 7.4360.15 4h 7.4360.05

7.4560.12 7.2460.09 a,b

7.4460.04 7.4160.04

7.3960.09 7.3660.03 a,c

PaO2 (mmHg) Baseline 117631 4h 115632

95638 99627

108624 106615

98620 101618

PaCO2 (mmHg) Baseline 3667 4h 3865

4269 50610

3764 3763

50610 4463

Values given represent a P,0.05 compared to b P,0.05 compared to c P,0.05 compared to

significantly compared to the corresponding values in NC group at the end of experiment. In the HB group, only arterial pH was significantly improved compared to the NB group, and improvement in mean arterial pressure and base excess did not reach a statistical significance.

3.2. Bilirubin, glucose and lactate concentration in the blood and brain In bilirubin infusion (NB and HB) groups, blood bilirubin levels increased abruptly after bolus infusion, and remain elevated throughout the experiment. There were no significant inter-group differences in blood and brain bilirubin levels between NB and HB groups during the experiment (Table 2). Blood glucose and lactate level in the NC group did not change significantly during the experiment. Although baseline glucose levels were not different and the same amount of glucose (5 mg / kg / min) was given in each experimental group during the experiment, blood glucose levels in HC, NB and HB groups became significantly higher compared to the corresponding values in NC group at the end of experiment (Table 2). In the NB group, blood-to-brain glucose ratio became significantly decreased, and lactate level in the blood and brain became significantly increased compared to corresponding values in the NC and HC groups, and these abnormalities were significantly attenuated in the HB group.

mean6S.D. NC. HC. NB.

3. Results

3.3. Biochemical data in the cerebral cortex

3.1. Physiologic variables

In the HC group, cerebral cortical cell membrane Na 1 ,K 1 –ATPase activity was slightly but significantly decreased compared to corresponding values in the NC group (Fig. 1). In the NB group, cerebral cortical cell membrane Na 1 ,K 1 –ATPase activity decreased, and levels of lipid peroxidation products (conjugated dienes) increased significantly compared to the NC group. The abnormalities observed in the NB group were significantly attenuated in the HB group. Brain ATP and phosphocreatine levels were significantly reduced in the NB group compared to the corresponding

The mean physiologic variables from the four experimental groups were summarized in Table 1. In the NC group, there were no significant changes in the values of physiologic variables including heart rate, arterial blood gases and mean arterial blood pressure during the experiment. In hypothermic (HC and HB) groups, heart rate became significantly lower than the corresponding values in the NC group at 4 h of hypothermia. In the NB group, mean arterial pressure, base excess and pH decreased

Table 2 Glucose, lactate and bilirubin levels in the blood and brain at 4 h of experiment in each group of newborn piglets

Blood glucose (mg / dl) Brain glucose (mmol / kg) Blood lactate (mmol / l) Brain lactate (mmol / kg) Blood bilirubin (mg / dl) Brain bilirubin (nmol / g) Values given represent a P,0.05 compared to b P,0.05 compared to c P,0.05 compared to

mean6S.D. NC. HC. NB.

NC (n59)

HC (n57)

NB (n511)

HB (n510)

78.00617.00 3.8760.95 1.1560.66 3.3561.09 0.3560.05 0.4560.17

107.00613.00 a 4.3360.73 0.2960.29 1.7361.81 0.3760.05 0.6360.43

128.00630.00 a 3.4261.60 3.7960.41 a,b 13.5668.26 a,b 27.1065.20 a,b 8.0363.05 a,b

112.00614.00 a 5.3061.14 a,c 1.8460.52 b,c 3.5263.86 c 25.8061.70 a,b 8.1363.73 a,b

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Fig. 1. Activity of Na 1 ,K 1 –ATPase (A) and concentrations of conjugated dienes (B) in the cerebral cortex harvested at the end of experiment in each group of newborn piglets. (a) P,0.05 compared to NC. (b) P,0.05 compared to HC. (c) P,0.05 compared to NB.

values in the NC and HC groups (Fig. 2). The decreased levels of cerebral high-energy phosphate compounds observed in the NB group were significantly improved in the HB group.

4. Discussion In the present study, we used the newborn piglet as an animal model of neonatal bilirubin encephalopathy because the piglet brain is comparable in energy metabolism and maturity to human brain at birth [14,16]. The amount of bilirubin given to the piglet in this study was sufficient enough to achieve significant bilirubin deposition in the cerebral cortex [2,20]. Although bilirubin is deposited in even higher concentrations in brain regions other than the cerebral cortex [6], and the main neurologic signs in kernicterus are extrapyramidal, if bilirubin has neurotoxic effects it may affect the cerebral cortex as well as other brain regions. Therefore, our data of alterations in cerebral cortical cell membrane function and energy metabolism

279

Fig. 2. Concentrations of ATP (A) and phosphocreatine (B) in the cerebral cortex harvested at the end of experiment in each group of newborn piglets. (a) P,0.05 compared to NC. (b) P,0.05 compared to HC. (c) P,0.05 compared to NB.

observed in the bilirubin infusion groups should reflect bilirubin deposition. Reduction in cerebral ATP and phosphocreatine levels observed in the NB group support the assumption that mitochondrial dysfunction is an important element of the toxic effects of bilirubin on neurons [29,30]. In accordance with our data, impaired cerebral energy metabolism has also been documented in kernicteric gunn rats [32] and newborn piglets [20,23]. However, similar experiments done in guinea-pig [11], gunn rats [26] and newborn piglet [2] failed to document a significant change in brain glucose metabolism or oxidative phosphorylation. These discrepancies are difficult to explain. Further studies will be necessary to determine whether mitochondrial injury is the primary pathway of bilirubin toxicity in the intact brain. In the present study, hypothermia significantly attenuated the bilirubin-induced depletion of cerebral ATP and phosphocreatine levels. Maintenance of cerebral energy stores with hypothermia can be achieved by reducing cerebral metabolic rate. As a large portion of

280

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basal energy production is required for maintenance of normal Na 1 ,K 1 –ATPase activity [1], the bulk of energy saving may be derived from active suppression of this enzyme activity. Our data of slightly but significantly decreased Na 1 ,K 1 –ATPase activity observed in the HC group support this assumption. The blockage of bilirubin-induced neuronal injury by the administration of the N-methyl-D-aspartate receptor antagonist MK-801 in the Gunn rat [28] suggests that excitotoxicity plays a key role in bilirubin-induced brain injury. Since reuptake of excitotoxic amino acids is energy dependent [33], bilirubin-induced depletion in cerebral energy stores would lead to the accumulation of these toxic amino acids and result in neuronal death. Thus preservation of cerebral energy stores by hypothermia observed in this study would reduce the release of excitotoxins [21,34], thereby attenuating the bilirubin-induced cytotoxic brain damage. Decreased blood-to-brain glucose ratio and increased lactate level in the blood and brain observed in the NB group, indicative of secondarily increased anaerobic glycolysis to compensate for the bilirubin-induced mitochondrial damage and energy depletion, was significantly attenuated by hypothermia. Significantly higher blood glucose level in the HC group compared to the NC group indicates suppressed cerebral metabolism and glucose utilization during hypothermia [19]. Reduced brain cell membrane Na 1 ,K 1 –ATPase activity observed in the NB group might be caused by reduced cerebral energy stores [13], increased lipid peroxidation products [37] or direct bilirubin-induced changes in the physical state of the lipids in the cell membrane [20,25,35]. Since hypothermia also has been known to suppress this enzyme activity to preserve cerebral energy stores and brain bilirubin level between the NB and HB groups was not significantly different, slight but significant increase in Na 1 ,K 1 –ATPase activity observed in the HB group might be secondary to a general down-modulation of degradation of the cell membrane structure by lipid peroxidation with hypothermia treatment. Since this enzyme maintains transmembrane gradients of sodium and potassium ions, decreased Na 1 ,K 1 –ATPase activity in the NB group would result in brain cell membrane dysfunction ultimately leading to osmotic cell swelling and neuronal death [27]. However, hypothermia prevented intracellular ion and water entry and consequent cell swelling, in vitro, when the ATP-dependent Na 1 ,K 1 –ATPase was inhibited by ouabain [38]. These findings suggest that hypothermia can intervene at steps distal to decreased Na 1 ,K 1 –ATPase activity, specifically slowing the rate of ion leakage [19,38]. These findings are also of interest in view of the action of hypothermia to preserve the membrane potential for a longer time, thus delaying the onset of cytotoxic edema and attenuating the ensuing neuronal injury during neonatal bilirubin encephalopathy. In contrast to the previous studies [2,20], significant

decrease in mean arterial blood pressure, base excess and pH was observed in the NB group, and among these abnormalities, only arterial pH was significantly improved in the HB group. The cause of these abnormalities might be due to the generalized toxic effects of bilirubin on cellular functions including disturbances of mitochondrial function and energy metabolism [7,18,23,24,29,30,32], with resultant vascular energetic and contractile failure, secondary anaerobic glycolysis and lactic acidosis. As metabolic acidosis has been known as an important risk factor for the development of neonatal bilirubin encephalopathy [5,15], the lactic acidosis observed in the NB group would have aggravated neuronal damage, and significant attenuation of metabolic acidosis by hypothermia might have contributed to ameliorating bilirubin-induced brain injury. Comparable brain bilirubin level between the NB and HB groups indicates that metabolic acidosis per se does not promote brain bilirubin uptake in rats [3]. In summary, hypothermia significantly attenuated the bilirubin-induced alterations in brain cell membrane function and energy metabolism without major side effects in experimental bilirubin encephalopathy in the newborn piglet. These findings suggest the possibility that hypothermia could be a good neuroprotective therapeutic modality in neonatal bilirubin encephalopathy.

Acknowledgements This study was supported by a grant from the Korea Research Foundation (KRF-99-031-F00179-F1208).

References [1] J. Astrup, P. Sorensen, H. Sorensen, Oxygen and glucose consumption related to Na 1 –K 1 transport in canine brain, Stroke 12 (1981) 726–730. [2] B.S. Brann, B.S. Stonestreet, W. Oh, W.J. Cashore, The in vivo effect of bilirubin and sulfisoxazole on cerebral oxygen, glucose, and lactate metabolism in newborn piglets, Pedaitr. Res. 22 (1987) 135–141. [3] D. Bratlid, W.J. Cashore, W. Oh, Effect of acidosis on bilirubin deposition in rat brain, Pediatrics 73 (1984) 431–434. [4] D. Bratlid, A. Winsnes, Determination of conjugated and unconjugated bilirubin by methods based on direct spectrophotometry and chloroform extraction. A reappraisal, Scand. J. Clin. Lab. Invest. 28 (1971) 41–48. [5] R. Brodersen, Binding of bilirubin to albumin, Crit. Rev. Clin. Lab. Sci. 11 (1980) 305–399. [6] G.H. Burgess, B.S. Stonestreet, W.J. Cashore, W. Oh, Brain bilirubin deposition and brain blood flow during acute urea-induced hyperosmolarity in newborn piglets, Pediatr. Res. 19 (1985) 537–542. [7] W.J. Cashore, The neurotoxicity of bilirubin, Clin. Perinatol. 17 (1990) 437–447. [8] Y.S. Chang, W.S. Park, M. Lee, K.S. Kim, S.M. Shin, J.H. Choi, Effect of hyperglycemia on brain cell membrane function and

W.S. Park et al. / Brain Research 922 (2001) 276 – 281

[9]

[10]

[11] [12]

[13]

[14] [15] [16]

[17]

[18] [19] [20]

[21]

[22]

[23]

energy metabolism during hypoxia–ischemia in newborn piglets, Brain Res. 798 (1998) 271–280. M. Chopp, R. Knight, C. Tidwell, J. Helpern, E. Brown, K. Welch, The metabolic effects of mild hypothermia on global cerebral ischemia and recirculation in the cat: comparison to normothermia and hyperthermia, J. Cereb. Blood Flow Metab. 9 (1989) 141–148. G.L. Clifton, J.Y. Jiang, B.G. Lyeth, L.W. Jenkins, R.J. Hamm, R.L. Hayes, Marked protection by moderate hypothermia after experimental traumatic brain injury, J. Cereb. Blood Flow Metab. 11 (1991) 114–121. I. Diamond, R. Schmid, Oxidative phosphorylation in experimental bilirubin encephalopathy, Science 155 (1967) 1288–1289. W.D. Dietrich, O. Alonso, R. Busto, M.Y.T. Globus, M.D. Ginsberg, Post-traumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat, Acta Neuropathol. 87 (1994) 250–258. J.E. DiGiacomo, C.R. Pane, S. Gwiazdowski, O.P. Mishra, M. Delivoria-Papadopoulos, Effect of graded hypoxia on brain cell membrane injury in newborn piglets, Biol. Neonate 61 (1992) 25–32. J. Dobbing, J. Sands, Comparative aspects of the brain growth spurt, Early Hum. Dev. 3 (1979) 79–83. E.F. Eriksen, H. Danielsen, R. Brodersen, Bilirubin–liposome interaction, J. Biol. Chem. 256 (1981) 4269–4274. P.A. Flecknell, R. Wootton, M. John, Accurate measurement of cerebral metabolism in the conscious unrestrained neonatal piglet, Biol. Neonate 41 (1982) 221–226. T.W. Hansen, Bilirubin in the brain: distribution and effects on neurophysiological and neurochemical processes, Clin. Pediatr. 33 (1994) 452–459. T.W.R. Hansen, D. Bratlid, Bilirubin and brain toxicity, Acta Paediatr. Scand. 75 (1986) 513–522. P. Hochachka, Defense strategies against hypoxia and hypothermia, Science 231 (1986) 234–241. D.J. Hoffman, S.A. Zanelli, J. Kubin, O.P. Mishra, M. DelivoriaPapadopoulos, The in vivo effect of bilirubin on the N-methyl-Daspartate receptor / ion channel complex in the brains of newborn piglets, Pediatr. Res. 40 (1996) 804–808. J.E. Irazuzta, J. Olson, M.P. Kiefaber, H. Wong, Hypothermia attenuates excitatory neurotransmitter release in meningitis, Brain Res. 847 (1999) 143–148. J.E. Irazuzta, R. Pretzlaff, M. Rowin, K. Milam, F.P. Zemlan, B. Zingarelli, Hypothermia as an adjunctive treatment for severe bacterial meningitis, Brain Res. 881 (2000) 88–97. N.K. Ives, N.M. Bolas, R.M. Gardiner, The effects of bilirubin on brain energy metabolism during hyperosmolar opening of the blood–brain barrier: an in vivo study using 31 P nuclear magnetic resonance spectroscopy, Pediatr. Res. 26 (1989) 356–361.

281

[24] W.B. Karp, Biochemical alterations in neonatal hyperbilirubinemia and bilirubin encephalopathy: a review, Pediatrics 64 (1979) 361– 368. [25] S. Kashiwamata, M. Asai, R.K. Semba, Effect of bilirubin on the Arrhenius plots of Na,K–ATPase activities of young and adult rat cerebra, J. Neurochem. 36 (1981) 826–829. [26] R. Katoh, S. Kashiwamata, F. Niwa, Studies on cellular toxicity of bilirubin: effect on the carbohydrate metabolism in the young rat brain, Brain Res. 83 (1975) 81–92. [27] G.J. Lees, Inhibition of sodium–potassium–ATPase: a potentially ubiquitous mechanism contributing to central nervous system neuropathology, Brain Res. Brain Res. Rev. 16 (1991) 283–300. [28] J.W. McDonald, S.M. Shapiro, F.S. Silverstein, M.V. Johnston, Role of glutamate receptor mediated excitotoxicity in bilirubin-induced brain injury in the gunn rat model, Exp. Neurol. 150 (1998) 21–29. [29] M.G. Mustafa, M.L. Cowger, T.E. King, Effects of bilirubin on mitochondrial reactions, J. Biol. Chem. 244 (1969) 6403–6414. [30] B.A. Noir, A. Boveris, A.M.G. Pereira, A.O.M. Stoppani, Bilirubin: a multi-site inhibitor of mitochondrial respiration, FEBS Lett. 27 (1972) 270–274. [31] W.S. Park, Y.S. Chang, M. Lee, Effect of induced hyperglycemia on brain cell membrane function and energy metabolism during the early phase of experimental meningitis in newborn piglets, Brain Res. 798 (1998) 195–203. [32] S. Schenker, D.W. McCandless, P.E. Zollman, Studies of cellular toxicity of unconjugated bilirubin in kernicteric brain, J. Clin. Invest. 45 (1966) 1210–1213. [33] R.A. Swanson, J. Chen, S.H. Graham, Glucose can fuel glutamate uptake in ischemic brain, J. Cereb. Blood Flow Metab. 14 (1994) 1–6. [34] M. Thoresen, S. Satas, M. Puka-Sundvall, A. Whitelaw, A. Hallstrom, E. Loberg, U. Ungerstedt, P. Steen, H. Hagberg, Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins, NeuroReport 8 (1997) 3359–3362. [35] S. Tsakiris, Na1, K(1)–ATPase and acetylcholinesterase activities: changes in postnatally developing rat brain induced by bilirubin, Pharmacol. Biochem. Behav. 45 (1993) 363–368. [36] G.D. Williams, B.J. Dardzinski, A.R. Buckalew, M.B. Smith, Modest hypothermia preserves cerebral energy metabolism during hypoxia–ischemia and correlates with brain damage: a 31 P nuclear magnetic resonance study in unanesthetized neonatal rats, Pediatr. Res. 42 (1997) 700–708. [37] P. Yeagle, Lipid regulation of cell membrane structure and function, FASEB J. 3 (1989) 1833–1842. [38] G. Zeevalk, W. Nicklas, Hypothermia and metabolic stress: narrowing the cellular site of early neuroprotection, J. Pharmacol. Exp. Ther. 279 (1996) 332–339.