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. Author manuscript; available in PMC: 2025 Jan 25.
Published in final edited form as: Anat Rec (Hoboken). 2008 Oct;291(10):1271–1277. doi: 10.1002/ar.20743

All-trans retinoic acid and intra-amniotic endotoxin mediated effects on fetal sheep lung

BW Kramer 1,5, KH Albertine 2, TJM Moss 4, I Nitsos 4, A Ladenburger 5, CP Speer 5, JP Newnham 4, AH Jobe 3
PMCID: PMC11759063  NIHMSID: NIHMS195387  PMID: 18727105

Abstract

All-trans retinoic acid (RA) is a potent modulator of lung development. Chorioamnionitis, which is frequently associated with preterm birth, causes fetal lung inflammation and improves lung function but also results in alveolar simplification and microvascular injury. Endotoxin-mediated chorioamnionitis reduces RA concentration in the fetal lung to 16% of control values. We hypothesized that administration of RA to the fetus before induction of chorioamnionitis would preserve septation of the distal airspaces. Time-mated ewes with singletons were assigned to receive a fetal intramuscular treatment with 20,000 IU of RA in olive oil (or olive oil only) 3h prior to intra-amniotic injection of endotoxin (20 mg, E. coli 055:B5) or saline, at 124d gestational age and 7d after the fetal treatment. The right cranial lung lobe was processed for morphometric analysis. RA treatment did not affect chorioamnionitis-induced fetal and systemic inflammation or interleukin-8 concentrations in lung tissue. RA administration alone did not alter lung structure. Relative to control lungs (5±3ml/kg), lung volume increased similarly with endotoxin (22±4ml/kg) or RA plus endotoxin (20±3ml/kg; p<0.05). Alveolar wall thickness was 4.2±0.3μm after endotoxin-induced chorioamnionitis, 6.0±0.4μm in controls (p<0.05 versus endotoxin) and 5.5±0.2μm after RA and endotoxin (p<0.05 versus control, n.s. versus endotoxin). The ratio of airspace versus tissue was 4.6±0.3 in endotoxin-induced chorioamnionitis, 2.1±0.3 in controls and 4.1±0.5 after RA and endotoxin. We conclude that fetal treatment with RA did not prevent inflammation-induced alveolar simplification.

Keywords: Antenatal inflammation, alveolar development, chorioamnionitis, bronchopulmonary ysplasia

Introduction

Multiple factors contribute to the development of bronchopulmonary dysplasia (BPD) in preterm infants who are born before the onset of the alveolar stage of lung development (Speer, 2006). Chronic exposure to antenatal and/or postnatal inflammation caused by chorioamnionitis, ventilation-mediated lung injury, oxygen toxicity or sepsis may injure the lung and affect subsequent lung growth (Speer, 2006). BPD is primarily a delay in alveolar development (Husain et al., 1998) with reduced numbers of alveoli and reduced microvasculature development (Bhatt et al., 2001; Lassus et al., 2001). The development of BPD is associated with low concentrations of vitamin A in preterm babies (Shenai et al., 1985; Shenai et al., 1987; Shenai et al., 1990; Bland et al., 2003) and postnatal treatment with vitamin A (retinol) administered intramuscularly reduced the incidence of BPD by 7% in a multicenter trial (Tyson et al., 1999).

Vitamin A (retinol) and its most important metabolite, all-trans retinoic acid (RA), are modulators for the transcription of genes that are involved in both prenatal and postnatal lung development (Maden and Hind, 2004; Cho et al., 2005). RA binds to nuclear and cytosolic receptors that are expressed in the lung early in gestation and regulate postnatal growth of alveoli (Maden and Hind, 2003). The role of retinoic acid and its metabolites on alveolar septation was demonstrated in rodents after delaying alveolar development by dexamethasone (Massaro and Massaro, 1996). Addition of RA induced catch-up septation during the alveolar growth stage in the offspring of RA deprived animals. In addition, RA supplementation decreased the inhibition of postnatal alveolarisation by glucocorticoid treatment in mice (Massaro and Massaro, 1996). There also may be links between RA and inflammation in the developing lung. RA supplementation can ameliorate the septation abnormalities induced by hyperoxia in rats (Veness-Meehan et al., 2002). Perinatal overexpression of the pro-inflammatory cytokine interleukin-1 beta resulted in lung morphology similar to BPD and reduced expression of some RA receptors in mice (Bry and Lappalainen, 2006). We have used a fetal sheep model of endotoxin-induced chorioamnionitis to study fetal inflammation and its effects on the saccular and early alveolar stage of lung development (Jobe et al., 2000; Willet et al., 2000a). Induction of chorioamnionitis with a single dose of intra-amniotic endotoxin initiated a cascade of lung injury, inflammation, apoptosis, proliferation and increased expression of surfactant proteins (Kallapur et al., 2001; Kramer et al., 2001; Kramer et al., 2002; Kunzmann et al., 2007). The fetal lung clinically matured after a 7 day exposure to intra-uterine inflammation, with an increase in alveolar surfactant pool size and thinning of the alveolar walls, but alveolar septation was decreased and microvascular injury occurred (Willet et al., 2000a; Kallapur et al., 2004). This chorioamnionitis-induced lung injury resembles the histologic phenotype of mild BPD (Husain et al., 1998). We hypothesized that the administration of retinoid acid to the fetus before the initiation of chorioamnionitis would alter the inflammation-associated alveolar simplification. We gave a single dose of retinoic acid to the fetus prior to the injection of endotoxin into the amniotic fluid and we evaluated lung inflammation, morphology and maturation.

Material and Methods

Animals

Animal protocols were approved by the Cincinnati Children’s Hospital, Ohio, USA, and the Western Australian Department of Agriculture, Perth, Australia. Time mated ewes with singleton pregnancies were assigned to groups of 5 to 7 lambs. Lambs received a fetal intramuscular treatment with 20,000 IU of all-trans retinoic acid (RA; Sigma Chemicals, St. Louis MO) in olive oil in utero as previously described (Willet et al., 2000b). This dose of retinoic acid increased the level of retinol and metabolites by 2- to 3-fold but did not affect lung function or lung structure, as assessed by measurements of alveolar number, size and wall thickness (Willet et al., 2000b). To reduce the number of animals, the group with RA treatment only was not redone. Injections were given intramuscularly, using ultrasound guidance 3 hours prior to the intra-amniotic injection of endotoxin (20 mg, E. coli 055B5, Sigma, St. Louis, MO). Seven animals received RA treatment before the induction of chorioamnionitis. Six animals received vehicle before the intra-amniotic endotoxin injection. Control lambs received intra-amniotic saline and intramuscular olive-oil injections (n=5). All procedures were done at 117 days gestation and delivery was at 124 days gestational age for all three groups (term is 150 days).

Assessments of fetal inflammation

Each lamb was delivered by caesarean section and sacrificed with a lethal dose of barbiturate. Fetal blood was collected from umbilical cord vessels before the chest was opened. The trachea was cannulated and the deflation limb of a pressure volume curve was recorded of which the lung gas volume at 40 cm H2O pressure is reported (Kramer et al., 2001).

The right cranial lobe was inflation fixed at 30 cm H2O pressure with 4% formalin and used for morphometric analysis. Tissue from the right caudal lobe was snap frozen for analysis. The left lung was lavaged with saline in a standardized fashion (Jobe et al., 2000) and neutrophils in the bronchoalveolar lavage fluid (BALF) were counted (Kramer et al., 2001). A sheep-specific ELISA was used to measure interleukin (IL)-8 concentrations in BALF (Kramer et al., 2001). White blood cell counts were measured from the cord blood (Kramer et al., 2001).

All-trans retinoic acid in lung

Quantification of all-trans retinoic acid was performed as previously described (Willet et al., 2000b). In summary, a piece of lung was homogenized and the lipids extracted. Retinoic acid was measured by column chromatography and fluorometric analysis according to the method of Thompson et al. (Thompson et al., 1971). Results were calculated per kg body weight. Supplemental measurements of RA concentrations in the fetal lungs were done for lambs that were exposed to intra-amniotic endotoxin-induced chorioamnionitis for 5 hours, 1 day, 3 days or 7 days prior to delivery at 124 days gestational age. Physiologic, histologic and inflammatory information for these lambs were previously reported (Kramer et al., 2001).

Elastin fibers

Elastin fibers were stained with a Hart’s resorcin-fuchsin solution (Bland et al., 2003). In short, 5 μm paraffin slides were hydrated and stained in a resorcin-fuchsin solution with hydrochloric acid and counterstained with a tartrazine solution. Elastin fibers were stained in black and all other tissue elements in yellow (Bland, 2005).

Morphometric analyses

We used morphometric approaches to quantify alveolar wall thickness and airspace and tissue volume density as maturational attributes of the lung parenchyma. Tissue sampling followed the principles of design-based stereology (Bolender et al., 1993) which incorporates uniformity and randomness into the unbiased sample selection process. The fixed cranial lobe of the right lung was cut parasagittally into uniform, serial slices (~3 mm thick). A random-number generator was used to determine the first slab to embed, from which every third slab also was embedded. Once the embedded tissue blocks were faced to obtain full profile of the embedded tissue, a random-number generator was used again to determine the first tissue section to collect, after which the blocks were serially sectioned to 50 μm to collect another tissue section. In this manner, we collected four adjacent tissue sections, spaced at 50 μm intervals, per tissue block (3–4 blocks per lung). The collected tissue sections were stained with hematoxylin and eosin.

We measured the width of the alveolar walls to quantify thinning of the mesenchyme. This measurement was performed by drawing a line perpendicularly across the distal airspace walls, at the midpoint between the junctions of adjacent airspace walls. We made 60 measurements per tissue section (5 width measurements were made in each of 15 calibrated fields, using a checker board sampling scheme to avoid overlapping fields of analysis). The average width of the distal airspaces per tissue section was calculated, and then the average measurements for each of the 3–4 tissue sections per lung were averaged. The coefficient of variation for measurements among tissue sections per lung was <15%. Airspace area and tissue area also were measured as indices of parenchymal development. Both measurements were done by color thresholding, by which the calibrated pixel area over tissue or airspace was automatically determined on 15 calibrated fields per tissue section (3–4 tissue sections/lung). Tissue fields were excluded if they contained airways, arteries, veins, or visceral pleura. Average measurements were calculated per lung as described for alveolar wall thickness. The average results are expressed as the ratio of airspace area/tissue area and airspace area/airspace + tissue areas as measurements of airspace and tissue volume density. We calculated both ratios because they provide different information. The area ratio for airspace/tissue estimates the compartmental distribution of the lung parenchyma but may be influenced by differences in tissue volume in the fixed and embedded tissue blocks. That influence is corrected for by using a reference space that includes both compartments (area of the airspaces and tissue).

Statistical analysis

Results are given as mean ± SEM. Comparisons between endotoxin-treated groups and untreated controls were by analyses of variance with Student-Newman-Keuls tests used for post-hoc analyses. Statistical significance was accepted at p<0.05.

Results

Reduction of RA in the fetal lungs by chorioamnionitis

We used tissue from lambs previously reported (Kramer et al., 2001) to demonstrate that endotoxin induced chorioamnionitis depleted RA in the fetal lung within 1 day of the induction of chorioamnionitis (figure 1A). The concentration of RA in the fetal lung decreased to less than 16% of the concentration in control lambs on days 1 and 3 and remained low on day 7. A single fetal injection of 20,000 IU of all-trans retinoic acid prevented the decrease in all-trans retinoic acid in the fetal lung 7 days after endotoxin-induced chorioamnionitis in the lambs used for this study (figure 1B).

Figure 1.

Figure 1

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Concentration of all-trans retinoic acid (RA) in fetal lungs after induction of chorioamnionitis by intra-amniotic endotoxin injection. A: The concentration of all-trans retinoic acid was decreased in the fetal lung 24 hours after induction of chorioamnionitis. Concentrations remained low for three days and partially recovered by seven days. Measurements were done in lung tissue from lambs previously reported (Kramer et al., 2001). B: The intra-amniotic endotoxin decreased RA levels at seven days in the lambs studied for this protocol. Treatment with 20,000 IU of all-trans RA in olive oil increased the concentration of RA in the fetal lung after seven days of chorioamnionitis to control levels (* p<0.05 versus control). Data are mean ± SEM.

Systemic effect of RA

We reported previously that the very immature lambs after RA treatment had open eyes while the eyes of the controls were shut (Willet et al., 2000b). We recorded that all animals that had received antenatally RA had open eyes whereas in the control group and endotoxin group together only 3 animals had open eyes (p<0.05).

Chorioamnionitis-induced inflammation

Chorioamnionitis induced by endotoxin resulted in mild systemic inflammation as indicated by a greater than 3-fold increase in umbilical blood white cell counts (figure 2A). Pre-treatment with retinoic acid did not alter the increased white blood count. Pulmonary inflammation was assessed by the concentration of interleukin (IL)-8 protein in the lung tissue and the number of neutrophils in the BALF. IL-8 concentration was increased after endotoxin-induced chorioamnionitis (figure 2B) and was not significantly changed by pre-treatment with RA (p=0.09). The number of neutrophils in the BALF was increased greatly by intra-amniotic endotoxin and RA pre-treatment had no effect on the neutrophils in the BALF (figure 2C).

Figure 2.

Figure 2

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Assessment of inflammation seven days after induction of chorioamnionitis by intra-amniotic endotoxin injection. A: Endotoxin-induced chorioamnionitis caused leucocytosis in cord blood irrespective of retinoic acid pre-treatment. B: Interleukin (IL)-8 concentrations in fetal lung tissue were greater with chorioamnionitis but were not different after RA pretreatment. C: Number of neutrophils in BALF also were greater with chorioamnionitis but were not different after RA pretreatment (* p<0.05 versus control). Data are mean ± SEM.

Lung gas volume

The chorioamnionitis resulted in a 5-fold increase in lung gas volume measured at 40 cm water pressure increasing from 5 ± 3 ml/kg to 22 ± 4 ml/kg and 20 ± 3 ml/kg in the Endo versus Endo+RA group respectively (both p<0.05 versus control). The increase in lung gas volume after endotoxin induced chorioamnionitis was not changed by pre-treatment with RA.

Lung morphometry

An example of the immature lung structure of control lambs is shown in figure 3A. Endotoxin-induced chorioamnionitis decreased the thickness of the distal airspace walls (figure 3B, C). Similar changes were observed after pre-treatment with RA (figure 3C). Elastin was located in control animals primarily at the distal tips of secondary crests (figure 3A). In contrast, the elastic fibers in animals exposed to chorioamnionitis were scattered along the wall with lack of definition of secondary crests. This disorganization of elastin fibers also was seen with the RA supplemented animals (figure 3B and C).

Figure 3.

Figure 3

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Elastin fiber deposition and lung morphology. A: Elastin, stained in black, was located primarily at the distal tips of secondary crests in control animals. Concentrated deposition was marked by circles. In contrast, the elastic fibers in animals exposed to chorioamnionitis were scattered along the wall with lack of definition of secondary crests marked by arrows (B: endotoxin-induced chorioamnionitis; C: endotoxin-induced chorioamnionitis and RA pretreatment). Endotoxin-induced chorioamnionitis decreased the thickness of the distal airspace walls (panel B, C) in comparison to control animals (panel A). Similar changes were observed after pre-treatment with RA (panel C). Representative sections are shown. Magnification 320×.

By morphometric analysis, endotoxin-induced chorioamnionitis reduced the distal airspace wall thickness by 30% in comparison to control lambs (figure 4). The effect of RA treatment in chorioamnionitis appeared to be inconsistent. The ratio of airspace to tissue in control lambs was 2.0 and increased to more than 4.6 in lambs exposed to endotoxin-induced chorioamnionitis (figure 4B). The ratio of airspace to tissue also was increased to 4.0 after pre-treatment of RA. The airspace as part of the whole lung was calculated by dividing airspace by the sum of air space plus tissue (figure 4C). The fraction of airspace increased from 0.70 in control lambs to 0.78 in lungs after endotoxin-induced chorioamnionitis. The fraction in RA pre-treated lambs was similar to the endotoxin only group (0.79).

Figure 4.

Figure 4

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Morphologic analysis of lung structure. A: The alveolar wall thickness was decreased in endotoxin-exposed lambs. The effect was not significant for lambs previously exposed to retinoid acid. B: The airspace-to-tissue ratio was greater after endotoxin-induced chorioamnionitis but was not different from the endotoxin group with pre-exposure to retinoic acid. C: The proportion of airspace in the whole tissue was greater after endotoxin exposure but was not different after exposure to retinoic acid (*p<0.05 versus control). Data are mean ± SEM.

Discussion

The postnatal administration of vitamin A being metabolized to RA to preterm babies at risk for developing BPD is the only validated pharmacologic treatment that is available for the primary prevention of BPD (Van Marter, 2006). Inflammation associated with chorioamnionitis, oxygen exposure, and mechanical ventilation is a common factor associated with BPD (Speer, 2006). Preterm babies have low concentrations of retinol (Shenai et al., 1985) and exposure to perinatal inflammation further aggravates this deficiency in mice and sheep (Bry and Lappalainen, 2006). In this study, we tested the hypothesis that the prenatal administration of retinoic acid could prevent the development of lung structural deficits induced by chorioamnionitis. Fetal sheep exposed to endotoxin-induced chorioamnionitis develop a mild histologic phenotype of mild BPD together with clinical lung maturation (Willet et al., 2000a). We previously reported that fetal treatment of sheep with all-trans RA increased RA levels in the fetus but had no effect on baseline gestational age-specific lung maturation (Willet et al., 2000b). We now find that RA pre-treatment did not affect the pulmonary inflammation, lung maturation, or lung structural changes induced by intra-amniotic endotoxin. A link between inflammation, RA metabolism and subsequent lung maturation has been identified (Bry and Lappalainen, 2006). Endotoxin has a half-life of about 30 h in the amniotic fluid and indicators of chronic inflammation can persist for weeks (Moss et al., 2002; Newnham et al., 2003; Kallapur et al., 2005). The fetus is, however, capable of controlling the inflammation (Kramer and Jobe, 2005) and abnormalities in lung structure do not persist at term in fetal sheep (Kallapur et al., 2005). In a transgenic mouse model with inducible overexpression of IL-1 beta in the lung, early prenatal and postnatal overexpression of IL-1 beta resulted in a lung phenotype similar to BPD with pulmonary inflammation and a deficiency in alveolar secondary septation (Bry and Lappalainen, 2006). The cytosolic and nuclear receptors for RA were differentially affected by IL-1 beta overexpression with a decrease in the expression of RA receptor (RAR) gamma 2 in inflamed lungs but the expression of other subtypes of RA receptors was not affected (Bry and Lappalainen, 2006). In another mouse model, deletion of RAR gamma 2 resulted in decreased numbers of alveoli, increased alveolar size and decreased elastin content (McGowan et al., 1995; McGowan et al., 2000; Hind et al., 2002; Snyder et al., 2005). In the current study, antenatal administration of a large single intramuscular dose of RA 3 h before endotoxin administration increased RA levels in the fetal lung but did not prevent changes in lung structure induced by antenatal inflammation.

Our experimental protocol is considerably different from the clinical use of 5,000 IU vitamin A given three times a week by intra-muscular injection to very preterm infants to decrease the incidence of BPD (Tyson et al., 1999). This chronic use is postulated to modulate the exposure of the preterm lung to oxygen, mechanical ventilation, and other factors such as postnatal sepsis thought to promote BPD (Akram Khan et al., 2006). In animal models, vitamin A was shown to minimize lung structural changes after exposure to toxic concentrations of oxygen (Veness-Meehan et al., 2002; Cho et al., 2005; Ozer et al., 2005). In our sheep model, RA supplementation did increase lung retinoid levels but did not modulate lung inflammation or anatomic changes in response to the acute stimulus of endotoxin given by intra-amniotic injection. Our study is limited by the use of a single dose of RA and the short interval between treatment and evaluation. In addition, we did not evaluate if RA would modulate more chronic effects of chorioamnionitis.

In the conditional IL-1beta transgenic mouse model, over-expression of IL-1beta decreased expression of mRNA for cellular retinoic acid binding protein-1 and the RA receptor gamma-2 (Bry and Lappalainen, 2006). The lack of response to RA supplementation in the endotoxin-exposed fetal sheep may be due to decreased expression of these proteins required for cellular responses to RA. Unfortunately, no reagents are available in sheep to address this question.

The mechanisms by which the inflammation induced by chorioamnionitis may alter fetal lung structure may differ from the postnatal abnormalities where oxidant pathways may predominate.

Acknowledgments

Funded by: NIH HL-65397, HD-12714, and HL62875; IZKF University of Würzburg, grant Z08

The excellent help of Daniele Herbst, Michaela Kapp, Silvia Seidenspinner, and Mar Janna Dahl is gratefully appreciated.

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