The early fetal brain is susceptible to teratogens in the maternal circulation, because brain mic... more The early fetal brain is susceptible to teratogens in the maternal circulation, because brain microvessel expression of drug efflux transporter, P-glycoprotein (P-gp), is very low. However, there is a dramatic up-regulation of brain microvessel P-gp in late gestation. This study investigated the role of cortisol and dexamethasone in this up-regulation of fetal brain microvessel P-gp expression. Primary brain endothelial cell (BEC) cultures derived from gestational d (GD)40, GD50, GD65 (term, ∼68 d) and postnatal d 14 male guinea pigs were treated with varying doses (10(-8) to 10(-5) m) of cortisol, dexamethasone, and aldosterone. After treatment, P-gp function was assessed using calcein-acetoxymethyl ester (P-gp substrate; 1 μm for 1 h) and measuring BEC accumulation of calcein. Corticosteroid treatment of BECs derived from postnatal d 14 resulted in increased P-gp activity. BECs derived from GD65 (near term) responded similarly, but these cells were extremely sensitive to the effects of mineralocorticoid receptor agonists (cortisol and aldosterone). BECs derived from GD50 displayed dose-dependent increases in P-gp function with dexamethasone (P < 0.05) and a trend towards increased function with cortisol. Cells derived from GD40 were unresponsive to all treatments. In conclusion, P-gp function in BECs is more responsive to glucocorticoids (GCs) in late gestation. Therefore, the late gestational surge in fetal plasma GCs, which parallels the increase in brain microvessel P-gp expression, may contribute to this P-gp up-regulation. Further, synthetic GCs (administered to pregnant women at risk of preterm delivery) may increase the protective capacity of the developing fetal blood-brain barrier, depending on the timing of GC exposure.
Neonates have been shown to have a heightened sensitivity to the central depressive effects of op... more Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain. Postmortem cortex samples from gestational age (GA) 20- 26 weeks, GA 36-40 weeks, postnatal age (PNA) 0-3 months, PNA 3-6 months, and adults were immunostained for P-gp. The intensity of P-gp staining in adults was significantly higher compared to at GA 20-26 weeks (p<0.05), GA 36-40 weeks (p<0.05), and PNA 0-3 months (p<0.05). P-gp intensity at GA 20-26 weeks (p<0.05), GA 36-40 weeks (p<0.05), and PNA 0-3 months (p<0.05) was significantly lower compared to at PNA 3-6 months. P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches adult levels at approximately 3-6 months of age. Given the immaturity of BBB P-gp after birth, morphine may concentrate in the brain. This provides mechanistic support to life threatening opioid toxicity seen with maternal codeine use during breastfeeding.Pediatric Research (2015); doi:10.1038/pr.2015.119.
Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucoco... more Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucocorticoids (sGC) to promote fetal lung development. Studies have indicated that prenatal sGC therapy modifies hypothalamic-pituitary-adrenal (HPA) function in first-generation (F 1 ) offspring. The objective of this study was to determine whether differences in HPA function and behavior are evident in the subsequent (F 2 ) generation. Pregnant guinea pigs (F 0 ) received betamethasone (BETA; 1 mg/kg) or saline on gestational d 40/41, 50/51, and 60/61. F 1 females were mated with control males to create F 2 offspring. HPA function was assessed in juvenile and adult F 2 offspring. Locomotor activity was assessed in juvenile offspring. Analysis of HPA-related gene expression was undertaken in adult hippocampi, hypothalami, and pituitaries. Locomotor activity was reduced in F 2 BETA males (P Ͻ 0.05). F 2 BETA offspring displayed blunted cortisol response to swim stress (P Ͻ 0.05). After dexamethasone challenge, F 2 BETA males and females displayed increased and decreased negative feedback, respectively. F 2 BETA females had reduced pituitary levels of proopiomelanocortin (and adrenocorticotropic hormone), and corticotropin-releasing hormone receptor mRNA and protein (P Ͻ 0.05). F 2 BETA males displayed increased hippocampal glucocorticoid receptor (P Ͻ 0.001), whereas in BETA females, hippocampal glucocorticoid receptor and mineralocorticoid receptor mRNA were decreased (P Ͻ 0.05). In conclusion, prenatal BETA treatment affects HPA function and behavior in F 2 offspring. In F 2 BETA females, pituitary function appears to be primarily affected, whereas hippocampal glucocorticoid feedback systems appear altered in both F 2 BETA males and females. These data have clinical implication given the widespread use of repeat course glucocorticoid therapy in the management of preterm labour. (Endocrinology 153: 3295-3307, 2012)
Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. Th... more Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. This protection declines with advancing gestation leaving the fetus and fetal brain vulnerable to these compounds and potential teratogens in maternal circulation. This vulnerability may be more pronounced in pregnancies complicated by infection, which is common during pregnancy. Pro-inflammatory cytokines (released during infection) have been shown to be potent inhibitors of P-gp, but nothing is known regarding their effects at the developing blood-brain barrier (BBB). We hypothesized that P-gp function and expression in endothelial cells of the developing BBB will be inhibited by proinflammatory cytokines. We have derived brain endothelial cell (BEC) cultures from various stages of development of the guinea pig: gestational day (GD) 50, 65 (term ,68 days) and postnatal day (PND) 14. Once these cultures reached confluence, BECs were treated with various doses (10 0 -10 4 pg/mL) of pro-inflammatory cytokines: interleukin-1b (IL-1b), interleukin-6 (IL-6) or tumor necrosis factor-a (TNF-a). P-gp function or abcb1 mRNA (encodes P-gp) expression was assessed following treatment. Incubation of GD50 BECs with IL-1b, IL-6 or TNF-a resulted in no change in P-gp function. GD65 BECs displayed a dose-dependent decrease in function with all cytokines tested; maximal effects at 42%, 65% and 34% with IL-1b, IL-6 and TNF-a treatment, respectively (P,0.01). Inhibition of P-gp function by IL-1b, IL-6 and TNF-a was even greater in PND14 BECs; maximal effects at 36% (P,0.01), 84% (P,0.05) and 55% (P,0.01), respectively. Cytokineinduced reductions in P-gp function were associated with decreased abcb1 mRNA expression. These data suggest that BBB P-gp function is increasingly responsive to the inhibitory effects of pro-inflammatory cytokines, with increasing developmental age. Thus, women who experience infection and take prescription medication during pregnancy may expose the developing fetal brain to greater amounts of exogenous compounds -many of which are considered potentially teratogenic. Citation: Iqbal M, Ho HL, Petropoulos S, Moisiadis VG, Gibb W, et al. (2012) Pro-Inflammatory Cytokine Regulation of P-glycoprotein in the Developing Blood-Brain Barrier. PLoS ONE 7(8): e43022.
The early fetal brain is susceptible to teratogens in the maternal circulation, because brain mic... more The early fetal brain is susceptible to teratogens in the maternal circulation, because brain microvessel expression of drug efflux transporter, P-glycoprotein (P-gp), is very low. However, there is a dramatic up-regulation of brain microvessel P-gp in late gestation. This study investigated the role of cortisol and dexamethasone in this up-regulation of fetal brain microvessel P-gp expression. Primary brain endothelial cell (BEC) cultures derived from gestational d (GD)40, GD50, GD65 (term, ∼68 d) and postnatal d 14 male guinea pigs were treated with varying doses (10(-8) to 10(-5) m) of cortisol, dexamethasone, and aldosterone. After treatment, P-gp function was assessed using calcein-acetoxymethyl ester (P-gp substrate; 1 μm for 1 h) and measuring BEC accumulation of calcein. Corticosteroid treatment of BECs derived from postnatal d 14 resulted in increased P-gp activity. BECs derived from GD65 (near term) responded similarly, but these cells were extremely sensitive to the effects of mineralocorticoid receptor agonists (cortisol and aldosterone). BECs derived from GD50 displayed dose-dependent increases in P-gp function with dexamethasone (P < 0.05) and a trend towards increased function with cortisol. Cells derived from GD40 were unresponsive to all treatments. In conclusion, P-gp function in BECs is more responsive to glucocorticoids (GCs) in late gestation. Therefore, the late gestational surge in fetal plasma GCs, which parallels the increase in brain microvessel P-gp expression, may contribute to this P-gp up-regulation. Further, synthetic GCs (administered to pregnant women at risk of preterm delivery) may increase the protective capacity of the developing fetal blood-brain barrier, depending on the timing of GC exposure.
Neonates have been shown to have a heightened sensitivity to the central depressive effects of op... more Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain. Postmortem cortex samples from gestational age (GA) 20- 26 weeks, GA 36-40 weeks, postnatal age (PNA) 0-3 months, PNA 3-6 months, and adults were immunostained for P-gp. The intensity of P-gp staining in adults was significantly higher compared to at GA 20-26 weeks (p<0.05), GA 36-40 weeks (p<0.05), and PNA 0-3 months (p<0.05). P-gp intensity at GA 20-26 weeks (p<0.05), GA 36-40 weeks (p<0.05), and PNA 0-3 months (p<0.05) was significantly lower compared to at PNA 3-6 months. P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches adult levels at approximately 3-6 months of age. Given the immaturity of BBB P-gp after birth, morphine may concentrate in the brain. This provides mechanistic support to life threatening opioid toxicity seen with maternal codeine use during breastfeeding.Pediatric Research (2015); doi:10.1038/pr.2015.119.
Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucoco... more Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucocorticoids (sGC) to promote fetal lung development. Studies have indicated that prenatal sGC therapy modifies hypothalamic-pituitary-adrenal (HPA) function in first-generation (F 1 ) offspring. The objective of this study was to determine whether differences in HPA function and behavior are evident in the subsequent (F 2 ) generation. Pregnant guinea pigs (F 0 ) received betamethasone (BETA; 1 mg/kg) or saline on gestational d 40/41, 50/51, and 60/61. F 1 females were mated with control males to create F 2 offspring. HPA function was assessed in juvenile and adult F 2 offspring. Locomotor activity was assessed in juvenile offspring. Analysis of HPA-related gene expression was undertaken in adult hippocampi, hypothalami, and pituitaries. Locomotor activity was reduced in F 2 BETA males (P Ͻ 0.05). F 2 BETA offspring displayed blunted cortisol response to swim stress (P Ͻ 0.05). After dexamethasone challenge, F 2 BETA males and females displayed increased and decreased negative feedback, respectively. F 2 BETA females had reduced pituitary levels of proopiomelanocortin (and adrenocorticotropic hormone), and corticotropin-releasing hormone receptor mRNA and protein (P Ͻ 0.05). F 2 BETA males displayed increased hippocampal glucocorticoid receptor (P Ͻ 0.001), whereas in BETA females, hippocampal glucocorticoid receptor and mineralocorticoid receptor mRNA were decreased (P Ͻ 0.05). In conclusion, prenatal BETA treatment affects HPA function and behavior in F 2 offspring. In F 2 BETA females, pituitary function appears to be primarily affected, whereas hippocampal glucocorticoid feedback systems appear altered in both F 2 BETA males and females. These data have clinical implication given the widespread use of repeat course glucocorticoid therapy in the management of preterm labour. (Endocrinology 153: 3295-3307, 2012)
Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. Th... more Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. This protection declines with advancing gestation leaving the fetus and fetal brain vulnerable to these compounds and potential teratogens in maternal circulation. This vulnerability may be more pronounced in pregnancies complicated by infection, which is common during pregnancy. Pro-inflammatory cytokines (released during infection) have been shown to be potent inhibitors of P-gp, but nothing is known regarding their effects at the developing blood-brain barrier (BBB). We hypothesized that P-gp function and expression in endothelial cells of the developing BBB will be inhibited by proinflammatory cytokines. We have derived brain endothelial cell (BEC) cultures from various stages of development of the guinea pig: gestational day (GD) 50, 65 (term ,68 days) and postnatal day (PND) 14. Once these cultures reached confluence, BECs were treated with various doses (10 0 -10 4 pg/mL) of pro-inflammatory cytokines: interleukin-1b (IL-1b), interleukin-6 (IL-6) or tumor necrosis factor-a (TNF-a). P-gp function or abcb1 mRNA (encodes P-gp) expression was assessed following treatment. Incubation of GD50 BECs with IL-1b, IL-6 or TNF-a resulted in no change in P-gp function. GD65 BECs displayed a dose-dependent decrease in function with all cytokines tested; maximal effects at 42%, 65% and 34% with IL-1b, IL-6 and TNF-a treatment, respectively (P,0.01). Inhibition of P-gp function by IL-1b, IL-6 and TNF-a was even greater in PND14 BECs; maximal effects at 36% (P,0.01), 84% (P,0.05) and 55% (P,0.01), respectively. Cytokineinduced reductions in P-gp function were associated with decreased abcb1 mRNA expression. These data suggest that BBB P-gp function is increasingly responsive to the inhibitory effects of pro-inflammatory cytokines, with increasing developmental age. Thus, women who experience infection and take prescription medication during pregnancy may expose the developing fetal brain to greater amounts of exogenous compounds -many of which are considered potentially teratogenic. Citation: Iqbal M, Ho HL, Petropoulos S, Moisiadis VG, Gibb W, et al. (2012) Pro-Inflammatory Cytokine Regulation of P-glycoprotein in the Developing Blood-Brain Barrier. PLoS ONE 7(8): e43022.
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