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1 Department of Pediatrics, Women and Infants' Hospital of Rhode Island, Brown University Medical School, 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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In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain to plasma, and brain volume deceases. The brain responds to this stress by gaining osmotically active solutes, which limit water loss. This phenomenon is termed brain volume (water) regulation. We tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. Brain water responses to acute hyperosmolality were measured in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs, young lambs at 20-30 days of age, and adult sheep. After exposure of the sheep to increases in systemic osmolality with mannitol plus NaCl, brain water content and electrolytes were quantified. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. There were significant differences from an ideal osmometer in the cerebral cortex of fetuses at 90% of gestation, cerebral cortex, and cerebellum of newborn lambs, and cerebral cortex, cerebellum, and medulla of young lambs and adult sheep; however, there were no differences in the brain regions of fetuses at 60% of gestation and premature lambs, cerebellum and medulla of fetuses at 90% of gestation, and medulla of newborn lambs. We conclude that 1) brain water loss is maximal and brain volume regulation impaired in most brain regions of fetuses at 60 and 90% of gestation and premature lambs; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs, and then it develops in the medulla of the lambs at 20-30 days of age; 3) brain water loss is limited and volume regulation present in the brain regions of young lambs and adult sheep; and 4) the ability of the brain to exhibit volume regulation develops in a region- and age-related fashion.
brain volume regulation; dehydration; development; electrolytes; maturation; sheep
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MAINTENANCE OF BRAIN CELL volume is essential for normal central nervous system functioning (13). The permeability properties of the cerebral capillary endothelium or the blood-brain barrier are closer to those of cell membranes than systemic capillaries. This endothelium is permeable to water but highly impermeable to most other plasma constituents. Osmotic forces dominate water flux across the blood-brain barrier, and the volume of the brain is a function of the osmolyte content of the tissue (4). In adult rats, when plasma osmolality increases, water flows across the blood-brain barrier down its concentration gradient from brain tissue to plasma, and brain volume deceases (5). The brain responds to this stress by gaining osmotically active solutes, which serve to limit water loss from brain tissue (5). This phenomenon has been termed brain volume (water) regulation (5).
An ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress (5). Although some reports have suggested that the brain responds as a "perfect osmometer" and that brain volume regulation is absent in adult rabbits exposed to acute hyperosmolality (8), quantitative investigations have shown that the adult rat brain does not behave as an ideal osmometer in response to acute hyperosmolality and that water loss and electrolyte gain occur simultaneously, such that brain volume regulation stabilizes within 30 min (5). Consequently, the adult brain shrinks less than predicted on the basis of an ideal osmometer, and the brain exhibits volume regulation (5). Although brain volume regulation has been extensively studied in the adult (7, 8), the ability of the brain to exhibit volume regulation has not been systematically examined during development, particularly in fetuses and newborns of a large mammal such as the sheep. Brain volume regulation is particularly important in premature and newborn subjects because they are potentially at high risk for water and electrolyte imbalance and for dehydration (20). Clinical magnetic resonance imaging studies in humans suggest that water diffusion is highly dependent on age and brain location (9) and considerably higher in the gray and white matter in newborns than adults (19).
Keep et al. (12) have been able to demonstrate a marked improvement in brain volume regulation in rats around the time of birth. In addition, Trachtman et al. (31) demonstrated that the cerebral cell volume regulatory response was sufficiently developed in young animals to limit brain water loss and maintain brain volume. Nonetheless, the effect of an acute osmotic stress on brain volume regulation has not been systematically examined during development in the fetus.
Given the above considerations, we tested the hypothesis that brain volume regulation is more effective in young lambs and adult sheep than in fetuses, premature lambs, and newborn lambs. To study this, we administered infusions of mannitol plus NaCl to produce graded increases in systemic osmolality and measured brain water and electrolyte responses to acute hyperosmolality in the cerebral cortex, cerebellum, and medulla of fetuses at 60 and 90% of gestation, ventilated premature lambs at 90% of gestation, newborn lambs, young lambs at 20-30 days of age, and adult sheep. We studied the ventilated premature lambs because ventilated premature infants are often at high risk for dehydration, hypernatremia, and hyperglycemia, which can result in hyperosmolality.
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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 Committee of Brown University and Women and Infants' Hospital of Rhode Island and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Animal preparation. Surgery was performed under 0.75-2.0% halothane anesthesia, as previously described in detail (24). Briefly, in the fetuses at 90% of gestation, polyvinyl catheters were placed into a brachial vein for mannitol plus NaCl or placebo (0.9% NaCl) administration, and they were placed into a 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 to correct for fetal arterial pressures. The surgery was modified slightly in the fetuses at 60% of gestation. The catheters were placed in the subclavian vein and artery and advanced to the thoracic aorta. A femoral artery catheter was placed in the ewes.
In the premature lambs, the surgery and study preparation were performed as previously described in detail (25). The lambs at 90% of gestation were designated as premature lambs. Briefly, in the fetuses, polyvinyl catheters were placed into a brachial vein for mannitol plus NaCl or placebo infusion, and they were placed into 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 skin of the fetus. After the forelimb had been replaced into the uterus, the head and neck were exposed through the same incision. An endotracheal tube then was placed into the trachea to facilitate immediate suctioning and ventilation at delivery. The endotracheal tube remained open in the amniotic cavity to allow for lung liquid egress. 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 and adult sheep 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 administration and into a brachial artery for study sampling. The regional brain water and electrolyte measurements in this study were obtained in part from animals enrolled in a series of studies to examine the effects of ontogeny, hyperosmolality, and ventilation on blood-brain barrier function in sheep (25-27).Study groups.
After recovery from surgery, the 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, lambs at 20-30
days of age, and adult sheep were randomly assigned 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
the plasma sodium and chloride concentrations from decreasing secondary
to mannitol administration (5). The groups, number of
animals in each group, duration of recovery from surgery, and age at
study are summarized in Table 1.
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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 under intravenous ketamine anesthesia (15-40 mg/kg). The ewes then 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), hand ventilated, 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 as needed with a respiratory rate of 18-71 breaths/min, a peak inspiratory pressure of 14-43 mmHg, and a positive-end expiratory pressure of 2-10 mmHg to achieve an initial arterial oxygen tension of 50-190 Torr, and a carbon dioxide tension of 23-36 Torr. The premature lambs were studied 2 h after they were stabilized on the ventilator with the blood-gas values as outlined above. The newborn lambs were studied while blindfolded and quietly resting in a sling, and the adult sheep were studied while standing quietly in a cart.
After baseline determinations, 20% mannitol and 0.5 M NaCl or placebo (0.154 M NaCl) was administered as initial rapid intravenous injections followed by a continuous infusion to achieve graded increases in systemic plasma osmolality within each group. The osmolar loads (Table 2) were selected to produce both a wide range of osmolalities for the subjects within each group and a steady-state increase in plasma osmolality within each subject for the duration of the study. In the fetal groups, mannitol plus NaCl or placebo infusions were administered to both the fetuses and ewes because in the initial experiments we found that a given osmolality could not be maintained in the fetuses unless the ewes also received mannitol, presumably because of fluid shifts among the fetuses, amniotic fluid, fetal membranes, and ewes (3). The rapid intravenous injections followed by a continuous infusion of mannitol plus NaCl or placebo was administered directly to the premature, newborn lambs, lambs, and adult sheep. Plasma osmolalities were obtained at baseline and at 5, 10, 20, 30, 40, 50, and 60 min of study, and pH, blood gases, heart rate, and mean arterial blood pressure were obtained at baseline and 30 and 60 min of study. Arterial blood plasma samples were taken after 60 min of the study for plasma sodium, potassium, and chloride concentrations. The 60-min study interval and exposure to hyperosmolality were considered sufficient for the brain volume to stabilize on the basis of previous work in adult rats (5). After exposure to the graded infusions of mannitol or placebo (Table 2), in the fetal groups the ewe was 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 intact and decapitated immediately (25, 27, 28). The ewe was then euthanized with pentobarbital sodium (100-200 mg/kg). A similar procedure was used in the lambs and adult sheep.
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Brain tissue water content, and brain and plasma electrolyte concentrations. The brain was removed within 8-10 min for regional brain tissue samples of the cerebrum, cerebellum, and medulla. Triplicate 1-g sections of cerebrum, cerebellum, and medulla were obtained for tissue water content and placed in weighed dry glass vials as previously described (5). Tissue samples were dried to a constant weight at 90°C for 72 h to determine sample water content. The dried samples were extracted with 0.75 N HNO3 for 72 h at room temperature. The acid extracts and arterial plasma samples were analyzed for sodium and potassium by atomic absorption spectroscopy (model 2380, Perkin-Elmer, Ueberlingen, Germany) and plasma for chloride by coulometric titration (model CMT10 chloride titrator, Radiometer, Copenhagen, Denmark). Chloride concentrations were not available on the brain tissue extract because of technical difficulties at the onset of the studies and therefore are not reported.
Analytic methods. 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) at 39.5°C in fetuses, 39°C in lambs, and 38.5°C in ewe and adult sheep. Plasma osmolality was measured in duplicate on a vapor pressure osmometer (Vapro model 5520, Wescor, Logan, UT).
Calculations and statistical analysis. The osmolar load to the ewe represented the total amount of solute (mosmol) administered to the ewe including both the initial rapid injection and the continuous infusion. Similarly, the osmolar load to the fetus was the total amount of solute (mosmol) administered to the fetus as the rapid injection and a continuous infusion. The total osmolar load was the sum of the osmolar load to the ewe and fetus. In the lambs and adult sheep, the total osmolar load also included the initial injection and the continuous infusions given directly to the lambs or adult sheep (Table 2).
The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress (5). In the absence of brain volume (water) regulation, the brain behaves on the basis of ideal osmotic behavior, and brain solute (VC) content remains constant as C changes, where V is volume or regional water content (ml/g dry wt brain) and C is the plasma osmolality (mosmol/kgH2O) at the end of the study. Total brain solute content under the acute hyperosmotic stress was calculated from the relationship where VC is the total regional brain solute (mosmol/g dry wt brain), and it was plotted against the plasma osmolality (see Figs. 3-5). Plasma osmolality was the final value measured at the end of the study. To calculate total solute (VC), it was assumed, based on previous work (5), that equilibrium had been reached at end of the study, where plasma C equals tissue C. When osmotic behavior is ideal, VC has to be constant as osmolality increases. When osmotic behavior is nonideal, the volume remains greater than expected for the ideal osmometer. Therefore, regional brain volume (water) regulation was considered to be present when VC was not constant; i.e., there was a significant positive correlation between osmotically active brain solute and plasma osmolality at the end of the study. When volume regulation is present (see Figs. 3-5), it may be easily shown that the actual water loss, as a percentage of water loss expected under ideal osmotic behavior, may be calculated as
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Weight increased with age as expected (Table 2), but it did not differ between the fetuses and premature lambs at 90% of gestation. Total osmolar load, calculated as the osmolar concentration corrected for the total amount of solution administered to the fetuses plus ewes, lambs, and adult sheep, is summarized in Table 2. Arterial pH, PO2, PCO2, and mean arterial blood pressure values were within the normal range for fetal, newborn, and adult sheep (data not shown) (24, 25, 27-29).
Brain water content (Fig. 1) in the
placebo-infused sheep decreased with age in the cerebral cortex,
cerebellum, and medulla, with the largest decreases between the fetuses
at 60 and 90% of gestation. Similarly, regional brain sodium content
in the placebo-infused sheep decreased (P < 0.05) with
age, with the largest decline between 60 and 90% of gestation without
further decreases after birth (data not shown). Brain potassium content
demonstrated similar, more modest (P < 0.05) decreases
with age (data not shown). The sodium-to-potassium ratio also decreased
(P < 0.05) with age. The regional brain electrolyte
pattern changed from a higher sodium concentration at 60% of gestation
to relatively higher potassium content at 90% of gestation. The ratio
of sodium to potassium was <1 in the fetuses at 90% of gestation,
lambs, and adult sheep.
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Plasma osmolality plotted against study time in minutes increased over
time and reached a plateau by 20 min of study (Fig. 2). Although for simplicity the graph
illustrates the means ± SE of the plasma osmolality values for
each age group, the individual animals within each group increased over
time in a similar manner. Moreover, within each age
group, there was a wide range of osmolar values, as shown by the individual plasma
osmolality values in Figs. 3-5.
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Regional brain water decreased monotonically with increasing plasma
osmolality in all groups (data not shown). The osmotically active
solute (mosmol/g dry wt) values demonstrated direct linear correlations
with plasma osmolality (mosmol/kgH2O) in the medulla (Fig.
3) of lambs at 20-30 days of age (r = 0.54, n = 29, P = 0.005) and adult sheep
(r = 0.71, n = 19, P = 0.007), but they were not demonstrated in fetuses at 60% of gestation
(r = 0.04, n = 24, P = 0.82), 90% of gestation (r =0.20, n = 27, P = 0.32), premature lambs (r = 
0.006, n = 24, P = 0.98), or newborn
lambs (r = 0.37, n = 23, P = 0.093). Direct linear correlations with plasma
osmolality were shown in the cerebellum (Fig. 4) of newborn lambs
(r = 0.67, n = 22, P = 0.0007), lambs at 20-30 days of age (r = 0.67, n = 25, P = 0.003), and adult sheep
(r = 0.73, n = 19, P = 0.0004), but they were not shown in fetuses at 60% of gestation
(r = 0.11, n = 26, P = 0.62), fetuses at 90% of gestation (r = 0.26, n = 27, P = 0.19), or premature lambs
(r = 0.38, n = 24, P = 0.07). Direct linear correlations with plasma osmolality were
demonstrated in the cerebral cortex (Fig. 5) of the fetuses at 90% of
gestation (r = 0.39, n = 27, P = 0.045), newborn lambs at 1-5 days of age
(r = 0.48, n = 22, P = 0.024), lambs at 20-30 days (r = 0.40, n = 25, P = 0.05), and adult sheep
(r = 0.57, n = 19, P = 0.012), but they were not demonstrated in fetuses at 60% of gestation
(r = 0.38, n = 26, P = 0.053) or premature lambs (r = 0.028,
n = 24, P = 0.9).
Inspection of Figs. 3-5 reveals that the range of plasma osmolalities that were achieved was relatively narrow in the fetuses at 60% of gestation compared with the other age groups, despite a wide range of osmolar loads (Table 2), and that the osmotically active solute values (y-axis) varied in response to the narrow range of osmolalities. However, the mean osmotically active solute values for the normoosmotic and hyperosmotic fetuses did not differ in the medulla or cerebellum (medulla: 2.14 ± 0.06 vs. 2.16 ± 0.03 mosmol/g dry wt, P = 0.82 and cerebellum: 2.67 ± 0.07 vs. 2.66 ± 0.04 mosmol/g dry wt, P = 0.88; normoosmotic vs. hyperosmotic), but were higher (P = 0.04) in cerebral cortex of the hyperosmotic (3.03 ± 0.04 mosmol/g dry wt) than normoosmotic (2.89 ± 0.03 mosmol/g dry wt) fetuses.
The actual water loss in the brain regions that exhibited volume
regulation (Figs. 3-5) as a percentage of water loss expected under ideal osmotic behavior is summarized in Table
3. This value represents the actual
percentage of water loss of that predicted on the basis of ideal
osmotic behavior in response to acute hyperosmolality in each brain
region.
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Brain sodium (meq/kg dry brain wt) values of the medulla did not
demonstrate correlations with plasma osmolality
(mosmol/kgH2O) in any of the age groups of the sheep,
except for a negative correlation in the medulla of the adult sheep
(medulla sodium content = 0.30 × plasma osmolality + 286.6; r = 0.49, n = 19, P = 0.032). A direct linear correlation with plasma
osmolality was shown in the cerebellum of the lambs at 20-30 days
of age (cerebellar sodium content = 0.47 × plasma
osmolality + 78.85; r = 0.56, n = 26, P = 0.003), but it was not shown in the fetuses at
60% of gestation (r = 0.052, n = 26, P = 0.79), fetuses at 90% of gestation
(r = 0.045, n = 27, P = 0.83), premature lambs (r = 0.16, n = 24, P = 0.46), newborn lambs (r = 0.30, n = 22, P = 0.18), or adult sheep
(r = 0.06, n = 19, P = 0.80). Brain sodium values demonstrated a direct linear correlation
with plasma osmolality in the cerebral cortex of fetuses at 60% of
gestation (cerebral cortical sodium content = 1.33 × plasma
osmolality + 436.78; r = 0.50, n = 26, P = 0.0093) and lambs at 20-30 days of age
(cerebral cortical sodium content = 0.48 × plasma
osmolality + 118.99; r = 0.54, n = 26, P = 0.0045), but this was not demonstarted in
fetuses at 90% of gestation (r = 0.14,
n = 27, P = 0.48), premature lambs (r = 0.04, n = 24, P = 0.85), newborn lambs (r = 0.22, n = 22, P = 0.3), or adult sheep (r = 0.23, n = 19, P = 0.34). Potassium content in the medulla demonstrated a negative correlation with plasma
osmolality in the adult sheep (medulla potassium content = 0.40 × plasma osmolality + 417.3; r = 0.55, n = 19, P = 0.015), but this
was not demonstrated in any other brain region or age group of sheep,
except for a positive correlation in the cerebellum (cerebellar
potassium content = 0.29 × plasma osmolality + 338.1; r = 0.42, n = 25, P = 0.035) with plasma osmolality in the lambs at 20-30 days of age.
Cerebral cortical potassium content did not show correlations with
plasma osmolality in any of the age groups of sheep. The plasma sodium,
potassium, and chloride concentrations in the mannitol- and
placebo-infused groups at 60 min of study are summarized in Table
4.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of our study was to examine the ability of the immature sheep brain to exhibit volume regulation in response to an acute hyperosmolar stress. The major findings of our study were that 1) the immature brain behaves similar to that predicted on the basis of perfect osmotic behavior [i.e., there is no volume regulation (5), and water loss is maximal in most regions of fetuses at 60 and 90% of gestation and premature lambs at 90% of gestation]; 2) brain volume regulation develops first in the cerebral cortex of the fetuses at 90% of gestation and in the cerebral cortex and cerebellum of newborn lambs and then in the medulla of lambs at 20-30 days of age; 3) the brain exhibits nonideal osmotic behavior and shrinks less than that predicted on the basis of ideal osmometer in all brain regions of the lambs at 20-30 days of age and adult sheep, and, consequently, brain volume regulation is present in lambs at 20-30 days of age and adult sheep; and 4) the ability of the brain to shrink less than predicted on the basis of ideal osmotic behavior and exhibit volume regulation develops in a region and age-related fashion.
The design of our study was to produce both a wide range of osmolalities for the subjects within each group and a steady-state increase in plasma osmolality within each subject for the duration of the study. This was achieved by a combination of a rapid injection of mannitol plus NaCl followed by a continuous infusion. In the case of the fetal subjects, we found it necessary to administer the infusions to both the fetuses and ewes to achieve stable elevations in plasma osmolality over the 1-h study, presumably because of fluid shifts among the fetus, placenta, amniotic fluid, fetal membranes, and ewes (3). Although there were small differences among the curves for plasma osmolality plotted against time (Fig. 2) in the different age groups, it is important to point out that the adult sheep that exhibited volume regulation had a plasma osmolality curve that was the third lowest among the six groups. Therefore, the differences among the plasma osmolality curves over time most likely did not contribute to differences in the ability of the age groups to exhibit volume regulation, particularly because volume regulation was examined separately within each group.
Consistent with previous findings in sheep (17), brain water content decreased with age in the isosmotic sheep, with the greatest decline between 60 and 90% of gestation. Similar to our findings, clinical magnetic resonance imaging studies suggest that water diffusion is highly dependent on age and brain region (9) and is higher in newborns than adults (19). The reduction in brain water with development most likely represents increased cellular growth and myelination (15, 17). Consistent with findings in postnatal rats, the decline in water content was accompanied by reductions in brain sodium and potassium concentrations (31). The largest decline in sodium concentration occurred between 60 and 90% of gestation, with no further decrease after birth. The decreases in potassium concentrations were less dramatic, suggesting that the decrease in the extracellular cation was greater than that in the intracellular cation. This is consistent with the decrease in the extracellular fluid space of the brain with maturation (2, 14). Similar to other species (15), the brain electrolyte pattern shifted from a proportionately higher sodium content at 60% of gestation to a higher potassium content, such that the ratio of sodium to potassium was <1 in the fetuses at 90% of gestation, lambs, and adult sheep.
The capillary endothelium that forms the blood-brain barrier is impermeable to solutes but is permeable to water (4). Therefore, osmotic forces dominate water flux across the blood-brain barrier, and brain volume is determined by the osmolyte content of the tissue (4). In the adult rat during acute hyperosmolality, water loss from the brain is only about one-third of that predicted on the basis of ideal osmotic behavior, revealing the presence of volume-regulatory mechanisms (5). However, by comparison, in the adult sheep during acute hyperosmolality, water loss from the cerebral cortex is about three-fourths of that predicted on the basis of ideal osmotic behavior. This suggests that, although the volume regulatory response is present in the sheep cortex, it may be less effective than in the adult rat brain. Nonetheless, we have used the concepts outlined by Cserr et al. (5) to determine when, during development in sheep, the brain would begin to exhibit volume regulation in response to acute hyperosmolality.
The response of the immature brain to hyperosmolality is particularly important in premature and newborn subjects because their smaller size and larger surface area render them susceptible to dehydration and hyperosmolality (20). Although the ability of the brain to exhibit volume regulation has not been systematically examined during development, brain volume regulation improves around the time of birth in the rat such that, for a similar increase in plasma osmolality, fetuses lost more brain water than neonates during an acute osmotic stress, and neonatal and adult rats both exhibited brain volume regulation to a similar extent (12). In young rats after 48 h of hypernatremia, the cerebral cell volume regulatory response was sufficiently developed to limit brain water loss (31). In our study, the absence of brain volume regulation, i.e., perfect osmotic behavior, was represented by a nonsignificant slope, indicating no change in the amount of the total brain solute over the range of plasma osmolalities examined (Figs. 3-5). This approach had the advantage that we did not have to correct for the higher water content in the immature animals (12, 31) and also examined the presence or absence of volume regulation within each age group separately. Nonideal osmotic behavior with the presence of brain volume regulation was indicated by a significant change in the amount of osmotically active brain solute over the range of experimental osmolalities. The volume regulatory response appeared to be effective earliest in the cerebral cortex of the fetuses at 90% of gestation and with increasing maturation was effective in more of the brain regions, such that, in the 20- to 30-day-old lambs and adult sheep, all regions exhibited volume regulation. The volume regulatory response also appeared to become more effective with age because the actual percentage of water loss represented a smaller component of that predicted on the basis of ideal osmotic behavior in the adult sheep, which suggests improving volume-regulatory mechanisms with maturation (Table 3).
The regulatory response was effective earlier in development in the cerebrum and cerebellum than in the medulla, which is the brain region with the largest amount of white matter. The water content of gray matter is higher than that of white matter, and gray and white matter have different properties because of their different cell compositions (10, 23). Gray matter is composed of glial cell processes interweaving complexly with neurons. The cells have low compliance such that formation of the interstitial fluid does not distort the complex composition. In contrast, white matter has a higher compliance because of its looser structure. Therefore, it remains possible that the different water contents and structure of the white matter and gray matter might have contributed to the differences in the maturational volume regulatory responses between the cerebrum, cerebellum, and medulla in the sheep.
The brain volume regulatory response of the fetus at 60% of gestation deserves comment. Inspection of Figs. 3-5 reveals that the range of plasma osmolalities that were achieved was relatively narrow compared with the other groups, despite a wide range of osmolar loads (Table 2). In all other groups, there was variability in the response of plasma osmolality to the osmolar loads. Although we cannot determine the reason that the fetus at 60% of gestation did not demonstrate variability in the response of plasma osmolality to the osmotic load similar to that of the other age groups, we speculate that the large amount of total body water and extracellular fluid at this early time in gestation might have contributed to the relatively narrow plasma osmolality response to the osmolar load. In addition, the osmotically active solute values (y-axis, vertical range) varied in response to the narrow range of plasma osmolalities. In contrast, the osmotically active solutes in the brain regions of the other groups showed either a linear relationship or a horizontal range, indicating a direct correlation or no change with changes in plasma osmolalities. Although we cannot be certain why the fetuses at this very early time in gestation demonstrated a wide range of osmotically active solute values for the narrow range of osmolalities, the average values of the osmotically active solute in the medulla and cerebellum of the normoosmotic and hyperosmotic fetuses did not differ, further suggesting that brain volume regulation was most likely absent in these regions. However, we cannot rule out the possibility that brain volume regulation might have been evident in the cerebral cortex, if we had been able to achieve a larger range of plasma osmolalities in the fetuses at this early time in gestation, particularly because the mean osmotically active solute values were higher in the hyperosmotic than the normoosmotic group in this region.
The fetal sheep at 90% of gestation exhibited volume regulation in the cerebral cortex, whereas the premature lambs that were identical in age to these fetuses did not exhibit volume regulation in any brain region. The basis for the ability of the fetuses but not the premature lambs to exhibit volume regulation in the cerebral cortex is not clear. However, the fetal sheep remain in utero with the placenta, amniotic membranes, and amniotic fluid intact. In contrast, the premature lambs were delivered, given surfactant, and placed on a ventilator before the studies. Although it cannot be determined whether any of these factors contributed to the lack of volume regulation in the brain regions of the ventilated premature lambs, the lack of regulation in the premature lambs raises concern because the situation is analogous to premature neonates who require ventilatory support.
In the adult rat, brain volume is regulated during acute hyperosmolal states, in part on the basis of tissue electrolyte gain (6, 7). However, inorganic ions cannot account for the entire volume regulatory response of the brain during hyperosmotic stress, and other solutes, such as amino acids, methylamines, and polyols, also serve to offset water loss from the brain (11, 18, 30). There are regional and developmental differences in the organic osmolytes in the brain (16, 18, 22). As an initial step in our study, we examined regional brain electrolyte content as a function of plasma osmolality 60 min after the onset of hyperosmolality (5). Although we detected a direct correlation between sodium content in the cerebral cortex, and sodium and potassium content in the cerebellum and plasma osmolality in the 20- to 30-day-old lambs, which exhibited volume regulation in these regions, we also found a direct correlation between cerebral cortical sodium content and plasma osmolality in the cortex of the fetuses at 60% of gestation, which most likely did not exhibit volume regulation. In addition, we did not find correlations in the regions of other age groups that exhibited volume regulation. Therefore, regional brain volume regulation cannot be accounted for on the basis of tissue electrolyte gain in our study. These findings are consistent with those of Cserr et al. (5) in which, during hypernatremic hyperosmolality, brain volume regulation could be explained on the basis of brain electrolyte accumulation, but during mannitol-induced hyperosmolality, electrolyte accumulation could not account for the volume regulation (5). It also is important to point out that the 20- to 30-day-old lambs that demonstrated significant sodium accumulation in the cerebral cortex and cerebellum also demonstrated marked increases in plasma sodium concentrations (Table 4).
The significant negative correlation between sodium and potassium content in the medulla of the adult sheep and plasma osmolality is also consistent with findings by Cserr et al. (5), who found a negative correlation between brain potassium content and plasma osmolality during mannitol and sucrose hyperosmolality. The decrease in the electrolyte content and concomitant increased water loss in the medulla might have served to offset the effectiveness of the volume regulatory response in this brain region in the adult sheep.
In young rats, taurine appears to be an important organic osmolyte component of the volume regulatory response (31). Moreover, taurine, which is the most important organic osmolyte before birth, decreases after postnatal day 7, when myoinositol, glutamine, creatine, and glutamate increase (18). We cannot comment on the role that these organic osmolytes might have had in the volume regulatory response in our sheep because they were not measured. However, because taurine appears to be the most important organic osmolyte before birth, myoinositol, glutamine, creatine, and glutamate increase after birth and creatine and glutamate predominate in the adult brain, and regional brain differences in these osmolytes have been reported, we speculate that the changes in these organic osmolytes might have contributed to the developmental and regional volume regulatory responses that we observed in sheep (16, 18, 22, 31).
Recent reports suggest that water homeostasis in the brain is maintained by regulatory processes that, by control of aquaporin expression and distribution, induce and organize water movements (1). Although changes in aquaporin expression might be involved in brain volume regulation in our sheep, our laboratory's recent work suggests that ontogenic increases in mRNA aquaporin 4 expression are not observed under basal conditions in sheep of similar ages (21). Nonetheless, we did not measure aquaporins during mannitol-induced hyperosmolality and cannot comment on their relative importance in brain volume regulation in sheep.
In our study, we examined the volume regulatory response of the brain in response to mannitol-induced hyperosmolality. In the adult rat, the brain water loss is similar after 30 min of hypernatremia and mannitol-induced hyperosmolality (5). In contrast, water loss for mannitol-induced hyperosmolality was greater than for hypernatremia after 120 min of exposure (5). The further reduction in brain volume after 120 min of exposure to mannitol was probably secondary to a decrease in tissue electrolyte content (5). Therefore, because we found a negative correlation between sodium and potassium content and plasma osmolality in some brain regions, it is likely that the water loss in our sheep might have been smaller after exposure to hypernatremia than mannitol-induced hyperosmolality.
In summary, brain water loss is limited and volume regulation is present in the brain regions of young lambs and adult sheep; and the ability of the brain to exhibit volume regulation develops in a region and age-related fashion.
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ACKNOWLEDGEMENTS |
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We express our appreciation to the late Helen F. Cserr. We acknowledge and are grateful for her scientific guidance at the onset of these studies. We also acknowledge the excellent technical assistance of Christopher M. Elitt, John Kazianis, Michael McManus, Christopher B. Reilly, and Mary Sehl.
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FOOTNOTES |
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This work was supported by National Institute of Child Health and Human Development, Diabetes Center, Grants P50 HD-11343 and R01-HD-34618.
Address for reprint requests and other correspondence: B. S. Stonestreet, Brown University School of Medicine, Dept. of Pediatrics, Women and Infants' Hospital of Rhode Island, 101 Dudley St., Providence, RI 0290-240 (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. Section 1734 solely to indicate this fact.
First published December 27, 2002;10.1152/japplphysiol.00923.2002
Received 8 October 2002; accepted in final form 13 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P < 0.05 vs. fetuses at 60% of gestation.
, Placebo-infused sheep; 
,
mannitol-infused sheep. The y-axis represents the total
regional brain solute, and the x-axis presents the plasma
osmolality after at least 60 min of exposure for each animal. Fetuses
at 60% of gestation: r = 0.04, n = 24, P = 0.82. Fetuses at 90% of gestation:
r = 0.20, n = 27, P = 0.32. Preterm lambs at 90% of gestation: r = 
