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

Abstract

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.

Figure 1

Occludin protein expression. Panel A shows a representative Western immunoblot of occludin protein expression in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation of the placebo (P) and dexamethasone (D) treated ewes and of the placebo and dexamethasone treated newborn and adult sheep. Panel B illustrates the occludin protein expression plotted as a ratio of the experimental densitometry values to the internal control values in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn and adult sheep. Open bars represent fetuses of the placebo ewes and closed bars the fetuses of the dexamethasone treated ewes. Bar designations are identical for the newborn and adult sheep.

Figure 2

Claudin-1 and claudin-5 expression. Panel A shows a representative Western immunoblot of claudin-1 protein expression in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation of the placebo and dexamethasone treated ewes and of the placebo and dexamethasone treated newborn and adult sheep. Designation legends as for figure 1. The bar graph illustrates the claudin-1 protein expression plotted as described for occludin in figure 1 in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn, and adult sheep. Bar legends as for figure 1. ^a P<0.05 versus fetuses of the placebo treated ewes at 60% of gestation; ^b P<0.05 versus fetuses of the placebo treated ewes at 70% of gestation; ^c P<0.05 versus fetuses of the placebo treated ewes at 90% of gestation. Panel B shows a representative Western immunoblot of claudin-5 protein expression in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation of the placebo and dexamethasone treated ewes and of the placebo and dexamethasone treated newborn and adult sheep. Designation legends as for figure 1. Bar graph illustrates the claudin-5 protein plotted as described for occludin in figure 1. Bar legends as for figure 1. ^a P<0.05 versus fetuses of the placebo treated ewes at 60% of gestation; ^b P<0.05 versus fetuses of the placebo treated ewes at 90% of gestation; ^c P<0.05 versus fetuses of the placebo treated ewes at 70% of gestation; ^d P<0.05 versus placebo treated newborn lambs.

Figure 3

ZO-1 protein expression. Panel A shows a representative Western immunoblot of ZO-1 protein expression in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation of the placebo and dexamethasone treated ewes and of the placebo and dexamethasone treated newborn and adult sheep. Designation legends as for figure 1. Note that two bands were apparent in some but not all of the age groups. Panel B illustrates the ZO-1 protein expression plotted as for occludin in figure 1 in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn and adult sheep. Bar designations as for figure 1. ^a P<0.05 versus fetuses of the placebo treated ewes at 60% of gestation; ^b P<0.05 versus fetuses of the placebo treated ewes at 70% of gestation; ^c P<0.05 versus fetuses of the placebo treated ewes at 90% of gestation; ^d P<0.05 versus placebo treated newborn lambs.

Figure 4

ZO-2 protein expression. Panel A shows a representative Western immunoblot of ZO-2 protein expression in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation of the placebo and dexamethasone treated ewes and of the placebo and dexamethasone treated newborn and adult sheep. Designation legends as for figure 1. Note three bands of varying expression intensity are present in all age groups. Panel B illustrates the ZO-2 protein expression of the upper band plotted as described for occludin in figure 1 in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn, and adult sheep. Bar legends as for figure 1. Panel C illustrates the ZO-2 protein expression of the middle band plotted as described for occludin in figure 1 in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn, and adult sheep. Bar legends as for figure 1. ^a P<0.05 versus fetuses of the placebo treated ewes at 70% of gestation; ^b P<0.05 versus fetuses of the placebo treated ewes at 60% of gestation; ^c P<0.05 versus fetuses of the placebo treated ewes at 90% of gestation. Panel D illustrates the ZO-2 protein expression of the lower band plotted as described for occludin in figure 1 in the cerebral cortex of the fetuses at 60%, 70%, and 90% of gestation, newborn and adult sheep. Bar legends as for figure 1. ^a P<0.05 versus fetuses of the placebo treated ewes at 60% of gestation; ^b P<0.05 versus fetuses of the placebo treated ewes at 70% of gestation; ^c P<0.05 versus placebo treated newborn lambs; ^d P<0.05 versus fetuses of the placebo treated ewes at 90% of gestation.

Figure 5

Panel A shows ZO-1 protein expression in the cerebral cortex of the fetuses of the placebo treated ewes at 60%, 70%, and 90% of gestation plotted against gestational age and plasma cortisol concentrations. Panel B shows ZO-2 protein expression in the cerebral cortex of the fetuses of the placebo treated ewes at 60%, 70%, and 90% of gestation plotted against gestational age and plasma cortisol concentrations. A composite value for the three ZO-2 bands was used for the correlational analyses.