Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain.

Hypoxic-ischemic brain injury is a leading cause of neurodevelopmental morbidities in preterm and full-term infants. Blood-brain barrier dysfunction represents an important component of perinatal hypoxic-ischemic brain injury. The extracellular matrix (ECM) is a vital component of the blood-brain barrier. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are important ECM components. They contribute to brain development, blood-brain barrier maintenance, and to regenerative and repair processes after hypoxic-ischemic brain injury. We hypothesized that ischemia at different durations of reperfusion affects the ECM protein composition of MMPs and TIMPs in the cerebral cortex of fetal sheep. Cerebral cortical samples were snap-frozen from sham control fetuses at 127 days of gestation and from fetuses after exposure to 30-min carotid occlusion and 4-, 24-, and 48-h of reperfusion. Protein expression of MMP-2, -8, -9, and -13 and TIMP-1, -2, -3, and -4 was measured by Western immunoblotting along with the gelatinolytic activity of MMP-2 and MMP-9 by zymography. The expression of MMP-8 was increased (Kruskal-Wallis, p = 0.04) in fetuses 48 h after ischemia. In contrast, changes were not observed in the protein expression of MMP-2, -9, or -13. The gelatinolytic activity of pro-MMP-2 was increased (ANOVA, p = 0.02, Tukey HSD, p = 0.05) 24 h after ischemia. TIMP-1 and -3 expression levels were also higher (TIMP-1, ANOVA, p = 0.003, Tukey HSD, p = 0.01; TIMP-3, ANOVA, p = 0.006, Tukey HSD, p = 0.01) 24 h after ischemia compared with both the sham controls and with fetuses exposed to 4 h of reperfusion. The changes in the expression of TIMP-1, -2, and -3 correlated with the changes in the MMP-8 and -13 protein expression. We speculate that regulation of MMP-8, MMP-13, and TIMPs contributes to ECM remodeling after is chemic-reperfusion injury in the fetal brain.

Neutralizing anti-interleukin-1ß antibodies reduce ischemia-related interleukin-1ß transport across the blood-brain barrier in fetal sheep.

Hypoxic ischemic insults predispose to perinatal brain injury. Pro-inflammatory cytokines are important in the evolution of this injury. Interleukin-1ß (IL-1ß) is a key mediator of inflammatory responses and elevated IL-1ß levels in brain correlate with adverse neurodevelopmental outcomes after brain injury. Impaired blood-brain barrier (BBB) function represents an important component of hypoxic-ischemic brain injury in the fetus. In addition, ischemia-reperfusion increases cytokine transport across the BBB of the ovine fetus. Reducing pro-inflammatory cytokine entry into brain could represent a novel approach to attenuate ischemia-related brain injury. We hypothesized that infusions of neutralizing IL-1ß monoclonal antibody (mAb) reduce IL-1ß transport across the BBB after ischemia in the fetus. Fetal sheep were studied 24-h after 30-min of carotid artery occlusion. Fetuses were treated with placebo- or anti-IL-1ß mAb intravenously 15-min and 4-h after ischemia. Ovine IL-1ß protein expressed from IL-1ß pGEX-2T vectors in Escherichia coli (E. coli) BL-21 cells was produced, purified, and radiolabeled with 125I. BBB permeability was quantified using the blood-to-brain transfer constant (Ki) with 125I-radiolabeled-IL-1ß. Increases in anti-IL-1ß mAb were observed in the brain of the mAb-treated group (P<0.001). Blood-to-brain transport of 125I-IL-1ß was lower (P<0.04) across brain regions in the anti-IL-1ß mAb-treated than placebo-treated ischemic fetuses. Plasma 125I-IL-1ß counts were higher (P<0.001) in the anti-IL-1ß mAb- than placebo-treated ischemic fetuses. Systemic infusions of anti-IL-1ß mAb reduce IL-1ß transport across the BBB after ischemia in the ovine fetus. Our findings suggest that conditions associated with increases in systemic pro-inflammatory cytokines and neurodevelopmental impairment could benefit from an anti-cytokine therapeutic strategy.

Ischemia reduces inter-alpha inhibitor proteins in the brain of the ovine fetus.

Hypoxic-ischemic (HI) brain injury is a major cause of neurological abnormalities in the perinatal period. Inflammation contributes to the evolution of HI brain injury. Inter-alpha inhibitor proteins (IAIPs) are a family of proteins that are part of the innate immune system. We have reported that endogenous IAIPs exhibit developmental changes in ovine brain and that exogenous IAIP treatment reduces neuronal death in HI neonatal rats. However, the effects of HI on endogenous IAIPs in brain have not been previously examined. In this study, we examined the effects of ischemia-reperfusion on endogenous IAIPs levels in fetal sheep brain. Cerebral cortex, cerebellum, cervical spinal cord, choroid plexus, and CSF were snap frozen from sham control fetuses at 127 days gestation and after 30-min of carotid occlusion and 4-, 24-, and 48-h of reperfusion. IAIP levels were determined by Western immunoblot. IAIP expressions of the 250 kDa Inter-alpha inhibitor (IaI) and 125 kDa Pre-alpha inhibitor (PaI) in cerebral cortex and PaI in cerebellum were reduced (p < 0.05) 4-h after ischemia compared with controls and returned toward control levels 24- and 48-h after ischemia. CSF PaI and IaI were reduced 48 h after ischemia. We conclude that IAIPs in cerebral cortex and cerebellum are reduced by brain ischemia, and return toward control levels between 24 and 48 h after ischemia. However, changes in CSF IAIPs were delayed, exhibiting decreases 48 h after ischemia. We speculate that the decreases in endogenous IAIPs reflect increased utilization, potentially suggesting that they have endogenous neuroprotective properties. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 726-737, 2017.

Interleukin-1ß transfer across the blood-brain barrier in the ovine fetus.

Pro-inflammatory cytokines contribute to hypoxic-ischemic brain injury. Blood-brain barrier (BBB) dysfunction represents an important component of hypoxic-ischemic brain injury in the fetus. Hypoxic-ischemic injury could accentuate systemic cytokine transfer across the fetal BBB. There has been considerable conjecture suggesting that systemic cytokines could cross the BBB during the perinatal period. Nonetheless, evidence to support this contention is sparse. We hypothesized that ischemia-reperfusion increases the transfer of systemic interleukin-1ß (IL-1ß) across the BBB in the fetus. Ovine fetuses at 127 days of gestation were studied 4 hours after 30 minutes of bilateral carotid artery occlusion and compared with a nonischemic group. Recombinant ovine IL-1ß protein was expressed from an IL-1ß pGEX-2 T vector in E. coli BL-21 cells and purified. The BBB function was quantified in 12 brain regions using a blood-to-brain transfer constant with intravenous (125)I-radiolabeled IL-1ß ((125)I-IL-1ß). Interleukin-1ß crossed the intact BBB in nonischemic fetuses. Blood-to-brain transport of (125)I-IL-1ß was higher (P<0.05) across brain regions in fetuses exposed to ischemia-reperfusion than nonischemic fetuses. We conclude that systemic IL-1ß crosses the intact fetal BBB, and that ischemia-reperfusion increases transfer of this cytokine across the fetal BBB. Therefore, altered BBB function after hypoxia-ischemia facilitates entry of systemic cytokines into the brain of the fetus.

Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.

Neutralizing anti-interleukin-1ß 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-1ß monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1ß protein. This antibody also neutralizes the effects of interleukin-1ß protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1ß 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-1ß antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1ß 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-1ß protein and anti-interleukin-1ß 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-1ß protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1ß protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1ß monoclonal antibody infusions, plasma anti-interleukin-1ß monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1ß monoclonal antibody levels were higher (P<0.03), and interleukin-1ß 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-1ß monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1ß monoclonal antibody infusions after ischemia result in brain anti-interleukin-1ß antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1ß protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1ß, contributes to impaired blood-brain barrier function after ischemia in the fetus.

Ischemia/Reperfusion-induced neovascularization in the cerebral cortex of the ovine fetus.

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

Ontogeny of inter-alpha inhibitor proteins in ovine brain and somatic tissues.

Inter-alpha inhibitor proteins (IAIPs) found in relatively high concentrations in human plasma are important in inflammation. IAIPs attenuate brain damage in young and adult subjects, decrease during sepsis and necrotizing enterocolitis in premature infants, and attenuate sepsis-related inflammation in newborn rats. Although a few studies have reported adult organ-specific IAIP expression, information is not available on age-dependent IAIP expression. Given evidence suggesting IAIPs attenuate brain damage in young and adult subjects, and inflammation in newborns, we examined IAIP expression in plasma, cerebral cortex (CC), choroid plexus (CP), cerebral spinal fluid (CSF), and somatic organs in fetal, newborn, and adult sheep to determine the endogenous expression patterns of these proteins during development. IAIPs (enzyme-linked immunosorbent assay) were higher in newborn and adult than fetal plasma (P < 0.05). Western immunoblot detected 125 kDa PaI (Pre-alpha Inhibitor) and 250 kDa IaI (Inter-alpha Inhibitor) in plasma, CNS, and somatic organs. PaI expression in CC and CP was higher in fetuses than newborns and adults, but IaI expression was higher in adults than fetuses and newborns. Both PaI and IaI were higher in fetal than newborn CSF. IAIPs exhibited organ-specific ontogenic patterns in placenta, liver, heart, and kidney. These results provide evidence for the first time that plasma, brain, placenta, liver, heart, and kidney express IAIPs throughout ovine development and that expression patterns are unique to each organ. Although exact functions of IAIPs in CNS and somatic tissues are not known, their presence in relatively high amounts during development suggests their potential importance in brain and organ development.

Effects of interleukin-6 on the expression of tight junction proteins in isolated cerebral microvessels from yearling and adult sheep.

OBJECTIVES: The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of tight junctions. We hypothesized that interleukin-6 (IL-6) downregulates key protein constituents of endothelial tight junctions (e.g. occludin and claudin-5). METHODS: We examined the effects of IL-6 on tight junction protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate-buffered saline), 1, 10, and 100 ng/ml for 24 h. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. RESULTS: Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (p < 0.05) occludin expression in microvessels from yearling and adult sheep and claudin-5 in microvessels from adult sheep. However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in tight junction proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 preincubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. CONCLUSION: We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep.

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

Interleukin (IL)-1ß and IL-6 have been implicated in brain development, injury progression, and fetal/maternal immune interactions. We examined IL-1ß 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 72h of reperfusion after ischemia. Protein expression was determined by Western immunoblot. In non-ischemic CC, IL-1ß 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-1ß did not differ. In CC, IL-1ß 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-1ß was higher in ewes at 135-137 than 87-90 days of gestation, but IL-6 did not differ. Forty-eight and 72h after ischemia, CC IL-1ß was higher than in non-ischemic fetuses. Seventy-two hours after ischemia, IL-1ß and IL-6 were higher in WM than CC. In conclusion, IL-1ß 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-1ß but not IL-6 increases after ischemia in CC.

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 (K(i)) in immature fetuses, premature, and newborn lambs. K(i) increased as a function of increases in osmolality. A segmented regression model described the relationship between K(i) and osmolality. At lower osmolalities, changes in K(i) were minimal but after a threshold, increases were linear. We examined responses of K(i) 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, α(1)-expression was higher at 90% gestation than the other ages; α(2)-expression was higher in newborns than fetuses; α(3)-expression was higher in newborns than 60% gestation; ß(1)-expression was higher in newborns than the other ages, and ß(2)-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 α(1)-expression higher in fetuses of dexamethasone- than placebo-treated ewes at 60% and 80% gestation. Renal cortex Na(+),K(+)-ATPase activity and α(1)-expression were higher in fetuses of dexamethasone- than placebo-treated ewes at each gestational age, and ß(1)-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, α(1)-expression, ß(1)-expression, and renal cortex α(1)-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.

Ovine proinflammatory cytokines cross the murine blood-brain barrier by a common saturable transport mechanism.

OBJECTIVES: The cytokines interleukin (IL)-1beta 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-1beta and IL-6 in the mouse. METHODS: IL-1beta or IL-6 was radioactively labeled with (131)I and injected into the jugular vein at time zero. A subset of mice received 1 or 3 microg/mouse of an unlabeled ovine or murine cytokine (IL-1beta 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 (131)I-IL-1beta and (131)I-IL-6 between brain/serum ratios and exposure time, indicating BBB permeability. Inclusion of 3 microg/mouse unlabeled ovine IL-1beta or IL-6 significantly reduced the transport of ovine (131)I-IL-1beta or (131)I-IL-6, respectively, across the BBB. Transport of both ovine (131)I-IL-1beta and (131)I-IL-6 was significantly inhibited by 1 microg/mouse of murine IL-1beta or IL-6, respectively. In contrast, 1 microg/mouse of unlabeled ovine IL-1beta or IL-6 did not inhibit the transport of murine (131)I-IL-1beta or (131)I-IL-6. CONCLUSIONS: Ovine IL-1beta 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.

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.

Ontogeny and the effects of exogenous and endogenous glucocorticoids on tight junction protein expression in ovine cerebral cortices.

Maternal glucocorticoid treatment reduces blood-brain permeability early, but not late in fetal development, and pretreatment with glucocorticoids does not affect barrier permeability in newborn lambs. In addition, endogenous increases in plasma cortisol levels are associated with decreases in blood-brain barrier permeability during normal fetal development. Therefore, we tested the hypotheses that development as well as endogenous and exogenous glucocorticoids alters the expression of tight junction proteins in the cerebral cortex of sheep. Cerebral cortices from fetuses at 60%, 70%, and 90% of gestation, newborn and adult sheep were snap frozen after four 6-mg dexamethasone or placebo injections were given over 48-h to the ewes and adult sheep. Lambs were treated similarly with 0.25 mg/kg-dexamethasone or placebo. Tight junction protein expression was measured by Western immunoblot. Claudin-1 was higher (P<0.05) in fetuses at 60% of gestation than in newborn and adult sheep. Claudin-5 was higher at 60% than 70% of gestation, and than in newborn and adult sheep. ZO-1 was higher in newborn than adult sheep. ZO-2 was higher at 90% gestation, in newborn and adult sheep than 60% gestation. Claudin-5 was higher in dexamethasone than placebo-treated lambs, and ZO-2 was higher in fetuses of dexamethasone than placebo-treated ewes at 90% gestation. ZO-2 expression demonstrated a direct correlation with increases in plasma cortisol during fetal development. We conclude that claudin-1, claudin-5, ZO-1, and ZO-2 expression exhibit differential developmental regulation, exogenous glucocorticoids regulate claudin-5 and ZO-2 in vivo at some, but not all ages, and increases in endogenous fetal glucocorticoids are associated with increases in ZO-2 expression, but not with occludin, claudin-1, claudin-5 or ZO-1 expression in ovine cerebral cortices.

Na+, K+-ATPase activity and subunit isoform protein abundance: effects of antenatal glucocorticoids in the frontal cerebral cortex and renal cortex of ovine fetuses.

We examined the effects of single and multiple maternal glucocorticoid courses on cerebral cortical (CC) and renal cortical (RC) Na(+),K(+)-ATPase activity and protein isoform abundance in fetal sheep. Ewes received four dexamethasone or placebo injections in the single course (SC) groups, and the same treatment once a week for five-weeks in the multiple course (MC) groups. CC Na(+),K(+)-ATPase a(2)-abundance was higher (P<0.05) and beta(2)-abundance lower in the SC dexamethasone than placebo group, but Na(+),K(+)-ATPase activity did not change. CC Na(+),K(+)-ATPase activity, a(1)-, beta(1) -, and beta(2)-abundance were lower in the MC dexamethasone than placebo group, but a(2)- and a(3)-abundance did not change. Both dexamethasone courses did not affect CC cell number. RC Na(+),K(+)-ATPase activity, a(1)- and beta(1) -abundance were higher in the MC dexamethasone than placebo group, but did not change in the SC dexamethasone group. We conclude MC, but not a SC of dexamethasone, affect fetal cerebral and renal Na(+),K(+)-ATPase, and MC result in differential effects on Na(+),K(+)-ATPase in these organs.

Ontogeny of connexin 32 and 43 expression in the cerebral cortices of ovine fetuses, newborns, and adults.

Gap junctions are specialized membrane structures that mediate intercellular communication and facilitate passage of ions and small molecules between adjacent cells. Connexins comprise a multigene family of transmembrane proteins that form gap junctions. Connexin-32 and connexin-43 are among the most abundant connexins in brain and are highly expressed during development. Connexin-32 is expressed primarily in oligodendrocytes and connexin-43 in astrocytes in adult brain. However, both connexins are expressed in neurons during development. We examined the effects of ontogeny on connexin-32 and connexin-43 protein abundance in cerebral cortices of sheep during development. Western immunoblot was used to measure connexin-32 and connexin-43 expression in cerebral cortices of fetuses at 60%, 80%, and 90% of gestation, in newborn lambs and adult sheep. Values were expressed as ratios to a single adult control cerebral cortical sample. Connexin-32 abundance was higher (P<0.05) in cerebral cortices of fetuses at 60% of gestation (3.0+/-0.68, mean+/-SD), than in those at 90% of gestation (1.7+/-0.3), in newborn (1.8+/-0.55), and adult sheep (0.84+/-0.19), respectively. In contrast, connexin-43 abundance was higher (P<0.05) in cerebral cortices of fetuses at 90% of gestation (0.44+/-0.17), newborn (0.69+/-0.12) and adult sheep (1.14+/-0.13), than in those at 60% of gestation (0.05+/-0.01). We conclude that (1) connexin-32 and connexin-43 protein are expressed early in fetal life and throughout development, (2) each connexin displays a unique pattern of change with development, (3) connexin-43 exhibited ontogenic increases in protein abundance, whereas, connexin-32 exhibited reciprocal decreases in abundance late in fetal development, in newborn and adult sheep.

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.

Effects of maternal treatment with corticosteroids on tight junction protein expression in the cerebral cortex of the ovine fetus with and without exposure to in utero brain ischemia.

Maternal treatment with corticosteroids reduces blood-brain barrier permeability in premature ovine fetuses and the incidence of intraventricular hemorrhage in premature infants. We tested the hypothesis that maternally administered corticosteroids increase the expression of tight junction (TJ) proteins in the cerebral cortex of ovine fetuses with and without exposure to in utero brain ischemia. Fetuses at 80% of gestation were studied 18 h after the last of four 4-6 mg dexamethasone or placebo injections were given over 48 h to ewes. Groups were placebo/control, dexamethasone/control, placebo/ischemic, and dexamethasone/ischemic. Ischemia consisted of 30 min of fetal carotid artery occlusion and 72 h of reperfusion. Cerebral cortex was snap frozen. Western immunoblot was used to measure the protein expression of occludin, claudin-1, claudin-5, zonula occludens (ZO)-1, and ZO-2, and a TJ accessory protein annexin-ll. Occludin and annexin-ll protein expression were 48% and 58% higher (P<0.05) in the dexamethasone/ischemic than placebo/control group, respectively. Claudin-5 protein expression was 69% and 73% higher (P<0.05) in the placebo/ischemic and dexamethasone/ischemic than placebo/control group. Claudin-1 expression did not differ among groups. ZO-1 protein expression was 25%, 40%, and 55% lower in the dexamethasone/control, placebo/ischemic, and dexamethasone/ischemic than placebo/control group, respectively. ZO-2 expression was 45% and 70% lower (P<0.01) in the placebo/ischemic and dexamethasone/ischemic than placebo/control group. We conclude that maternal corticosteroid treatment differentially regulates the expression of component proteins of TJs in the cerebral cortex of fetuses exposed to brain ischemia. The functional significance of this differential regulation warrants further investigation.

Effects of postnatal steroids on Na+/K+-ATPase activity and alpha1- and beta1-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.