Ovine Fetal Lung Research Articles

Mechanism of arginine vasopressin suppression of ovine fetal lung fluid secretion: Lack of V2‐receptor effect

Fetal lung liquid production is essential for in utero pulmonary development, and the resorption of lung liquid at birth facilitates neonatal transition. Lung liquid also contributes importantly to amniotic fluid volume. Factors that influence lung liquid production/resorption may, therefore, impact fetal pulmonary growth and development as well as amniotic fluid homeostasis. Arginine vasopressin (AVP) inhibits fetal lung liquid production and facilitates lung liquid resorption. In view of studies administering fetal AVP or the V2 receptor agonist, 1 desamino 8-D AVP (dDAVP) for the regulation of amniotic fluid volume, we sought to determine the impact of AVP V2 receptor stimulation on fetal lung liquid production. Eight near-term ovine fetuses (130 ± 2 d; term = 145 d) were prepared with hind limb vascular catheters, a bladder catheter, and a tracheal catheter. Each ewe received vascular and amniotic catheters. After 5 days of recovery, the animals were studied for a 2-hr control period and for 2 hr following intravenous dDAVP injection (1 ng/kg). Lung liquid and urine flow rates and plasma electrolyte and osmolality composition were determined at 30-min intervals. To confirm fetal lung liquid suppression in response to AVP, two animals received an intravenous injection of AVP (2 ng/kg) 24 hr after the first experiment. dDAVP administration had no effect on lung liquid production (3.4 ± 0.4 ml/kg/h), electrolyte concentrations, or osmolality. Fetal urine electrolyte concentrations and osmolality (154 ± 24–295 ± 22 mOsm/kg H2O) increased in response to dDAVP, whereas urine flow decreased (9.4 ± 2.5–4.2 ± 1.5 ml/kg/h). In the fetuses exposed to AVP, lung liquid production decreased (3.3–1.6 ml/kg/h). As AVP, although not dDAVP, inhibited fetal lung liquid production, these results indicate that AVP effects on lung liquid are likely mediated via the AVP V1 receptor, not the V2 receptor. The use of fetal administration of dDAVP for the regulation of urine production and amniotic fluid volume will not adversely impact lung liquid production. J. Matern.–Fetal Med. 7:177–182, 1998. © 1998 Wiley-Liss, Inc.

Demonstration of a capsule on Mycoplasma ovipneumoniae

Abstract Objective To examine Mycoplasma ovipneumoniae for presence of a capsule and its potential role in adherence. Sample Population 17 isolates of M ovipneumoniae and 2 isolates of M arginini, recovered from sheep with respiratory tract disease. Procedure Mycoplasmas were cultured in modified Friis broth medium, ovine fetal lung cells, or ovine tracheal ring explants. Pelleted mycoplasmas or ring cultures infected with mycoplasmas were treated with ruthenium red or polycationic ferritin and visualized by transmission electron microscopy. Reactivity of several lectins with the mycoplasmas was studied by use of a microtitration plate agglutination test. Results Electron microscopy revealed a large number of M ovipneumoniae cells covered with an electron dense-stained amorphous material suggesting that it was a capsule. Multiple passages of the microorganisms in modified Friis broth medium decreased thickness of the capsule, but not percentage of cells encapsulated. Marked differences were observed when M ovipneumoniae isolates grown in modified Friis broth medium or co-cultured with ovine fetal lung cells were compared for capsular thickness or percentage of encapsulation. In thin sections of ruthenium red-stained tracheal ring cultures, the mycoplasmas appeared to be in close contact with cilia through their capsule. All isolates of M ovipneumoniae reacted strongly with wheat germ agglutinin lectin. Conclusions Mycoplasma ovipneumoniae produces a polysaccharide capsule with variable thickness that is dependent on culture conditions and strain. Morphologic observations suggest that this capsule facilitates adherence of the organism to ciliated epithelium. (Am J Vet Res 1998;59:557–562)

Increased lung preproET-1 and decreased ETB-receptor gene expression in fetal pulmonary hypertension

Endothelin (ET)-1, a potent vasoconstrictor and smooth muscle mitogen, is produced from its precursor, preproET-1, by endothelin-converting enzyme (ECE)-1 activity. ET-1 may bind to two receptors, ETA and ETB, that mediate vasoconstriction and vasodilation in the ovine fetal lung, respectively. ET-1 contributes to high pulmonary vascular resistance in experimental perinatal pulmonary hypertension induced by ligation of the ductus arteriosus in the fetal lamb. Physiological studies in this model have demonstrated enhanced ETA- and diminished ETB-receptor activities and a threefold increase in lung immunoreactive ET-1 protein content. We hypothesized that increased ET production and an imbalance in receptor expression would favor vasoconstriction and smooth muscle cell hypertrophy in pulmonary hypertension and may be partially due to alterations in gene expression. To test this hypothesis, we studied lung mRNA expression of preproET-1, ECE-1, and the ETA and ETB receptors in normal and hypertensive fetal lambs. Total RNA was isolated from whole lung tissue in normal late-gestation fetuses (135 +/- 3 days; 147 days = term) and from animals with pulmonary hypertension after ductus arteriosus ligation for 8 days (134 +/- 4 days). Ductus arteriosus ligation increased right ventricular hypertrophy [control 0.56 +/- 0.02 vs. hypertension 0.85 +/- 0.05; right ventricle/(left ventricle + septum); P < 0.05]. Northern blot analysis was performed using cDNA probes and was normalized to the signal for 18S rRNA. We found a 71 +/- 24% increase in steady-state preproET-1 mRNA (P < 0.05) and a 62 +/- 5% decrease in ETB mRNA (P < 0.05) expression in ductus arteriosus ligation. ECE-1 and ETA-receptor mRNA expression did not change. We conclude that chronic intrauterine pulmonary hypertension after ductus arteriosus ligation increases steady-state preproET-1 mRNA and decreases ETB-receptor mRNA without changing ECE-1 mRNA or ETA-receptor mRNA expression. These findings suggest that increased ET-1 production and decreased ETB-receptor expression may contribute to increased vasoconstrictor tone in this experimental model of neonatal pulmonary hypertension.

Role of inducible nitric oxide synthase in regulation of pulmonary vascular tone in the late gestation ovine fetus.

Nitric oxide (NO) produced by NO synthase (NOS) modulates fetal pulmonary vascular tone and contributes to the fall in pulmonary vascular resistance (PVR) at birth. Although the inducible (type II) NOS isoform is present in human and rat fetal lungs, it is uncertain whether type II NOS activity contributes to vascular NO production in the fetal lung. To determine whether type II NOS is present in the ovine fetal lung and to study the potential contribution of type II NOS on the regulation of basal PVR in the fetus, we measured the hemodynamic effects of three selective type II NOS antagonists: aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT), and S-ethylisothiourea (EIT). Studies were performed after at least 72 h of recovery from surgery in 19 chronically prepared fetal lambs (133+/-3 d; 147 d, term). Brief intrapulmonary infusions of AG (140 mg), AMT (0.12 mg), and EIT (0.12 mg) increased basal PVR by 82, 69, and 77%, respectively (P < 0.05). The maximum increase in PVR occurred within 20 min, but often persisted up to 80 min. These agents also increased mean aortic pressure but did not alter the pressure gradient between the pulmonary artery and aorta, suggesting little effect on tone of the ductus arteriosus. Acetylcholine-induced pulmonary vasodilation remained intact after treatment with selective type II NOS antagonists, but not after treatment with the nonselective NOS blocker, nitro-L-arginine. Using Northern blot analysis with poly(A)+ RNA, we demonstrated the presence of two mRNA transcripts for type II NOS (4.1 and 2.6 kb) in the fetal lung. We conclude that the type II NOS isoform is present in the ovine fetal lung, and that selective type II NOS antagonists increase PVR and systemic arterial pressure in the late-gestation fetus. We speculate that type II NOS may play a physiological role in the modulation of vascular tone in the developing fetal lung.

Endothelin blockade augments pulmonary vasodilation in the ovine fetus

The physiological role of endothelin-1 (ET-1) in regulation of vascular tone in the perinatal lung is controversial. Recent studies suggest that ET-1 contributes to high basal pulmonary vascular resistance in the normal fetus, but its role in the modulation of pulmonary vascular tone remains uncertain. We hypothesized that high ET-1 activity opposes the vasodilator response to some physiological stimuli such as increased pressure. To test the hypothesis that ET-1 modulates fetal pulmonary vascular responses to acute and prolonged physiological stimuli, we performed a series of experiments in the late-gestation ovine fetus. We studied the hemodynamic effects of two ET-1 antagonists, BQ-123 (a selective ETA-receptor antagonist) and phosphoramidon (a nonselective ET-1-converting enzyme inhibitor) during mechanical increases in pressure due to partial ductus arteriosus compression in chronically prepared late-gestation fetal lambs. In control studies, partial ductus arteriosus compression decreased the ratio of pulmonary arterial pressure to pulmonary artery flow in the left lung 34 +/- 6% from baseline. Intrapulmonary infusions of BQ-123 (0.5 microgram/min for 10 min; 0.025 microgram/min for 2 h) or phosphoramidon (1.0 mg/min for 10 min) augmented the peak vasodilator response during ductus arteriosus compression (52 +/- 3 and 49 +/- 6% from baseline, respectively, P < 0.05 vs. control). In addition, unlike the transient vasodilator response to ductus arteriosus compression in control studies, ET-1 blockade with BQ-123 or phosphoramidon prolonged the increase in flow caused by ductus arteriosus compression. In summary, ETA-receptor blockade and ET-1-converting enzyme inhibition augment and prolong fetal pulmonary vasodilation during partial compression of the ductus arteriosus. We conclude that ET-1 activity modulates acute and prolonged responses of the fetal pulmonary circulation to changes in vascular pressure. We speculate that ET-1 contributes to regulation and maintenance of high pulmonary vascular resistance in the normal ovine fetal lung.

Contribution of ovine fetal lung fluid to amniotic fluid technetium-99m (Tc-99m) activity.

Contribution of ovine fetal lung fluid to amniotic fluid technetium-99m (Tc-99m) activity.

Chronic intrauterine pulmonary hypertension alters endothelin receptor activity in the ovine fetal lung.

Although endothelin (ET) contributes to the regulation of pulmonary vascular tone in the normal fetus, little is known about its role in pulmonary hypertension in the perinatal period. To examine the role of the ETB receptor in the normal ovine fetal lung, we studied the hemodynamic effects of ET-3 (a selective ETB receptor agonist) before and after RES-701 (a selective ETB receptor antagonist). RES-701 (10 mu g/min for 10 min) did not change basal pulmonary tone and blocked pulmonary vasodilation to ET-3 (500 ng/min for 10 min). To examine the effects of experimental perinatal pulmonary hypertension on activity of the ETA and ETB receptors, we studied the hemodynamic effects of ET-3, ET-1 (a nonselective ETA and ETB receptor agonist), and BQ 123 (a selective ETA receptor antagonist) in 12 chronically prepared late gestation fetal lambs after partial ligation of the ductus arteriosus. Serial changes in the pulmonary vascular effects of these agents were measured early (1-3 d) and late (7-10 d) after partial ductus arteriosus ligation. Left lung total pulmonary resistance in the normal late-gestation fetus was 0.62 +/- 0.01 mm Hg/ml/min (n = 4). After partial ductus arteriosus ligation, total pulmonary resistance increased to 1.2 +/- 0.3 (early; p < 0.05 versus normal), and progressively rose to 1.9 +/- 0.2 mm Hg/ml/min (late; p < 0.05 versus early). Intrapulmonary infusion of ET-3 (500 ng/min for 10 min) increased pulmonary blood flow from 94 +/- 11 to 183 +/- 17 mL/min in the normal fetus, but had no effect during late pulmonary hypertension. Infusions of ET-1 (50 ng/min for 30 min) caused transient pulmonary vasodilation followed by vasoconstriction during early pulmonary hypertension. During late pulmonary hypertension, however, infusion of ET-1 caused predominantly vasoconstriction. Pulmonary vasodilation to BQ 123 (100 mu g/min for 10 min) was greater during late than early pulmonary hypertension (43 versus 21%; p < 0.05). After 10 d of ductus arteriosus ligation, immunoreactive ET-1 content in whole lung tissue was 3-fold higher in hypertensive (n = 7) than control (n = 10) lungs (p < 0.05). We conclude that the ETB receptor contributes little to regulation of basal vascular tone in the normal ovine fetal lung and that chronic intrauterine pulmonary hypertension causes the loss of ETB-mediated vasodilation, progressive ETA-mediated vasoconstriction, and increased lung ET-1 content. We speculate that diminished ETB receptor-mediated vasodilation in combination with enhanced ETA receptor-mediated vasoconstriction and increased ET-1 production contributes to high pulmonary vascular resistance in perinatal pulmonary hypertension.

Ontogeny of 11 beta-hydroxysteroid dehydrogenase in ovine fetal kidney and lung.

Eleven-beta-hydroxysteroid dehydrogenase (11 beta-HSD) is an enzyme which degrades 11-hydroxycorticosteroids to biologically inactive 11-oxocorticosteroids (cortisone and 11-dehydrocorticosterone). In some tissues, the activity of this enzyme prevents binding of cortisol to mineralocorticoid receptors. The present experiments were designed to test the hypothesis that the fetal kidney contains 11 beta-HSD, that the activity of 11 beta-HSD in fetal kidney increases near term, and that the fetal lung does not contain significant 11 beta-HSD activity. In kidney and lung tissue from 23 fetal sheep ranging in age between 86 and 145 days' gestation, we measured 11 beta-HSD activity. We found significant activity in fetal kidney (14-85% conversion from cortisol to cortisone) but no measurable activity in fetal lung (0-9%). The activity of 11 beta-HSD was significantly related to fetal gestational age (r = 0.76, n = 14). We conclude that 11 beta-HSD activity in the fetal kidney develops as a function of fetal gestational age, and that activity cannot be demonstrated in fetal lung. We speculate 11 beta-HSD in the fetus might function to alter the sensitivity of target organs to glucocorticoids, as well as to mineralocorticoids, and that the absence of activity in the lung allows a high sensitivity of pulmonary tissue to cortisol at the end of gestation.

Content, in vivo release, and bioactivity of fetal pulmonary immunoreactive adrenocorticotropin

We hypothesized that fetal lung contains and releases immunoreactive (ir) adrenocorticotropin (ACTH). Fetal sheep lung (89-145 days gestation, n = 13) irACTH content was 4,100 +/- 672 (SE) pg/g lung wet wt compared with 5,425 +/- 1,403 (SE) pg/g in adults (n = 5) when measured by radioimmunoassay (RIA). irACTH values normalized to protein content were 158 +/- 18 and 95 +/- 22 (SE) ng/g protein for fetuses and adults, respectively. Grouped by age, < 90 days (n = 4), 131-139 days (n = 5), and 143-145 days (n = 4), the values for irACTH were 184 +/- 28, 173 +/- 33, and 114 +/- 33 (SE) ng/g protein, respectively, and declined significantly from < 90 days to adulthood. RIA serial dilution curve slopes were different from standard in 7 of 13 fetuses and in all adults, suggesting that irACTH structure differs from ACTH-(1-39). Acute in vivo fetal pulmonary artery and vein sampling (n = 5, 120-128 days gestation) revealed a mean difference of -609 +/- 476 (SE) pg/ml, a statistically significant release of irACTH across the lung. Western blotting revealed that pulmonary irACTH migrates in multiple immunostaining bands between molecular weight 14,000 and 46,000 and does not colocalize with ACTH-(1-39). Adrenal cell bioassay revealed that pulmonary irACTH is not corticotropic. We concluded that ovine fetal lung contains large-molecular-weight irACTH, that content declines from 90 days gestation to adulthood, and that, under acute conditions, the fetal lung releases irACTH substances in sufficient quantity to contribute to circulating hormone concentrations of irACTH.

Late Gestation Alterations in Fetal Pulmonary Lactate Metabolism in Vivo

We have previously shown that lactate is produced by the ovine fetal lung. Inasmuch as factors that might affect lactate production, such as pulmonary glucose and oxygen uptake, change late in gestation we investigated whether pulmonary lactate metabolism also changes. Eleven chronically catheterized fetal lambs were studied over 119-141 d gestation. Lactate, glucose, and oxygen concentrations were measured in the pulmonary artery (PA) and vein while lung blood flow was determined using labeled microspheres. Between early studies (less than or equal to 127 d) and studies near term (greater than or equal to 134 d) PA lactate levels did not change, but due to increasing pulmonary blood flow, lung lactate delivery rose 51% (p less than 0.05). Because of a decline in PA glucose, lactate also made up a larger fraction of the major nonnitrogenous substrate in PA blood near term (p less than 0.001). Despite this, no net pulmonary uptake of lactate occurred. Lactate production continued, but decreased by 80% between early and late studies (p less than 0.05) and the maximum fraction of glucose uptake that could be accounted for by lactate production dropped from 0.78 to 0.20 (p less than 0.025). Correlations were found between lung lactate production and glucose uptake also correlated with PA glucose (p less than 0.05). No relationships were observed between lactate production and PA oxygen content, oxygen delivery, lactate concentration, or lactate delivery. The decreasing fraction of glucose uptake explained by lactate production suggests that metabolism of pulmonary glucose is altered near term. The correlation between decreasing glucose delivery and declining lactate production also suggests that glucose itself influences this change.

Synergistic hormonal effects on lung maturation in fetal sheep.

Cortisol has minimal effects on lung maturation in fetal sheep before 130 days gestation. To test whether there is enhancement of cortisol action by other hormones, cortisol (F), triiodothyronine (T3), epinephrine (E), prolactin (PRL), and epidermal growth factor (EGF), alone or in combination, were infused into fetal sheep for 84 h between 124 and 128 days gestation. A mixture of F + T3 + PRL, but not any combination of two hormones, increased both distensibility [1.71 +/- 0.12 (SE) ml of air/g wet wt at 40 cmH2O, V40] and stability (1.16 +/- 0.09 ml of air per g wet wt at 5 cmH2O, V5) to near full-term values, above values resulting from treatment with F alone (0.91 +/- 0.12 and 0.43 +/- 0.09 ml/g, P less than 0.01). Only F had an effect when given alone, V40 increasing (P less than 0.05). Treatment with F + T3 (0.81 +/- 0.18 ml/g) and F + E (0.77 +/- 0.07 ml/g) increased V5 above values obtained with F alone (P less than 0.05). Alveolar saturated phosphatidylcholine (SPC) was higher after treatment with F + T3 (161 +/- 52 micrograms/g), F + T3 + PRL (156 +/- 53 micrograms/g, P less than 0.05), and F + E (113 +/- 40 micrograms/g, P = 0.07) than after F (12 +/- 3 micrograms/g). We conclude that F, T3, and PRL have a synergistic effect on the development of distensibility and stability of the ovine fetal lung.

Propagation and quantitation of animal herpesviruses in eight cell culture systems

A comparative study was carried out to determine the relative sensitivities of eight different cell culture systems to six different herpesviruses of animals. The cells used were: OFL (ovine fetal lung), ML (mink lung), FK (ferret kidney), PTK-2 (potoroo kidney), TEK (turkey embryo kidney), ED (equine dermal), BT (bovine turbinate), and PK 15 (porcine kidney). The viruses tested were: PRV (pseudorabies) of swine, CPHV (caprine herpesvirus), IBRV (infectious bovine rhinotracheitis virus), DN-599 strain of bovine herpesvirus type 4, EHV-1 (equine herpesvirus), and CHV (canine herpesvirus). On the basis of virus titers obtained and the time of appearance of CPE (cytopathic effects), ML cells were found to be the most useful because of their sensitivity to all six viruses tested. BT and OFL cells were also found to be highly sensitive to all viruses with the exception of CHV.

Effect of thyroid status on lung and heart beta-adrenergic receptors in fetal and newborn sheep.

The effect of altered thyroid status on the development of beta-adrenergic receptor (BAR) density and affinity was investigated in ovine fetal and newborn heart and lung. Fetal (119-121 days gestation) and newborn (2-3 days of age) sheep underwent either thyroidectomy alone, thyroidectomy plus infusion of a large dose of T3 or sham operation. Eight days later BAR were measured in heart and lung using the tritiated radioligand dihydroalprenolol. There was no apparent effect of altered thyroid status on fetal heart or lung BAR density or affinity. In contrast, the newborns thyroidectomized and infused with T3 had a 96% increase in heart BAR density and an 83% increase in lung BAR compared to the thyroidectomized only or sham-operated newborns which were similar. These results suggest that in the near term ovine fetus heart and lung tissue BAR are neither dependent on nor responsive to thyroid hormones, whereas in the newborn period heart and lung BAR are highly responsive to thyroid hormones.

The estimation of elastin in fetal tissues by radioimmunoassay of isodesmosine.

A radioimmunoassay was developed for the determination of isodesmosine as the tetraacetyl derivative. Isodesmosine tetraacetate conjugated with bovine albumin was injected into rabbits which developed useful titers of antibodies after five months. The radioligand for the assay was prepared by acetylating isodesmosine with [3H] acetic anhydride. The bound ligand was separated from free ligand by coprecipitation with human gamma-globulin in 46% saturated ammonium sulfate solution. The sensitivity of the assay was 2 ng isodesmosine. The antiserum was specific for isodesmosine tetraacetate and only desmosine tetraacetate gave appreciable cross-reactivity (4%). The assay was found to be suitable for the accurate estimation of elastin in small samples (5 mg dry weight) of rat and ovine fetal lung tissue and for elastin degradation products in amniotic fluid (0.5 ml).

The effect of thyroxine (T 4) on ovine fetal lung maturation

The effect of thyroxine (T 4) on ovine fetal lung maturation

The Effect of Fetal Thyroidectomy on Ovine Fetal Lung Maturation

Summary: Studies were conducted to assess the effect of fetal thyroidectomy early in the third trimester on lung growth and surfactant maturation in the sheep. At 95–99 days of gestation, a fetal thyroidectomy was performed. The preparation was killed 36–45 days postthyroidectomy. The mean weights of the cerebrum, cerebellum, and combined heart and lungs were significantly smaller and the pituitary gland significantly greater in the thyroidectomized fetus. The mean tracheal fluid lecithin to sphingomyelin (L/S) ratio was significantly lower in the athyrotic fetus; however, there was no significant difference in the mean cortisol concentration or mean whole lung homogenate phosphatidic acid phosphohydrolase (PAPase) specific activity between the two groups. There was a significant direct correlation between the serum cortisol concentration and tracheal fluid L/S ratio and between the whole lung homogenate PAPase specific activity and serum cortisol level in the euthyroid group, which were not present in the thyroidectomized group. The mean lung DNA concentration was significantly increased and mean lung protein/DNA ratio significantly decreased in the thyroidectomized fetus. On histologic examination, the lung from the athyrotic fetus was hypercellular with thickened alveolar septae. The type II pneumocyte in the hypothyroid lung was less mature with fewer lamellar bodies or its precursors. These results suggest that in the ovine fetus, thyroid hormone deficiency during the last trimester of intrauterine development impairs lung growth and surfactant maturation.Speculation: The present results suggest that thyroid hormones are necessary for normal lung growth and surfactant maturation. In the human preterm infant, idiopathic respiratory distress syndrome is a major cause of morbidity and mortality. Stimulation of the hypothalamic-pituitary-thyroid axis in the fetus at high risk for developing idiopathic respiratory distress syndrome before delivery may accelerate surfactant maturation and decrease the sequelae from this syndrome.

The relationship of cortisol and cortisone to saturated lecithin concentration in ovine amniotic fluid and fetal lung liquid.

Cortisol and cortisone concentrations in ovine amniotic fluid and fetal lung liquid were analyzed with respect to gestational age and fetal lung maturation as reflected by saturated lecithin concentration. In amniotic fluid, the cortisol concentration rises as a function of gestational age while that of cortisone falls (P less than 0.001). The ratio of cortisol/cortisone is significantly related to gestational age (r=0.693, P less than 0.001), and shows an even stronger correlation with saturated lecithin concentration (r=0.832, P less than 0.001). When examined independently of gestational age, the correlation of the cortisol/cortisone ratio to saturated lecithin concentration remains highly significant (r=0.641, P less than 0.001). Similar findings were observed in fetal lung liquid, except that in this fluid both the saturated lecithin concentration and the ratio of cortisol/cortisone are significantly higher than simultaneously determined values in amniotic fluid. These observations provide further evidence that the process of fetal lung maturation is closely linked to the fetal hormonal milieu.