Grazyna B. Sadowska

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.

Antenatal dexamethasone: effect on ovine placental 11beta-hydroxysteroid dehydrogenase type 2 expression and fetal growth.

Antenatal glucocorticoids are routinely given to women at risk for preterm delivery. The fetus is protected from excessive glucocorticoids by the placental enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2), which catalyzes the conversion of cortisol to its biologically inactive metabolite, cortisone. We examined the effects of antenatal dexamethasone on the expression of placental 11β-HSD-2 in fetal sheep. Ewes were randomized to receive repeated or single courses of dexamethasone or placebo beginning at 76-78 or 104-106 d of gestation, respectively. In the single course group, the ewes received dexamethasone (6 mg, n = 7) or placebo (n = 6) as four intramuscular injections over 48 h up to 18 h before placental harvest. In the repeated course group, the ewes received the same treatment (dexamethasone, n = 10, or placebo, n = 9) once a week for 5 consecutive weeks starting at 76–78 d of gestation. Placental harvest occurred at 106–108 d of gestation in the four groups. By semi-quantitative RT-PCR, we found that placental 11β-HSD-2 expression was lower in the fetuses of ewes exposed to a single course of dexamethasone than placebo (p < 0.05). Placental 11β-HSD-2 expression did not differ significantly between fetuses of ewes treated with repeated courses of dexamethasone compared with placebo, or a single course of dexamethasone. Fetuses of dexamethasone treated ewes weighed less than those of placebo treated ewes (ANOVA, main effects for dexamethasone versus placebo treatment:F = 14.5, p = 0.007). Fetuses of ewes exposed to repeated courses of dexamethasone weighed less than those of ewes exposed to placebo or a single course of dexamethasone (p < 0.05). We conclude that maternal antenatal dexamethasone treatment reduces placental 11β-HSD-2 expression and fetal weight at mid-gestation in the ovine pregnancy.

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.

Ischemia-reperfusion impairs blood-brain barrier function and alters tight junction protein expression in the ovine fetus

The blood-brain barrier is a restrictive interface between the brain parenchyma and the intravascular compartment. Tight junctions contribute to the integrity of the blood-brain barrier. Hypoxic-ischemic damage to the blood-brain barrier could be an important component of fetal brain injury. We hypothesized that increases in blood-brain barrier permeability after ischemia depend upon the duration of reperfusion and that decreases in tight junction proteins are associated with the ischemia-related impairment in blood-brain barrier function in the fetus. Blood-brain barrier function was quantified with the blood-to-brain transfer constant (K(i)) and tight junction proteins by Western immunoblot in fetal sheep at 127 days of gestation without ischemia, and 4, 24, or 48 h after ischemia. The largest increase in K(i) (P<0.05) was 4 h after ischemia. Occludin and claudin-5 expressions decreased at 4 h, but returned toward control levels 24 and 48 h after ischemia. Zonula occludens-1 and -2 decreased after ischemia. Inverse correlations between K(i) and tight junction proteins suggest that the decreases in tight junction proteins contribute to impaired blood-brain barrier function after ischemia. We conclude that impaired blood-brain barrier function is an important component of hypoxic-ischemic brain injury in the fetus, and that increases in quantitatively measured barrier permeability (K(i)) change as a function of the duration of reperfusion after ischemia. The largest increase in permeability occurs 4 h after ischemia and blood-brain barrier function improves early after injury because the blood-brain barrier is less permeable 24 and 48 than 4 h after ischemia. Changes in the tight junction molecular composition are associated with increases in blood-brain barrier permeability after ischemia.

Maternal glucocorticoid exposure alters tight junction protein expression in the brain of fetal sheep.

We examined the expression of tight junction (TJ) proteins in the cerebral cortex, cerebellum, and spinal cord of fetuses after maternal treatment with single and multiple courses of dexamethasone. Ewes received either single courses of four 6-mg dexamethasone or placebo injections every 12 h for 48 h between 104 and 107 days or the same treatment once a week between 76-78 and 104-107 days of gestation. TJ protein expression was determined by Western immunoblot analysis on tissue harvested at 105-108 days of gestation. Blood-brain barrier permeability has been previously quantified with the blood-to-brain transfer constant (K(i)) with alpha-aminoisobutyric acid (39). After a single course of dexamethasone, claudin-5 increased (P < 0.05) in the cerebral cortex, occludin and claudin-1 increased in the cerebellum, and occludin increased in the spinal cord. After multiple dexamethasone courses, occludin and zonula occludens (ZO)-1 increased in the cerebral cortex, and occludin and claudin-1 increased in the cerebellum. Junctional adhesion molecule-A and ZO-2 expressions did not change. Linear regression comparing K(i) to TJ proteins showed inverse correlations with claudin-1 and claudin-5 in the cerebral cortex after a single course and ZO-2 in the spinal cord after multiple courses and direct correlations with ZO-1 in the cerebellum and spinal cord after multiple courses. We conclude that maternal glucocorticoid treatment increases the expression of specific TJ proteins in vivo, patterns of TJ protein expression vary after exposure to single and multiple glucocorticoid courses, and decreases in blood-brain barrier permeability are associated with increases in claudin-1, claudin-5, and ZO-2 expression and decreases in ZO-1 expression. In utero glucocorticoid exposure alters the molecular composition of the barrier and affects fetal blood-brain barrier function.

Ovine Proinflammatory Cytokines Cross the Murine Blood-Brain Barrier by a Common Saturable Transport Mechanism

Objectives: The cytokines interleukin (IL)-1β and IL-6 are modulators of the neuroimmune axis and have been implicated in neuronal cell death cascades after ischemia or infection. Previous work has shown that some cross-species conservation exists between human and rodent blood-brain barrier (BBB) transport systems. To further assess cross-species conservation of cytokine transport across the BBB, the current studies investigated permeability and inhibition of ovine IL-1β and IL-6 in the mouse. Methods: IL-1β or IL-6 was radioactively labeled with 131I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 µg/mouse of an unlabeled ovine or murine cytokine (IL-1β or IL-6) to assess self- and/or cross-inhibition of transport. Permeability was assessed using multiple-regression analysis. Results: There was a significant linear relationship for both ovine 131I-IL-1β and 131I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 µg/mouse unlabeled ovine IL-1β or IL-6 significantly reduced the transport of ovine 131I-IL-1β or 131I-IL-6, respectively, across the BBB. Transport of both ovine 131I-IL-1β and 131I-IL-6 was significantly inhibited by 1 µg/mouse of murine IL-1β or IL-6, respectively. In contrast, 1 µg/mouse of unlabeled ovine IL-1β or IL-6 did not inhibit the transport of murine 131I-IL-1β or 131I-IL-6. Conclusions: Ovine IL-1β and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

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.

Neutralizing anti-interleukin-1β antibodies modulate fetal blood-brain barrier function after ischemia.

We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0.03), and interleukin-1β protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus.

Effects of Maternal Antenatal Glucocorticoid Treatment on Apoptosis in the Ovine Fetal Cerebral Cortex

We examined the effects of single and multiple maternal glucocorticoid courses on apoptosis in the cerebral cortices of ovine fetuses (CC). Ewes received single dexamethasone or placebo courses at 104–106 or 133–135 days or multiple courses between 76–78 and 104–106 days gestation. In the single‐course groups, ewes received four 6 mg dexamethasone or placebo injections every 12 hr for 48 hr. Multiple‐course groups received the same treatment once per week for 5 weeks. Neuronal and nonneuronal apoptotic cell numbers per square millimeter were determined with TUNEL and NeuN staining and with caspase‐3 enzyme activity on CC tissues harvested at 106–108 (70%) or 135–137 (90%) days of gestation. Apoptotic cell numbers and caspase‐3 activity were 50% lower (P < 0.02) after single placebo courses at 90% than 70% gestation; 90% of apoptotic cells were (P < 0.01) nonneuronal at both ages. Nonneuronal apoptotic cells and caspase‐3 activity were 40% and 20% lower (P < 0.02) after single dexamethasone than placebo courses at 70%, but not 90%, gestation. Caspase‐3 activity was 20% lower (P < 0.01) after multiple dexamethasone than placebo courses, but apoptotic cell number did not differ. We conclude that nonneuronal apoptosis represents the major form of apoptosis in the CC at both 70% and 90% of gestation. Apoptosis in nonneuronal cells decreases with maturity and after a single course of dexamethasone at 70%, but not at 90%, gestation and not after multiple courses at 70% gestation. We speculate that a single course of glucocorticoids exerts maturational changes on the rate of apoptosis in the cerebral cortex of preterm ovine fetuses.

Ontogeny and the effects of in utero brain ischemia on interleukin-1β and interleukin-6 protein expression in ovine cerebral cortex and white matter.

Interleukin (IL)‐1β and IL‐6 have been implicated in brain development, injury progression, and fetal/maternal immune interactions. We examined IL‐1β and IL‐6 protein expression in cerebral cortex (CC) and white matter (WM) from non‐ischemic ovine fetuses at 87–90, 122–127, and 135–137 days of gestation, pregnant ewes at 87–90 and 135–137 days of gestation, and fetuses exposed to 48 or 72 h of reperfusion after ischemia. Protein expression was determined by Western immunoblot. In non‐ischemic CC, IL‐1β was higher (P < 0.05) in adult sheep and fetuses at 135–137 than 87–90 and 122–127 days, and IL‐6 higher at 122–127 than 87–90 days, and in adults than fetuses at 87–90, 122–127, and 135–137 days of gestation. In non‐ischemic fetal WM, IL‐6 was higher at 135–137 than 87–90 days, but IL‐1β did not differ. In CC, IL‐1β was higher in ewes at 135–137 than 87–90 days and IL‐6 at 135–137 days and in non‐pregnant adults than ewes at 87–90 days of gestation. In WM, IL‐1β was higher in ewes at 135–137 than 87–90 days of gestation, but IL‐6 did not differ. Forty‐eight and 72 h after ischemia, CC IL‐1β was higher than in non‐ischemic fetuses. Seventy‐two hours after ischemia, IL‐1β and IL‐6 were higher in WM than CC. In conclusion, IL‐1β and IL‐6 exhibit developmental regulation in fetal brain, change during gestation in brains of pregnant ewes, show regional differences in normal brains of fetuses and ewes, demonstrate differential responses after ischemia in CC and WM, and IL‐1β but not IL‐6 increases after ischemia in CC.