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In a study on the tissue sources of neonatal water loss in newborn rabbit pups Coulter and Avery(1) demonstrated a paradoxical reduction in the water content of lean body mass, skin, and skeletal muscle with increasing fluid intake and weight gain. The observation was interpreted to indicate the presence of a cellular water reservoir at birth that served to protect circulation and to maintain tissue perfusion when fluid intake was restricted. In response to adequate fluid administration, however, the superfluous cellular water was rapidly released, presumably through a mechanism that involved endocrine regulation(1, 2).

We have extended these observations to suggest that, irrespective of its location in the intra- or extracellular compartment, the physical state of neonatal body water should also be considered(3), and evidence has been provided that the motionally constrained bound water fraction might function as a tissue water reservoir. Water bound to macromolecules can be stored or released according to the actual need of maintaining plasma volume(4).

In contrast to that of the skin and skeletal muscle, water content of the brain was found to correlate directly with fluid intake and weight gain. It was assumed, therefore, that brain water accretion is related to the process of growth, and it cannot be mobilized to supply water for maintaining circulation(1).

With respect to the organ-specific differences in regulating tissue hydration, the present study using H1 NMR relaxation measurements, was undertaken to assess motionally distinct water fractions, i.e. free, loosely bound, and tightly bound water fractions in the brain of newborn rabbits during the first 4 d of life. Furthermore, an attempt was made to obtain information on the mechanism of brain water regulation by comparing physical compartments of brain water in rabbit pups nursed conventionally and in those deprived completely of fluid intake.

METHODS

Experimental animals, procedures, and H1 NMR measurements, described previously(4), are as follows. Experiments were performed in 45 newborn New Zealand White rabbits at the postnatal age of 0-1, 24, 48, 72, and 96 h. They were born at term (after 32-d gestation) by spontaneous vaginal delivery. The newborn rabbit pups were randomly selected for one of the following two groups. Group I consisted of pups housed with their mothers. The littermates were nursed as described by Coulter and Avery(1) and provided water ad libitum. Group II were pups separated from their mothers and completely withheld from fluid intake.

Experimental procedure. For measurements of tissues hydration and H1 NMR relaxation, the animals were killed with a lethal dose of pentobarbital, and forebrain samples of about 200 mg were obtained at each study age. To prevent postmortem changes in water compartments, NMR measurements were done immediately after sacrificing the animals. Body weight as an estimate of fluid balance was measured before tissue sampling.

The tissue water content was determined as the difference between the wet and desiccated tissue weights. Tissues were dried at 105 °C until no further decrease in weight could be detected. At each study age five animals were analyzed for tissue water content and H1 NMR times in both groups.

NMR measurements. Tissues samples were placed in 5-mm diameter NMR glass tubes with the use of a plastic straw and incubated at 40 °C for 5 min to reach thermal equilibrium with the magnet temperature.

MINISPEC PC 140 NMR system (Bruker, Germany) was used for measuring the T1 and T2 relaxation times. Operating frequency was 40 MHz. A 386 AT personal computer was used as a storage scope to adjust the 90 ° and 180 ° pulses. T1 relaxation time was measured by an inversion recovery method with eight different time intervals between the 180 ° and 90 ° pulses. Repetition time was 5 times T1. T2 relaxation time was obtained by using Carr-Purcell-Meiboom-Gill sequence; 1000 echoes with 1-ms echo time were applied. Each point was the average of five measurements. The data were transferred to a PC for storage and analysis.

Mathematical analysis. To approximate quantitatively tissue water fractions according to their mobility, multicomponent analysis of the T2 relaxation decay curves was applied(5). The free induction decay of the proton relaxation process follows an exponential function. This function can be described by a multiexponential equation provided that in the tissues studied there are water compartments with different rates of relaxation, and these compartments are not interdependent at the time of measurements.

The process of proton T2 relaxation can be derived from the following expression: where k1,k2, and kn represent the relative contribution of each set of protons; T21, T22, and T2n are the relaxation times of the different components.

Data are expressed as mean ± SEM. For statistical analysis the nonparametric Wilcoxon test was used.

RESULTS

Table 1 shows that brain water content remained practically unchanged in the suckling pups, whereas it decreased significantly from 89.4 ± 0.5% at birth to 87.7 ± 0.1% at the age of 96 h(p < 0.05) in pups withheld from suckling. T1 relaxation time did not change with age in either groups, and there was no consistent change in T2 relaxation time in pups of group I, whereas it decreased significantly in pups of group II by the age of 72 h (188 ± 12versus 178 ± 4 ms, p < 0.05) and 96 h (171± 6 ms, p < 0.05). As a result, T2 values proved to be about 10% lower in group II than in group I, in contrast to only a 1% difference in the brain water between the two groups at the corresponding ages.

Table 1 Water content (%), T1,and T2 relaxation times (ms) of the brain in newborn rabbits aged 0-96 h (mean ± SE)
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Biexponential analysis of the T2 relaxation curves made possible to distinguish the fast (T21) and slow (T22) components that corresponded to the bound and and free water fractions. Relaxation times and partition of brain water into bound and free fractions as derived from biexponential fits of T2 decay curves are shown in Figure 1.

Figure 1
figure 1

Relaxation times and partition of brain water fractions according to their mobility as derived from the biexponential analysis of T2 decay curves in newborn rabbits (□, fast-bound; ▪, slow-free components). Upper panels represent absolute values; lower panels represent percent distribution of different water fractions. Data are given as mean ± SE.

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It can be seen that in the suckling pups T21 and T22 values were nearly identical during the first 48 h of life. Later on, T22 remained at about the same levels; T21, however, declined steadily to 153 ± 32 ms (p < 0.05) and 94 ± 6 ms (p< 0.05) at the age of 72 and 96 h, respectively. As a consequence, the percent contribution of bound water fraction represented by T21 fell progressively from 61% at birth to 3% at the age of 72-96 h (p < 0.05).

In the starving pups of group II, T21 fell rapidly from the initial value of 190 ± 12 ms at birth to 73 ± 24 ms at the age 24 h(p < 0.06) without further significant changes during the rest of the study. In contrast there was only a slight decrease in T22 during the whole study period.

The percent T21, i.e. the bound fraction of tissue water, accounted for 61 ± 15% of total brain water at birth, and in response to complete withholding of fluid intake there was a prompt decrease to a value of as low as 4 ± 2% (p < 0.05) as early as the first day of life, and it remained at this depressed level thereafter.

Using triexponential analysis further partition of the T2 relaxation curve was made, and the fast (T31), the middle (T32), and the slow (T33) components could be distinguished corresponding to the tightly bound, the loosely bound, and the free water fractions.Figure 2 demonstrates that most of the brain water was loosely bound (T32, 48-94%) followed by the free (T33, 3-49%) and tightly bound water fractions (T31, 3-29%). This pattern of distribution appeared to be profoundly influenced by fluid intake. Pups of group II responded to fluid deprival with a 3-6-fold decrease in the tightly bound water (T31) and with a simultaneous 4-fold increase in the free water fraction (T33).

Figure 2
figure 2

Relaxation times and partition of brain water fractions according to their mobility as derived from the triexponential analysis of T2 decay curves in newborn rabbits (□, fast-tightly bound; ▪, middle-loosely bound; □, slow-free components). Upper panels represent absolute values; lower panels represent percent distribution of different water fractions. Data are given as mean ±SE.

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DISCUSSION

The present study using H1 NMR relaxation measurements provided evidence that in newborn rabbit pups brain water can be partitioned into fractions with different mobility. Accordingly, free, loosely bound, and tightly bound water fractions have been established. During the first 4 d of life, bound (biexponential analysis) and tightly bound (triexponential analysis) water fractions decline progressively with age, and this decline is further accelerated in pups deprived of fluid intake. The changes that occur in water mobility greatly exceed the moderate reduction in brain hydration, indicating that brain water should be restructured when newborn pups are challenged by fluid deprivation.

The H1 NMR technique has been proposed to quantitate the distribution of tissue water among physical compartments by measuring the proton relaxation rate. As proton relaxation decay follows a multiexponential pattern, components of proton pools relaxing at different rate can be derived from the measured multicomponent decay curves(6, 7). Slowly relaxing components are thought to be attributed to the relatively free, bulk water fraction, whereas the macromolecular bound water contributes to the quickly relaxing components(8).

During the perinatal period, only limited data are available on body composition determined by H1 NMR method. Fried at al.(9) have recently demonstrated that in rabbits the T1 and T2 relaxation times for cardiac and skeletal muscles declined steadily during the late fetal and early postnatal life and concluded that these developmental changes were due to the reduction in total tissue water and to the relative increase in the highly ordered, motionally restricted cellular water. The latter appeared to be related to the increase in cell protein content and to the increase of water-macromolecular interaction(9). The same group conducted another H1 NMR study to reveal developmental changes in water content of specified brain areas and found that during the first 30-d postnatal period T1 and T2 relaxation times decreased progressively with advancing age in all sampled brain areas. The shortening of relaxation times closely related to postnatal brain dehydration and induction of cytotoxic edema with triethyltin resulted in prolongation of T1 and T2 relaxation times that paralleled the increases of water content of the respective brain areas(10). These findings seem to suggest that the major factor determining the rate of water proton relaxation is the hydration of brain tissue. It is to be noted, however, that the relationship of T2 to brain water was less apparent than that of T1, and in the white matter the changes in T1 and T2 were not proportional to the increase in water content(10). Taking all this into account, it is tempting to assume that, in addition to brain dehydration, other factors might also contribute to the postnatal reduction of relaxation times. Macromolecular components of the CNS may play a role, although brain protein content was found to fall at this critical period of shortening proton relaxation, and the rapid phase of myelination occurred only after the 12th postnatal day(11).

Our present results are also favor the contention that in the immediate postnatal period factors other than brain water content may be responsible for the alterations in the relaxation rates. In newborn rabbits subjected to complete withdrawal of fluid intake, the 0.8 and 1.0% reduction in brain water was associated with an 8 and 12% decrease in T2 relaxation times at the respective ages of 72 and 96 h, compared with the normal suckling pups. Furthermore, the fast components of the T2 decay curves identified by biexponential fit proved to be 45-62% shorter in the starving than in the suckling pups irrespective of age and total brain water. Similarly, in starving animals there was a 52 and 62% decrease in the fast components of the T2 decay curve at the age of 24 and 48 h, respectively, when they were derived by using triexponential fits. All these observations are consistent with the notion that in response to withholding fluid intake marked changes occur in the physical state of brain water without or with only a minor reduction in its total water content. The possible reorganization of brain water is further supported by the fact that the 3-6-fold decrease in the tightly bound water fraction is associated with a 4-fold increase in the free water fraction when the newborn animals are challenged with fluid deprival.

The underlying mechanism(s) of regulating water proton mobility in the neonatal brain is not known. It is likely to be accounted for by quantitative or qualitative alterations in the macromolecular constituents of the immature brain(12), including proteins, lipids, and glycosaminoglycans, particularly hyaluronic acid(13–16). Hyaluronic acid is known to have a great number of hydrophilic residues to bind water, and its role as a determinant of neonatal tissue hydration has been established(17–19). One-fourth of tissue hyaluronic acid is exchangeable(20), it may undergo enzymatic degradation with subsequent release of the previously bound water fraction(21). This process may be controlled by hormonal factors(22).

In conclusion, the present H1 NMR study provided quantitative estimate of the free and bound water fractions of brain tissue in newborn rabbit pups suckling ad libitum and in those completely deprived of fluid intake. Withholding fluid was associated with a marked reduction in the bound and a simultaneous rise in the free water fraction. This observation is regarded to indicate that some of the brain water functions as a tissue water reservoir from which the stored water can be released to supply free water for brain volume regulation. The regulation and clinical significance of this process remains to be elucidated.