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1 Department of Pediatrics, Women and Infants Hospital of Rhode Island, Brown University School of Medicine, Providence, Rhode Island 02905; and 2 Department of Surgery, State University of New York at Stony Brook, Stony Brook, New York 11794-8191
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We previously reported
decreases in blood-brain barrier permeability in the ovine fetus at
80% of gestation after antenatal corticosteroids and shown that
permeability is not reduced in newborn lambs after postnatal
corticosteroids. We now test the hypotheses that exogenous antenatal
corticosteroids decrease blood-brain barrier permeability at 60% but
not 90% of gestation in ovine fetuses and that endogenous increases in
plasma cortisol concentrations are associated with ontogenic decreases
in barrier permeability during gestation. Chronically instrumented
ovine fetuses were studied 12 h after the last of four 6-mg
dexamethasone or placebo injections were given 12 h apart over
48 h to ewes. Fetuses at 80% of gestation from placebo-treated
ewes studied under the same protocol were also included. Blood-brain
barrier function was quantified with the blood-to-brain transfer
constant (Ki) to 
-aminoisobutyric acid.
Ki values were lower in cerebral cortex, caudate
nucleus, hippocampus, superior colliculus, thalamus, medulla, and
cervical spinal cord in fetuses of dexamethasone- than placebo-treated ewes at 60% but not 90% of gestation. Regional brain
Ki values demonstrated inverse correlations with
increases in gestation and plasma cortisol concentrations in most brain
regions. We conclude that maternal treatment with exogenous
corticosteroids was associated with decreases in blood-brain barrier
permeability at 60% but not 90% of gestation and that increases in
gestation and endogenous cortisol concentrations were associated with
ontogenic decreases in barrier permeability during fetal development.
alpha-aminoisobutyric acid; brain; cortisol; gestation; maturation; sheep; steroids
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MATERNALLY ADMINISTERED ANTENATAL corticosteroids have been used widely to reduce the incidence of respiratory distress syndrome in low birth weight infants (9). This therapy also has been shown to facilitate the transition from fetal to neonatal life by beneficial effects on other organ systems (1, 19, 26, 27, 31). Antenatal steroid administration has been reported to have an important role in lowering the risk of early onset and severe intraventricular hemorrhage in premature infants (13, 21, 30). These effects might be explained in part by accelerated vascular maturation.
The blood-brain barrier is composed of a continuous layer of cerebrovascular endothelial cells connected by intercellular tight junctions (2, 4, 6, 15). This specialized barrier serves as an interface among the circulating blood, brain interstitium, and parenchyma, isolating brain tissue from blood constituents. Therefore, the blood-brain barrier maintains central nervous system (CNS) homeostasis by preventing entry of substances that might alter neuronal function in the CNS.
The development of the blood-brain barrier is an important component of
brain maturation. The blood-brain barrier is a selectively permeable
barrier, which may potentially change during development. We previously
have demonstrated ontogenic decreases in blood-brain barrier
permeability to -aminoisobutyric acid (AIB) from 60% of fetal
gestation through the neonatal period up to maturity in adult sheep
(33). We also have shown that the blood-brain barrier is
relatively impermeable to AIB in most brain regions of fetuses and
lambs (33). Because the blood-brain barrier is relatively
impermeable in the fetus and newborn, the barrier also protects the
developing brain from factors that could impair neuronal function.
Evidence in adult subjects suggests that the blood-brain barrier is under hormonal control (16, 20, 24, 40). In adult rats adrenalectomy increases blood-brain barrier permeability, and corticosterone replacement reverses this effect on the barrier (20). These findings suggest that the pituitary-adrenal cortical axis may function as a physiological regulator of barrier function (20). In addition, in adult rodents, pharmacological doses of dexamethasone have been reported to reduce barrier permeability (16, 28, 40).
In the prenatal and perinatal periods, damage to the blood-brain barrier might potentially result from hypoxic-ischemic brain injury and/or intraventricular hemorrhage. In addition, in this setting blood-brain barrier damage might potentially render premature infants with hyperbilirubinemia more susceptible to brain injury. Consequently, we examined the effects of maternal treatment with antenatal steroids on blood-brain barrier function in the fetus (34). Consistent with findings in adult rodents, we recently have reported decreases in blood-brain barrier permeability in the ovine fetus at 80% of gestation after antenatal corticosteroids had been administered to pregnant ewes (34). In contrast, we have shown that postnatal corticosteroids do not reduce blood-brain barrier permeability in newborn lambs (35). It remains to be determined whether the decreases in blood-brain barrier permeability after maternal treatment with antenatal corticosteroids are present throughout fetal development or whether there is a time before birth when the barrier is no longer responsive to this maternal treatment. It is well known that the pituitary-adrenal cortical axis matures during fetal development and that endogenous cortisol concentrations increase particularly in the latter part of gestation (39). Based on the findings in adult rodents suggesting that the pituitary-adrenal cortical axis may function as a physiological regulator of the blood-brain barrier, it is also likely that developmental changes in this axis might affect barrier function in the fetus.
Given the above considerations, there is evidence to suggest that maternally administered exogenous corticosteroids might exert age-related differential effects on the developing barrier and that the pituitary-adrenal cortical axis may function as a physiological regulator of barrier function in the fetus. Therefore, in the present study, we tested the hypotheses that 1) antenatal corticosteroids decrease blood-brain barrier permeability in early and not in late gestation ovine fetuses and 2) increases in endogenous plasma cortisol concentrations are associated with developmental decreases in blood-brain barrier permeability in the fetus. To test these hypotheses, we examined the effects of maternal antenatal corticosteroid treatment on blood-brain barrier permeability early (60%) and late (90% of gestation) in ovine fetal development. We also examined the association between increases in endogenous plasma cortisol concentrations and decreases in barrier permeability during fetal development.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was conducted after approval by the Institutional Animal Care and Use Committees of Brown University and Women and Infants Hospital of Rhode Island and according to the Guide For the Care and Use of Laboratory Animals, National Institutes of Health.
Animal preparation. As previously described in detail (32, 33), surgery was done under 1-2% halothane anesthesia on 19 mixed breed ewes at 82-83 days, 6 at 112-113 days, and 17 at 128-129 days of gestation. Singleton and twin pregnancies were included. When a twin gestation was present, the surgery and study were performed on one fetus. Briefly, in the fetuses, polyvinyl catheters were placed into a brachial vein for isotope administration and into the thoracic aorta via the brachial artery for blood sample withdrawal and heart rate and blood pressure monitoring. The surgery was modified slightly in the fetuses at 60% of gestation. In this group, catheters were placed in the subclavian vein and artery and advanced to the thoracic aorta (33). An amniotic fluid catheter was placed for pressure monitoring and to correct fetal arterial blood pressures. A femoral artery catheter was also placed in the ewes.
The six fetuses operated on at 112-113 days of gestation were part of a previous report (34). They have been included here to examine the association between increases in endogenous corticosteroids and barrier development in the fetus (see Figs. 3-5). This was done to limit unnecessary animal usage and was justified because those studies were performed within the same time frame as the ones in the current report using identical methods and study design. After 2-6 days of recovery from surgery, the ewes were randomly assigned to receive either four 6-mg doses of dexamethasone (4 mg/ml; Fujsawa, Deerfield, IL) or placebo (0.9% NaCl) given as intramuscular injections every 12 h for 48 h. The final injection was given 12 h before the onset of the studies. The percent of gestation and treatment groups, number of animals in each group, and duration of recovery from surgery to dexamethasone or placebo treatment are summarized in Table 1.
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Experimental protocol and methodology.
Four to eight days after recovery from surgery at 87-90 days or
60%, 117-120 days or 80%, 135-137 days or 90% of
gestation, the studies were performed. The fetuses were studied while
the ewes were standing quietly in a cart after being acclimatized to
the laboratory for 2 h. Blood-brain barrier function was measured in the fetuses with [14C]AIB (Dupont-NEN, Boston, MA).
The blood-to-brain transfer constant (Ki) was
measured as previously described (10, 25,
33, 34). After baseline physiological
determinations were obtained, [14C]AIB was rapidly
injected intravenously and arterial plasma concentrations were obtained
at fixed times before and after injection as follows: 
1, 0.5, 1, 2, 3, 5, 7, 15, 30, 45, 60 min and at termination within 8-10 min
after the end of the study. On the basis of our previous analysis of
rate constants and exposure times for tracers in adult rats
(10) and mathematical analysis of AIB in fetal sheep, the
~60-min interval and this sampling regimen were determined to
accurately characterize the plasma profile needed for calculation of
the blood-to-brain Ki (33,
34). Brain parenchymal tracer concentration was determined
at the end of the experiment. Knowledge of the plasma concentration
profile and the concentration of tracer in the parenchyma allows
calculation of the blood-to-brain Ki as
described by Ohno et al. (25). In our experiments, the
unidirectional Ki was quantified for
[14C]AIB in the fetal sheep. Brain vascular volume was
determined by giving [14C]polyethylene glycol (PEG;
Amersham, UK) 2 min before the end of the experiment to fetuses of
separate dexamethasone- and placebo-treated ewes at each gestational
age examined. The brain vascular volume values did not differ between
the fetuses of dexamethasone- and placebo-treated ewes and were similar
to our previous values (33).
1 · min1) is given by
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Vpcp, where Vp is the plasma volume
of brain tissue (µl/g) and cp is the concentration of
tracer in the terminal plasma sample (dpm/g). Vp = AmH/cpH, where AmH and
cpH have the same definitions as
Am and cp above except that they apply to
[14C]PEG (10).
Arterial pH, blood gases, hematocrit, heart rate, and mean arterial
blood pressure were measured on the fetuses and ewes at baseline, 30 min, and 50 min of study. Arterial plasma osmolality, glucose, insulin,
and cortisol concentrations were measured on the fetuses and ewes
before the end of the study. Blood removed for study sampling was not
replaced because the maximum amount of blood withdrawn for any study
was <6% of the fetal blood volume in the three age groups. Heart
rates, mean arterial blood, and amniotic fluid pressures in fetal
sheep, and heart rates and mean arterial blood pressures in the ewes
were measured with pressure transducers (model 1280 C; Hewlett Packard,
Lexington, MA) and recorded on a polygraph (model 17758 B series,
Hewlett Packard). Blood gases and pH were measured on a Corning blood
gas analyzer (model 238; Corning Scientific, Medford, MA) at 39.5°C
in fetuses and 38.5°C in ewes. Hematocrit was measured in duplicate
by the microhematocrit method. Plasma osmolality was measured in
duplicate on a vapor pressure osmometer (Vapro model 5520; Wescor,
Logan, UT), glucose on a glucose analyzer (YSI 2300; STAT, Yellow
Springs, OH). Insulin concentrations were measured in duplicate using
the Coat-A-Count Insulin, a solid phase
125I-radioimmunoassay (DPC, Los Angelas, CA). The
Coat-A-Count Insulin antiserum exhibits 100% cross-reactivity with
insulin. The observed coefficient of variation for inter- and
intra-assay precision were 6.1 and 6.3%, respectively. Cortisol
concentrations were measured in duplicate using Clinical Assays
GammaCoat Cortisol 125I-radioimmunoassay (DiaSorin,
Stillwater, MN). The GammaCoat antiserum exhibits 100%
cross-reactivity with cortisol. The observed coefficient of variation
for inter- and intra-assay precision were 10.1 and 7.9%, respectively.
Statistical analysis.
All results were expressed as means ± SD. Repeated measures ANOVA
for three factors was used to compare regional brain permeability among
the fetuses of the dexamethasone- and placebo-treated ewes at 60 and
90% of gestation. The three factors were 1) gestational age, 2) dexamethasone or placebo treatment, and
3) brain regions. A repeated measures ANOVA was applied to
the brain regions because multiple regions were examined within the
same brain. To further describe and enhance the statistically
significant treatment by regions and/or group interactions, separate
ANOVAs for repeated measures were performed within the fetuses of
dexamethasone- and placebo-treated ewes at 60 and 90% of gestation. If
a significant difference was present by ANOVA, the regional
permeability of the two groups was further compared by the Newman-Keuls
post hoc test. Serial measurements of physiological variables were
compared by two-factor ANOVA for repeated measures. Two-way ANOVA was
used to compare fetal weights and physiological and hormonal variables among the fetuses of the dexamethasone- and placebo-treated ewes and
the ewes at 60 and 90% of gestation with treatment and age as factors (Tables 2 and
3). If a significant difference
was found by ANOVA, the Newman-Keuls post hoc test was used to compare the fetuses of the dexamethasone- and placebo-treated ewes and the ewes
at 60 and 90% of gestation. To reduce inter-animal variability a log
transformation was performed on the hormonal values before analysis.
2 Analysis was used to compare the number of twin
pregnancies between the fetuses of the dexamethasone- and
placebo-treated ewes and the ewes at 60 and 90% of gestation.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The fetuses of the dexamethasone- and placebo-treated ewes were
88 ± 1 and 88 ± 1 days, and 136 ± 1 and 137 ± 1 days of gestation at the time of study, respectively. The weights of
the fetuses of the dexamethasone-treated ewes did not differ from those
of the placebo-treated ewes at 60% (0.59 ± 0.11 vs. 0.63 ± 0.09 kg) or 90% (4.23 ± 0.72 vs. 3.66 ± 0.63 kg) of
gestation. In the dexamethasone-treated ewes 1 of 9 and in the
placebo-treated ewes 2 of 10 pregnancies were twins at 60% of
gestation [2, not significant (ns)]. In the
dexamethasone-treated ewes 4 of 9 and in the placebo-treated ewes 5 of
8 pregnancies were twins at 90% of gestation (2, ns).
The numbers of twin pregnancies also did not differ between the ewes at
60 and 90% of gestation (2, ns). None of the treated
ewes developed premature labor.
Arterial pH, oxygen tension, carbon dioxide tension, base excess, hematocrit, plasma osmolality, and cortisol concentrations did not differ between the fetuses of dexamethasone- and placebo-treated ewes at 60 or 90% of gestation (Table 2). Heart rate was higher in the fetuses of dexamethasone- than the placebo-treated ewes at 90% but not 60% of gestation. Mean arterial blood pressure was higher in the fetuses of dexamethasone- than the placebo-treated ewes at 60% but not 90% of gestation. Arterial plasma glucose and insulin concentrations were higher in the fetuses of the dexamethasone- than the placebo-treated ewes at 60 and 90% of gestation. Arterial pH oxygen tension, heart rate, and plasma glucose and insulin concentrations were higher, and hematocrit, mean arterial blood pressure, and plasma cortisol concentration were lower in the fetuses of the placebo-treated ewes at 60 than at 90% of gestation (Table 2).
Arterial oxygen tension, carbon dioxide tension, hematocrit, heart rate, mean arterial blood pressure, and plasma osmolality did not differ between the dexamethasone- and the placebo-treated ewes at 60 or 90% of gestation (Table 3). Whereas, the arterial pH and plasma glucose concentrations were higher and the plasma cortisol concentrations were lower in the dexamethasone- than the placebo-treated ewes at 60 and 90% of gestation. The insulin concentration was higher in the dexamethasone- than the placebo-treated ewes at 60 but not 90% of gestation. The arterial base excess was higher in the dexamethasone- than the placebo-treated ewes at 90% of gestation. Heart rate was lower, and plasma glucose and insulin concentrations were higher in the placebo-treated ewes at 60% than at 90% of gestation. The arterial pH, oxygen tension, carbon dioxide tension, base excess, hematocrit, heart rate, and mean arterial blood pressure values of fetuses and ewes did not change during the 1-h study in any group.
The regional brain Ki values for AIB in fetuses
of the dexamethasone- and placebo-treated ewes are illustrated at 60 (Fig. 1A) and 90% (Fig.
1B) of gestation. The Ki values in
the cerebral cortex, caudate nucleus, hippocampus, thalamus, superior
colliculus, medulla, and cervical spinal cord were lower at 60% of
gestation in the fetuses of the dexamethasone- than in the
placebo-treated ewes (ANOVA, main effects for dexamethasone treatment:
F = 6.17, P < 0.025, Fig.
1A). The magnitude of the decreases in permeability was not
uniform across the brain regions. In contrast, the
Ki values did not differ in the brain regions at
90% of gestation between the fetuses of the dexamethasone- and the
placebo-treated ewes (ANOVA, main effects for dexamethasone treatment:
F = 0.73, P = 0.41, Fig.
1B). The Ki values were also lower in
the parietal cortex at 60% of gestation in the fetuses of the
dexamethasone- than the placebo-treated ewes (ANOVA, main effects for
dexamethasone treatment: F = 13.88, P < 0.01, Fig. 2). The magnitude of the decreases in permeability was also not uniform across the cerebral cortical regions. In contrast, the Ki values did
not differ in the cerebral cortical regions at 90% of gestation
between the fetuses of the dexamethasone- and the placebo-treated ewes
(ANOVA, main effects for dexamethasone treatment: F = 0.41, P = 0.53).
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Endogenous plasma cortisol concentrations were (ANOVA:
F = 19.60, P < 0.01) higher at 90%
than at 60 and 80% of gestation in the fetuses of the placebo-treated
ewes (Fig. 3). The
Ki values for AIB in the fetuses of the
placebo-treated ewes demonstrated inverse relationships with both
increases in gestation and endogenous plasma cortisol concentrations in
the cerebral cortex, cerebellum, medulla, caudate nucleus, hippocampus,
thalamus, superior colliculus, inferior colliculus, pons, and cervical
spinal cord. Ki values for AIB in the fetuses of
the placebo-treated ewes are plotted against gestational age and plasma
cortisol concentrations for the cerebral cortex (Fig.
4A), cerebellum (Fig.
4B), and medulla (Fig. 4C) and for the
hippocampus (Fig. 5A),
thalamus (Fig. 5B), and cervical spinal cord (Fig.
5C). The Ki values for AIB for the
brain regions that are not illustrated demonstrated similar inverse
relationships with both increases in gestation and endogenous plasma
cortisol concentrations: caudate nucleus (gestation: r = 0.39, n = 24, P = 0.06; plasma
cortisol concentrations: r = 0.53, n = 24, P < 0.01), superior colliculus (gestation:
r = 0.56, n = 24, P < 0.01; plasma cortisol concentrations: r = 0.65, n = 24, P < 0.01), inferior colliculus
(gestation: r = 0.47, n = 24, P < 0.02; plasma cortisol concentrations:
r = 0.65, n = 24, P < 0.01), and pons (gestation: r = 0.42,
n = 24, P < 0.05; plasma cortisol
concentrations: r = 0.56, n = 24, P < 0.01).
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In Table 4 the multiple regression correlation coefficients "R" and the partial correlation coefficients are summarized for regional brain Ki values vs. gestational age and plasma cortisol concentrations. The multiple regression partial correlational analysis compared the relative strength of the association between the increases in gestation and plasma cortisol concentrations and the decreases in regional brain Ki values. This analysis demonstrated relatively equal strengths of association between the increases in gestational age and plasma cortisol concentrations and the decreases in Ki.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The novel findings of the present study are that maternally administered exogenous antenatal corticosteroids reduced blood-brain permeability early but not late in fetal development and that increases in endogenous plasma cortisol concentrations were associated with decreases in regional blood-brain barrier permeability during fetal development. The present findings combined with those in our previous report (34) can be interpreted to suggest that there is an age-related differential responsiveness of the fetal blood-brain barrier to maternally administered corticosteroids.
Dexamethasone was used because it is one of the most extensively studied corticosteroids for accelerating fetal maturation, has been used widely in studies of the CNS, and is also used to treat CNS disorders (5, 12, 16, 18, 23, 24, 40). The dose of dexamethasone was based on current recommendations for fetal maturation in pregnant women who are in premature labor (23). Although optimal effects from a complete course of antenatal corticosteroids begin 24 h after the last dose, even short-term exposure to fetal or maternal treatment has been shown to alter cardiovascular and renal function in preterm sheep (1, 27). In addition, women in premature labor often deliver less than 24 h after a complete course of corticosteroids. Therefore, the dexamethasone dose and treatment regimen in this study were similar to those used in pregnant women for fetal maturation and were selected to achieve near maximal corticosteroid effects, while minimizing the risk of premature labor (11, 19, 23).
Maternal antenatal dexamethasone treatment affected the hormonal and metabolic homeostasis of the ewes and the biochemical, metabolic, hormonal, and hemodynamic homeostasis of the fetal sheep. In the ewes, the lower plasma cortisol and higher glucose concentrations suggest that dexamethasone had suppressed the adrenocortical axis and caused glucose intolerance both at 60 and 90% of gestation (7, 34). Likewise, in the fetuses, the higher glucose and insulin concentrations also suggest glucocorticoid-induced glucose intolerance was present at both gestational ages. The dysregulation of fetal glucose homeostasis could have been a result of corticosteroid-induced insulin resistance, hepatic gluconeogenesis, and/or the elevated glucose concentrations in the ewes (37).
Antenatal corticosteroids have been shown to enhance cardiovascular stability and elevate blood pressure in premature lambs (1, 27). Continuous direct infusions of corticosteroids also increase blood pressure in fetal sheep (11, 36). The mean arterial blood pressure values in the fetuses of the dexamethasone-treated ewes were higher than those of the placebo-treated ewes at 60% but not 90% of gestation. The increase in blood pressure observed at 60% of gestation is consistent with previous reports using other antenatal and fetal corticosteroid regimens but differ from our findings at 80% of gestation in which fetal blood pressure was not elevated after the same treatment (1, 11, 27, 34). The increase in blood pressure at 60% and not 90% of gestation and the increase in heart rate at 90% and not 60% of gestation suggest that the fetal hemodynamic responses to exogenous corticosteroids are dependent on the specific hemodynamic measure, treatment regimen, and time in gestation when corticosteroids are given (1, 11, 27, 34). Moreover, at least for the dose and treatment regimen of dexamethasone in this study, age-related differential responses were observed with regard to cardiovascular effects and decreases in regional blood-brain barrier permeability (Figs. 1 and 2), but not for dysregulation of fetal glucose homeostasis (Table 2). These findings emphasize that the effects of maternally administered exogenous corticosteroids may differ depending on the fetal response evaluated and the time in gestation when they are given.
It is also important to point out that although acute hypertension has been reported to result in increases in blood-brain barrier permeability, the increase in blood pressure at 60% of gestation was relatively small and not in the range that would be expected to affect barrier permeability (14).
The findings of the present study suggest that a course of antenatal corticosteroids, similar to that which is used to treat pregnant women in premature labor, reduced blood-brain permeability early but not late in fetal development. Considered together with our previous work (34), these findings suggest that antenatal corticosteroids reduce blood-brain barrier permeability at 60 and 80% of gestation but not in near-term fetal sheep at 90% of gestation. Therefore, our results support the concept that exogenous corticosteroids enhanced cerebral microvascular maturation earlier but not later in gestation.
Although maternal antenatal corticosteroid treatment reduced blood-brain barrier permeability in the fetuses at 60% of gestation, the decreases in barrier permeability were not large. Several factors related to our dexamethasone treatment regimen may have influenced the effects of exogenous corticosteroids on barrier function in the fetus. It remains possible that the fetal dexamethasone plasma concentrations might not have been high enough or elevated for long enough to have had a maximal impact on the fetal barrier, the effect of dexamethasone administration to the ewe might differ from that of direct fetal administration, and the effect of dexamethasone on the fetal barrier is most likely mediated via glucocorticoid receptors, whereas the effects of endogenous corticosteroids might have been mediated by glucocorticoid and mineralocorticoid receptors. Nevertheless, we have confirmed that antenatal corticosteroid treatment reduced barrier permeability earlier but not later in gestation. Similar to our findings at 80% of gestation, the effects of antenatal corticosteroids were not uniform in all brain regions at 60% of gestation (34). The reasons for these differential effects on barrier function might relate to the nonuniform distribution of glucocorticoid receptors in the brain (22).
The findings in this study are important for several reasons. Our earlier work examined blood-brain barrier permeability to AIB at 80% of gestation, which was not comparable to the time in gestation when human premature infants are at the highest risk for intraventricular hemorrhage (34). The infants who are at the greatest risk for this complication of prematurity are the most immature infants at 23-25 wk of gestation, which is 57-63% of the human gestation. Although our findings in the sheep fetus cannot be directly applied to the human fetus, our results can be interpreted to suggest that maternally administered antenatal corticosteroids reduce blood-brain barrier permeability at the earliest time in gestation, when chronic catheterization of the ovine fetus is feasible (33). These findings at 60% of gestation imply that this phenomenon occurs at a very early time in gestation, which might be comparable to the time when very low birth weight infants are at the highest risk for intraventricular hemorrhage.
The findings that maternal antenatal corticosteroid treatment did not influence blood-brain barrier permeability in near-term ovine fetuses are consistent with our recent results in newborn lambs (35). Blood-brain barrier permeability was not reduced even when a dose of 0.5 mg/kg was given directly to the lambs in the same regimen as used for the ewes (35). However, it should be pointed out that pharmacological doses of dexamethasone have been reported to reduce blood-brain barrier permeability even in adult rodents (16, 28, 40). Although the differences between our findings in fetuses at 90% of gestation and in newborn lambs compared with those in adult rodents (16, 28, 40) might be related to differences in species and treatment regimen, we cannot rule out the possibility that a higher dose of dexamethasone administered directly to the late gestation fetus may have reduced blood-brain barrier permeability. Therefore, our recent work in newborn lambs (35), earlier findings in fetal sheep (34), and current results suggest that corticosteroids reduce blood-brain barrier permeability in fetuses at 60 and 80% of gestation but not in the near-term fetuses or newborn lambs. Consequently, there is a time in gestation when the blood-brain barrier vasculature is responsive to exogenous antenatal corticosteroids and a time when this responsiveness is no longer present.
The reason for this age-related differential responsiveness cannot be determined from our study. Although the concentration of glucocorticoid binding sites have been reported to remain relatively constant in the fetus and newborn (29), the brain is a heterogeneous tissue and the majority of glucocorticoid receptors are most likely present on fibroblasts, astrocytes, neurons, and blood vessels rather than endothelial cells. Therefore, we cannot rule out the possibility that changes in regional brain endothelial cell corticosteroid receptors might have occurred during fetal maturation. It is also important to point out that the studies of glucocorticoid binding sites were done in fetuses with intact adrenal glands (8, 29). Because activated glucocorticoid receptors do not rebind steroids, simple binding characteristics do not necessarily reveal anything about receptor availability or action (8). Therefore, conclusions regarding the contribution of changes in regional brain endothelial cell glucocorticoid receptors await studies that focus on the fetal maturation of these receptors specifically within brain endothelial cells.
In the present study, we have demonstrated inverse correlations between increases in endogenous cortisol concentrations and decreases in Ki values to AIB in most brain regions. These findings strongly suggest that endogenous increases in cortisol concentrations could be in part responsible for the ontogenic decreases in blood-brain barrier permeability that occur during normal fetal life (33). However, because endogenous plasma cortisol concentrations increase during gestation, we used multiple regression partial correlational analysis to examine the relative strength of the associations between the increases in gestation and plasma cortisol concentrations and the decreases in regional permeability. Results of this analysis suggested that the strengths of these associations were relatively equal.
Our findings that maternal treatment with exogenous corticosteroids reduces blood-brain barrier permeability early but not later in gestation and that ontogenic decreases in barrier permeability correlate with elevations in endogenous corticosteroids are consistent with the view that early in gestation the barrier is still responsive to exogenous corticosteroids because it has not yet been exposed to endogenous elevations in corticosteroids. Later in gestation, increases in gestational age and endogenous cortisol concentrations are associated with reductions in barrier permeability; thus the barrier is no longer responsive to exogenous corticosteroids. Therefore, the age-related differential responsiveness of the fetal blood-brain barrier to maternally administered exogenous corticosteroids is most likely related to the effects of endogenous corticosteroids on barrier maturation.
The site of action of the exogenous and endogenous corticosteroids on the blood-brain barrier in the fetus cannot be ascertained with certainty from our studies (34). However, in an in vitro model of the blood-brain barrier, physiologically relevant hydrocortisone concentrations were found to increase transendothelial resistance and decrease permeability to sucrose (17). Therefore, it is probable that the site of action of corticosteroids on the blood-brain barrier is the intercellular tight junctions of cerebrovascular endothelial cells.
In summary, maternal treatment with a corticosteroid regimen similar to that used in the clinical setting reduced blood-brain barrier permeability in the ovine fetus at 60% but not 90% of gestation. Both increases in gestation and endogenous plasma cortisol concentrations are associated with the developmental decreases in permeability.
Perspectives
Antenatal corticosteroid therapy is used widely to treat pregnant women in premature labor. The relatively low dose and treatment regimen given in this and our former study (34) were similar to those used in the clinical setting. This antenatal corticosteroid treatment regimen appears to accelerate a vital component of brain maturation in the fetus at 60 and 80% but not 90% of gestation (34). The findings of this study also suggest that at an early time in gestation, which is similar to the time when the most immature infants are at highest risk for intraventricular hemorrhage, antenatal corticosteroids reduce blood-brain barrier permeability. In addition, our findings support the more general concept that the effects of exogenous steroids may differ depending on the stage of maturation, when they are given, and the aspect of fetal development examined.| |
ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Jesse M. Allen, Christopher M. Ellit, Joshua E. Markowitz, and Aimee Till, and the expert statistical advice of Richard H. Lavoie, PhD, Professor of Mathematics, Providence College, Providence, RI.
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FOOTNOTES |
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This work was supported by the National Institute of Child Health and Human Development Grant R01-HD-34618.
Address for reprint requests and other correspondence: B. S. Stonestreet, Brown Univ. School of Medicine, Dept. of Pediatrics, Women and Infants Hospital of Rhode Island, 101 Dudley St., Providence, RI 02905 (E-mail: bstonest{at}wihri.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 1 December 1999; accepted in final form 25 February 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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