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The DA is widely patent in the fetus and closes rapidly after birth, in about 15 h in human infants(1). PGE is responsible for fetal patency of the DA(1,2). Observations suggest that other factors also dilate the fetal and neonatal DA, inasmuch as the effect of indomethacin on closing PDA in premature babies is not consistent(3,4). Clinically, the constricting response of the fetal DA depends on fetal gestational age and does not occur before 24 wk of age(5–7), although the fetal DA is histologically mature earlier(8). The same phenomenon is observed in experimental studies, in which the fetal DA does not constrict in response to maternally administered indomethacin in preterm rats on the 19th day of gestation (term, 21.5 d)(9). Recently, Coceani et al.(10) and Clyman et al.(11) reported production of NO in a ductal strip of the near-term lamb at 132-146 d of gestation and preterm fetal lamb at 60-104 d of gestation (term, 145 or 147 d). NO production was larger under conditions of high Po2 (106-276 mm Hg, or 30% inspired O2) in both studies. With low, Po2 similar to the fetal ductal environment, NO production was half that of production under high Po2 in the former study(10) or null in the latter study(11). Fox et al.(12) studied the effect of inhibition of NO in near-term fetal lambs at 120 to 123 d of gestation in in vivo experiments, and their results suggested that inhibition of NO production did not result in constriction of the fetal DA. Therefore, the role of NO in fetal ductal patency is highly controversial. We studied the physiologic role of NO in dilation of the DA of preterm and near-term fetal rats.

METHODS

Animals. Forty-nine virgin Wistar rats (pregnancy period, 21.5 d) were mated overnight from 1700 to 0900 h, and the presence of sperm in vaginal smears was used to determine d 0 of pregnancy. These rats were fed commercial solid food and water. Treatment of rats conformed to the guiding principle of the American Physiological Society. The experiment was approved by the Ethical Committee of Animal Experiments of the institute.

Administration of L-NAME, L-arginine, and indomethacin. NO synthase inhibitor, L-NAME(13) (Sigma Chemical), was dissolved in 1 mL of physiologic saline and injected into the dorsum of pregnant rats at 0900 h. L-Arginine hydrochloride (Morishita-Russel, Osaka, Japan; 10% solution) was injected intraperitoneally at 0900 h. Indomethacin (Research Biochemicals International, Natick, MA) was diluted 400-fold with lactose, suspended in 1 mL of water, and administered through an orogastric tube to the pregnant rat at 0900 h.

Study of in situ diameter of the fetal DA.. To study the in situ morphology of the fetal DA, a rapid whole-body freezing method was used as described in an earlier study(14). Fetuses were delivered by caesarean section with atlas dislocation of the mother, and frozen immediately in acetone cooled to -80°C by dry ice. Body weight of the frozen fetuses was measured. Seven to eight fetuses were studied per litter. The frozen thorax was cut on a freezing microtome in the frontal plane, and the inner diameters of the ascending aorta, the main pulmonary artery, and the DA were measured with a microscope and a micrometer every 100 µm in 10 to 15 planes. Constriction of the DA was not uniform, but was most severe at the middle or aortic end(15). The narrowest diameter of the DA was used to get the DA/PA. In some experiments, additional longitudinal sections of the DA were photographed as follows. The frozen thorax was cut in the left sagittal plane, and cardiac and ductal cross-sections were photographed with a binocular stereoscopic microscope (Wild M 400 Photomacroscope, Wild Heerbrugg Ltd., Heerbrugg, Switzerland) using color film (Reala, Fuji Film, Tokyo, Japan).

Protocol. In total, 20 studies were performed as listed in Table 1. Studies in near-term rats were performed on the 21st day of gestation. Drugs were administered at 0900 h, and fetuses were fixed at 1300 h. Studies in preterm rats were performed on the 19th day. Drugs were administered at 0900 h, and fetuses were fixed 1, 2, 4, or 7 h later.

Table 1 Protocol for studying physiologic role of NO in dilating fetal DA in near-term and preterm rats
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Fetal viability. Viability of the fetus was assessed at birth as follows. Dead fetuses were pale and motionless, and these were not frozen. Only live fetuses were frozen for subsequent study. Circulatory collapse was recognized on cross-section on the freezing microtome by a compressed aorta, pulmonary artery, and superior vena cava.

Statistics. Results were expressed as mean ± SEM. The statistical significance of differences between group means was determined separately by ANOVA and Bonferroni methods(16). The accepted level of significance was 5%.

RESULTS

Most fetuses were alive and showed evidence of an intact circulation. Only one fetus was dead (study 5), and four fetuses showed vascular collapse; one in study 2, one in study 10, and two in study 17. All other fetuses had anatomically intact heart and vessels.

In near-term rats, indomethacin (1 mg/kg, 4 h) induced moderate constriction of the fetal DA, and DA/PA decreased to 0.65 (Table 2). L-NAME in moderate dose (10 mg/kg) induced minimal constriction of the fetal DA, with DA/PA ratio 0.97. A large dose of L-NAME (100 mg/kg) induced only mild constriction, with DA/PA ratio 0.83. Combined administration of indomethacin and a moderate dose of L-NAME induced severe constriction of the DA, inasmuch as DA/PA decreased to 0.26 (Table 2).

Table 2 Results of fetal ductal constriction by L-NAME and indomethacin in near-term and preterm fetal rats
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In preterm rats, indomethacin (1 mg/kg, 4 h) induced only minimal constriction of the ductus, and DA/PA ratio decreased to 0.86. A moderate dose of L-NAME (10 mg/kg) induced severe constriction of the DA, with DA/PA ratio 0.32 in 4 h (Figs. 1 and 2). The time course of fetal DA constriction with this dose of L-NAME is shown in Figure 3. Fetal DA constriction was maximal 4 h after injection and persisted more than 7 h. Larger doses (100 mg/kg) of L-NAME (Fig. 4) induced a small and statistically insignificant increase in DA constriction compared with moderate doses (10 mg/kg).

Figure 1
figure 1

Left sagittal cross-sections of the heart and the DA of the preterm fetus on the 19th day. A, Fetus with no drug. The DA is widely patent. B, Fetus 4 h after injection of 10 mg/kg L-NAME. The DA is severely constricted. A, anterior; AAo, ascending aorta; DAo, descending aorta; LB, left bronchus; LPA, left pulmonary artery; I, inferior; LA, left atrium; LSVC, left superior vena cava; MPA, main pulmonary artery; P, posterior; RA, right atrium; S, superior.

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Figure 2
figure 2

Frontal sections of the DA in preterm fetuses on the 19th day. A, Fetus with no drug. The DA is widely patent. B, Fetus 4 h after administration of 1 mg/kg indomethacin. The DA is constricted slightly. C, Fetus 4 h after injection of L-NAME (10 mg/kg). The DA is constricted severely. AoA, aortic arch; L, left; R, right; RPA, right pulmonary artery; T, trachea. Other abbreviations as in Figure 1.

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Figure 3
figure 3

Time course of fetal ductal constriction after injection of 10 mg/kg L-NAME into the dorsum of pregnant rats on the 19th day.

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Figure 4
figure 4

Dose-response curve of fetal ductal constriction after injection of L-NAME into the dorsum of pregnant rats on the 19th day.

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The effect of L-NAME on fetal DA constriction was partially and dose-dependently attenuated by simultaneous administration of L-arginine in studies 19 and 20 (Table 2).

The inner diameters of the ascending aorta, the main pulmonary artery, and the DA in studies 1, 2, 4, 8, 9, and 15 are shown in Table 3. In addition to the remarkable change in ductal diameter, a small but significant decrease of the main pulmonary artery diameter occurred after administration of L-NAME.

Table 3 Inner diameters of the ascending aorta, main pulmonary artery, and DA after administration of L-NAME and indomethacin in near-term and preterm fetal rats
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DISCUSSION

The main finding in this study is the major role of NO in ductal patency in preterm fetal rats at the 19th day of gestation. This statement assumes placental transfer of L-NAME and its direct effect to the fetus. Recently, the fetal effects of long-term administration of L-NAME were studied(17–19), but no data were available as to placental transfer of L-NAME to the fetus. However, transfer of L-NAME to the fetus is very likely because of its closely related biochemical structure to L-arginine. Direct measurement of L-NAME concentrations in fetal blood and its renal and hepatic clearance from the maternal and fetal compartments remain to be studied.

Another possible indirect effect of L-NAME on the fetal DA is fetal asphyxia because of uterine contraction and uterine artery constriction. The fetal DA markedly constricts at 5-8 mm Hg of Po2(20). However, severe fetal asphyxia was unlikely in this study with L-NAME, because the majority of fetuses survived without circulatory collapse after its administration.

The present study has shown a changing role of NO and prostaglandins in fetal ductal patency in rats. Pregnancy duration in rats is 21.5 d. Cardiovascular morphogenesis is completed and the embryonic stage ends at 14th day of gestation(21), with duration of the fetal stage of only 7 d, from the 15th day through the 21st day of gestation. Because of a shorter fetal stage in rats, our findings cannot be extrapolated to the human or sheep fetus.

Near-term fetuses are sensitive to maternally administered indomethacin, responding with ductal constriction. The indomethacin-sensitive period in rats is only 2 d, beginning on the 20th day of gestation(9). The indomethacin-sensitive period begins at the 24th week in the human fetus(5–7), and persists for 4 mo. In the sheep, the indomethacin-sensitive period begins at 103 d of gestation, persisting until term (147 d)(1,22). Our study has shown for the first time that NO is the major factor in ductal patency in early fetal life before the indomethacin-sensitive period. Clyman et al.(11) showed the presence of intimal NO synthase in both the early and late fetal DA in lambs. They also showed increased sensitivity of ductal strips to a dilating effect of NO in early fetal stages and decreasing sensitivity in late stages in the lamb(11). These findings are concordant with the results of our present study. Fox et al.(12) reported that ductal production of NO is decreased by half in low Po2, compared with NO production under high Po2. Coceani et al.(10) showed a reduced ductus-dilating mechanism by NO at low Po2 compared with high Po2. Clyman et al.(11) reported almost no production of ductal NO under low Po2. The present study is in agreement with the results of Fox et al.(12) and Coceani et al.(10).

The second finding in this study is that a minor but definite role of NO in ductal patency exists in the near-term fetus. Every previous study showed that prostaglandins had a major role in ductal patency in near-term fetuses in humans(5–7), lambs(22), and rats(9). In the present study, a large dose of indomethacin caused severe ductal constriction in near-term fetuses, showing that NO is not sufficient to maintain ductal patency at this stage. NO inhibition alone produced minimal ductal constriction in this study, concordant with the early in vivo study by Fox et al.(12) and in vitro studies by Coceani et al.(10) and Clyman et al.(11). However, some role for NO is indicated by the combined administration of L-NAME and indomethacin, which markedly increased constriction of the fetal DA. We speculate that dilating prostaglandins and NO work together in maintaining fetal ductal patency in near-term fetuses, although NO has only a minor role at this stage.

The mechanism for the decreased effectiveness of NO in dilating the DA in the near-term fetus is not known. NO works to inhibit migration and proliferation of vascular endothelial and smooth muscle cells(23), which are important mechanisms of anatomic ductal closure after functional closure in the neonate. The shift from NO to prostaglandins in mediation of fetal ductal dilation may be a preparative process for postnatal closure. The mechanism for the switch is not clear, but Yallampalli et al.(24) recently reported on uterine NO production in the rat, suggesting that progesterone increases NO formation during pregnancy and that a rise in estrogen at term inhibits NO to initiate labor. This mechanism may be responsible for the functional change from NO to prostaglandins in the fetal DA.

The diameter of the main pulmonary artery decreased about 10% after administration of L-NAME, indicating that the fetal pulmonary artery is a reactive vessel and that NO is dilating the fetal pulmonary artery, confirming the earlier report by Fox et al.(12).