INTRODUCTION

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There is evidence to suggest that there may be delays of fetal lung maturation associated with congenital diaphragmatic hernia (CDH) (1). We have reported that there are low amniotic fluid concentrations of surfactant protein A (SP-A) and saturated phosphatidylcholine (Sat PC) in human pregnancies with fetuses in which CDH was diagnosed (4). However, these findings may reflect quantitative abnormalities of surfactant secondary to fetal lung hypoplasia, or changes in amniotic fluid volume.

Nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether) is a herbicide that can cause fetal lung hypoplasia and diaphragmatic defects when fed to pregnant rats on Days 9 or 10 of gestation (5). Several investigators have reported decreases of total phospholipids and Sat PC in the lungs of Nitrofen-exposed rat fetuses with CDH (6, 7). In one of these studies no changes of the expression of surfactant proteins between control fetuses and those with CDH were detected, although only one gestational age was examined, and actual quantitative assessments of those proteins were not performed (6).

In the present study, we used this rat model to test the hypothesis that the expression of SP-A is altered in lungs from fetuses with CDH. In the rat, SP-A is initially detectable around 18 d of gestation and continues to increase until term (8). For this reason, we studied several time points during late gestation of the rat.

	METHODS

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The study protocol was approved by the institutional review board for animal research at the University of Texas Southwestern Medical Center. Timed-pregnant Sprague-Dawley rats received orogastrically on Day 9 of pregnancy either 100 mg of Nitrofen (Rohm and Haas Co., Spring House, PA) suspended in 1 ml of olive oil, or 1 ml of olive oil only, under light ether anesthesia (9). The day following overnight mating was designated as Day 0 (Day 22 = term). The fetuses were delivered by hysterotomy on Days 19, 20, 21, and 22 of gestation and were examined under a dissecting microscope for the presence or absence of CDH. Three groups were formed: a "CDH group," consisting of fetuses with CDH; a "NO CDH group," which included fetuses exposed to Nitrofen, but without CDH; and a "control group" of fetuses whose mothers received olive oil only. Apical sections of the lungs were used for immunocytochemistry for SP-A. The remaining portions of the lungs were rapidly frozen in liquid N₂ an stored at 80° C until used for other determinations. Total protein was determined by the method of Lowry and coworkers (10).

Immunocytochemistry for SP-A

Formalin-fixed, paraffin-embedded sections of the fetal lungs were deparaffinized, rehydrated, and incubated at room temperature with rabbit polyclonal antiserum against human SP-A and then with biotinylated secondary antibody and alkaline-phosphatase-coagulated streptavidin (Biogenex, San Ramon, CA). Negative controls were run concurrently. The quantity and distribution of immunoreactivity for SP-A were qualitatively estimated by two independent observers (L.R.M., M.A.J.) who were blinded to group assignment and gestational age.

Immunoblot for SP-A

The relative amounts of SP-A in the fetal lung samples were analyzed using a rabbit antihuman SP-A polyclonal antibody followed by peroxidase-conjugated goat antirabbit IgG antibody binding as previously described (11). Detection of the relative amounts of SP-A was visualized by enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). Quantitation was performed using a computerized densitometer (Molecular Dynamics).

Northern Analysis for SP-A

Total RNA was obtained from the fetal lung homogenates using a single extraction step with an acid guanidinium thiocyanate-phenol-chloroform mixture. Northern analysis for SP-A was performed as previously described by Boggaram and colleagues (12) using a ³²P-labeled cDNA insert for human SP-A (gift of Dr. Carole Mendelson, UT Southwestern Medical Center at Dallas). The blots were subjected to autoradiography using intensifying screens, followed by densitometric measurements for quantification of mRNA. A probe for 18s RNA was used to correct for the amount of total RNA loaded.

Statistical Analysis

Comparisons between groups were done by analysis of variance. Repeated-measures analyses of variance were used to compare changes within each group over time. A p < 0.05 was considered significant. For all determinations fetal lung samples from a minimum of three to four separate litters were included.

	RESULTS

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Fifty-eight percent of fetuses exposed to Nitrofen had diaphragmatic hernias, whereas none of those exposed to olive oil only had this defect.

Immunocytochemistry for SP-A

This was performed on 96 sections of lungs from fetuses at 19 to 22 d of gestation. Trace staining for SP-A was detectable at 20 d of gestation lining the distal air-space lumens of lungs from the three groups, with no distinct differences between them. With advancing gestation, more abundant immunoprecipitates were evident lining a greater number of air spaces. Alveolar epithelium showed some granular cytoplasmic staining. In three of six litters at 22 d of gestation, less immunostaining for SP-A was present in the lungs of fetuses with CDH than in NO CDH littermates or control animals (Figure 1).

View larger version (154K): [in this window] [in a new window]   Figure 1.   Immunocytochemisry staining for SP-A (dark pink areas) is seen lining the surface of the air spaces. Note increased staining (arrows) in lung tissue from a term fetus without CDH (A) compared with a littermate with CDH (B).

Immunoblot and Northern Analysis for SP-A

SP-A was barely detectable by immunoblotting prior to Day 19 of gestation. Small but measurable amounts of SP-A were detected at this point of gestation, with no differences between groups. There was a severalfold increase in SP-A by Day 21 of gestation in the three groups of fetuses, but the relative amount of SP-A in lungs from fetuses with CDH was significantly lower than in the other groups (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 2.   Immunoblot for SP-A using a rabbit antihuman SP-A antibody in lung tissue from 21-d fetuses from the control (CON) and Nitrofen-exposed groups with CDH (CDH) or without CDH (NO CDH) (A). Cumulative results for four different experiments are shown as percent of the control values on Day 19 (B). Similar amounts of total protein were loaded per each group and time point. There was a significant decrease of the relative amount of SP-A in lungs from fetuses with CDH at 21 d of gestation.

A very small amount of mRNA for SP-A could be detected on Day 19 of gestation in fetal lungs from all groups. The relative amount of mRNA for SP-A increased severalfold on subsequent days in all groups. On Day 20 of gestation there were decreased levels of mRNA for SP-A in lungs from Nitrofen-exposed rat fetuses with and without CDH relative to the control fetuses (Figure 3). There was great variability in mRNA levels on Day 21, but no differences between groups were found.

View larger version (36K): [in this window] [in a new window]   Figure 3.   Northern blotting for SP-A using a cDNA for human SP-A in lung tissue from fetuses from the control (CON) and Nitrofen-exposed groups with CDH (CDH) or without CDH (NO CDH). In the CDH groups: LL = left lung and RL = right lung from fetuses with a left CDH (A). Cumulative results of four different experiments are shown as percent of values from the control group on Day 20 of gestation corrected for total 18S RNA (B). There were decreased mRNA levels for SP-A in Nitrofen-exposed fetuses with and without CDH at 20 d of gestation. No significant differences were found on Day 21.

	DISCUSSION

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We used the Nitrofen-induced CDH rat model to investigate whether the expression of SP-A is altered in the presence of a CDH. We focused on this protein since it reaches its maximal expression during late gestation, and we had previously detected quantitative abnormalities of SP-A in amniotic fluid from human pregnancies with fetuses in which CDH had been diagnosed (4, 8). Because most previous reports using this rat model studied only one point in gestation, we sought to compare several measurements of SP-A over a much broader period of late gestation (3, 6, 13).

Our findings show that the expression of SP-A is decreased in the presence of a CDH in this rat model. Using a primarily qualitative technique such as immunocytochemistry, decreases of SP-A were observed in fetuses with CDH only at term, compared with those exposed to Nitrofen without CDH or with the control group. However, when we utilized more quantitative methods such as immunoblotting, lower amounts of SP-A in the CDH group were detectable on Day 21 of gestation. This correlates with the lower expression of the mRNA for SP-A found on Day 20 of gestation in fetal rats exposed to Nitrofen with and without CDH. These findings suggest that alterations in the expression of SP-A in rat fetuses with CDH become more marked when the normal process of lung maturation ensues during late gestation. Furthermore, these abnormalities may be quite variable just like our findings in amniotic fluid from human pregnancies with fetuses with CDH (4). Prior to our study there was very limited information on SP-A or other surfactant proteins in models of CDH. Wilcox and colleagues (3) reported decreased amounts of SP-A in lung lavage from term fetal lambs with surgically induced CDH. Investigators from the same group suggested that those abnormalities may reflect impairments of the post-translational processing of surfactant proteins (14). However, only term fetal lambs were studied. Suen and colleagues (6) determined the expression of SP-A, SP-B, and SP-C in rat fetuses exposed to Nitrofen with and without CDH after prenatal dexamethasone therapy, but they did not report the developmental changes of any of these proteins compared with fetal rats not exposed to Nitrofen. Moreover, actual measurements of surfactant proteins were not performed. These investigators were unable to identify changes in mRNA for endothelial nitric oxide synthase between fetuses with and without CDH, whereas we have reported marked differences in the expression of this enzyme in lung tissue from rat fetuses with and without CDH (9). Brandsma and colleagues (13) did not find abnormalities of SP-A in bronchoalveolar lavage of fetal rats with CDH induced by Nitrofen using a dot blot assay. However, they examined only one gestational age and analyzed a surfactant pellet fraction normalized for the amount of phospholipids. Because surfactant phospholipids are also decreased in this model, lower amounts of SP-A may have gone undetected.

The mechanisms responsible for a decreased expression of SP-A in fetal rats with CDH are unclear. However, these may relate at least in part to the chronic compression of the lungs, which may limit the growth-promoting stimulus of lung stretch and fetal breathing movements (15). Nitrofen interferes with receptor binding of triiodothyronine, but this hormone does not seem to play a major role in stimulation of the expression of SP-A in the developing lung (16). Although we did not use either antibodies or a cDNA specific for rat SP-A, the developmental changes of SP-A and its mRNA that we observed in our control fetuses are in agreement with previous work by other investigators using specific probes for rat SP-A (8). Moreover, there is a substantial homology between SP-A and its genetic sequence from different species (17).

In summary, we found a decreased expression of SP-A in the lungs of fetuses with Nitrofen-induced CDH. This finding may explain the amniotic fluid abnormalities of SP-A previously reported by us. A deficiency of surfactant components may play a role in the pathophysiology of CDH.