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.

In-vitro Validation of Cytokine Neutralizing Antibodies by Testing with Ovine Mononuclear Splenocytes

“…In the present study, we further documented that specific anti-IL-1β and anti-IL-6 mAbs neutralize the stimulatory effects of ovine IL-1β and IL-6 proteins in isolated splenic mononuclear cells. It is well known that IL-1β and IL-6 mediate their actions mainly by binding to their receptors and increasing expression of the NF-κβ and STAT-3 transcription factors through the TNF and Janus kinase (JAK)/STAT signalling pathways, respectively (Kitamura et al , 2005; Albrecht et al , 2007; Carey et al , 2008; Duncan et al , 2009). Compared with control experiments using the non-specific mouse anti-sheep IgG1, treatment with specific IL-1β antibody attenuated the increased NF-κβ protein expression after exposure to ovine IL-1β protein, which supports the contention that IL-1β mAb is a neutralizing antibody.…”

Ontogeny of tight junction protein expression in the ovine cerebral cortex during development

“…We also previously reported differential developmental regulation of occludin, claudin-1 and -5 and zonula occludens (ZO)-1 and ZO-2 in sheep cerebral cortex (Duncan et al, 2009). …”

“…Glucocorticoids have been shown to have an important role in modulating blood-brain barrier function during development and affecting the expression of some tight junction proteins during normal and pathological conditions (Stonestreet et al, 2000, Stonestreet et al, 2003, Malaeb et al, 2007, Duncan et al, 2009). …”

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

“…Our laboratory has previously described the use of the IC sample as a reference standard for immunoblots to ensure consistent quality of loading and transferring, and to control for variability across gels [4,[34][35][36][37]. We employed this method in our laboratory because our initial studies showed that most of the traditional housekeeping protein standards are affected by development and ischemic injury in sheep, thereby necessitating an alternative normalization method.…”

“…Experimental densitometry values for gelatinolytic activity in each study group were normalized to the average of 3 adult cerebral cortical samples. The densitometry readings of the protein expression were normalized to the average protein expression of the 3 IC values on each immunoblot, as previously described [4,34,35,40]. The final values represent an average of the IOD values obtained from at least 2 different immunoblots and are shown as a ratio to IC.…”