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Am J Physiol Regul Integr Comp Physiol 291: R1031-R1039, 2006. First published May 11, 2006; doi:10.1152/ajpregu.00883.2005
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Effects of acute hyperosmolality on blood-brain barrier function in ovine fetuses and lambs

Barbara S. Stonestreet,1 Grazyna B. Sadowska,1 Joanne Leeman,2 R. Choudary Hanumara,2 Katherine H. Petersson,1 and Clifford S. Patlak3

1Department of Pediatrics, Brown University Medical School, Women and Infants' Hospital of Rhode Island, Providence, Rhode Island; 2Department of Computer Science and Statistics, University of Rhode Island, Kingston, Rhode Island; and 3Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 15 December 2005 ; accepted in final form 28 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the effects of hyperosmolality on blood-brain barrier (BBB) permeability during development to test the vulnerability of the immature barrier to stress. The BBB response to hyperosmolality was quantified using the blood-to-brain transfer constant (Ki) with {alpha}-aminoisobutyric acid in fetuses at 60% and 90% gestation, premature, newborn, and older lambs. Ki plotted against osmolality increased as a function of increases in osmolality in all groups and brain regions. The relationship was described (P < 0.05) by a segmented regression model. At lower osmolalities, changes in Ki were minimal, but after a break point (threshold) was reached, the increase (P < 0.05) was linear. We examined the responses of Ki to hyperosmolality within each brain region by comparing the thresholds and slopes of the second regression segment. Lower thresholds and higher slopes imply greater vulnerability to hyperosmolality in the younger groups. Thresholds increased (P < 0.05) with development in the thalamus, superior colliculus, pons, and spinal cord, and slopes of the second regression segment decreased (P < 0.05) in the cerebellum, hippocampus, inferior colliculus, medulla, and spinal cord. BBB resistance to hyperosmolality increased (P < 0.05) with development in most brain regions. The pattern of the Ki plotted against osmolality was (P < 0.05) heterogenous among brain regions in fetuses and premature and newborn lambs, but not in older lambs. We conclude that 1) BBB permeability increased as a function of changes in osmolality, 2) the barrier becomes more resistant to hyperosmolality during development, and 3) the permeability response to hyperosmolality is heterogenous among brain regions in fetuses and premature and newborn lambs.

{alpha}-aminoisobutyric acid; brain; barrier; development; immature; lambs; mannitol; sheep

THE BLOOD-BRAIN BARRIER IS composed of a continuous layer of cerebrovascular endothelial cells connected by tight intercellular junctions (1, 4). This specialized barrier serves as an interface between the circulating blood, brain interstitium, and parenchyma, isolating brain tissue from such blood constituents as ions, hormones, and neurotransmitters. The blood-brain barrier maintains central nervous system homeostasis and prevents entry of substances that might alter neuronal function. The adult cerebral capillary endothelial barrier is characterized by tight junctions that exclude most lipid-insoluble substances from the brain (2). We have previously examined the ontogeny of blood-brain barrier function measured quantitatively with a small hydrophilic molecule {alpha}-aminoisobutyric acid (AIB) from the last 2 mo of fetal and first month of neonatal development up to 3 years of age in normal sheep (27). We have shown that, although the blood-brain barrier exhibited ontogenic decreases in permeability from 60% of gestation to maturity, the barrier is relatively impermeable to AIB in all age groups (27). We also have shown that there is heterogeneity in the barrier permeability among brain regions, which is accentuated at 60% of gestation, but also present in fetuses at 90% of gestation, lambs, and adult sheep (27).

The phenomenon of hyperosmotic opening of the blood-brain barrier has been investigated extensively in the adult (18). Hypertonic solutions appear to open the barrier by shrinking endothelial cells and widening the tight junctions. The degree of barrier opening varies both with the concentration of the solute and the type of solute used. Generally, the degree of barrier opening is increased as a function of increasing osmolality. In contrast to the high osmolalities associated with the intracarotid injection method used by Rapoport et al. (18), Cserr et al. (8) demonstrated barrier opening with moderate increases in plasma osmolality associated with systemic administration of hyperosmolar solutions. Systemic administration of solute allows the relationship between barrier opening and plasma osmolality to be determined quantitatively (8).

There is very little information regarding the effect of factors that might adversely affect blood-brain barrier function during development (23). In addition, the likelihood exists that the effects of a particular stress on barrier function could vary at different times during development (23). In this study, we examined the effects of exposure to hyperosmolality as a means to test the relative vulnerability of the immature blood-brain barrier to a specific stress. It is important to point out that the experiments of Cserr et al. (8) showed barrier opening with degrees of hyperosmolality that may be encountered clinically in immature subjects (11, 14, 29). The effects of a hyperosmotic stress on fetal and neonatal blood-brain barrier function have not been previously investigated.

In this study, we tested the hypotheses that 1) blood-brain barrier permeability measured by the blood-to-brain transfer constant with AIB increases as a function of changes in systemic plasma osmolality in immature subjects, 2) the blood-brain barrier becomes more resistant to the effects of a systemic hyperosmotic stress during development, and 3) the heterogeneity in the barrier in response to hyperosmolality among the brain regions decreases during development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 Care and Use of Laboratory Animals" of the National Institutes of Health.

Animal preparation. Surgery was performed under 1.0–2.0% halothane anesthesia, as previously described (27), on fetuses at 60 and 90% of gestation (full-term gestation in sheep is 150 days), premature lambs at 90% of gestation, newborn lambs at 2–5 days of age, and lambs at 20–30 days of age. For the purpose of this study, the premature lambs at 90% of gestation are designated as premature lambs, and the lambs at 20–30 days of age as older lambs.

Briefly, in the fetuses at 90% of gestation, polyvinyl catheters were placed into a brachial vein for mannitol plus sodium or placebo (0.9% NaCl) administration and brachial artery and advanced to the thoracic aorta for blood sample withdrawal, heart rate, and blood pressure monitoring. An amniotic fluid catheter was placed for pressure monitoring as a referent for fetal arterial blood pressures. In the fetuses at 60% of gestation, the catheters were placed in the subclavian vein and artery and advanced to the thoracic aorta. Catheters were placed in a femoral artery and vein in the ewes.

In the premature lambs, the surgery and study preparation were performed as previously described (25). The surgery on the premature lambs was performed on the fetuses in utero to avoid the stress of surgery on the day of study. Briefly, in the fetuses, polyvinyl catheters were placed into a brachial vein for mannitol plus NaCl or placebo infusion, and a brachial artery and advanced to the thoracic aorta for blood sample withdrawal and blood pressure monitoring. After insertion, the catheters were closed and attached to the fetal skin. When the forelimb had been replaced into the uterus, the head and neck were exposed through the same incision. An endotracheal tube was placed into the trachea to facilitate immediate suctioning and ventilation at delivery. The endotracheal tube remained open in the amniotic cavity to allow the egress of lung liquid. The uterus and abdomen of the ewe were then closed. In the fetal and hysterotomy-delivered lambs, singleton and twin pregnancies were included. When a twin pregnancy was present, only one fetus was catheterized and studied.

The lambs were intubated under ketamine (10 mg/kg) and maintained with 0.75–1.5% halothane anesthesia. Catheters were placed into a brachial vein for mannitol plus NaCl or placebo infusion and brachial artery for sampling.

The blood-brain barrier permeability measurements in this study were obtained, in part, from animals in a series of studies to examine the effects of ontogeny and ventilation on blood-brain barrier function, and the effects of hyperosmolarity on brain volume regulation in sheep (25–27).

Study groups. The fetuses at 60% and 90% of gestation, premature, newborn, and older lambs were assigned randomly to receive mannitol (20% mannitol and 0.5 M NaCl) or placebo (0.154 M NaCl) infusions. The NaCl was added to the mannitol solution to prevent sodium chloride loss because of the mannitol administration (7). The groups, number of animals in each group, duration of recovery from surgery, and age at the time of study are summarized in Table 1.


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  		Table 1. Study groups

 
The premature lambs at 90% of gestation were studied for two reasons. After the initial studies, we observed that the fetal sheep at 90% of gestation developed more severe acidosis than the sheep in the other age groups. The premature lambs were enrolled as an age-matched ex utero comparison group for the in utero fetal sheep at 90% of gestation. In addition, we studied the ventilated premature lambs, because ventilated premature infants are often at risk for dehydration and hyperglycemia, which can result in hyperosmolality.

Experimental protocol and methodology. The fetuses were studied while the ewes were standing quietly in a cart. The premature lambs at 90% of gestation were studied after delivery by hysterotomy with the ewes under intravenous ketamine anesthesia (15–40 mg/kg). Then, the ewes were killed with an overdose of pentobarbital sodium (100–200 mg/kg). Immediately after delivery, the premature lambs were suctioned via the endotracheal tube, treated with surfactant (100 mg/kg, Survanta, Beractant, Ross Products, Columbus, OH), stabilized, and placed on a positive-pressure ventilator (Bio-Med Devices, Flow-Disc MVP-10, Pediatric Respirator, Stamford, CT) (25). Ventilation was begun on room air or oxygen and the premature lambs were studied 2 h after delivery and stabilization on the ventilator, as previously described (25). The newborn and older lambs were studied while blindfolded and quietly resting in a sling.

After baseline determinations, an initial intravenous infusion of 20% mannitol and 0.5 M NaCl or placebo (0.154 M NaCl) was given followed by a continuous infusion for the duration of the osmolar exposure within each group. The timing of the initial and continuous mannitol infusions, and [14C]-labeled AIB administration are shown in Fig. 1. The osmolar loads were selected to produce both a wide range of osmolalities for the subjects within each group and a steady-state elevation in plasma osmolality within each subject for the duration of the study (Fig. 1). In the fetal groups, mannitol plus NaCl or placebo infusions were administered to both the fetuses and ewes, because in initial experiments, we found that a given osmolality could not be maintained in the fetuses unless the ewes also were given mannitol presumably because of fluid shifts among the fetuses, amniotic fluid, fetal membranes, and ewes (3). The initial intravenous infusion followed by a continuous infusion of mannitol and 0.5 M NaCl or placebo was given directly to the premature, newborn, and older lambs.


Figure 1
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  		Fig. 1. Plasma osmolality (mosmol/kgH2O) plotted against study time in minutes for the mannitol-infused fetuses at 60% of gestation (bullet), fetuses at 90% of gestation ({circ}), preterm lambs at 90% of gestation ({blacktriangleup}), newborn lambs at 3–5 days of age ({blacksquare}), and lambs at 27–30 days of age ({square}). The mannitol infusions and [14C]-labeled {alpha}-aminoisobutyric acid (AIB) administration are schematically shown. Values are means ± SE. *P < 0.05 ANOVA: main effects for age vs. 60% of gestation; +P < 0.05 ANOVA: main effects for age vs. fetuses at 90% of gestation; and {ddagger}P < 0.05 ANOVA: interactions for age over time vs. fetuses at 90% of gestation.

 
Plasma osmolalities were obtained at baseline, before the onset of the initial mannitol infusions, and 5, 10, 20, 30, 40, 50, and 60 min after the end of the initial mannitol infusion and during the continuous infusion. Blood gases, pH, heart rate, and arterial blood pressure were measured at baseline, 30, and 60 min, and plasma sodium, potassium, and chloride concentrations were measured at baseline and 60 min of study.

Analytic methods. Blood-brain barrier function was measured in the fetuses and premature, newborn, and older lambs with [14C]-labeled AIB (Dupont-New England Nuclear, Boston, MA), as previously described (8, 15, 24, 25, 28). Briefly, after baseline physiological determinations were obtained and the initial intravenous infusion of mannitol and 0.5 M NaCl or placebo (0.154 M NaCl) were given, [14C]-labeled AIB was rapidly injected intravenously (Fig. 1). Arterial plasma concentrations of [14C]-labeled AIB then were obtained at fixed times before and after the injection, as previously reported (27). Brain vascular volume was determined in three additional separate fetuses, and lambs in each group were treated under the same protocol with [14C]-labeled polyethylene glycol (PEG; Amersham, England), which was injected intravenously 2 min before the end of the experiment (25). The brain vascular volume values did not differ between mannitol and 0.5 M NaCl or placebo groups and were similar to our previous values (27). Brain parenchymal tracer concentration was determined at the end of the experiment.

AIB is a synthetic amino acid that is not present in mammalian tissues. This amino acid has been used extensively to measure the total and regional blood-brain barrier permeability in a variety of mammals including sheep (2, 25, 27, 28). Arterial blood samples were withdrawn from the thoracic aorta. Intermittent samples were withdrawn, so that the plasma radioactivity profile could be compared among the mannitol and 0.5 M NaCl and placebo, and different age groups to ensure the accuracy of the methodology (27).

Knowledge of the plasma concentration profile and the concentration of tracer in the parenchyma allows calculation of the blood-to-brain transfer constant Ki (15). At the end of the studies in the fetal groups, the ewes were given ketamine (15–40 mg/kg) intravenously to achieve a surgical plane of anesthesia. A hysterotomy was performed, and the fetus was withdrawn from the uterus and decapitated immediately (25, 27). The brain was removed within 3–5 min for regional brain tissue samples. The ewe was then euthanized with pentobarbital sodium (100–200 mg/kg). A similar procedure was used in the lambs.

The brains were dissected into the following regions: cerebral cortex, hippocampus, cerebellum, thalamus, superior colliculus, inferior colliculus, pons, medulla, and cervical spinal cord. Tissue samples were treated as previously described (8, 25, 27, 28). All samples were corrected for background, sample spillover, and quenching. The plasma from the arterial blood samples was measured into scintillation vials, and scintillation cocktail was added to each vial. Plasma and tissue radioactivity were quantified as previously described (8, 25, 27, 28).

The blood-to-brain transfer constant Ki (µl·g brain–1·min–1) is given by Eq. 1

Formula 1(1)

where Abr is the amount of tracer that crossed the blood-brain barrier from blood to brain during the tracer study [disintegrations/min (dpm)/g], and cp is the tracer concentration in plasma (dpm/µl) at the time t (min). Abr is obtained by correcting the total amount of isotope measured in the tissue Am (dpm/g) for that residual part remaining in the brain vasculature space, which is measured by [14C]-labeled PEG. Thus Abr = Am – Vpcp, where Vp is the plasma volume of brain tissue (microliters per gram) and cp is the concentration of tracer in the terminal plasma sample (dpm/g). Vp = A{dagger}m/c{dagger}p, where A{dagger}m and c{dagger}p have the same definitions as Am and cp above, except that they apply to [14C]-labeled PEG (8).

Heart rate, mean arterial blood pressure, and amniotic fluid pressures were measured with pressure transducers (model 1280 C; Hewlett-Packard; Lexington, MA) and recorded on a polygraph (model 17758 B Series; Hewlett-Packard). Arterial pH and blood gases were measured on a Corning blood gas analyzer (model 238; Corning Scientific, Medford, MA) and corrected to 39.5°C in fetuses, 39°C in lambs, and 38.5°C in the ewes. Hematocrit was measured by the microhematocrit method. Plasma osmolality was measured on a vapor pressure osmometer (Vapro model 5520; Wescor, Logan, UT). Plasma sodium and potassium were measured by flame photometry (480 Flame Photometer; Ciba Corning Diagnostic, Medfield, MA) and chloride by coulometric titration (model CMT10 chloride titrator; Radiometer, Copenhagen, Denmark).

Calculations. The osmolar load to the ewe represented the total amount of solute administered to the ewe, including the initial and continuous infusions. Likewise, the osmolar load to the fetus was the total amount of solute administered to the fetus as the initial and continuous infusions. The total osmolar load was the sum of the osmolar load to the ewe and fetus. In the lambs, the total osmolar load was calculated in the same manner (Table 2).


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  		Table 2. Weight and total osmolar load in the mannitol-infused subjects by age group

 
Statistical analysis. One-way ANOVA was used to compare the weight of the sheep and the total osmolar load among the groups (Table 2). Serial measurements were compared among the mannitol-infused groups (Fig. 1), and among the mannitol- and placebo-infused groups (Tables 3 and 4) by three-factor ANOVA for repeated measures with time, age groups, and mannitol or placebo infusions as the factors. If a significant difference was found by ANOVA, the Newman-Keuls post hoc test was used. All data were expressed as means ± SE. P < 0.05 was considered statistically significant unless otherwise specified.


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  		Table 3. Arterial pH, blood gases, hematocrit, heart rate, and mean arterial blood pressure by age group

 

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  		Table 4. Plasma electrolyte concentrations at baseline (–30 min) and 60 min of study by age group

 
Modeling and comparison of models of the blood-to-brain transfer constant vs. plasma osmolality. In a preliminary analysis, we found that the time-averaged plasma osmolality values represented a statistically higher correlation with the Ki values than the final plasma osmolality values. The time-averaged plasma osmolality values were calculated individually for each animal during the continuous infusions (Fig. 1). The scattergrams (Figs. 2–5) represent the blood-to-brain transfer constant (Ki) plotted on the y-axis against the time-averaged plasma osmolality values for the duration of the study for each animal on the x-axis. The scattergrams showed an increase in the Ki values as plasma osmolarity increased. However, the rate of change in Ki did not remain constant throughout the range of values. Therefore, we investigated both quadratic and segmented regression models. The coefficient of determination as a measure of the goodness of fit was higher in most cases for segmented regression model than the quadratic model. In the segmented regression model, we fit a linear regression model for each segment with a separate slope, and an unknown break point between the two lines. For the purpose of this study, the estimated breakpoint between the lines is termed the "threshold" (8).


Figure 2
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  		Fig. 2. The open circles represent the placebo-infused and the closed-circles the mannitol-infused sheep. The y-axis represents the blood-to brain transfer constant (Ki), and the x-axis represents the time-averaged plasma osmolality values for the duration of the study. Ki plotted against plasma osmolality in the cerebrum for fetuses at 60% of gestation, fetuses at 90% of gestation, preterm lambs at 90% of gestation, newborn lambs at 2–5 days of age, and lambs at 20–30 days of age. The lines represent the first and second segment from the segmented regression model, as described under statistical analysis, and the point between the two lines represents the threshold.

 

Figure 5
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  		Fig. 5. Ki plotted against plasma osmolality in the medulla. Groups, symbol legends, and description of the lines as for Fig. 2.

 
Equation 2 written below in a convenient way for computational purposes gives the segmented regression model for Ki (y-axis) vs. plasma osmolality (x-axis)

Formula 2(2)

where al and am are the smallest and largest values of plasma osmolality, respectively, b1 and b2 are the coefficients of the slopes of the first and second segments, "a" is the threshold between the two segments, and c is a constant (22). Because the threshold has to be estimated, the model is nonlinear, and we used the NONLIN Program in SAS to fit the models (21). The program gives SEs for parameter estimates, which were used for comparison of thresholds and slopes for any two age groups within the same brain region.

We examined the relationship between blood-brain barrier permeability (Ki) and plasma osmolality during development by statistically comparing the thresholds and slopes of the second regression segment among the fetuses and lambs within each brain region. The lower thresholds in the younger age groups and higher slopes of the postthreshold segments after the threshold has been reached would imply a greater vulnerability to hyperosmotic stress in the younger age groups within a given brain region.

Pairwise comparisons of the thresholds and slopes were made for the five age groups of fetuses and lambs within each brain region using an approximate two-sample t-test adjusting for alpha values as suggested by Holm and summarized by Glantz (10). Within each brain region (Figs. 2–5, Table 5), there were 10 pairwise comparisons for the age groups; therefore, a modified Bonferroni-type procedure proposed by Holm was used to determine the critical t values for each comparison. The smallest computed t value in the 10 pairwise comparisons was compared against the t value from the tables at the alpha = 0.05 level. The second-smallest computed t value was compared against the table t value at alpha = 0.05·(1/2) = 0.025 value, etc., and the largest t value was compared against the table t value at alpha = 0.05·(1/10) = 0.005. Holm suggested that this adjustment of alpha value, instead of using the 0.005 level for each test as in the Bonferroni method, has a larger power. Therefore, the comparisons of thresholds and slopes within each brain region were adjusted as suggested by Holm.


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  		Table 5. Plasma osmolality values of the estimated thresholds in the brain regions of the sheep by age group

 
Quadratic model comparing the heterogeneity among brain regions in response to hyperosmolality within each age group. We used a multivariate procedure referred to as the M-test (12) to examine the heterogeneity among the brain regions in the response to hyperosmolality within each group. Although a segmented regression model may be fitted for the data on each brain region, the comparison of such models in a fixed age group could not be done using an F-test similar to the one described above. This is because of the dependence of the data among the multiple brain regions within each age group. Hence, we fitted a quadratic model for each brain region (Eq. 3)

Formula 3(3)

For any two brain regions 1 and 2, the hypothesis tested was that f10=f20, f11=f21, f12=f22. The SAS program computes the M-test value, and the corresponding P value. The Holm procedure was used to find which pairwise comparisons among the brain regions were significant (10).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Weight increased with age as expected (Table 2). The total osmolar load given to the fetuses plus ewes, and lambs are summarized in Table 2.

Plasma osmolality plotted against study time in minutes remained relatively stable during the studies (Fig. 1). Although for simplicity Fig. 1 illustrates the means ± SE of the plasma osmolality values in each age group, the individual animals within each group demonstrated similar stable elevations in plasma osmolality during the studies. Moreover, there was a wide range of osmolar values within each age group, as shown by the individual plasma osmolality values in Figs. 2–5.

Table 3 contains the physiological variables of the study groups before (baseline) and during the mannitol and placebo infusions. Arterial pH decreased in all age groups during the mannitol infusions. Small decreases also were observed in arterial oxygen tension in the fetuses at 90% of gestation, newborn, and older lambs, and increases in carbon dioxide tension in the fetuses at 60% and 90% of gestation, newborn, and older lambs. Heart rate increased in the fetuses at 90% of gestation and older lambs during the mannitol infusions. Mean arterial blood pressure did not change in any group. Arterial plasma sodium concentrations increased in the fetuses at 60% of gestation and in newborn lambs, potassium decreased in the preterm lambs, and chloride increased in the fetuses at 60% of gestation, preterm lambs, newborn, and older lambs during the mannitol infusions (Table 4).

The Ki increased as a nonlinear function of plasma osmolality in all of the brain regions and age groups. At the lower osmolality values, the change in Ki was minimal, but after a threshold was reached, the increase (P < 0.05) in Ki was linear. The slopes of the first regression segment did not differ among any of the age groups in any brain region.

Figure 2 illustrates the pattern of change in Ki as plasma osmolality increased in the cerebral cortex. There were no significant differences in the threshold values or slopes of the second segments among any of the age groups. Figure 3 shows the pattern of change in Ki as plasma osmolality increased in the hippocampus. The slope of the second regression segment was significantly greater in the fetuses at 60% of gestation than in the premature lambs. Figure 4 summarizes the changes in the cerebellum. In the cerebellum, the slope of the second regression segment was significantly greater in the fetuses at 60% of gestation than in the newborn and older lambs, likewise, in the fetuses at 90% of gestation than in the premature, newborn, and older lambs, and in the premature than in the older lambs. Figure 5 shows the changes in the medulla. The slope of second regression segment was significantly greater in the fetuses at 60% of gestation than in the premature, newborn, and older lambs; and in the fetuses at 90% of gestation than in the premature, newborn, and older lambs.


Figure 3
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  		Fig. 3. Ki plotted against plasma osmolality in the hippocampus. Groups, symbol legends, and description of the lines as for Fig. 2.

 

Figure 4
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  		Fig. 4. Ki plotted against plasma osmolality in the cerebellum. Groups, symbol legends, and description of the lines as for Fig. 2.

 
Similar patterns of change in Ki vs. plasma osmolality were observed in the other brain regions, including the thalamus, pons, superior colliculus, inferior colliculus, and cervical spinal cord (data not shown). In the thalamus, the slope of the second regression segment was significantly greater in the fetuses at 90% of gestation than in the fetuses at 60% of gestation, as well as in the fetuses at 90% of gestation than in the preterm lambs. In the superior colliculus, the slope of second regression segment was significantly greater in the fetuses at 60% and 90% of gestation than in the premature and older lambs. In the inferior colliculus, the slope of the second regression segment was significantly greater in the fetuses at 60% of gestation than in the fetuses at 90% of gestation, premature, newborn, and older lambs. In the cervical spinal cord, the slope of the second regression segment was significantly greater in the fetuses at 60% of gestation than in the premature lambs.

Table 5 contains the thresholds by age group and brain region. Significant increases in the thresholds with development were observed in the thalamus, superior colliculus, pons, and cervical spinal cord.

The pattern of the Ki values plotted against plasma osmolality exhibited significant heterogeneity among the brain regions in response to the hyperosmotic stress within the fetuses at 60% (F = 18.4, P < 0.0001) and 90% (F = 170.0, P < 0.0001) of gestation, premature (F = 98.4, P < 0.0001), and newborn (F = 91.5, P < 0.0001) lambs, but not within the older lambs (F = 1.81, P = 0.53).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of our study was to examine the relative vulnerability of the immature blood-brain barrier to stress during development. We examined the effects of a hyperosmolar stress on blood-brain barrier function measured quantitatively from the last two months of fetal up to the first month of neonatal development in chronically catheterized fetal sheep and lambs. There are three major findings in this study. First, blood-brain barrier permeability measured by the blood-to-brain transfer constant increases as a function of changes in systemic plasma osmolality in fetal sheep and lambs, but the rate of change in Ki was not constant throughout the range of plasma osmolalities and is described best by a segmented regression model. Second, there is developmental regulation of blood-brain barrier function because the barrier becomes more resistant to the effects of a hyperosmotic stress during development in sheep. Third, there is in a pronounced heterogeneity in barrier permeability among brain regions during a hyperosmotic stress in the fetuses and premature and newborn lambs, but not in older lambs.

Arterial pH decreased within most age groups during the study, most likely because of renal sodium bicarbonate losses during the mannitol infusions (16) and the development of a mild respiratory acidosis in the fetuses and newborn and older lambs. Acidosis was most severe in the fetuses at 90% of gestation and newborn lambs.

Hypercapnia has been shown to increase the penetration of sucrose into the brain of fetal and newborn sheep (9) and adult rabbits (6), suggesting that hypercapnia increases blood-brain barrier permeability. However, in those studies (6, 9) the levels of carbon dioxide far exceeded the increases in carbon dioxide tension that we observed during the mannitol infusions (Table 3). Further, we previously have shown that asphyxia (carbon dioxide tensions >60 mmHg) in newborn piglets does not result in increases in blood-brain barrier permeability (24). The increases in arterial carbon dioxide tension during the mannitol infusions were ~19, 15, 13, and 12 mmHg in the fetuses at 60% and 90% of gestation, newborn, and older lambs, respectively. However, it is important to point out that the premature lambs were unique as the initial arterial carbon dioxide tensions were lower than in the other groups. Furthermore, in contrast to the other groups, they did not exhibit increases in carbon dioxide during hyperosmolality, most likely because they were ventilated during the studies. As a result, the effect was that the carbon dioxide tensions in the other four groups are 20 to 30 mmHg higher than in the premature lambs during hyperosmolality. Therefore, the differences in the response of the blood-brain barrier to hyperosmolality in premature lambs compared with those of the fetal sheep at 90% of gestation and the other groups could be partly attributable to the effects of the higher carbon dioxide tensions during the hyperosmotic stress. However, the relative increases in the arterial carbon dioxide tensions in the fetuses at 60% and 90% of gestation (15 and 14 mmHg, respectively) were similar to those of the newborn and older lambs (14 and 11 mmHg, respectively). Nonetheless, the blood-brain barrier of the newborn and older lambs exhibited greater resistance to the effects of hyperosmotic stress compared with those of the fetal groups.

We have previously shown that the blood-brain barrier demonstrates ontogenic decreases in permeability in normoosmotic ovine fetuses from 60% of gestation up to 3 years of age in the adult (27). The current study extends our previous work by examining developmental regulation of the blood-brain barrier to hyperosmotic stress. Many previous studies have established the ability of hyperosmolality to increase the permeability of the blood-brain barrier in adult subjects (5, 8, 17–20, 30). In previous studies of the relationship between hypertonicity and permeability of the cerebral vasculature, hyperosmotic solutions were typically applied by a bolus injection into the carotid artery or by topical application to the pia-arachnoid (8, 17–20, 30). In contrast, the study by Cserr et al. (8) examined the effects of more moderate increases in systemic osmolality produced by intraperitoneal administration and showed that, with an increase in plasma osmolality to values above 385 mosmol/kgH2O, there was an abrupt change in barrier function with increases in permeability measured for sodium and mannitol (8). We administered hyperosmotic solutions by intravenous infusions and in the case of the fetal sheep by combined maternal and fetal infusions. In most age groups and brain regions in sheep, we did not identify as abrupt an increase in barrier permeability in response to increases in systemic osmolality as identified in the adult rats (8). However, several differences exist between our work and the former study in rats (8); there are species and age differences, and, importantly, we examined barrier permeability in individual brain regions rather than in the whole brain (8).

We examined the effects of exposure to hyperosmolality to test the vulnerability of the developing blood-brain barrier to a specific stress. We examined the responses of the blood-brain barrier to hyperosmolality for any two age groups within each brain region by comparing the thresholds between the two lines and the slopes of the second regression segment. Although there was some variability in the relationship between Ki and increases in osmolality within each age group and brain region, the statistical analysis supported the contention that the barrier became more resistant to the effects of osmotic stress with advancing age. The estimated osmotic threshold (8) of the blood-brain barrier increased with development in some brain regions. The analysis of the postthreshold slopes of the second regression segment describes the rate of change in Ki after the osmotic threshold was reached (8). Higher rates of change in Ki vs. plasma osmolality, that is, greater slopes of the second regression segment, in the younger age groups imply a greater sensitivity to hyperosmotic stress. A relatively greater sensitivity to hyperosmotic stress in the younger compared with the older age groups was observed in several brain regions, including the hippocampus, cerebellum, thalamus, superior colliculus, inferior colliculus, medulla, and cervical spinal cord. Conversely, decreases in the postthreshold slope with increasing age imply greater resistance of the barrier to osmotic stress in the newborn and older lambs compared with the premature lambs and fetuses. In general, our analyses suggested greater sensitivity to the hyperosmotic stress at the more immature ages and increasing resistance to this stress with development. Our findings also are consistent with the view that hyperosmotic stress affects blood-brain barrier function to some extent in all age groups. However, similar to our report in normoosmotic sheep (27) and work examining breakdown of the blood-brain barrier to proteins in white matter during postnatal development of rats and opossums (23), the increases in barrier permeability are not attributable to a generalized immaturity of the barrier (27). Rather, consistent with recent findings (23), it appears that there is an age-related increased vulnerability of the immature barrier to hyperosmotic stress.

In our study, hypertonicity did not appear to abolish barrier function in the fetuses and lambs. Osmotic modification of the blood-brain barrier did not appear uniformly throughout the brain regions, it differentially affected barrier function to varying extents in different age groups and brain regions (13). Although our study did not address the mechanism(s) by which osmotic stress increased blood-brain barrier permeability, previous work suggests that increases in blood-brain barrier permeability after osmotic stress are based upon diffusion through pores that have been created between adjacent endothelial cells that are formed by the opening of the tight junctions that form the blood-brain barrier (30).

We have previously shown that there is a pronounced regional heterogeneity in barrier permeability in normoosmolar fetuses, lambs, and adult sheep, and that this pattern of regional brain heterogeneity is accentuated in the younger age groups (27). The current study extends these findings and further demonstrates that there is heterogeneity in blood-brain barrier permeability among brain regions during osmotic stress in fetuses at 60% and 90% of gestation, in premature and newborn lambs, but not in the older lambs.

In summary, blood-brain barrier permeability measured by the blood-to-brain transfer constant increases as a function of changes in plasma osmolality in fetal sheep and lambs. The blood-brain barrier becomes relatively more resistant to the effects of a systemic hyperosmotic stress during development. Our findings suggest that the blood-brain barrier of the immature fetus may be more vulnerable to the effects of an osmotic stress compared with that of older subjects.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Child Health and Human Development Grants P50-HD-11343 and R01-HD-34618.


   ACKNOWLEDGMENTS
 
We acknowledge and are thankful to the late Helen F. Cserr for her superb scientific guidance at the onset of these studies. We thank Karen D. Pettigrew for expert statistical analysis during the initial phase of the study. We thank Jini Han, Timothy Lee, Maricruz Merino, John A. Kazianis, Christopher B. Reilly, and Cheryl Smith for excellent technical assistance.


   FOOTNOTES
 

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 02905–240 (e-mail: bstonestreet{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. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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