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During fetal life the potential air space of the lung is filled with fluid, which is generated by continuous epithelial secretion driven by active chloride transport. As parturition approaches, the lung fluid secretion decreases, and sodium transport–dependent reabsorption begins. This switch in transepithelial water movement culminates at birth and results in rapid clearance of lung fluid, which is critical for successful transition to air breathing (1–4).

The osmotic water permeability of the alveolar epithelium is developmentally regulated by and related to the expression of water-transporting proteins, called AQPs. Three AQPs have been identified in the lung: AQP1 in the capillary endothelium, AQP4 in the basolateral membrane of the airway epithelium, and AQP5 in the apical membrane of alveolar epithelial type I cells (5–8).

The specific localization of AQPs to endothelial and epithelial cells suggests water movement between the air space and the interstitial and capillary compartments. It takes place through individual water channels. The mRNA and protein expression of AQPs appears late in gestation, increases markedly at birth, and remains elevated during early postnatal life (9, 10). This developmental pattern of AQP expression parallels perinatal changes in the osmotic water permeability of the lung and the rate of lung water removal that occurs around birth (11).

In addition to active ion transport–driven AQP-mediated water transport, the physical state of tissue water is also an important determinant of perinatal lung water clearance. Namely, a fraction of tissue water is motionally constrained, i.e. bound to macromolecules and thus made unavailable for immediate transport across alveolar, microvascular, and airway barriers (12).

H1-NMR relaxation measurement is a unique method of investigating the physical state of tissue water compartments of the fetal lung even without changes in the total water content of the lung. To our knowledge, the contribution of motionally distinct water fractions to the maturity-related alterations in the fetal and neonatal lung water metabolism has not yet been characterized. By applying the H1-NMR technique, the behavior of water molecules, i.e. the water–macromolecular interaction, can be assessed quantitatively, and information can be obtained about the dynamics of the reduction in fetal lung water (13).

HA, the major macromolecule that limits water movements, has a great number of hydrophilic residues that bind water and form viscous hydrated gels, and it also has the ability to immobilize water molecules by osmotic forces (14–17). As a large amount of HA has been detected in fetal and neonatal tissues, it was considered of importance to investigate the role of HA in the control of lung tissue water during the perinatal period.

The present study, using in vitro H1-NMR relaxometry, was therefore performed to determine simultaneously total lung water, the motionally distinct water fractions, and HA concentration in lungs of preterm and term rabbit pups. Attempts were also made to establish the relationship of HA to total lung water and also to the motionally constrained bound water fraction.

METHODS

Experiments were performed in fetal Pannon white rabbit pups at gestational ages of 25 (n = 17), 27 (n = 14), 29 (n = 17), and 31 d (n = 12). An additional group of 18 full-term (31 d) newborn rabbits was studied at a postnatal age of 4 d. These animals were nursed by their mothers and suckled ad libitum. The rabbit pups were born of does with known conception times by elective cesarean section under epidural anesthesia with 1.5–2.0 mL of 0.125% bupivacaine after pretreatment with 15–20 mg/kg ketamine.

Experimental procedures.

Immediately after delivery, the animals were killed by an overdose of s.c. pentobarbital, and the lungs were removed as quickly as possible. Lung samples were separated by lobes and used to determine the tissue water content, NMR relaxation times, and HA concentration. To prevent influence of postmortem changes in water compartments, NMR measurements were made promptly after killing the animals, and samples for HA determinations were kept frozen at −70o until analyzed.

HA determination.

The lung tissue was weighed and frozen and then freeze-dried for 74 h. The dried lung was reweighed, and the water content was calculated as a wet to dry weight ratio. The dried lung tissue was then digested with pronase (protease p-5147, Streptomyces griseus, Sigma Chemical Co., St. Louis, MO, U.S.A.) in a 2.5 U/mL buffer solution [0.05 M Tris (hydroxymethyl) aminomethane, 0.01 M CaCl2, pH 7.2] at 55°C overnight (>16 h). One unit of enzyme was added for each 10 mg of dry lung material. The digestion was terminated by heating to 100°C for 10 min in a water bath. The HA content was determined with a radiometric assay kit [HA 50, Pharmacia, Uppsala, Sweden; (18)].

Magnetic resonance spectroscopy.

Tissue samples of approximately 200 mg were placed in 5-mm-diameter NMR glass tubes and incubated at 40°C for 5 min to reach thermal equilibrium with the magnet temperature. MRS was performed on a Bruker Minispec PC 140 portable MR spectroscope (Bruker Co., Karlsruhe, Germany) operating at 0.96 T (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 × T1. T2 relaxation time was obtained by using the Carr-Purcell-Meiboom-Gill sequence; 1000 echoes with an echo time of 1 ms was applied. Each point was the average of five measurements. The data were transferred to the PC for storage and analysis.

Mathematical analysis.

To approximate tissue water fractions quantitatively according to their mobility, multicomponent analysis of the T2 relaxation decay curves was applied by a nonlinear least-squares method (19). 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 tissue studied there are water compartments with different rates of relaxation and that these compartments are not interdependent at the time of the measurements. The multiexponential nature of the T2 decay curve indicates that protons of water molecules do not undergo rapid exchange between the compartments. In contrast, when water proton relaxation follows a pattern of monoexponential decay, there is a rapid exchange of protons between tissue water and macromolecules, proving that the water compartments are interdependent (20, 21).

We applied biexponential fitting for estimation of the bound and free water compartments. Two components of the T2 relaxation curve were derived from the following expression:MATH

where F is the function of T2 relaxation, k1 and k2 represent the relative contributions of the two sets of protons, and T21 and T22 are the relaxation times of the different components.

Statistical analysis.

Data are expressed as mean ± SD of the mean. For statistical analysis, the Mann-Whitney test and least-squares linear regression analysis were used. The study was approved by the Institutional Ethics Committee on Animal Experiments.

RESULTS

Lung water expressed as wet-to-dry weight ratio did not change with advancing gestation from 25 d (8.9 ± 0.5) to 29 d (8.8 ± 0.4). It then started to decline, reaching 7.7 ± 0.7 (p < 0.001) at 31 d, and this was followed by a marked fall to 5.9 ± 0.3 (p < 0.001) at 4 d postnatally. The trends and time courses of T1 and T2 relaxation times proved to be quite similar; there was a transient rise from their initial values of 1300 ± 100 ms and 191.5 ± 25.8 ms (25 d) to 1430 ± 80 ms (p < 0.01) and 248.5 ± 19.2 ms (p < 0.001), respectively, at 27 d of gestation. Later on, a steady decline occurred in both values, but the extent and rate of the decline appeared to be more pronounced for T2 than for T1 relaxation time (10%versus 33% at a gestational age of 31 d and 28%versus 56% 4 d after birth at term).

Using biexponential analysis of the T2 relaxation curves, distinction could be made between their fast (T21) and slow (T22) components that corresponded to the bound and free water fractions, respectively. Both T21 and T22 increased initially up to the 27th day of gestation and then decreased progressively during the rest of the age period studied. As a result, the relative contribution of the bound water fraction represented by T21 amounted to 31% to 34% of the total lung water in the period between 25 and 29 d of gestation, decreased significantly before delivery (21.6 ± 8.0% at 31 d;p < 0.01), and decreased even further (16.6 ± 9.4%) until a postnatal age of 4 d after birth at term (p < 0.001). The maturity-related changes in the HA concentration in the immature lung are also shown in Table 1. The highest HA concentrations were observed in the most immature animals, and with advancing gestational age, there was a progressive reduction from 870.8 ± 205.2 μg/g dry weight at 25 d to 162.6 ± 32.4 μg/g dry weight at 31 d of gestation (p < 0.001). It is of interest that the HA concentration increased 2-fold to 347.3 ± 708 μg/g dry weight postnatally (p < 0.001) in spite of the concomitant abrupt decrease in lung tissue water, especially in the bound fraction. (Table 1).

Table 1 Lung water and HA content, T1 and T2 relaxation times, and the fast (T21) and slow (T22) components of T2 relaxation time in fetal and neonatal rabbit pups (mean ± SD) *p < 0.01. †p < 0.001.
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To analyze the influence of total lung water on the relaxation times and the components of T2 relaxation, the lung water content was studied as a function of T1 and T2 relaxation times and also of the fast and slow components of T2 relaxation. Lung water was found to correlate positively with T1 (r = 0.87, p < 0.001) and T2 relaxation times (r = 0.93, p < 0.001) and also with the T2-derived bound water fraction (r = 0.67, p < 0.001;Fig 1). As shown in Figure 2, the HA concentration correlated with the lung water content (r = 0.39, p < 0.001) but not with the T1 (r = 0.15, p > 0.05) or T2 (r = 0.22, p > 0.05) relaxation times. The fast component of T2, representing the bound water fraction, also proved to be independent of lung HA (r = 0.24, p > 0.05).

Figure 1
figure 1

Relationship of lung water content to T1 relaxation time, T2 relaxation time, and the fast component of T2 relaxation time in preterm and term rabbit pups studied at birth and in term rabbit pups studied at 4 d postnatally.

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Figure 2
figure 2

Relationship of lung HA concentration to brain water content, T1 and T2 relaxation times, and the fast component of T2 relaxation time in preterm and term rabbit pups studied at birth and in term rabbit pups studied at 4 d postnatally.

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When the association of tissue HA with lung water characteristics determined by H1-NMR relaxometry was analyzed separately at different study ages, again no significant relationship was found.

DISCUSSION

The present study confirms previous observations that the removal of fluids from the fetal lung begins before birth to permit effective pulmonary gas exchange in postnatal life. Furthermore, our results provide new insight into the changes in physical properties of lung water by demonstrating quantitatively a perinatal reduction in the bound fractions of lung tissue water and a simultaneous increase in the free fractions. Additionally, this study has shown a significant positive correlation between lung water content and tissue HA concentration, but has failed to demonstrate an association of HA with the motionally constrained bound water fraction. These data suggest a macromolecule-related reorganization of lung water in which process HA is not the only factor that may play a role.

H1-NMR relaxation measurements have already been applied to analyze the physical state of tissue water in experimental lung injury induced by oxygen, endotoxin, irradiation, and norepinephrine administration. Lung edema formation has been found to be associated with prolongation of T1 and T2 relaxation times (22–25).

T1 relaxation time is assumed to be related to the tissue water content, whereas T2 relaxation time is mainly influenced by water–macromolecular interaction (26). Changes in T2 relaxation therefore suggest an alteration in the free and bound fractions of lung tissue water.

In a very recent study using time domain reflectometry, Miura et al. (27) investigated the dynamic structure of tissue water in normal lungs and in pulmonary edema induced by oleic acid in rats. They found that free water accounted for 59% of the total tissue water in the normal lung and that there was an increase in free water to 76% in pulmonary edema. Interestingly, free water in the lung tissue had the same physical properties as pure water when assessed by relaxation time measurements.

In our H1-NMR study, decomposition of T2 relaxation curve into components with fast and slow relaxing rates yielded a reliable estimate of the free water fraction. The relative contribution of free water to the total lung water was smaller than that to the brain water (28), but considerably larger than the relative contributions to the water contents of the skin, skeletal muscle, and liver in newborn rabbits (12).

An important finding in our experiment is that the maturity-related reduction in the lung water is accompanied by a paradoxical increase in the free water fraction from 67% at 25–29 d of gestation to 78% at 31 d and 83% at a postnatal age of 4 d after birth at term. The increased free water in the face of the physiologic dehydration of the lung tissue can be interpreted as indicating reorganization of lung water, possibly as a result of quantitative or qualitative changes in tissue macromolecules.

Our experimental model does not allow anatomic localization of tissue water fractions and does not reveal the underlying mechanism of fetal lung water elimination. H1-NMR investigation of oleic acid–induced pulmonary edema with intraalveolar exudates, however, has shown significant prolongation of both the fast and slow components of the T2 decay curves, which has been claimed to be accounted for by cellular, interstitial, and alveolar fluid accumulation (29). Fetal lung dehydration, on the other hand, is associated with shortening of both components of T2 relaxation, with a more pronounced reduction in the fast component. This observation can be interpreted as indicating that water molecules are first liberated from the tissue store and set free for subsequent transport across the pulmonary membranes irrespective of their tissue localization. Furthermore, the marked postnatal rise in the free water fraction is accompanied by a simultaneous increase in pulmonary blood flow, and intravascular water has been shown to contribute to the augmentation of the slow component of T2 in the lung (29).

HA has been claimed to be the major macromolecular compound controlling water mobility and water balance in the lung. Hällgren et al. (30) observed accumulation of HA in the small airways in patients with adult respiratory distress syndrome and proposed that it contributed to water immobilization and generation of interstitial and alveolar edema. In agreement with this contention, a strong positive correlation has been reported between tissue HA and the water content of the lung (31, 32).

During fetal and neonatal life, higher HA concentrations have been found in the lung tissue of preterm rabbits (33) and preterm infants (34) than in rabbits and infants born at term, and the role of HA as a determinant of the tissue water content during pulmonary adaptation has been established. In this context, it is worthy to note that an increased lung HA concentration and water content have been demonstrated in neonatal respiratory distress syndrome (35, 36) and in preterm and term rabbit pups during exposure to oxygen breathing (37).

The developmental changes in lung HA observed in this study are almost identical to those described by Allen et al. (33) and seem to support their conclusion that, during gestation, the major function of HA is to facilitate morphogenesis, whereas in early postnatal life it is involved in the regulation of lung fluid balance. However, the apparent dissociation of tissue HA and lung water, in particular the bound fraction of tissue water, argues against this conclusion. To reconcile these seemingly conflicting contentions, one may assume that during the perinatal period HA may undergo still only partly defined molecular alterations related to its water-binding capacity. These may include maturity-related changes in HA molecular size (38), changes in its electrical charge, variations in its conformation with subsequent (un)covering of active, polar sites of the molecular surface (39), and also hormonally regulated enzymatic degradation (40, 41). It is possible, furthermore, that the relative importance of other macromolecules in reorganizing lung water becomes more significant. In this context, it is of interest that the intermittent mechanical strain that may be associated with fetal breathing movements differentially regulates gene and protein expression of extracellular matrix molecules (laminin, procollagen I, collagen type I and IV, biglycan, metalloproteinases 1, 2, and 3, and tissue inhibitor of metalloproteinase) in fetal lung cells (42).

In conclusion, the physiologic lung dehydration that occurs during late fetal and early postnatal life is a complex process, one which involves water movements that take place through specific epithelial and endothelial channels and are driven by active ion transport and generation of free water. Our H1-NMR relaxation study has demonstrated that elimination of lung fluid is associated with an increase in free water at the expense of the bound water fraction. The underlying mechanisms of the release of water molecules from macromolecular bindings remain to be established. HA does not appear to be directly involved in this process.