Gregory D. Sysyn

Ontogeny and the effects of corticosteroid pretreatment on aquaporin water channels in the ovine cerebral cortex

The aim of the present study was to determine the ontogeny and effects of corticosteroid pretreatment on aquaporin 4 (AQP4) channel mRNA and protein expression in the cerebral cortex of sheep during development. A portion of the cerebral cortex was snap-frozen from fetuses of dexamethasone- and placebo-treated ewes at 60%, 80% and 90% of gestation, dexamethasone- and placebo-treated newborn lambs and adult sheep. Cerebral cortical samples were obtained 18 h after the last of four 6 mg dexamethasone or placebo injections were given over 48 h to the ewes and adult sheep. Lambs were treated with 0.01 mg kg(-1) dexamethasone or placebo in the same schedule as the ewes and adult sheep. Amplification of an ovine AQP4 cDNA fragment was accomplished by reverse transcription-polymerase chain reaction using primers based on a homologous bovine sequence. The resulting cDNA was used to determine AQP4 channel mRNA expression by Northern hybridisation using phosphorimaging. The relative abundance of AQP4 mRNA was normalised to the ovine ribosomal gene L32. A portion of the frontal cortex was also analysed for AQP4 protein expression by Western immunoblot. Densitometry was performed and the results expressed as a ratio to an adult brain pool. Aquaporin 4 channel mRNA and protein were detectable as early as at 60% gestation. There were no changes in AQP4 mRNA expression among the fetal, newborn and adult groups or after dexamethasone pretreatment in any age group. The expression of the AQP4 protein was higher (P < 0.05) in fetuses at 80% and 90% of gestation (2.9- and 3.3-fold, respectively), in lambs (3.2-fold) and in adult sheep (3.8-fold) compared with fetuses at 60% of gestation, as well as in adult sheep (1.3-fold) compared with fetuses at 80% of gestation. Dexamethasone pretreatment resulted in decreases (P < 0.05) in AQP4 protein expression in the lambs and adult sheep, but not in the fetal groups. We conclude that: (1) AQP4 mRNA and protein were expressed early in fetal and throughout ovine development; (2) protein, but not mRNA, expression increased between 60% and 80% of gestation and did not differ from adult levels by 90% of gestation; and (3) dexamethasone pretreatment resulted in decreases in AQP4 protein expression in lambs and adult sheep, but not in fetuses. The maturational increases in AQP4 protein expression and dexamethasone-related decreases in expression were post-transcriptional, because changes in AQP4 mRNA expression were not observed.

Effects of postnatal steroids on Na+/K+-ATPase activity and α1- and β1-subunit protein expression in the cerebral cortex and renal cortex of newborn lambs

Na(+)/K(+)-ATPase is a membrane-bound enzyme responsible for Na(+)/K(+) translocation across cell membranes. It is essential for the generation of electrochemical gradients, which control the ionic environment necessary for electrical activity and water and electrolyte balance. Newborn infants who are at risk of developing bronchopulmonary dysplasia (BPD) are frequently treated with corticosteroids. Although these infants are at risk for neurological, water and electrolyte abnormalities, there is little information regarding the effects of clinically relevant doses of corticosteroids on Na(+)/K(+)-ATPase activity and protein isoform expression in the brain and kidney of newborns. In the present study, we examined the effects of dexamethasone on cerebral cortical and renal cortical Na(+)/K(+)-ATPase activity and alpha1- and beta1-protein isoform expression in newborn lambs. Lambs were given four injections of a placebo (n = 11) or one of three different doses of dexamethasone (0.01 mg kg(-1), n = 9; 0.25 mg kg(-1), n = 11; or 0.50 mg kg(-1), n = 9) 12 h apart on Postnatal Days 3 and 4 up to 18 h before harvest of the cerebral cortex and renal cortex. We selected doses in a range to approximate those used to treat infants with BPD. Na(+)/K(+)-ATPase activity was measured in membrane preparations as ouabain-sensitive inorganic phosphate liberation from ATP and alpha1- and beta1-subunit abundance by Western immunoblot. Postnatal treatment of lambs with dexamethasone resulted in a 21.4% increase in Na(+)/K(+)-ATPase activity and a 30.4% increase in catalytic alpha1-protein expression in the cerebral cortex at a dose of 0.50 mg kg(-1) dexamethasone, but not at the lower doses. Dexamethasone treatment was not associated with changes in beta1-isoform expression in the cerebral cortex. In the kidney, dexamethasone treatment was not associated with significant changes in Na(+)/K(+)-ATPase activity or alpha1- or beta1-isoform expression for the doses we examined. Therefore, clinically relevant corticosteroid treatment exerts dose-related, differential organ-specific effects on Na(+)/K(+)-ATPase activity and protein isoform expression in newborn lambs.

Prognostic Significance of Early Elevated Serum Uric Acid Concentrations in Low Birthweight Infants 1149

Uric acid (UA) concentrations can be elevated after ischemia-reperfusion injury and high levels in the first day of life have been associated with intracranial pathology in low birthweight infants (Pediatr Res 1996; 39: 238A). To determine the prognostic significance of high serum UA concentrations in the first 24 hours after birth, the records of 154 inborn infants who weighed 7.5 mg/dl and by univariate analysis was significantly related to gestational age, peak serum creatinine, serum HCO3 and base deficit, the presence of pregnancy-induced hypertension (PIH), intrauterine growth retardation and how long after birth the sample was obtained. By multiple logistic regression, peak creatinine concentrations, base deficit and time of sample were risk factors for high serum UA concentrations. There were no significant differences in UA concentrations between those with any IVH or PVL(n = 41, UA=5.35 ± 0.32 mg/dl) and those with none (n=97, UA=5.88± 0.22 mg/dl, p > 0.05), nor did UA concentrations differ between the survivors vs the 21 who died (5.73 ± 0.19 vs 5.45 ± 0.43 mg/dl, p > 0.05). Elevated UA concentrations were not found in those with more severe intracranial pathology (Grades III and IV IVH and/or PVL) nor in the group defined as bad outcome (died or had IVH/PVL). Of the 132 infants eligible for follow-up, 79 (60%) were seen at a median age of 11 months(range 5-29 mo). The 20 infants with neurodevelopmental or neuromotor abnormalities had a first day UA concentration of 5.38 ± 0.38 compared with 6.03± 0.33 for those with a normal exam (p > 0.05). Elevated serum UA concentrations in the first 24 hr of life do not relate to intracranial pathology, death or abnormal neurological outcome. Since the concentrations are higher in the presence of maternal hypertension, serum creatinine and how long after birth the sample is obtained, it is possible that any detection of elevated serum UA from ischemia-reperfusion injury in the infant is obscured by transplacental passage of UA from the mother (serum UA is higher in mothers with PIH) and from a longer half-life for UA in the infants circulation during the first day of life.

Effect of Steroid Administration on N-methyl-D-aspartate (NMDA) Receptor Function in Immature Brain |[diams]| 342

Corticosteroid administration in the fetus and newborn enhances organ maturation and may decrease perinatal brain injury. To test the hypothesis that dexamethasone pretreatment alters development of the NMDA receptor, a mediator of perinatal hypoxic-ischemic brain injury, NMDA receptor ion channel function was investigated in 6 groups: fetal lambs at 84d gestation (n=6), fetal lambs at 120d gestation after maternal pretreatment with either placebo(n=4) or dexamethasone (n=4), and 5d-old term (154d) newborn lambs treated with placebo (PD, n=3), or low-dose (LD, n=5) or high-dose (HD, n=5) dexamethasone. Fetal treatment consisted of dexamethasone, 6 mg IM, or placebo every 12 h × 4 doses to pregnant ewes. Lambs received dexamethasone, 0.02 mg/kg (LD), 0.5 mg/kg (HD), or saline (PD) every 12 h × 4 doses from postnatal day 3. Postnatal doses were chosen to match fetal exposure to maternal steroids (LD) or doses used in premature infants (HD). Tissue was obtained 12 hr after the 4th dose in all animals. Saturation binding assays were performed using 3H-MK801, a noncompetitive NMDA receptor antagonist, and Bmax (apparent number of receptors) and Kd (dissociation constant) were calculated. Bmax (pmoles/mg protein) increased from 0.60± 0.09 at 84d to 0.97 ± 0.12 at 120d and 1.91 ± 0.32 in 5-day-old lambs (p<0.05). Kd did not change significantly with maturation(5.1 ± 0.4 nM at 84d, 4.9 ± 0.8 at 120d, and 5.5 ± 0.4 in lambs). Steroid pretreatment had no effect on Bmax or Kd at 120d. However, Bmax decreased significantly in treated lambs, to 1.65 ± 0.08 and 1.62± 0.06 in LD and HD, respectively (p<0.05 for both vs PD). Kd was also significantly lower in both LD and HD compared to PD (4.4 ± 0.4 and 4.0 ± 0.3, respectively, vs 5.5 ± 0.4 nM), indicating a 20% increase in affinity of the MK-801 binding site within the NMDA receptor ion channel. Previous studies in several species have shown that Bmax peaks in the perinatal period, then declines to adult levels. Thus the decrease in Bmax after postnatal steroid treatment suggests that dexamethasone accelerates the maturational decrease in number of NMDA receptors in the term lamb. The change in Kd associated with steroid treatment could be due to modification of cell membrane lipid composition, leading to altered conformation of the receptor ion channel. These results suggest that steroid administration alters NMDA-type glutamate receptor function in immature brain, and that the effect is dependent on the degree of brain development at the time of administration.

Effect of Postnatal Steroids on Blood-Brain Barrier Permeability in Newborn Lambs. |[dagger]| 1150

Antenatal steroids given to enhance pulmonary maturity have been reported to exert a wide range of extrapulmonary effects including a reduction in the incidence of intraventricular hemorrhage. Effects of perinatal steroids on the developing brain have not been well documented. We have recently shown that antenatal steroids decrease blood-brain barrier (BBB) permeability in ovine fetuses at 80% gestation. The effect of steroids on BBB function has not been evaluated in the newborn of any species. We hypothesize that postnatal steroids decrease BBB permeability in newborn lambs. Three groups were studied after birth: placebo and two dexamethasone (DEX) injected groups. DEX was given as either a low dose (LD), 0.02 mg/kg, selected to mimic the estimated fetal dose received in our previous work; or a high dose (HD), 0.5 mg/kg, chosen to simulate the doses used to attenuate bronchopulmonary dysplasia. Lambs were instrumented on day 2 of life and allowed to recover. Beginning on day 3 of life, four intramuscular doses were given every 12 hours for 48 hours. BBB permeability was measured in awake unanesthetized animals 12 hours after the final dose. Regional barrier permeability was quantified with the blood-to-brain transfer constant, Ki(μL/g Brain/min). Ki was measured using the integral technique with 14C-α-aminoisobutyric acid. Regional brain Ki values are summarized in the Table. The Ki values in the LD group were higher(*ANOVA, F=9.6, p<0.05) than that of the placebo and (+ F=5.7, p<0.05) that of the HD group. There was no difference (F=0.3, p=0.58) in Ki values between HD and placebo treated lambs. Steroid treatment did not reduce BBB permeability in newborn lambs. We conclude that effects of steroid pretreatment on BBB permeability in newborn lambs are unique and different from that of fetal sheep and adult rats, in which steroids reduce BBB permeability. We speculate that, during the early postnatal period, the BBB is highly sensitive to modulation by the adrenocortical axis by either endogenous or exogenous corticosteroids and that the stress of birth may modify the BBBs response to steroids. Table: Ki(M±SEM μL/g Brain/min).