Joanna Kubin

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets

Bilirubin neurotoxicity can be mediated by numerous mechanisms due to its increased permeability in neuronal membranes. The present study tests the hypothesis that a prolonged bilirubin infusion modifies theN- methyl-D-aspartate (NMDA) receptor/ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in seven control and six bilirubin-exposed piglets, 2-4 d of age. Piglets in the bilirubin group received a 35 mg/kg bolus of bilirubin followed by a 4-h infusion (25 mg/kg/h) of a buffer solution containing 0.1 N NaOH, 5% human albumin, and 0.055 Na2HPO4 with 3 mg/mL bilirubin. The final mean bilirubin concentration in the bilirubin group was 495.9 ± 85.5 μmol/L (29.0± 5.0 mg/dL). The control group received a bilirubin-free buffer solution. Sulfisoxazole was administered to animals in both groups. P2 membrane fractions were prepared from the cerebral cortex. [3H]MK-801 binding assays were performed to study NMDA receptor modification. TheBmax in the control and bilirubin groups were 1.20 ± 0.10 (mean ± SD) and 1.32 ± 0.14 pmol/mg protein, respectively. The value for Kd in the control brains was 6.97± 0.80 nM compared with 4.80 ± 0.28 nM in the bilirubin-exposed brains (p < 0.001). [3H]Glutamate binding studies did not show a significant difference in the Bmax andKd for the NMDA-specific glutamate site in the two groups. The results show that in vivo exposure to bilirubin increases the affinity of the receptor (decreasedKd) for [3H]MK-801, indicating that bilirubin modifies the function of the NMDA receptor/ion channel complex in the brain of the newborn piglet. We speculate that the affinity of bilirubin for neuronal membranes leads to bilirubin-mediated neurotoxicity, resulting in either short- or long-term disruption of neuronal function.

Kainate receptor modification in the fetal guinea pig brain during hypoxia.

The present study tests the hypothesis that hypoxia alters the high-affinity kainate receptors in fetal guinea pig brain. Experiments were conducted in normoxic and hypoxic guinea pig fetus at preterm (45 days of gestation) and term (60 days of gestation). Hypoxia in the guinea pig fetus was induced by exposure to maternal hypoxia (FiO2=7%) for 60 min. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphorreatine. [3H]-Kainate binding characteristics (Bmax=number of receptors, Kd=dissociation constant) were used as indices of kainate receptor modification. P2 membrane fractions were prepared from the cortex of normoxic and hypoxic fetuses and were washed six times prior to performing the binding assays. [3H]kainate binding was performed at 0°C for 30 min in a 500 μl medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 μg protein and varying concentrations of radiolabelled kainate ranging from 1 to 200 nM. Non-specific binding was determined in the presence of 1.0 mM glutamate. During brain development from 45 to 60 days gestation, Bmax value increased from 330±16 to 417±10 fmoles/mg protein; however, the Kd was unchanged (8.2±0.4 vs 8.8±0.5 nM, respectively). During hypoxia at 60 days, the Kd value significantly increased as compared to normoxic control (15.5±0.7 vs 8.8±0.5 nM, respectively), whereas the Bmax was not affected (435±12 vs 417±10 fmol/mg protein, respectively). At 45 days, hypoxia also increased the Kd (11.9±0.6 vs 8.2±0.4 nM) without affecting the Bmax (290±15 vs 330±16 fmol/mg protein, respectively). The results show that the number of kainate receptors increase during gestation without change in affinity and demonstrate that hypoxia modifies the high-affinity kainate receptor sites at both ages; however the effect is much stronger at 60 days (term). The decreased affinity of the site could decrease the kainate receptor-mediated fast kinetics of desensitization and provide a longer period for increased Na+-influx, leading to increased accumulation of intracellular Ca2+ by reversal of the Na+−Ca2+ exchange mechanism. In addition, Kd values for kainate-type glutamate receptor sites are 30–40 fold lower (i.e. higher affinity) than those for NMDA-displaceable glutamate sites. The higher affinity suggests that the activation of the kainate-type glutamate receptor during hypoxia could precede initiation of NMDA receptormediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na+, K+-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO2 of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl2, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na+, K+-ATPase activity. Following peroxynitrite exposure, the activity of Na+, K+-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na+, K+-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na+, K+-ATPase. The results demonstrate that peroxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity.

Brain cell membrane Na+, K+-ATPase activity following severe hypoxic injury in the newborn piglet

This study tests the hypothesis that severe brain hypoxia causes decreased Na+,K(+)-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3)) were studied. Hypoxia was induced by lowering the FiO2 to maintain PCr/Pi ratio at 25% of baseline for 1 h as monitored by 31P-NMR spectroscopy. PCr/Pi returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na+,K(+)-ATPase activity was determined. Na+,K(+)-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na+,K(+)-ATPase activity of 58.3 +/- 1.3 microM Pi/mg protein/h (mean +/- S.E.M.). Na+,K(+)-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 +/- 1.3, 47.4 +/- 3.6, 48.7 +/- 2.9 microM Pi/mg protein/h) (P < 0.05 compared to NC). There was no difference in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxidative phosphorylation (PCr/Pi) by 48 h, there is a permanent decrease in Na+,K(+)-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na+,K(+)-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/Pi values cannot be used as an index of recovery of cell function.

The effect of hypothermia on neuronal viability following cardiopulmonary bypass and circulatory arrest in newborn piglets

OBJECTIVE To determine the effect of recovery with mild hypothermia after cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) on the activity of selected key proteins involved in initiation (Bax, Caspase-3) or inhibition of apoptotic injury (Bcl-2, increased ratio Bcl-2/Bax) in the brain of newborn piglets. METHODS The piglets were placed on CPB, cooled with pH-stat management to 18 degrees C, subjected to 30 min of DHCA followed by 1h of low flow at 20 ml/kg/min, rewarmed to 37 degrees C (normothermia) or to 33 degrees C (hypothermia), separated from CPB, and monitored for 6h. Expression of above proteins was measured in striatum, hippocampus and frontal cortex by Western blots. The results are mean for six experiments+/-SEM. RESULTS There were no significant differences in Bcl-2 level between normothermic and hypothermic groups. The Bax levels in normothermic group in cortex, hippocampus and striatum were 94+/-9, 136+/-22 and 125+/-34 and decreased in the hypothermic group to 59+/-17 (p=0.028), 70+/-6 (p=0.002) and 48+/-8 (p=0.01). In cortex, hippocampus and striatum Bcl-2/Bax ratio increased from 1.23, 0.79 and 0.88 in normothermia to 1.96, 1.28 and 2.92 in hypothermia. Expression of Caspase-3 was 245+/-39, 202+/-74 and 244+/-31 in cortex, hippocampus and striatum in the normothermic group and this decreased to 146+/-24 (p=0.018), 44+/-16 (p=7 x 10(-7)) and 81+/-16 (p=0.01) in the hypothermic group. CONCLUSION In neonatal piglet model of cardiopulmonary bypass with circulatory arrest, mild hypothermia during post bypass recovery provides significant protection from cellular apoptosis, as indicated by lower expression of Bax and Caspase-3 and an increased Bcl-2/Bax ratio. The biggest protection was observed in striatum probably by decreasing of neurotoxicity of striatal dopamine.

Modification of the glycine (co-activator) binding site of the N-methyl-d-aspartate receptor in the guinea pig fetus brain during development following hypoxia

The present study was designed to investigate the mechanism of NMDA receptor activation as a function of brain maturation by studying the development of the glycine binding site of the NMDA receptor and its modification by in-utero hypoxia in the guinea pig fetus brain during gestation. Measurements of Bmax (number of functional receptors) and Kd (apparent receptor affinity) of glycine binding sites of the NMDA receptor were performed in eleven (45 days, n = 5; 60 days, n = 6) synaptosomal membranes from normoxic (control) fetuses and ten (45 days, n = 4; 60 days, n = 6) synaptosomal membranes constituted the hypoxic (experimental) group. In the experimental group, fetuses were exposed to maternal hypoxia (FiO2 0.07) for 1 h. Synaptosomal membranes were prepared and strychnine-insensitive specific [3H]glycine binding was determined During development, the number of glycine binding sites increased (Bmax:392 +/- 30 vs. 583 +/- 30 fmol/mg protein at 45 and 60 days respectively, P < 0.05) where as the affinity remained unchanged (Kd: 190 +/- 9 vs. 211 +/- 30 nM at 45 and 60 days respectively). Following hypoxia, glycine binding sites increased at 45 days (Bmax:392 +/- 30 vs. 561 +/- 96 fmol/mg protein, P < 0.005) but decreased at 60 days (Bmax:583 +/- 85 vs. 411 +/- 65 fmol/mg protein, P < 0.005) with change in Kd only at 60 days (Kd:211 +/- 30 vs. 149 +/- 52 nM, P < 0.05). The data show that there are alterations in the characteristics of the glycine binding site during development and following hypoxia. We conclude that developmental changes in the glycine binding site might modulate NMDA receptor activation as a function of brain maturation. Furthermore, hypoxia-induced modification of the glycine binding site might be a potential mechanism of neurotoxicity and might increase susceptibility of the fetal brain to excitotoxicity at term.

Effect of granulocyte-colony stimulating factor on expression of selected proteins involved in regulation of apoptosis in the brain of newborn piglets after cardiopulmonary bypass and deep hypothermic circulatory arrest

OBJECTIVE The study objective was to investigate the effect of granulocyte-colony stimulating factor on the expression of proteins that regulate apoptosis in newborn piglet brain after cardiopulmonary bypass and deep hypothermic circulatory arrest. METHODS The newborn piglets were assigned to 3 groups: (1) deep hypothermic circulatory arrest (30 minutes of deep hypothermic circulatory arrest, 1 hour of low-flow cardiopulmonary bypass); (2) deep hypothermic circulatory arrest with prior injection of granulocyte-colony stimulating factor (17 μg/kg 2 hours before cardiopulmonary bypass); and (3) sham-operated. After 2 hours of post-bypass recovery, the frontal cortex, striatum, and hippocampus were dissected. The expression of proteins was measured by gel electrophoresis or protein arrays. Data are presented in arbitrary units. Statistical analysis was performed using 1-way analysis of variance. RESULTS In the frontal cortex, only Fas ligand expression was significantly lower in the granulocyte-colony stimulating factor group when compared with the deep hypothermic circulatory arrest group. In the hippocampus, granulocyte-colony stimulating factor increased Bcl-2 (54.3 ± 6.4 vs 32.3 ± 2.2, P = .001) and serine/threonine-specific protein kinase (141.4 ± 19 vs 95.9 ± 21.1, P = .047) when compared with deep hypothermic circulatory arrest group. Caspase-3, Bax, Fas, Fas ligand, death receptor 6, and Janus protein tyrosine kinase 2 levels were unchanged. The Bcl-2/Bax ratio was 0.33 for deep hypothermic circulatory arrest group and 0.93 for the granulocyte-colony stimulating factor group (P = .02). In the striatum, when compared with the deep hypothermic circulatory arrest group, the granulocyte-colony stimulating factor group had higher levels of Bcl-2 (50.3 ± 7.4 vs 31.8 ± 3.8, P = .01), serine/threonine-specific protein kinase (132.7 ± 12.3 vs 14 ± 1.34, P = 2.3 × 10(6)), and Janus protein tyrosine kinase 2 (126 ± 17.4 vs 77.9 ± 13.6, P = .011), and lower levels of caspase-3 (12.8 ± 5.0 vs 32.2 ± 11.5, P = .033), Fas (390 ± 31 vs 581 ± 74, P = .038), Fas ligand (20.5 ± 11.5 vs 57.8 ± 15.6, P = .04), and death receptor 6 (57.4 ± 4.4 vs 108.8 ± 13.4, P = .007). The Bcl-2/Bax ratio was 0.25 for deep hypothermic circulatory arrest and 0.44 for the granulocyte-colony stimulating factor groups (P = .046). CONCLUSIONS In the piglet model of hypoxic brain injury, granulocyte-colony stimulating factor decreases proapoptotic signaling, particularly in the striatum.

Effect of deep hypothermic circulatory arrest followed by low-flow cardiopulmonary bypass on brain metabolism in newborn piglets: comparison of pH-stat and α-stat management.

Objective: To compare the effects of pH-stat and &agr;-stat management before deep hypothermic circulatory arrest followed by a period of low-flow (two rates) cardiopulmonary bypass on cortical oxygenation and selected regulatory proteins: Bax, Bcl-2, Caspase-3, and phospho-Akt. Design: Piglets were placed on cardiopulmonary bypass, cooled with pH-stat or &agr;-stat management to 18°C over 30 mins, subjected to 30-min deep hypothermic circulatory arrest and 1-hr low flow at 20 mL/kg/min (LF-20) or 50 mL/kg/min (LF-50), rewarmed to 37°C, separated from cardiopulmonary bypass, and recovered for 6 hrs. Subjects: Newborn piglets, 2–5 days old, assigned randomly to experimental groups. Interventions: None. Measurements and Main Results: Cortical oxygen was measured by oxygen-dependent quenching of phosphorescence; proteins were measured by Western blots. The means from six experiments ± sem are presented as % of &agr;-stat. Significance was determined by Students t test. For LF-20, cortical oxygenation was similar for &agr;-stat and pH-stat, whereas for LF-50, it was significantly better using pH-stat. For LF-20, the measured proteins were not different except for Bax in the cortex (214 ± 24%, p = .006) and hippocampus (118 ± 6%, p = .024) and Caspase 3 in striatum (126% ± 7%, p = .019). For LF-50, in pH-stat group: In cortex, Bax and Caspase-3 were lower (72 ± 8%, p = .001 and 72 ± 10%, p = .004, respectively) and pAkt was higher (138 ± 12%, p = .049). In hippocampus, Bcl-2 and Bax were not different but pAkt was higher (212 ± 37%, p = .005) and Caspase 3 was lower (84 ± 4%, p = .018). In striatum, Bax and pAkt did not differ, but Bcl-2 increased (146 ± 11%, p = .001) and Caspase-3 decreased (81 ± 11%, p = .042). Conclusions: In this deep hypothermic circulatory arrest-LF model, when flow was 20 mL/kg/min, there was little difference between &agr;-stat and pH-stat management. However, for LF-50, pH-stat management resulted in better cortical oxygenation during recovery and Bax, Bcl-2, pAk, and Caspase-3 changes were consistent with lesser activation of proapoptotic signaling with pH-stat than with &agr;-stat.

Inositol 1, 4, 5 Triphosphate (IP 3 ) Receptor Modification During Hypoxia in Cerebral Cortical Nuclei of the Guinea Pig Fetus. ♦ 328

Inositol 1, 4, 5 Triphosphate (IP 3 ) Receptor Modification During Hypoxia in Cerebral Cortical Nuclei of the Guinea Pig Fetus. ♦ 328

Granulocyte colony-stimulating factor significantly decreases density of hippocampal caspase 3-positive nuclei, thus ameliorating apoptosis-mediated damage, in a model of ischaemic neonatal brain injury

OBJECTIVES Ischaemic brain injury is a major complication in patients undergoing surgery for congenital heart disease, with the hippocampus being a particularly vulnerable region. We hypothesized that neuronal injury resulting from cardiopulmonary bypass and associated circulatory arrest is ameliorated by pretreatment with granulocyte colony-stimulating factor (G-CSF), a cytokine and an anti-apoptotic neurotrophic factor. METHODS In a model of ischaemic brain injury, 4 male newborn piglets were anaesthetized and subjected to deep hypothermic circulatory arrest (DHCA) (cooled to 18°C, DHCA maintained for 60 min, rewarmed and recovered for 8-9 h), while 4 animals received G-CSF (34 µg/kg, intravenously) 2 h prior to the DHCA procedure. At the end of each experiment, the animals were perfused with a fixative, the hippocampus was extracted, cryoprotected, cut and the brain sections were immunoprocessed for activated caspase 3, a pro-apoptotic factor. Immunopositive neuronal nuclei were counted in multiple counting boxes (440 × 330 µm) centred on the CA1 or CA3 hippocampal regions and their mean numbers compared between the different treatment groups and regions. RESULTS G-CSF pretreatment resulted in significantly lower counts of caspase 3-positive nuclei per counting box in both the CA1 [52.2 ± 9.3 (SD) vs 61.6 ± 8.4, P < 0.001] and CA3 (41.2 ± 6.9 vs 60.4 ± 16.4, P < 0.00002) regions of the hippocampus as compared to DHCA groups. The effects of G-CSF were significant for pyramidal cells of both regions and for interneurons in the CA3 region. CONCLUSIONS In an animal model of ischaemic brain injury, G-CSF reduces neuronal injury in the hippocampus, thus potentially having beneficial effect on neurologic outcomes.