David J Hoffman

Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg(2+)-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO2 to 5-7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg(2+)-treated group received MgSO4 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg2+ concentrations increased from 1.6 +/- 0.1 mg/dl to 17.7 +/- 3.3 mg/dl. 3H-MK-801 binding was used as an index of NMDA receptor modification. The Bmax in control, hypoxic and Mg(2+)-treated hypoxic piglets was 1.09 +/- 0.17, 0.70 +/- 0.25 and 0.96 +/- 0.14 pmoles/mg protein, respectively. The Kd for the same groups were 10.02 +/- 2.04, 4.88 +/- 1.43 and 8.71 +/- 2.23 nM, respectively. The Bmax and Kd in the hypoxic group were significantly lower compared to the control and Mg(2+)-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg2+. The activity of Na+, K+ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg(2+)-treated hypoxic groups. These findings show that MgSO4 prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia-induced modification of the N-methyl-d-aspartate receptor in the brain of the newborn piglet

The effect of hypoxia on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex in the brain cell membrane of the newborn piglet was studied. Experiments were conducted on newborn piglets, 2-4 days of age, that were anesthetized and mechanically ventilated. Hypoxic hypoxia was induced in the experimental group by lowering the FiO2 to 5-7%. The control group was ventilated under normoxic conditions. Tissue hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine (PCr) in the hypoxic group (52% and 81% lower than the normoxic group, respectively). [3H]MK-801 binding characteristics (Bmax = number of receptors, Kd = dissociation constant) were used as an index of NMDA receptor modification. In hypoxic brains, Bmax decreased from the control level of 1.13 +/- 0.15 pmol/mg protein to 0.68 +/- 0.23 pmol/mg protein (P < 0.01) and the Kd value decreased (reflecting increased affinity) from 9.46 +/- 1.68 nM in the control brains to 4.87 +/- 1.42 nM (P < 0.01) in the hypoxic brains. The Na+,K(+)-ATPase activity, an index of brain cell membrane function, decreased from a control value of 46.5 +/- 0.4 to 40.5 +/- 2.3 mumol inorganic phosphate (Pi) mg protein/h (P < 0.005) during hypoxia. The results of this study indicate that hypoxia in newborn piglets modifies the NMDA receptor in the cerebral cortex, resulting in an increased affinity of the receptor channel. Hypoxia-induced modification of the NMDA ion/receptor complex may be a potential mechanism of cerebral excitotoxicity.

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.

Cytosolic and membrane-bound cerebral nitric oxide synthase activity during hypoxia in cortical tissue of newborn piglets.

To determine the role of nitric oxide production during hypoxia, the presence of two forms of neuronal nitric oxide synthase, cytosolic (cNOS) and membrane-bound (memNOS), in cortical tissue of newborn piglets and the effects of hypoxia on the activity of these enzymes were studied. Experiments were performed in 12 anesthetized and ventilated Yorkshire piglets, 2-4 days of age. Hypoxia was induced by decreasing the FiO2 to 0.07. The control group was ventilated maintaining normoxia. After 1 h of normoxic or hypoxic ventilation brain tissue was removed and frozen immediately in liquid nitrogen. Tissue hypoxia was confirmed by analysis of adenosine triphosphate (ATP) and phosphocreatine (PCr): ATP was reduced to 52% and PCr to 28% of control values. cNOS activity was 35.3 +/- 13.7 pmol/mg protein per min in the control group and 28.3 +/- 7.0 in the hypoxia group; memNOS activity was 10.5 +/- 4.5 and 12.0 +/- 3.9 pmol/mg protein per min in the control and hypoxia groups, respectively. Differences in cNOS and memNOS activity between control and hypoxic animals were not significant. The results indicate that both cNOS and memNOS are present in cortical tissue of newborn piglets and that the activity is unaffected by 1 h of tissue hypoxia. We suggest that production of nitric oxide and its derivative peroxynitrite during hypoxia may therefore be a potential mechanism for hypoxia-induced brain cell membrane lipid peroxidation.

Effect of Reoxygenation with 21% or 100% Oxygen on Free Radical Formation Following Hypoxia in the Cerebral Cortex of Newborn Piglets. |[dagger]| 166

The present study tests the hypothesis that reoxygenation after cerebral tissue hypoxia with 100% oxygen increases generation of oxygen free radicals compared to reoxygenation with room air. Studies were performed in anesthetized, ventilated newborn piglets subjected to 1 hr of hypoxia(FiO2 0.05-0.07, PaO2 < 20 mmHg). Following hypoxia, animals were randomized to Group 1 (reoxygenation with 21% O2, n=8) or Group 2(reoxygenation with 100% O2, n=8). Near infrared spectroscopy (NIRS) was used with wavelengths of 716 nm and 850 nm to monitor tissue oxygenation using a phase modulation spectrometer with an interprobe distance of 22 mm. A phase shift in the NIRS signal was observed at 50±10 sec following initiation of reoxygenation. Cortical biopsies were obtained via cranial windows for free radical measurements using electron spin resonance spectroscopy (ESR) immediately following appearance of the phase shift. ESR spectra of electron spin adducts were obtained in duplicate using a Varian-E-109 spectrometer. Signal heights were measured from the resultant graph and divided by tissue weight. Reference ESR signals were measured from tissues incubated at room temperature for 10 min to confirm the absence of free radicals. Tissue was homogenized in 100 mMα-phenyl-tert-butyl-nitrone (PBN) and extracted with 2:1 chloroform:methanol. Intensity of the ESR signal from the PBN extract was expressed as signal height (mm)/g tissue after subtracting the reference value determined in control animals. Free radical signal height was 130±30 mm/g brain in Group 1 and 230±90 mm/g brain in Group 2 (p <0.05). Reoxygenation with 100% O2 was associated with significantly more free radical generation than reoxygenation with 21% O2. We speculate that mitochondria which were anaerobic during the hypoxic phase produce a surge of free radicals during reoxygenation and that the amount of free radicals produced increases with increased oxygen concentration. (NIH#HD-20337 and AAP/AHA/Neonatal Resuscitation Program)

Pulmonary Function and Electrolyte Balance Following Spironolactone Treatment in Preterm Infants with Chronic Lung Disease. |[bull]| 321

Oral diuretic therapy is often required by premature infants with chronic lung disease (CLD). Although spironolactone (SP) is frequently used in combination with chlorthiazide (CT), the effects of SP on electrolyte losses, electrolyte supplementation and pulmonary function have not been evaluated. We tested the hypothesis that the addition of SP to CT administration will not improve pulmonary mechanics or minimize the need for dietary electrolyte supplementation. 16 infants received CT + placebo, and 17 infants received CT+ SP in a randomized, double-blind study. Baseline mean measurements in the two groups: BW 859 vs 837 gm, GA 26 vs 26 wks, PCA 32.5 vs 33 wks, lung compliance (CL)(1.15 vs 1.14 ml/cmH2O/kg), total lung resistance(RL) (56.8 vs 52.5 cmH2O/1/sec), FiO2 0.25 vs 0.27 and serum electrolytes were similar (p=ns). Mean data after 14 days of treatment:Table

The Effect of Reoxygenation with 21% or 100% Oxygen on Hypercapnic-Hypoxia Induced DNA Damage in Cerebral Cortices of Newborn Piglets |[dagger]| 1024

Previous studies have shown that both hypoxia, and hypoxia followed by reoxygenation with either 21% or 100% oxygen are associated with significant generation of free radicals in the cerebral cortices of newborn piglets. The present study investigates the DNA degradation pattern associated with cell death following cerebral hypercapnic hypoxia. Studies were performed on groups(n=4 each) of anesthetized and ventilated newborn piglets. Group 1 was a room air control, and Groups 2 and 3 were exposed to 1hr of hypercapnic-hypoxia followed by reventilation with either 21% or 100% oxygen for 45 minutes, respectively. Group 4 was made hypercapnic and hypoxic but was not reventilated. Hypercapnic hypoxia was achieved by slow inhalation of CO2 and a reduction in the FiO2 to 0.05-0.07. Prior to reventilation, mean values for pH, PaO2 and PaCO2 were 7.05, 20 mmHg and 82 mmHg respectively. After the period of reventilation, brain tissue was removed for analysis. ATP and phosphocreatine (PCr) concentrations were determined biochemically as indices of brain tissue energy metabolism. Mean concentrations of ATP in Grp 1, and the 21% (Grp 2) and 100% (Grp 3) reventilated groups were 4.26, 5.18 and 4.40 mmol/ g brain; PCr values were 3.71, 3.65 and 3.18 mmol/g brain, respectively. Brain cell nuclei and DNA were isolated and the pattern of DNA degradation was assessed using 1% agarose gel-electrophoresis. The gel pattern in Group 1 showed a single band of DNA. High-molecular weight DNA appeared as a large smear in Groups 2 and 3. Although the gel pattern in Group 4 revealed DNA degradation predominantly as a continuous smear, discrete bands of DNA fragments were identified in the lower molecular weight range (2,000-100 bp). The presence of a smear as opposed to a ladder-type pattern implies random fragmentation of DNA, possibly mediated by free radicals, and not due to the action of specific endonucleases. We speculate that irrespective of the O2 concentration used during reoxygenation, the molecular structure of the DNA is altered without being cleaved into small fragments.

The Effect of Reoxygenation with 21% and 100% Oxygen Following Hypercapnic Hypoxia on the Glutamate Site of the NMDA Receptor in the Brains of Newborn Piglets. |[dagger]| 904

The Effect of Reoxygenation with 21% and 100% Oxygen Following Hypercapnic Hypoxia on the Glutamate Site of the NMDA Receptor in the Brains of Newborn Piglets. † 904

EFFECT OF HYPERVENTILATION ON THE SPERMINE AND MAGNESIUM SITES OF THE N-METHYL-D-ASPARTATE RECEPTOR IN THE BRAINS OF NEWBORN PIGLETS. † 272

The present study tests the hypothesis that cerebral ischemia induced by severe hypocapnia results in tissue hypoxia and modifies the N-methyl-D-aspartate (NMDA) receptor/ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in a control(normoxic) group (n=4) and hypocapnic (brain tissue hypoxic) group (n=4) of anesthetized and ventilated newborn piglets. Hypocapnia was induced by hyperventilation (PaCO2 = 9-11 mmHg) for 1 hr. Brain tissue hypoxia was confirmed biochemically in the hypocapnic (ischemic) group by an 80% decrease in [phosphocreatinine]. 3[H]MK-801 binding studies, an index of NMDA receptor activation, was performed at 32°C for 3 hours in an assay medium containing 20mM HEPES (pH 7.0), 75μg protein, glycine (100μM) and glutamate (100μM). Nonspecific binding was measured in the presence of 10μM unlabeled MK-801. Spermine-dependent activation was determined by adding increasing [spermine] from 2.5 to 50μM. Mg++ dependent binding was assayed separately in the presence of [Mg2+] from 2.5 to 100 μM. Maximal spermine-dependent activation in the control and hypocapnic groups was 19.8 ± 4.3% (at 5μM) and 47.6 ± 23.7% (at 6.7μM) above baseline, respectively. Maximal 3[H]MK-801 binding in the control and hypocapnic groups occurred at a [Mg2+] of 25μM and 5.8μM, respectively (p < 0.001). The data show that hypocapnic-induced tissue hypoxia results in increased spermine-dependent maximal activation and increased sensitivity to Mg++, indicating modification of the polyamine and Mg++ sites of the NMDA receptor. We conclude that hypocapnia-induced modification of the spermine and magnesium sites leads to increased activation of the NMDA receptor, resulting in increased NMDA receptor-mediated neurotoxicity in the newborn brain. (NIH-HD20337, MOD-6FY940135, UCP-R50693).

EFFECT OF 100|[percnt]| OXYGEN AND 21|[percnt]| OXYGEN DURING RESUSCITATION ON CEREBRAL CELLULAR FUNCTION FOLLOWING NEONATAL ASPHYXIA. |[dagger]| 1279

Previous studies have shown that hypoxia and severe repeated asphyxia modify the N-methyl-D-aspartate (NMDA) receptor, decrease Na+,K+-ATPase activity, and increase lipid peroxidation and free radical production. The present study investigates the effect of hyperoxic and normoxic reventilation following asphyxia in piglets on NMDA receptor activation, lipid peroxidation and free radical generation. Asphyxia was induced in 8 anesthetized and paralyzed piglets by discontinuing mechanical ventilation for 6-12 min until the heart rate was 120mmHg and PO2<5mmHg). After asphyxia, animals in Grp 1 (n=4) and Grp 2 (n=4) were reventilated with 21% oxygen and 100% oxygen for 20 min. The cerebral cortices were removed for analysis.3[H]MK-801 binding, an index of NMDA receptor activation, showed the Bmax (number of receptors) in Grp 1 and Grp 2 to be 1.32±0.13 and 1.57±0.34 pmol/mg protein (mean±SD). Kd for the same groups were 4.45±1.45 and 3.79±0.96 nM. Bmax and Kd in controls were 1.23±0.06 pmol/mg protein and 5.8±0.5 nM. Na+,K+-ATPase activity in Grps 1 and 2 were 23.5±0.9 and 24.4±3.9 μmol Pi/mg protein/hr, as compared to control(49.9±0.5, p<0.05). Lipid peroxidation products, conjugated dienes(0.07 vs. 0.05 μmol/g brain) and fluorescent compounds (0.78 vs. 0.54 μg quinine sulfate/g brain) were similar. Electron spin resonance spectroscopy performed after trapping spin adducts in α-phenyl-N-tert-butylnitrone for the detection of alkoxyl radical showed similar spin adduct signal height intensity (10.39 vs. 13.68 mm/g tissue). The results show that NMDA receptor activation, lipid peroxidation. Na+,K+-ATPase activity and alkoxyl radical generation were similar following reventilation with 100% and 21% oxygen. These data did not reveal immediate cerebral cellular dysfunction with either normoxic or hyperoxic reventilation following asphyxia. Since previous data have shown a 48 h delay in the appearance of neuronal membrane injury, the long term effect of reventilation with oxygen on neuronal membrane function remains unknown. (Funded by NIH HD-20337, MOD-6FY940135, UCP#R-506-93).