Role of nitrite in regulation of fetal cephalic circulation in sheep.

Nitrite has been postulated to provide a reservoir for conversion to nitric oxide (NO), especially in tissues with reduced oxygen levels as in the fetus. Nitrite would thus provide local vasodilatation and restore a balance between oxygen supply and need, a putative mechanism of importance especially in the brain. The current experiments test the hypothesis that exogenous nitrite acts as a vasodilator in the cephalic vasculature of the intact, near term fetal sheep. Fetuses were first instrumented to measure arterial blood pressure and carotid artery blood flow and then studied 4-5 days later while in utero without anaesthesia. Initially l-nitro-arginine (LNNA) was given to block endogenous NO production. Carotid resistance to flow increased 2-fold from 0.54 ± 0.01 (SEM) to 1.20 ± 0.08 mmHg min ml(-1) (in 13 fetuses, P < 0.001), indicating NO tonically reduces cerebral vascular tone. Sodium nitrite (or saline as control) was then infused in increasing step-doses from 0.01 to 33 µm in half-log increments over a period of 2 h. Carotid artery pressure, blood flow and vascular resistance did not change compared to fetuses receiving saline, even at plasma nitrite concentrations two orders of magnitude above the physiological range. The results indicate that while cephalic vascular tone is controlled by endogenous nitric oxide synthase activity, exogenously administered nitrite is not a vasodilator at physiological concentrations in the vasculature served by the carotid artery of fetal sheep.

A "top-down-approach" to insert catheters into the circulation of fetal sheep using ultrasound B-scan.

OBJECTIVE: The objective of the study was the catheterization of vessels in fetal sheep under ultrasound control. STUDY DESIGN: A catheter with guiding wire was introduced into the external jugular vein or carotid artery and advanced caudally. RESULTS: The aorta and umbilical vein could be accessed. CONCLUSION: Major segments of the circulation are accessible from the neck for chronic instrumentation.

Fetal MRI in experimental tracheal occlusion.

Congenital diaphragmatic hernia (CDH) is associated with a high mortality, which is mainly due to pulmonary hypoplasia and secondary pulmonary hypertension. In severely affected fetuses, tracheal occlusion (TO) is performed prenatally to reverse pulmonary hypoplasia, because TO leads to accelerated lung growth. Prenatal imaging is important to identify fetuses with pulmonary hypoplasia, to diagnose high-risk fetuses who would benefit from TO, and to monitor the effect of TO after surgery. In fetal imaging, ultrasound (US) is the method of choice, because it is widely available, less expensive, and less time-consuming to perform than magnetic resonance imaging (MRI). However, there are some limitations for US in the evaluation of CDH fetuses. In those cases, MRI is helpful because of a better tissue contrast between liver and lung, which enables evaluation of liver herniation for the diagnosis of a high-risk fetus. MRI provides the ability to determine absolute lung volumes to detect lung hypoplasia. In fetal sheep with normal and hyperplastic lungs after TO, lung growth was assessed on the basis of cross-sectional US measurements, after initial lung volume determination by MRI. To monitor fetal lung growth after prenatal TO, both MRI and US seem to be useful methods.

The ductus venosus and intrahepatic venous system in Callithrix jacchus jacchus and Macaca fascicularis fetuses.

BACKGROUND: The ductus venosus (DV) and the intrahepatic branches of the portal vein are arranged as parallel vessels. Blood shunting through the DV ensures fetal survival during periods of stress. The availability of a suitable animal model with similar structure and function to the human fetus would greatly improve the understanding of DV function. The anatomical and histological structure of the DV has not been thoroughly investigated in non-human primate species. METHODS: Morphological investigations were performed on eight marmoset (Callithrix jacchus jacchus) at 112.5 +/- 5.5 days gestational age (mean +/- SEM) and four near-term (165 days) cynomolgus (Macaca fascicularis) fetuses. RESULTS: The DV drains into the collectus venosus. An asymmetrical muscular lip forms a contractile element of the isthmic portion of the DV. A spherical 'dividing' eminence was found on the dorsal wall of the venous collector just above the outlet of the DV in marmoset fetuses. CONCLUSIONS: Our findings regarding the structure of the DV in cynomolgus and marmoset fetuses were generally in agreement with previous descriptions of the morpho-histological structure of the DV in human fetuses.

Functional MR imaging: comparison of BOLD signal intensity changes in fetal organs with fetal and maternal oxyhemoglobin saturation during hypoxia in sheep.

PURPOSE: To compare relative changes in blood oxygen level-dependent (BOLD) signal intensity in the fetal brain, liver, heart, lungs, and cotyledon with maternal and fetal blood oxygenation during maternal hypoxia in sheep. MATERIALS AND METHODS: All experimental protocols were reviewed and approved by local authorities on animal protection. Six anesthetized ewes carrying singleton fetuses underwent magnetic resonance (MR) imaging with rapid single-shot echo-planar imaging BOLD sequence. BOLD imaging of the fetal brain, lungs, liver, heart, and cotyledon was performed during a control phase (ie, normoxia) and a hypoxic phase. Maternal oxyhemoglobin saturation was recorded continuously with pulse oximetry. Fetal blood samples were obtained with a carotid catheter at each phase. Regions of interest were placed in fetal organs. Normalized BOLD signal intensity was calculated with mean values of control and hypoxic plateaus. BOLD signal intensity was correlated with maternal oxyhemoglobin saturation and fetal oxyhemoglobin saturation; linear regression analysis was performed. RESULTS: Control maternal and fetal oxyhemoglobin saturation values were 97% (95% confidence interval [CI]: 95%, 100%) and 62% (95% CI: 51%, 73%), respectively. During hypoxia, maternal and fetal oxyhemoglobin saturation values decreased to 75% (95% CI: 65%, 85%) and 23% (95% CI: 17%, 29%), respectively. Fetal BOLD signal intensity decreased to 81% (95% CI: 73%, 88%) in the cerebrum, 78% (95% CI: 67%, 89%) in the cerebellum, 83% (95% CI: 80%, 86%) in the lungs, 58% (95% CI: 33%, 84%) in the liver, 53% (95% CI: 43%, 64%) in the heart, and 71% (95% CI: 48%, 94%) in the cotyledon. Correlation of fetal BOLD signal intensity was stronger with fetal (r = 0.91) than with maternal (r = 0.68) oxyhemoglobin saturation; however, the difference was not significant. The highest slope values were obtained for the heart: 1.68% BOLD signal intensity increase per 1% maternal oxyhemoglobin saturation (95% CI: 1.58, 1.77) and 1.04% BOLD signal intensity increase per 1% fetal oxyhemoglobin saturation (95% CI: 0.94, 1.13). CONCLUSION: BOLD MR imaging can be used to measure changes of oxyhemoglobin saturation in fetal organs during hypoxia. The liver and heart demonstrated the greatest signal intensity decreases during hypoxia.

Fetal sheep brains: findings at functional blood oxygen level-dependent 3-T MR imaging--relationship to maternal oxygen saturation during hypoxia.

PURPOSE: To quantify the dependence of the signal intensity (SI) at blood oxygen level-dependent (BOLD) magnetic resonance (MR) imaging of fetal sheep brains on maternal oxygen saturation and to investigate the influence of positions of regions of interest (ROIs). MATERIALS AND METHODS: All experimental protocols were reviewed and approved by the local authorities on animal protection. The brains of singleton fetuses of five anesthetized sheep were subjected to rapid sequences (single-shot echo-planar imaging) of BOLD measurements with a 3-T MR imaging unit. Maternal oxygen saturation and heart rate were recorded continuously. After a normoxic phase, hypoxia was induced by reducing the oxygen in a ventilated gas mixture. ROIs were placed in the cerebrum at a reference level and in the cerebellum. Normalized BOLD SI values were calculated from the mean values of steady-state BOLD SIs at the control (SI(c)) and hypoxic (SI(h)) plateaus as follows: normalized BOLD SI = (SI(h)/ SI(c)) x 100. Normalized BOLD SI values were correlated with maternal oxygen saturation, and linear regression (slope) analysis was performed. Additionally, ROIs were varied in section level and position. Differences in normalized BOLD SI values for ROI placements were calculated by using analysis of variance. A t test was performed to evaluate differences. RESULTS: Mean maternal oxygen saturation (as the percentage of oxygen in the blood) was 88% (95% confidence interval [CI]: 80%, 96%) in the control period. During hypoxia, it was reduced to 62% (95% CI: 50%, 75%), while fetal normalized BOLD SI decreased to 64% (95% CI: 44%, 85%) in the cerebrum and 56% (95% CI: 32%, 80%) in the cerebellum. Correlations between normalized BOLD SI values and maternal oxygen saturation were as follows: r2 = 0.84 and slope = 1.27 (95% CI: 1.17, 1.36) in the cerebrum and r2 = 0.83 and slope = 1.54 (95% CI: 1.44, 1.63) in the cerebellum. Normalized BOLD SI was 4% lower in the section above the reference level. Variations in normalized BOLD SI for different ROI positions ranged between 0% and 12%. CONCLUSION: The depletion of oxygen supply is reflected by decreases in fetal brain BOLD SIs that are more distinct in the cerebellum than in the cerebrum. Normalized BOLD SI is influenced only slightly by ROI position.

Structural evidence for mechanisms to redistribute hepatic and ductus venosus blood flows in nonhuman primate fetuses.

OBJECTIVE: The ductus venosus (DV) and the intrahepatic branches of the portal vein (BPV) play an important role in umbilical blood distribution to the fetal liver and the rest of the fetal circulation. Increased DV shunting is a major fetal survival mechanism during stress situations. The availability of a nonpregnant primate animal model with similar structure and function would greatly improve our understanding of DV function. However, the anatomic and histologic structure of the DV and the BPV have not been thoroughly investigated in any nonhuman primate species. METHODS: Anatomic and immunohistochemical (Masson's and alpha-smooth actin stains) investigations were performed on 17 baboon fetuses at 173 +/- 5 days' gestation (mean +/- SEM, term = 180 days) (Papio sp. ) and 3 near term rhesus (Macaca mulatta) fetuses. RESULTS: In both species the branchless, funnel-shaped DV coursed cranially, posteriorly, and slightly oblique to the left side. The DV and the efferent hepatic veins drained into a dilated ampullary area (the collectus venosus) that joined directly with the inferior cava. The length of the DV in baboons increased with gestational age ( r = 0.86, n = 16). In 4 baboon fetuses, we observed an asymmetrical muscular lip at the isthmic portion of the DV. The media of intrahepatic BPV contained more smooth muscle cells than the media of the DV. CONCLUSION: In nonhuman primate fetuses, the DV drains into a dilated ampullary area. An asymmetrical muscular lip forms a contractile element of the isthmic portion of the DV. The increased thickness of smooth muscle tissue in the DV isthmus and intrahepatic BPV in nonhuman primate fetuses support the concept of a general organization of a contractile apparatus that performs a sphincter-like function in the central venous hepatic system and plays a key role in blood flow redistribution.

Models of fetal growth restriction.

The growth of the fetus is determined by substrate supply mostly for mass accretion and energy gain, and by control systems. Experiments with whole animal models will face the following problems: (1) The fetus, like a three compartmental "Russian doll", is at the end of a long supply chain. There are interactions (e.g. hormones) and partitioning of substrates between the compartments. (2) The fetal organism is growing and differentiating at the same time and not in a steady-state. Experimental results thus depend on gestational age. (3) About 75% of animal experiments on fetal growth restriction have been performed in rats and mice. The possible experimental methods and the results depend on the species which include sheep, pigs, rabbits, guinea pigs, horses and non-human primates. Many experiments have clearly shown that restriction of substrate supply will usually impair fetal growth. Less is known about growth control mechanisms but recent studies in gene mutant mice have opened a new approach to study the effects of systemic and local controlling factors. It appears that insulin-like growth factors and their binding proteins may play an important role for fetal growth.

Differential effects of catecholamines on vascular rings from ductus venosus and intrahepatic veins of fetal sheep.

Ductus venosus (DV) sparing means the maintenance of blood flow through the DV following reduction of liver venous blood supply during fetal hypoxia. The present study compared the reactions of the isthmic portion of the DV and intrahepatic veins (IHVs) to catecholamines in vitro. Vessel rings of 1 mm width and 3 mm diameter were obtained from 17 fetal sheep (88-136 days gestational age, median 120 days). The immunohistochemical examination of the DV and IHV was performed in eight cases using an antibody against alpha-smooth muscle actin and an antibody against alpha-adrenergic receptors. Five vessel rings of the DV in early gestation (median 95 days) did not respond to KCl-induced depolarisation. Force development in response to KCl of both vessel types increased with gestational age (P < 0.05). The IHV required 4.1 +/- 0.8 min (mean +/- S.E.M.) and the DV 14.5 +/- 4.0 min to reach the maximum tension in response to KCl, which was 5.0 +/- 4.0 mN in the IHV and 2.2 +/- 1.9 mN in the DV (n = 12, P < 0.05). The maximum forces developed in response to noradrenaline (norepinephrine; 42 microM, n = 9) and adrenaline (epinephrine; 100 microM, n = 12) were about sixfold higher in the IHV rings than in the DV rings (P < 0.05). The EC50 values of the DV and the IHV rings to noradrenaline were 5.9 +/- 1.3 microM and 5.0 +/- 1.3 microM, respectively (P = 0.03). The EC50 values of the adrenaline responses were 2.5 +/- 0.5 microM for the DV and 2.2 +/- 0.7 microM for the IHV (not significant). The alpha-adrenergic receptors were present in the well-structured media of IHVs, but were less distinctive in the wall of the DV. DV sparing can be attributed to an increased resistance of IHVs to catecholamines compared with the DV. The different responses can be explained by different anatomical and functional properties of the two vessel types.

Pressure pulses and flow velocities in central veins of the anesthetized sheep fetus.

The pressure drop and pressure pulses in the isthmus of the ductus venosus (DV) in fetal sheep have not been measured directly and related to flow. In eight acutely anesthetized fetal sheep, a 3-Fr tip pressure transducer (TP) was inserted from the external jugular into the umbilical vein (UV). Ultrasound Doppler flow velocities, TP position, and intravenous pressures were recorded in the UV, DV, and inferior vena cava (VC) while the TP was withdrawn. Flow was steady in the UV, but small pressure fluctuations (<0.4 mmHg) could be detected. Time-averaged pressure dropped 1.9 mmHg (mean; 0.5-3.3 mmHg 95% confidence interval) across the DV isthmus. Pressure pulses increased from 1.7 mmHg (mean; 1.2-2.1 mmHg 95% confidence interval) in the DV to 3.9 mmHg (mean; 1.8-6.0 mmHg 95% confidence interval) in the inferior VC. The pressure wave from the heart arrived later [0.053 s (mean; 0.025-0.080 s 95% confidence interval)] in the isthmus of the DV than in the diaphragmatic inferior VC, indicating a wave velocity of approximately 1.1 m/s. At all locations, pressures and flow velocities were inversely related.