Katherine H. Petersson

Antenatal steroids decrease blood-brain barrier permeability in the ovine fetus

Antenatal corticosteroid therapy reduces the incidence of intraventricular hemorrhage in premature infants. Enhanced microvascular integrity might provide protection against intraventricular hemorrhage. In the adult, there is evidence to suggest that the blood-brain barrier may be under hormonal control. We hypothesized that antenatal corticosteroids decrease blood-brain barrier permeability in the preterm ovine fetus. Chronically instrumented 120-day-gestation fetuses were studied 12 h after the last of four 6-mg dexamethasone (n = 5) or placebo (n = 6) injections had been given over 48 h to the ewes. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (Ki) for alpha-aminoisobutyric acid (AIB). Ki was significantly lower across brain regions in the fetuses of ewes that received antenatal dexamethasone compared with placebo (ANOVA; interaction, F = 2.54, P < 0.004). In fetuses of dexamethasone- and placebo-treated ewes, Ki (microliter . g brain wt-1. min-1, mean +/- SD) was, respectively, 2.43 +/- 0.27 vs. 3.41 +/- 0.74 in the cortex, 4.46 +/- 0.49 vs. 5.29 +/- 0.85 in the cerebellum, and 3.70 +/- 0.49 vs. 5.11 +/- 0.70 in the medulla. We conclude that antenatal treatment with corticosteroids reduces blood-brain permeability in the ovine fetus.Antenatal corticosteroid therapy reduces the incidence of intraventricular hemorrhage in premature infants. Enhanced microvascular integrity might provide protection against intraventricular hemorrhage. In the adult, there is evidence to suggest that the blood-brain barrier may be under hormonal control. We hypothesized that antenatal corticosteroids decrease blood-brain barrier permeability in the preterm ovine fetus. Chronically instrumented 120-day-gestation fetuses were studied 12 h after the last of four 6-mg dexamethasone ( n = 5) or placebo ( n = 6) injections had been given over 48 h to the ewes. Blood-brain barrier function was quantified with the blood-to-brain transfer constant ( K i) for α-aminoisobutyric acid (AIB). K i was significantly lower across brain regions in the fetuses of ewes that received antenatal dexamethasone compared with placebo (ANOVA; interaction, F = 2.54, P < 0.004). In fetuses of dexamethasone- and placebo-treated ewes, K i(μl ⋅ g brain wt-1 ⋅ min-1, mean ± SD) was, respectively, 2.43 ± 0.27 vs. 3.41 ± 0.74 in the cortex, 4.46 ± 0.49 vs. 5.29 ± 0.85 in the cerebellum, and 3.70 ± 0.49 vs. 5.11 ± 0.70 in the medulla. We conclude that antenatal treatment with corticosteroids reduces blood-brain permeability in the ovine fetus.

White Matter Injury after Cerebral Ischemia in Ovine Fetuses

The effects of cerebral ischemia on white matter changes in ovine fetuses were examined after exposure to bilateral carotid artery occlusion. Fetal sheep were exposed to 30 min of ischemia followed by 48 (I/R-48, n = 8) or 72 (I/R-72, n = 10) h of reperfusion or control sham treatment (control, n = 4). Serial coronal sections stained with Luxol fast blue/hematoxylin and eosin were scored for white matter, cerebral cortical, and hippocampal lesions. All areas received graded pathologic scores of 0 to 5, reflecting the degree of injury where 0 = 0%, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, 4 = 76% to 95%, and 5 = 96% to 100% of the area damaged. Dual-label immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used to characterize white matter lesions. Basic fibroblast growth factor (FGF-2) was measured in the frontal cortex by ELISA. Results of the pathologic scores showed that the white matter of the I/R-72 (2.74 ± 0.53, mean ± SEM) was more (p < 0.05) damaged when compared with the control (0.80 ± 0.33) group. Cortical lesions were greater (p < 0.05) in the I/R-48 (2.12 ± 0.35) than the control (0.93 ± 0.09) group. White matter lesions were characterized by reactive GFAP-positive astrocytes and a loss of MBP in oligodendrocytes. The ratio of MBP to GFAP decreased (p < 0.05) as a function of ischemia, indicative of a proportionally greater loss of MBP than GFAP. FGF-2 concentrations were higher (p < 0.05) in the I/R-72 than the control group and there was a direct correlation between the pathologic scores (PS) and FGF-2 concentrations (FGF-2 = e(1.6 PS-0.90) + 743, n = 17, r = 0.73, p < 0.001). We conclude that carotid artery occlusion results in quantifiable white matter lesions that are associated with a loss of MBP from myelin, and that FGF-2, a purported mediator of recovery from brain injury in adult subjects, increases in concentration in proportion to the severity of brain damage in the fetus.

Effects of antenatal steroids on ischemic brain injury in near-term ovine fetuses

BACKGROUND Hypoxia/ischemia in utero can result in brain damage to the fetus and newborn. Antenatal steroids are a routine part of the management of women who develop premature labor. Pretreatment of young postnatal rats with dexamethasone before hypoxic/ischemic insults has been reported to attenuate brain injury. However, the effects of antenatal steroids on ischemic brain injury in fetuses have not been investigated. OBJECTIVE We examined the effects of maternally administered antenatal corticosteroids on ischemic brain injury in near-term ovine fetuses. METHODS Chronically instrumented fetuses at 122 days of gestation were studied 12 h after the last of four 4 mg dexamethasone, or placebo injections were given over 48 h to the ewes. Groups were dexamethasone/ischemic, placebo/ischemic and sham-treated control. Fetuses were exposed to 30 min of carotid occlusion (ischemia) or no occlusion (control) and 72 h of reperfusion. Whole brain coronal sections stained with Luxol fast blue-hematoxylin-eosin were scored for white matter and cerebral cortical lesions. Both areas received pathological scores of 0 to 5 reflecting the degree of injury (0=0%, 1=1-10%, 2=11-50%, 3=51-90%, 4=91-99% and 5=100%). Bilateral carotid blood flow also was measured before, during and after brain ischemia in the dexamethasone/ischemic and placebo/ischemic groups. RESULTS White matter (WM) and cerebral cortical scores did not differ between the dexamethasone/ischemic and placebo/ischemic (WM: 3.0+/-1.9 and 2.9+/-1.7; cortex: 3.1+/-1.7 and 2.6+/-1.8, mean+/-S.D.) groups. White matter and cerebral cortical scores were higher in the dexamethasone/ischemic (WM: 3.0+/-1.9, P<0.02; cortex: 3.1+/-1.7, P<0.005) and placebo/ischemic (WM: 2.9+/-1.7, P<0.006; cortex: 2.6+/-1.8, P<0.007) than control (WM: 0.2+/-0.4; cortex: 0.2+/-0.4) group. Carotid blood flow was relatively higher (P<0.05) after 24, 48 and 72 h of reperfusion in the dexamethasone/ischemic than placebo/ischemic group. CONCLUSIONS We conclude that maternal pretreatment with antenatal dexamethasone did not attenuate ischemic brain injury in the fetus, and that carotid blood flow was higher during reperfusion in fetuses of dexamethasone than placebo-treated ewes, most likely secondary to decreases in arterial oxygen tension.

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.

Effects of multiple courses of antenatal corticosteroids on blood-brain barrier permeability in the ovine fetus.

Objective: To test the hypothesis that multiple courses of antenatal corticosteroids accentuate the decreases in blood-brain barrier permeability observed after a single course of corticosteroids in pretern ovine fetuses. Methods: Chronically instrumented 106-day gestation ovine fetuses were studied after single and multiple courses of dexamethasone or placebo were given to ewes beginning at 104 to 106 or 76 to 78 days of gestation, respectively. In the single-course groups, the ewes received dexamethasone (6 mg, n = 6) or placebo (n = 6) as four intramuscular injections every 12 hours over 48 hours. In the multiple course groups, the ewes received the same treatment (dexamethasone, n = 9, or placebo, n = 8), once per week for 5 weeks starting at 76 to 78 days of gestation. Blood-brain barrier permeability was quantified with the blood-to-brain transfer constant (Ki) for α-aminoisobytyric acid (AIB) in the brain regions of the fetuses 12 hours after the last injection of dexamethasone was given to the ewes at 106 to 107 days of gestation. Results: Both single (analysis of variance [ANOVA]; main effects for dexamethasone treatment, F = 5.92, P <.04) and multiple (ANOVA; main effects for dexamethasone treatment, F = 4.74, P <.04) course of antenatal corticosteriods were associated with decreases in blood-brain barrier permeability in the brain regions of the ovine fetus. However, the multiple course did not accentuate (ANOVA; main effects for single versus multiple courses, F = 1.06, P = .32) the decreases in permeability observed after a single course. Conclusion: Contrary to our hypothesis, antenatal treatment with a 5-week course of corticosteroids did not accentuate the reductions in blood-brain barrier permeability that we observed after a single course of corticosteroids in the fetus.

Effects of exogenous glucose on brain ischemia in ovine fetuses

We examined the effects of prolonged moderate hyperglycemia with and without an additional rapid glucose injection on ischemic brain injury in the fetus. Twenty-five ewes (117–124 d of gestation) were assigned to one of four groups: 1) glucose-infused fetuses exposed to 30 min of carotid artery occlusion followed by 48 h of reperfusion (I/R-Glu, n = 8); 2) glucose-infused plus rapid glucose injection given 100 min before 30 min of occlusion followed by 48 h of reperfusion (I/R-GluR, n = 4); 3) placebo-infused exposed to 30 min of occlusion and 48 h of reperfusion (I/R-PL, n = 8); and 4) glucose-infused sham occlusion and 48 h of sham reperfusion (control, n = 5). After baseline measurements, fetuses were infused with glucose (9–16 mg/kg/min) for 48 h before and after carotid occlusion or sham treatment. The I/R-PL group received 0.9% NaCl. Brain pathologic outcome was determined. Serial sections stained with Luxol fast blue-hematoxylin and eosin were scored for white matter, cerebral cortical, and hippocampal lesions. These areas received graded pathologic scores of 0 to 5, reflecting the amount of injury, where 0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–95%, and 5 = 96–100% of the area damaged. Comparisons of the pathologic scores for cerebral cortex (CC), white matter (WM), and hippocampus (H) demonstrated that the I/R-GluR (CC: 4.56 ± 0.11, WM: 4.50 ± 0.11, H: 3.44 ± 0.48, mean ± SEM) had more (p < 0.05) damage than the I/R-Glu (CC: 2.46 ± 0.47, WM: 1.97 ± 0.37, H: 1.81 ± 0.36) and control (CC: 1.12 ± 0.13, WM: 0.82 ± 0.34, H: 0.80 ± 0.34) groups. The pathologic scores in the I/R-Glu were (p < 0.05) greater than the control, but not the I/R-PL (CC: 2.12 ± 0.35, WM: 2.20 ± 0.44, H: 1.59 ± 0.41) group. We conclude that exposure to prolonged moderate hyperglycemia before ischemia and during reperfusion does not affect the extent of brain injury, but exposure to an additional acute increase in plasma glucose concentration before ischemia is extremely detrimental to the fetal brain.

Effects of Antenatal Corticosteroids on Regional Brain and Non-neural Tissue Water Content in the Ovine Fetus

Objective: We examined the effects of maternal corticosteroid administration on water content in regional tissue in ovine fetuses at 60%, 80%, and 90% of gestation. Methods: After catheters were placed in the fetuses, the ewes were given four 6-mg doses of dexamethasone or placebo injections 12 hours apart over 48 hours. Water conent of fetal tissue was determined 18 hours after the last injection was given to the ewes. Tissue water ws determined by wet-to-dry weight ratio in brain (cerebral cortex, caudate nucleus, cerebellum, midbrain, and medulla) and non-neural tissues (kidney, liver, muscle, and skin) at each gestational age. Results: Water content (P < .05) in brain regions was lower in fetuses from dexamethasone-treated than placebo-treated ewes at 60% but not 80% or 90% of gestation and in non-neural tissues at each gestational age. Conclusion: Maternal treatmet with a contricosteroid regimen similar to that used in the clinical setting was associated with small decreases in brain water content early but not later in gestation. This corticosteroid treatment regimen was also associated with decreased regional non-neural tissue water content at 60%, 80%, and 90% of gestation.

Comparative effects of glucose- and mannitol-induced osmolar stress on blood-brain barrier function in ovine fetuses and lambs

We examined the effects of hyperglycemic hyperosmolality on blood-brain barrier (BBB) permeability during development. We hypothesized that the barrier becomes more resistant to hyperglycemic hyperosmolality during development, and the immature BBB is more resistant to glucose than to mannitol hyperosmolality. We quantified the BBB response to hyperosmolality with the blood-to-brain transfer constant (Ki) in immature fetuses, premature, and newborn lambs. Ki increased as a function of increases in osmolality. A segmented regression model described the relationship between Ki and osmolality. At lower osmolalities, changes in Ki were minimal but after a threshold, increases were linear. We examined responses of Ki to hyperglycemic hyperosmolality by comparing the thresholds and slopes of the second regression segments. Lower thresholds and steeper slopes indicate greater vulnerability to hyperosmolality. Thresholds increased (P < 0.05) during development in pons and superior colliculus. Thresholds were higher (P < 0.05) during glucose than mannitol hyperosmolality in thalamus, superior colliculus, inferior colliculus and medulla of premature lambs, and in cerebrum and cerebellum of newborns. We conclude that BBB permeability increased as a function of changes in glucose osmolality, the barrier becomes more resistant to glucose hyperosmolality in two brain regions during development, and the barrier is more resistant to glucose than to mannitol hyperosmolality in some brain regions of premature and newborn lambs.

Na+,K+-ATPase Activity and Subunit Protein Expression Ontogeny and Effects of Exogenous and Endogenous Steroids on the Cerebral Cortex and Renal Cortex of Sheep

We examined the effects of development, exogenous, and endogenous glucocorticoids on Na+,K+-ATPase activity and subunit protein expression in ovine cerebral cortices and renal cortices. Ewes at 60%, 80%, and 90% gestation, newborns, and adults received 4 dexamethasone or placebo injections. Cerebral cortex Na+,K+-ATPase activity was higher (P < .05) in placebo-treated newborns than fetuses of placebo-treated ewes and adults, α1-expression was higher at 90% gestation than the other ages; α2-expression was higher in newborns than fetuses; α3-expression was higher in newborns than 60% gestation; β1-expression was higher in newborns than the other ages, and β2-expression higher at 60% than 80% and 90% gestation, and in adults. Renal cortex Na+,K+-ATPase activity was higher in placebo-treated adults and newborns than fetuses. Cerebral cortex Na+,K+-ATPase activity was higher in dexamethasone- than placebo-treated adults, and α1-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na+,K+-ATPase activity and α1-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and β1-expression was higher in fetuses of dexamethasone- than placebo-treated ewes at 90% gestation and in dexamethasone- than placebo-treated adults. Cerebral cortex Na+,K+-ATPase activity, α1-expression, β1-expression, and renal cortex α1-expression correlated directly with increases in fetal cortisol. In conclusion, Na+,K+-ATPase activity and subunit expression exhibit specific developmental patterns in brain and kidney; exogenous glucocorticoids regulate activity and subunit expression in brain and kidney at some ages; endogenous increases in fetal cortisol regulate cerebral Na+,K+-ATPase, but exogenous glucocorticoids have a greater effect on renal than cerebral Na+,K+-ATPase.

The Duration of Positive-Pressure Ventilation Influences Blood-Brain Barrier Function in Preterm Lambs 233

The Duration of Positive-Pressure Ventilation Influences Blood-Brain Barrier Function in Preterm Lambs 233