Effect of hyperoxia on cortical neuronal nuclear function and programmed cell death mechanisms.

There is growing concern over detrimental neurologic effects to human newborns caused by increased inspired oxygen concentrations. We hypothesize that hyperoxia (FiO(2)>0.95) results in increased high-affinity Ca(2+)-ATPase activity, Ca(2+)-influx, and proapoptotic protein expression in cortical neuronal nuclei of newborn piglets. Neuronal cerebral energy metabolism was documented by determining ATP and phosphocreatine levels. Neuronal nuclear conjugated dienes and fluorescent compounds were measured as indices of lipid peroxidation. High-affinity Ca(2+)-ATPase activity and ATP-dependent Ca(2+)-influx were determined to document neuronal nuclear membrane function. Hyperoxia resulted in increases in lipid peroxidation, high-affinity Ca(2+)-ATPase activity, ATP-dependent Ca(2+)-influx, and Bax/Bcl-2 ratio in the cortical neuronal nuclei of newborn piglets. We conclude that hyperoxia results in modification of neuronal nuclear membrane function leading to increased nuclear Ca(2+)-influx, and propose that hyperoxia-induced increases in intranuclear Ca(2+) activates the Ca(2+)/calmodulin-dependent protein kinase pathway, triggering increased CREB protein-mediated apoptotic protein expression in hyperoxic neurons.

Effects of severe hypocapnia on expression of bax and bcl-2 proteins, DNA fragmentation, and membrane peroxidation products in cerebral cortical mitochondria of newborn piglets.

BACKGROUND: Hypocapnia occurs in the newborn infant inadvertently or as a therapeutic modality and may result in neuronal and mitochondrial alterations in the newborn brain. Since mitochondria regulate apoptosis, these alterations may initiate a cascade of reactions that lead to apoptotic cell death. OBJECTIVES: This study tests the hypothesis that hypocapnia results in increased expression of the pro-apoptotic protein Bax, fragmentation of DNA and membrane lipid peroxidation in cerebral cortical mitochondria (mt) of newborn piglets. METHODS: Studies were performed in three groups of anesthetized normoxic newborn piglets: hypocapnic (H, n = 5), ventilated at a PaCO(2) of 11-15 mm Hg; normocapnic (N, n = 5), ventilated at a PaCO(2) of 40 mm Hg; and corrected normocapnic (CN, n = 4), ventilated as H with CO(2) added to maintain normocapnia. Tissue ATP and phosphocreatine levels were determined. Mitochondrial membrane proteins were separated, transblotted and probed with antibodies to Bax and Bcl-2. Bands were detected by enhanced chemiluminescence and analyzed by imaging densitometry. mtDNA was isolated. Cell and mitochondrial membrane lipid peroxidation products were measured spectrofluorometrically. RESULTS: ATP and PCr concentrations were similar in the 3 groups. The ratio of Bax/Bcl-2 increased significantly in H compared to N and CN. mtDNA fragmentation was also significantly greater in H compared to N or CN. Membrane lipid peroxidation was higher in H than in N or CN; and in CN compared to N. CONCLUSIONS: The data demonstrate that severe hypocapnia results in increased Bax expression, DNA fragmentation, and membrane lipid peroxidation in mitochondria of cerebral cortical neurons of newborn piglets, and may result in apoptotic cell death.

Mechanisms of injury to the newborn brain.

Preterm and ill term infants are at risk for brain injury and subsequent neurodevelopmental delay as a result of many perinatal factors. Outlined in this article are the basic science mechanisms by which hypoxia, hypocapnia, and hypercapnia may result in neuronal injury in the newborn brain.

Effect of 7-nitroindazole sodium on the cellular distribution of neuronal nitric oxide synthase in the cerebral cortex of hypoxic newborn piglets.

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca(++)-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO(2) to 0.05-0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.

Effects of magnesium sulfate administration during hypoxia on CaM kinase IV and protein tyrosine kinase activities in the cerebral cortex of newborn piglets.

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca(2+)/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg(2+)-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg(2+)-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 +/- 49), Mg(2+)-Hx (317 +/- 82) and Hx (574 +/- 41, P < 0.05 vs. Nx and Mg(2+)-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 +/- 68), Mg(2+)-Hx (455 +/- 67) and Hx (922 +/- 66, P < 0.05 vs. Nx and Mg(2+)-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca(2+)-flux, magnesium sulfate administration inhibits the Ca(2+)/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.

Hypercapnia-induced modifications of neuronal function in the cerebral cortex of newborn piglets.

There is significant controversy over the effects of hypercapnia on the human newborn brain. Previous studies have shown that 1 h of an arterial CO2 pressure (Paco2) of 80 mm Hg alters brain cell membrane Na+K+-ATPase enzyme activity in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypercapnia (either a Paco2 of 65 or 80 mm Hg) results in decreased energy metabolism and alters neuronal nuclear enzyme activity and protein expression, specifically Ca++/calmodulin-dependent kinase (CaMK) IV activity, phosphorylation of cAMP response element binding protein (CREB), and expression of apoptotic proteins in cortical neuronal nuclei of newborn piglets. Studies were performed in 20 anesthetized normoxic piglets ventilated at either a Paco2 of 65 mm Hg, 80 mm Hg, or 40 mm Hg for 6 h. Energy metabolism was documented by ATP and phosphocreatine (PCr) levels. Results show ATP and PCr levels were significantly lower in the hypercapnic groups than the normocapnic. CaMK IV activity, phosphorylated CREB density, and Bax protein expression were all significantly higher in the hypercapnic groups than the normocapnic group. Bcl-2 protein was similar in all three groups, making the ratio of Bax/Bcl-2 significantly higher in the hypercapnic groups than in the normocapnic group. We conclude that hypercapnia alters neuronal energy metabolism, increases phosphorylation of transcription factors, and increases the expression of apoptotic proteins in the cerebral cortex of newborn piglets and therefore may be deleterious to the newborn brain.

The effect of moderate hypocapnic ventilation on nuclear Ca2+-ATPase activity, nuclear Ca2+ flux, and Ca2+/calmodulin kinase IV activity in the cerebral cortex of newborn piglets.

Previous studies have shown that hypocapnia results in fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. We tested the hypothesis that hypocapnia results in decreased ATP and phosphocreatine (PCr) levels and increased nuclear high-affinity Ca++-ATPase activity, intranuclear Ca++ flux, and CaM kinase IV activity in neuronal nuclei of piglets. Three groups of piglets were ventilated as either hypocapnic (a PaCO2 of 20 mm Hg), normocapnic (a PaCO2 of 40 mm Hg), or corrected hypocapnic (ventilated as hypocapnic but with CO2 added to maintain normocapnia) for 1 h. Tissue ATP levels were lower in the hypocapnic than in the normocapnic group. PCr levels were lower and 45Ca++-influx, Ca++-ATPase activity and CaM kinase IV activity were higher in hypocapnic than in normocapnic or corrected hypocapnic piglets. We conclude that hypocapnia alters nuclear membrane Ca++ flux mechanisms and may alter neuronal phosphorylation mechanisms in the cerebral cortex of piglets.

Nitric oxide-mediated modification of the glycine binding site of the NMDA receptor during hypoxia in the cerebral cortex of the newborn piglet.

This study tested the hypothesis that cerebral hypoxia results in nitric oxide (NO)-mediated modification of the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor. Glycine binding characteristics were determined in normoxic, hypoxic, and hypoxic with 7-nitroindazole (7-NINA)-pretreated newborn piglets. The role of nitration was evaluated by determining binding characteristics in non-nitrated and in-vitro nitrated membranes. Bmax and Kd values were 30% higher in the hypoxic group than the normoxic and 7-NINA pretreated hypoxic groups. Kd values in the in-vitro normoxic nitrated membranes were similar to the non-nitrated hypoxic group. Bmax values in the in-vitro) normoxic nitrated membrane samples were 16% lower than in the non-nitrated hypoxic group. We conclude cerebral hypoxia causes modification of the glycine-binding site of the NMDA receptor and this modification of the glycine-binding site may be NO mediated. We propose that NO-mediated modification of the glycine-binding site of the NMDA receptor regulates calcium influx through its ion-channel.

Expression of phosphorylation of N-methyl-D-aspartate receptor subunits during graded hypoxia in the cerebral cortex of newborn piglets.

The present study tests the hypothesis that during graded hypoxia, N-methyl-D-aspartate (NMDA) receptor expression and phosphorylation are altered in the cerebral cortex of newborn piglets. Studies were performed in anesthetized, ventilated piglets, 6 normoxic and 9 exposed to different lengths of decreased fractions of inspired oxygen to achieve varying biochemical levels of phosphocreatine (PCr). P(2) membrane proteins were immunoprecipitated with antiphosphoserine, antiphosphotyrosine, or antiphosphothreonine antibodies and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transblotted and probed with NMDA receptor subunit 1 (NR1), NR2A or NR2B antibodies. As tissue PCr levels decreased from 3.5 to 0.5 micromol/g brain during hypoxia, NR1, NR2A and NR2B protein increased by 84, 56 and 38%, respectively. Phosphorylated serine, tyrosine and threonine residues also increased during hypoxia on the three subunits. However, the increase in subunit protein exceeded the increase in phosphorylated residues for all three subunits. Therefore, the ratio of phosphorylated/dephosphorylated serine, tyrosine and threonine residues decreased with worsening hypoxia. We speculate that an alteration in the ratio of phosphorylated/dephosphorylated residues of the NMDA receptor may regulate receptor activation during hypoxia.

The effect of hypocapnia (PaCO2 27 mmHg) on CaM kinase IV activity, Bax/Bcl-2 protein expression and DNA fragmentation in the cerebral cortex of newborn piglets.

The present study tests the hypothesis that a PaCO(2) of 27 mmHg for 1 hr results in increased neuronal nuclear Ca(++)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity, pro-apoptotic protein expression and DNA fragmentation in the cerebral cortex of newborn piglets. Hypocapnic (HC) and normocapnic newborn piglets were studied. Tissue levels of ATP and phosphocreatine (PCr) were lower in the HC group. CaM kinase IV activity and Bax protein density were higher in the HC group. Bcl-2 protein density was the same in both groups, resulting in an increased ratio of Bax/Bcl-2 in the HC group. Density of nuclear DNA fragments was greater in the HC group and varied inversely with ATP and PCr levels. We conclude that hypocapnia (PaCO(2) 27 mmHg) results in increased expression of pro-apoptotic proteins and fragmentation of nuclear DNA in newborn piglets.

Nitric oxide-mediated Ca2+/calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei from newborn piglets.

The present study tested the hypothesis that hypoxia results in increased Ca(2+)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity and that inhibition of nitric oxide (NO) synthase by N-nitro-L-arginine (NNLA) prevents the hypoxia- induced increase in neuronal nuclear CaM kinase IV activity in newborn piglets. CaM kinase IV activity was determined in normoxic (Nx), hypoxic (Hx), and NNLA-pretreated Hx piglets. Cerebral hypoxia was confirmed biochemically. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (285.22+/-86.12), Hx (494.77+/-99.79, P<0.05 vs. Nx), and NNLA-pretreated Hx (249.55+/-53.85)(P=NS vs. Nx, P<0.05 vs. Hx) animals. The results demonstrate that the cerebral tissue hypoxia results in an increase in neuronal nuclear CaM kinase IV activity, and the hypoxia-induced increase in CaM kinase IV activity is NO-mediated.

Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets.

The present study tests the hypothesis that nitric oxide mediates the hypoxia-induced increase in expression of Bax and in DNA fragmentation in the cerebral cortex of newborn piglets, and that administration of N-nitro-L-arginine (NNLA), a nitric oxide synthase inhibitor, will prevent a change in hypoxia-induced expression of apoptotic genes and DNA damage. Piglets were assigned to normoxic, hypoxic, or NNLA-pretreated hypoxic groups. Cerebral tissue hypoxia was documented biochemically by measuring ATP and phosphocreatine (PCr) levels. Cerebral cortical neuronal nuclei were isolated and nuclear proteins were separated electrophoretically and probed with specific antibodies against Bcl-2 or Bax proteins. Neuronal nuclear DNA from normoxic, hypoxic, and NNLA-pretreated hypoxic animals was isolated, separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. Cerebral hypoxia resulted in an increase in nuclear membrane Bax protein levels from 121.33+/-47.7 optical density (OD)xmm(2) in normoxic to 273.67+/-67.3 ODxmm(2) in hypoxic group (P<0.05 vs. normoxic), but levels in NNLA-pretreated hypoxic group were 155.78+/-48.3 ODxmm(2) (P<0.05 vs. hypoxic, P=NS vs. normoxic). Similarly, cerebral hypoxia resulted in the density of DNA fragments increasing from 1530.3+/-309.8 OD/mm(2) in the normoxic group to 5383.3+/-775 OD/mm(2) in the hypoxic group (P<0.05), while levels in NNLA-pretreated hypoxic group were 3574.0+/-952 OD/mm(2) (P<0.05 compared to hypoxic and normoxic groups). The data show that NNLA-pretreatment prevents the hypoxia-induced increase in Bax expression and DNA fragmentation demonstrating that the hypoxia-induced Bax gene expression and the DNA fragmentation are NO-mediated.

NMDA receptor modification during graded hypoxia in the cerebral cortex of newborn piglets.

Previous studies have shown that the cerebral N-methyl-D-aspartate (NMDA) receptor is altered during hypoxia in newborn piglets. The present study tests whether modification of the glutamate and ion channel sites of the NMDA receptor correlates with the progressive decrease in cerebral energy metabolism induced by hypoxia. Degrees of cerebral hypoxia were attained by exposure of ventilated piglets to decreased oxygen at different concentrations and confirmed by tissue phosphocreatine levels. During graded hypoxia, the number of glutamate sites decreased, the affinity of the ion channel site increased, the inhibition by Zn(2+) increased, the activation by glutamate increased, and the activation by glycine decreased. Therefore, modification of the NMDA receptor correlates with the energy state of the tissue. Alterations in receptor phosphorylation may gradually modify the NMDA receptor and may be initiated by subtle decreases in tissue oxygenation in the newborn brain.