Abstract
Antenatal glucocorticoids are thought to be less effective when delivery occurs more than 7 d after initiation of treatment; therefore, repeat courses are often administered. We examined lung structure after single or repetitive antenatal glucocorticoid injections in fetal sheep. Pregnant ewes received single or repetitive doses of 0.5 mg/kg betamethasone at 7-d intervals by maternal or fetal injection, beginning at D104 or D114 with delivery at D125, D135, or D146 gestation (term = 150 d). Changes in lung structure were more pronounced after repetitive versus single injections. Repetitive fetal or maternal injections beginning at D104 (delivery at D125) resulted in comparable structural changes: alveolar volume increased by 50 to 80%, alveolar numerical density decreased by 30 to 40%, and pleural and interlobular septal volumes decreased by as much as 70%. Similar changes were seen in animals delivered at D135 after repetitive maternal injections beginning at D114. There were no structural differences between control and repetitive betamethasone animals when delivery was delayed until D146, indicating that betamethasone induced structural changes were reversible.
Antenatal glucocorticoids have maturational effects on the fetal lung, having been shown to promote an improvement in lung compliance in experimental animals (1, 2) and to reduce the incidence and severity of respiratory distress syndrome in preterm infants (3). It is common clinical practice to administer repeat courses of antenatal glucocorticoids when delivery is delayed by 7 d or more (4), although it is not known whether repetitive treatments offer additional benefit over single exposures. This practice stems from clinical studies in which infants delivered 7 days or more after initiation of treatment did not appear to benefit (5, 6).
In a previous study in preterm sheep, we found that repetitive maternal injections of betamethasone beginning at Day 104 of gestation (D104) (term = 150 d) did indeed offer additional benefit over a single exposure, however, sequential treatments were associated with progressive fetal growth retardation (7). This finding was at odds with an earlier study in which we found no evidence of growth retardation when fetuses received two fetal injections 6 d apart, beginning at D121 (80%) (8). More recently, we examined whether growth retardation may depend on either the route of administration (maternal versus fetal) or to the timing of initiation of treatment (9). We found that multiple fetal treatments did not inhibit fetal growth and did not improve lung function to the same extent as did multiple maternal treatments. Inhibition of fetal growth also occurred when maternal treatment was initiated later in gestation. The effect on fetal growth, but not the effect on lung function, persisted when delivery was delayed until near term. The improvement in lung function coincided with a large increase in both alveolar and lung tissue surfactant, indicating a potent effect of repetitive betamethasone treatment on the surfactant system. Previous studies in rats (10) and in nonhuman primates (11) have shown that repetitive daily exposures to glucocorticoids can lead to changes in lung structure that persist after cessation of treatment. We examined whether repetitive exposure at a more clinically relevant interval might also cause marked changes in lung structure. The physiologic and surfactant data from these animals were reported elsewhere (9). We describe the impact of repetitive weekly betamethasone treatment on lung structure.
Protocols were approved by the Animal Ethics Committees at the Children's Hospital Medical Center in Cincinnati and the Western Australian Department of Agriculture. Ewes were randomized to one of three protocols (9). Protocol 1 compared fetal and maternal treatment. All ewes were weighed and received 150 mg medroxyprogesterone acetate (kindly donated by Pharmacia and Upjohn, Rydalmere, Australia) by intramuscular injection at D98. Beginning at D104 each animal received a total of three injections at 7-d intervals. Maternal treatment was by intramuscular injection and dosing was based on maternal weight at D98. One group of ewes received 0.5 mg/kg betamethasone intramuscularly (Celestone Chronodose; Schering, NSW, Australia) at D104, D111, and D118 (three dose maternal), a second group received betamethasone at D104 and saline at D111 and D118 (one dose maternal), and a third group received saline at each time point (control). The remaining animals in this protocol received fetal treatment by ultrasound-guided intramuscular injection, and dosing was based on estimated fetal weights of 1.4 kg at D104, 1.9 kg at D111, and 2.2 kg at D118 (2). The three-dose fetal group received betamethasone at D104, D111, and D118, the one-dose fetal group received betamethasone at D104 and saline at D111 and D118, and the control group received saline at each time point. All animals were delivered at D125 (term = 150 d).
Protocol 2 evaluated whether the maturational and growth retardation effects occurred when treatment was initiated 10 d later and delivery occurred at D135. Ewes were weighed at D98 and received 150 mg medroxyprogesterone at D108. The three-dose group received maternal betamethasone intramuscularly at D114, D121, and D128, the one-dose group received betamethasone at D114 and saline at D121 and D128, and the control group received saline at each time point.
Protocol 3 examined whether the lung maturation and growth retardation caused by repetitive treatment early in gestation persisted when delivery was delayed until near term. Ewes were randomized to receive three doses intramuscularly of either betamethasone (three-dose maternal) or saline (control) at D104, D111, and D118. All fetuses were delivered at D146.
Fetuses were delivered by cesarean section and mechanically ventilated for 40 min to assess lung function, using a ventilation strategy designed to optimize ventilation and minimize the risk of ventilator-induced injury (2). Lung function and surfactant data for this group of animals have been reported elsewhere (9); however, a brief summary has been included for easy reference (Table 1).
| Gestational Age | Treatment | n | BW (kg) | PaCO2 (mm Hg) | Compliance (ml/cm H2O·kg) | V40(ml/kg) | Alveolar SatPC (μmol/kg) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 125 | Control (F/M) | 12 | 2.7 ± 0.1 | 85 ± 6 | 0.204 ± 0.018 | 13 ± 1 | 0.57 ± 0.24 | |||||||
| 1-dose (F) | 12 | 2.6 ± 0.1 | 86 ± 11 | 0.218 ± 0.029 | 20 ± 5 | 0.65 ± 0.19 | ||||||||
| 3-dose (F) | 12 | 2.6 ± 0.1 | 70 ± 7 | 0.271 ± 0.036 | 28 ± 7 | 1.22 ± 0.35 | ||||||||
| 1-dose (M) | 10 | 2.1 ± 0.2† | 71 ± 5 | 0.285 ± 0.022 | 24 ± 6 | 1.93 ± 0.46† | ||||||||
| 3-dose (M) | 10 | 2.1 ± 0.1† | 55 ± 4† | 0.399 ± 0.017† | 46 ± 4† | 5.7 ± 0.92† | ||||||||
| 135 | Control (M) | 11 | 3.5 ± 0.1† | 50 ± 2† | 0.338 ± 0.028† | 34 ± 5† | 2.6 ± 0.6† | |||||||
| 1-dose (M) | 10 | 3.1 ± 0.1 | 53 ± 3 | 0.417 ± 0.028 | 45 ± 5 | 6.4 ± 1.0‡ | ||||||||
| 3-dose (M) | 11 | 2.6 ± 0.1‡ | 45 ± 2 | 0.496 ± 0.037‡ | 72 ± 5‡ | 17.9 ± 2.4‡ | ||||||||
| 146 | Control (M) | 10 | 4.8 ± 0.2‡,† | 55 ± 3† | 0.532 ± 0.039‡,† | 73 ± 6‡,† | 28.0 ± 3.7‡,† | |||||||
| 3-dose (M) | 9 | 3.4 ± 0.2§ | 50 ± 3 | 0.710 ± 0.109 | 73 ± 4 | 28.1 ± 3.7 |
Preparation and sampling. As lung maturation is known to vary between regions of the lung (12, 13), all morphometric assessments were performed on the right cranial lobe. Lung samples were fixed overnight via bronchial instillation of 10% phosphate-buffered formalin at a distending pressure of 30 cm H2O. Morphometric assessments were performed blind. Measurements were made on all animals (n = 9 to 12 animals per group). Fixed lobe volume (Vlobe) was measured by volume displacement (14). Each lobe was then cut into 5-mm serial slices and three slices were randomly chosen for morphometric examination (15). Measurements were made on three 5-μm sections stained with Hematoxylin-eosin per lobe.
Volumes and volume fractions. Stained sections were enlarged and printed onto photographic paper at a linear magnification X16. Volume fraction of lung parenchyma (VVpar = alveoli and alveolar ducts), nonparenchyma (VVnp = conducting airways plus blood vessels), interlobular septa (VVils = interstitial tissue forming the distinct lobulation of the lungs), and pleura (VVpl) were estimated by superimposing a cycloid point counting grid (72 lines/144 points) onto each of the photographic images. Volume fraction is equal to Pi/Pt, where Pi represents the number of test points hitting the structure of interest (e.g., parenchyma) and Pt is the total number of points hitting the reference space (total of all compartments). Parenchymal volume per lobe (Vpar) was derived from fixed lobe volume as follows: Vpar = VVpar · Vlobe. Nonparenchymal (Vnp), pleural (Vpl), and interlobular septal (Vils) volumes were similarly estimated from the relevant volume fractions.
Parenchymal morphometry. Digitized images from 10 nonoverlapping parenchymal fields were captured from each 5-μm section using a Sony 3CCD Color video camera interfaced with a Leica DMLS microscope and a Macintosh 8100/80AV computer. Images were examined at a final magnification ×950. The number of points that fell on air space and on alveolar septal tissue and the number of air/tissue tissue/air intercepts were counted by superimposing a linear point-counting grid (21 lines/42 points). The surface area of alveoli and alveolar ducts per unit volume of alveolar tissue (surface fraction, SV) was determined using the formula SV = 2I0/Lr where I0 is the number of intercepts with the air tissue interface and Lr is the length of the test line within the reference volume. Total alveolar surface area (Salv) of the right cranial lobe was estimated by multiplying SV by Vpar. Alveolar wall thickness was determined as volume per unit area of alveolar surface according to the formula VVaw/SV, where VVaw is the volume fraction of alveolar wall tissue. Alveolar numerical density (number per unit volume, NV) was calculated according to the method of Weibel (16) using the equation:
where NA = number of alveoli per unit area and VVas = volume fraction of alveolar air space, both determined on digitized images (Mag ×950), B = shape constant describing alveolar shape (1.55) and D = distribution variable of the characteristic linear dimension of the alveoli, taken to equal 1. In transverse section, alveoli were identified as those structures opening onto a common air space (alveolar duct). In cross section, alveoli were identified on the basis of size, shape, and morphology. Ambiguous structures were rejected. Total number of alveoli (NT) in the right cranial lobe was calculated by multiplying Vpar by NV. Average alveolar volume (VA) was calculated by dividing total alveolar volume [Vpar · VVas] by total number of alveoli.
Changes with gestational age were examined in control animals using one-way analysis of variance. When the difference was found to be statistically significant, post-hoc pairwise comparisons were performed using the Tukey multiple comparison test. The effect of betamethasone treatment was separately examined at each gestational age by one-way ANOVA. When the difference between treatment groups was significant, post-hoc pairwise comparisons between the control group and individual treatment groups was performed using Dunnett's multiple comparison test. Statistical significance was accepted at p < 0.05.
In those animals delivered at D125, repetitive maternal betamethasone treatment led to a dramatic improvement in postnatal lung function and a 10-fold increase in alveolar surfactant, but birth weight decreased by more than 20% (Table 1). A single maternal injection also decreased birth weight by around 20%; however, the effects on lung function and surfactant pool size were less striking. Animals exposed to repetitive fetal betamethasone injections showed functional improvements similar to those exposed to a single maternal injection. A single fetal betamethasone injection at D104 had no effect on lung function or surfactant pool size. Findings were similar for D135 animals: repetitive treatment retarded fetal growth and led to more pronounced improvements in lung function than single treatment. The effect of repetitive maternal treatment on fetal growth, but not on lung function or surfactant, was still apparent when animals were delivered near term.
Lobar volumes. Fixed lobe volume (Vlobe) increased 2.5-fold between D121 and D146 (p < 0.001) (Table 2). Parenchymal (Vpar), nonparenchymal (Vnp), and pleural volumes (Vpl) also increased with advancing gestation. In contrast, interlobular septal volume (Vils) decreased between D135 and D146 (p < 0.05). When normalized to birth weight both Vlobe and Vpar increased progressively with advancing gestation, whereas Vils and Vpl decreased (data not shown).
| Gestational Age | Treatment | Vlobe(ml ) | Vpar(ml ) | Vnp(ml ) | Vils(ml ) | Vpl(ml ) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 125 | Control (F/M) | 17.1 ± 1.1 | 12.4 ± 1.1 | 2.8 ± 0.21 | 1.5 ± 0.14 | 0.36 ± 0.06 | ||||||
| 1-dose (F) | 18.0 ± 1.1 | 13.3 ± 1.2 | 2.7 ± 0.17 | 1.5 ± 0.23 | 0.42 ± 0.07 | |||||||
| 3-dose (F) | 19.2 ± 1.7 | 15.6 ± 1.6 | 2.9 ± 0.29 | 0.96 ± 0.13* | 0.34 ± 0.04 | |||||||
| 1-dose (M) | 14.9 ± 1.0 | 10.9 ± 0.8 | 2.4 ± 0.20 | 1.2 ± 0.19 | 0.42 ± 0.05 | |||||||
| 3-dose (M) | 19.0 ± 1.4 | 15.8 ± 1.3 | 2.6 ± 0.13 | 0.46 ± 0.09* | 0.19 ± 0.03† | |||||||
| 135 | Control (M) | 27.2 ± 2.3† | 21.2 ± 1.9† | 4.2 ± 0.39† | 1.2 ± 0.16 | 0.51 ± 0.10 | ||||||
| 1-dose (M) | 29.1 ± 1.7 | 23.5 ± 1.8 | 3.9 ± 0.25 | 1.3 ± 0.22 | 0.40 ± 0.06 | |||||||
| 3-dose (M) | 29.1 ± 2.1 | 24.5 ± 2.0 | 3.8 ± 0.40 | 0.54 ± 0.12‡ | 0.33 ± 0.06 | |||||||
| 146 | Control (M) | 43.8 ± 4.0‡,† | 38.2 ± 3.6‡ | 4.7 ± 0.50† | 0.48 ± 0.04‡,† | 0.45 ± 0.06 | ||||||
| 3-dose (M) | 38.1 ± 2.8 | 33.3 ± 2.1 | 4.6 ± 0.23 | 0.59 ± 0.09 | 0.39 ± 0.08 |
Parenchymal morphometry. Maturation of parenchymal architecture between D121 and D146 was characterized by thinning of alveolar walls, elongation of secondary alveolar septa, and increasing alveolar volume (Figure 1). Alveolar wall thickness decreased progressively from around 4 μm at D125 to less than 3 μm at D146 (p < 0.001) (Figure 2). Surface fraction (SV) did not change appreciably with advancing gestation; however, total surface area (Salv) increased dramatically paralleling the increase in Vpar (p < 0.001) (Figure 2). Numerical density of alveoli (NV) was similar in D125 and D135 animals, but was around 30% lower in D146 animals (Figure 3). Alveolar volume increased by 110% between D135 and D146 (p < 0.05) coinciding with a 50% increase in volume fraction of alveolar airspace. Total alveolar number increased by around 50% between D125 and D135, and by a further 25% between D135 and D146 (Figure 3).
Fig. 1. Hematoxylin-Eosin-stained 5-μm sections of lung parenchyma. Panel A: D125 control animal (#97-45-2). Panel B: D125 3-dose maternal betamethasone-treated animal (#97-40-3). Panel C: D135 control animal (#97-83-2). Panel D: D135 3-dose maternal betamethasone treated animal (#97-84-1). Panel E: D146 control animal (#97-56-3). Panel F: D146 3-dose maternal betamethasone-treated animal (#97-72-3). Gestational effects: Alveolar walls become progressively thinner with advancing gestation. Alveolar size increases between D135 and D146. Treatment effects: Repetitive maternal betamethasone led to an increase in alveolar volume and thinning of alveolar septa at D125 and an increase in alveolar volume at D135. No treatment effects were seen when animals were delivered at D146. Original magnification: ×320; bar = 50 μm.
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Fig. 2. Alveolar wall thickness and surface area. (Top panel ) Alveolar wall thickness. (Middle panel ) Surface volume fraction (SV). (Bottom panel ) Total alveolar surface area (Salv) of the right upper lobe. (F) = fetal treatment; (M) = maternal treatment. Group means with standard error bars are shown for control (open column), single dose (hatched column), and repetitive (closed column) betamethasone treatment groups. Gestational effects. Progressive decrease in alveolar wall thickness (p < 0.001) and increase in Salv (p < 0.001) with advancing gestation. Treatment effects. D125: Significant decrease in SV in 1-dose fetal betamethasone group and significant decrease in alveolar wall thickness in 3-dose maternal group. No significant differences between treated and control groups at D135 or D146; * p < 0.05 versus D125 control; ‡ p < 0.05 versus D135 control.
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Fig. 3. Alveolar number, volume, and volume fraction. (Top panel ) Alveolar numerical density. (Second panel ) Total alveolar number in the right upper lobe. (Third panel ) Average alveolar volume. (Bottom panel ) Alveolar air space volume fraction. (F) = fetal treatment; (M) = maternal treatment. Group means with standard error bars are shown for control (open column), single dose (hatched column), and repetitive (closed column) betamethasone treatment groups. Gestational effects. Increase in total alveolar number (p < 0.01), average alveolar volume (p < 0.001), and alveolar air space volume fraction (p < 0.001) and decrease in alveolar numerical density (p < 0.05) with advancing gestation. Treatment effects. D125: Decrease in alveolar numerical density and increase in alveolar volume after 3-dose maternal or fetal treatment. D135: Decreased alveolar numerical density and increased alveolar volume after repetitive treatment. D146: No treatment effect; * p < 0.05 versus D125 control; ‡ p < 0.05 versus D135 control.
[More] [Minimize]Lobar volumes. Repetitive fetal betamethasone injections led to a significant reduction in Vils, whereas repetitive maternal injections led to a decrease in both Vils and Vpl (Table 2). Vlobe, Vpar, and Vnp were unaffected by repetitive treatment. No volume changes were seen after single maternal or fetal betamethasone treatment. Both weight-corrected Vlobe and Vpar were significantly greater in the three-dose maternal betamethasone group compared with the control group, reflecting the significantly lower birth weight in this group (data not shown).
Parenchymal morphometry. The most striking change in parenchymal morphometry after repetitive betamethasone treatment was an increase in alveolar volume (Figure 1). Alveolar wall thickness and SV varied significantly between groups (p < 0.05 for each variable) (Figure 2), although there was no consistent effect of treatment on either parameter. Alveolar wall thickness decreased significantly in the three dose maternal betamethasone group, whereas SV decreased in the one-dose fetal betamethasone group compared with the control group (p < 0.05 for both variables). Salv varied between groups, but this was of borderline statistical significance only (p = 0.055). Alveolar numerical density was significantly reduced in both the three-dose maternal and three-dose fetal betamethasone groups (Figure 3). However, despite the marked decrease in NV, NT did not change significantly with betamethasone treatment. Alveolar volume increased by 50 to 80%, although VValv did not change (Figure 3).
Lobar volumes. The pattern of changes with betamethasone treatment in these animals closely resembled those in animals delivered at D125. Again Vlobe, Vpar, and Vnp were not significantly different in single or repetitive betamethasone-treated animals compared with control animals (Table 2). Both Vils and Vpl were reduced after repetitive betamethasone, although the decrease was significant only for Vils. When normalized to birth weight, both Vlobe and Vpar were significantly greater in the three-dose betamethasone group than in the control group, again reflecting the reduction in birth weight in this group (data not shown).
Parenchymal morphometry. Again the most striking effect of repetitive betamethasone injections was an increase in alveolar volume (Figure 1). There was no effect of single or repetitive betamethasone treatment on alveolar wall thickness, SV, or Salv (Figure 2). Repetitive betamethasone treatment led to a 70% increase in VA (p < 0.05) and a 30% decrease in NV (p < 0.05), but neither NT nor VVas changed (Figure 2).
There were no significant differences in lobar volumes or parenchymal morphometry between control and treated animals when delivery was delayed until D146 (Table 2 and Figures 1-3), suggesting that the structural changes observed when delivery occurred at D125 were remodeled and did not persist until term. As with animals delivered at D125 or D135, Vlobe and Vpar were significantly greater in repetitive betamethasone-treated animals than in control animals when normalized to birth weight.
This study examined changes in lung morphometry after repetitive antenatal glucocorticoid exposure in preterm sheep. We compared single versus repetitive treatment, fetal versus maternal route of administration, initiation of treatment early versus late in gestation, and residual effects on lung structure when delivery was delayed until term. In line with the pattern of changes in lung function (9), repetitive injections led to more pronounced structural changes than a single injection. Unlike the changes in lung function, however, maternal and fetal treatment routes led to comparable morphometric changes. The structural changes induced by betamethasone early in gestation appear to be reversible, as we found no evidence of morphometric differences between control and multiple-dose animals when delivery was delayed until near term. The structural changes promoted by repetitive treatment initiated at D104 (mid-canalicular stage) were similar in nature and magnitude to those induced by treatment initiated at D114 (late-canalicular/early saccular stage). The most notable change with repetitive treatment was an increase in alveolar volume. This coincided with a decrease in numerical density of alveoli, although total alveolar number did not change significantly. Repetitive treatment was also associated with a reduction in both interlobular septal and pleural volumes.
When normalized to birth weight, both fixed lobe volume (Vlobe) and parenchymal volume Vpar, increased progressively with advancing gestation; thus, during this gestational period the rate of increase in functional lung volume exceeds the rate of increase in body weight as term approaches. Both Vlobe and Vpar were also significantly greater in repetitive maternal betamethasone groups than in their control counterparts. However, in these animals, absolute Vlobe and Vpar did not change appreciably, but birth weight was significantly reduced by repetitive betamethasone injections. Clearly when treatment affects somatic growth, it may not be appropriate to normalize to birth weight. Lung weight was also significantly reduced in animals given repetitive betamethasone injections (9); therefore, this index may be inappropriate. The effect of repetitive treatment is not on lung volume per se, but rather on somatic growth, the outcome being that smaller animals are ventilated by lungs of a similar volume.
Findings from the present study, coupled with a number of previous studies by our group, suggest that glucocorticoids improve lung function both by accelerating structural maturation and by increasing production and secretion of surfactant, although the timing of these processes differ. We found that preterm sheep delivered 48 h after a single injection of betamethasone exhibit significant improvements in lung function (2, 17– 20). Alveolar septal thickness was markedly reduced at this time (21), whereas alveolar surfactant concentration was minimally affected (8, 19, 20). When delivery occurred 7 d after treatment, lung function was similarly improved (22), but at this time point there was a significant increase in surfactant pool size (8) and no apparent effect on alveolar wall thickness (Willet, unpublished observation). Similarly, fetuses exposed to two or more doses of glucocorticoids over a 3-wk period had sizeable increases in alveolar surfactant (20). These observations suggest a relatively rapid change in lung structure followed by a slower, perhaps longer-lasting, effect on the surfactant system. The predominant influence on lung function at the time of delivery therefore may vary depending on the treatment-to-delivery interval. In the present study, where delivery occurred 1 to 4 wk after cessation of treatment, influences on the surfactant system probably predominate. In support of this argument, the more pronounced improvement in lung function after maternal versus fetal betamethasone administration (9) coincides with a greater increase in alveolar surfactant pool size, but not with more pronounced structural maturation.
It is interesting to note that one of the most prominent structural differences between control and repetitive betamethasone animals occurred outside the parenchymal compartment, namely, a decrease in the volume of pleura and interlobular septa. It is not certain whether this effect occurred prenatally, or whether it relates to different rates of postnatal clearance of fetal lung liquid during mechanical ventilation. After birth, fetal lung liquid is rapidly absorbed from the alveolar lumen, temporarily pooling in the perivascular spaces, interlobular septa, and pleura before being cleared from the lung via the pulmonary circulation (23). Clearance of fetal lung liquid from the lung takes around 6 h in term lambs (24) and slightly longer in preterm animals (25). The effects of prenatal glucocorticoids on the rate of postnatal fluid clearance during mechanical ventilation are not known.
The marked reductions in pleural and interlobular septal volumes after repetitive betamethasone may also be due, at least in part, to a loss of interstitial proteins. In preterm sheep both the pleura and interlobular septa are rich in collagen fibers (26). Interlobular septal collagen accounts for around 60% of total interstitial (alveolar plus pleural plus interlobular septal) collagen and is significantly reduced in preterm sheep delivered 48 h after a single fetal injection of 0.5 mg/kg betamethasone. In contrast to collagen, around 60 to 70% of interstitial elastin is found in the alveolar region, and it is this elastin pool that is susceptible to antenatal betamethasone treatment (26). Alveolar elastin content was profoundly decreased in animals delivered at D121 and marginally increased in animals delivered at D135. Both collagen and elastin are important determinants of lung mechanics, and a change in content of either structural protein has the potential to significantly alter the mechanical behavior of the lungs (27).
Previous studies in rats and nonhuman primates have raised concerns that repetitive exposure to glucocorticoids may lead to impaired alveolarization (10, 11). Rhesus macaques exposed to triamcinolone during early gestation had delayed alveolarization (11). Rats exposed to repetitive glucocorticoids during alveolarization had precocious thinning of alveolar septa and markedly impaired septation (28, 29), an effect that appeared to be irreversible (10, 29). On the basis of these observations, it has been suggested that alveolar septation is possible only when the supporting septum is immature and that the ability to septate may be greatly diminished when exogenous glucocorticoids are administered before septation is complete (30). We found no evidence in the present study to suggest that repetitive weekly steroid injections inhibited alveolar formation. Despite a significant reduction in alveolar number per unit volume in steroid-treated animals delivered prematurely, total alveolar number remained the same. The reduction in alveolar number per unit volume probably reflected a greater degree of lung inflation during the process of fixation, as average alveolar volume was significantly increased in steroid-treated animals. Our data also suggest that repetitive steroid treatment promotes reversible changes in lung structure, as the differences we observed between control and repetitive steroid-treated animals at D125 were not seen when animals were delivered at D146. The difference between our findings and previous findings in neonatal rats and fetal macaques may relate to differences in the timing of alveolar development between the three species, to species differences in responsiveness to glucocorticoids, or to different dosing schedules (daily versus weekly treatments).
There are several potential methodologic limitations that should be considered when interpreting results from the present study. First, estimation of alveolar number from two-dimensional sections may be associated with errors relating to differences in alveolar size (31). The probability of a structure appearing in any 2-D plane is proportional to its size, such that the larger a structure the greater probability it will be encountered in the plane or image. In this way, counting is biased in favor of larger alveoli, and alveolar number may be overestimated in lungs with larger alveoli. Similarly, assumptions about alveolar shape may also lead to errors in estimates of alveolar number (16). The shape coefficient (β) relates volume to cross-sectional area, and is inversely related to ɛ , the ratio of diameter to length (height) (16). In immature (shallow) alveoli, where the ratio of diameter to height is large, β would be expected to be smaller. Using a shape coefficient devised for mature lungs in immature lungs may therefore lead to an overestimation of alveolar number.
It should also be stressed that morphometric analyses were restricted to the right cranial lobe. The cranial lobe is known to mature slightly earlier in gestation than the caudal lobe (12), although the magnitude of the discrepancy has never been carefully examined. In a recent study we demonstrated that the nature and magnitude of morphometric changes after antenatal glucocorticoid treatment is similar for fetal sheep delivered between D121 and D128. During this 7-d period the sheep lung undergoes profound structural changes, including a 40% increase in volume of lung parenchyma, a 50% increase in alveolar number, and a 25% reduction in alveolar wall thickness (26). Despite considerable differences in structural maturity in D121 and D128 lambs, the lung responds in a similar manner to antenatal glucocorticoid intervention. Structural differences between the cranial and caudal lobes during this gestational period appear to be of a lesser magnitude (32). On the basis of these observations, measurements made on the right cranial lobe would be expected to provide a reliable approximation of the pattern of changes occurring in the lung as a whole.
A final methodologic consideration is the impact of tissue shrinkage on volume estimates. Lum and Mitzner (33) estimated the degree of linear shrinkage from the unfixed condition to the stained section in adult sheep lungs, fixed at a distending pressure of 30 cm H2O, to be around 40%. The degree of shrinkage is species-dependent and may also vary with relative tissue composition (e.g., water content, degree of vascularization, and relative fixable connective tissue content) (33– 35). We did not measure tissue shrinkage in the present study, and no corrections have been applied to our reported data. Our interpretation of results is based on the assumption that linear shrinkage is comparable for different gestational and treatment groups.
We hypothesized that given the sizeable improvements in compliance and gas exchange after repetitive antenatal glucocorticoids, we might expect to find profound remodeling of lung structure. We found significant structural changes in both the parenchymal and nonparenchymal compartments, which could partly contribute to altered lung function. Our results are consistent with the idea that glucocorticoids promote functional maturation through effects on both lung structure and on the surfactant system. Furthermore, our data suggest that structural changes induced by clinically relevant, repetitive doses of betamethasone are reversible.
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