Antenatal fetal VEGF therapy to promote pulmonary maturation in a preterm rabbit model.

AIM: To assess the effects of fetal tracheal administration of VEGF on pulmonary maturation in a preterm rabbit model. METHODS: On day 26 (term=31days), fetal rabbits received recombinant rat VEGF (30microg in 70microL normal saline) or placebo (normal saline 70microL) intratracheally, with or without subsequent tracheal occlusion. Non-operated littermates served as internal controls. Fetuses were harvested on day 28 for morphometric study of the lungs or for mechanical ventilation and measurement of lung mechanics. In total, 96 fetuses from 42 does were used, 47 for ventilation and 49 for morphometry. RESULTS: In fetuses receiving intratracheal VEGF, an increase in immunoreactivity for Flk-1 was observed throughout the lung parenchyma. Tracheal occlusion (TO) adversely affected pulmonary mechanics as compared to un-occluded controls. That effect is partly reversed by intratracheal VEGF. Intratracheal injection of VEGF without tracheal occlusion improves lung mechanics but no more than what was observed in placebo injected controls. CONCLUSION: Antenatal intratracheal VEGF administration was associated with an increase in Flk-1 immunoreactivity. It also improves lung mechanics, however more so when the trachea is occluded. Without TO, the effects were comparable to placebo controls.

Ultrasound-guided injection and occlusion of the trachea in fetal sheep.

OBJECTIVES: To access the fetal sheep trachea by ultrasound-guided transthoracic injection in order to deliver gene therapy vectors or occlude the trachea with a detachable balloon. METHODS: Fetal sheep were operated on at a mean gestational age of 102 (range, 81-116) days (term = 145 days). Under ultrasound guidance, either a 20-G spinal (for vector delivery) or a 16-G Kellett (for placement of an occlusive balloon) needle was inserted via the fetal thorax into the fetal trachea. RESULTS: Using the 20-G spinal needle the trachea was accessed successfully in 33/36 fetuses, with 97% survival. Failure to inject was related to fetal position and gestational age. Blood vessel damage causing significant morbidity occurred in two fetuses (6%). Tracheal occlusion was achieved by puncturing the trachea with the 16-G needle and advancing an endoluminal balloon in three out of five attempts in a mean time of 17 (range, 16-19) min, with 100% survival. In one case, the balloon became sited within the accessory lobe bronchus and was not inflated. At postmortem examination 21 days later, all balloons remained inflated and occluded the trachea, and the lung-to-body weight ratio and airways morphometric indices were consistent with relative pulmonary hyperplasia in the obstructed lungs. CONCLUSIONS: Ultrasound-guided transthoracic tracheal puncture is a reliable technique in fetal sheep, with low morbidity and mortality. Using this technique, a detachable endotracheal balloon can be placed to provoke pulmonary growth. Advances in needle design and balloon size may improve the success rate.

Accuracy of magnetic resonance imaging for measuring fetal sheep lungs and other organs.

OBJECTIVE: Lung volume measurement by fetal magnetic resonance imaging (fetMRI) has been used to predict survival of fetuses with isolated congenital diaphragmatic hernia (CDH). So far, the accuracy and precision of fetMRI for volumetry of either the normal or hypoplastic developing lung has not been formally studied. METHODS: A total of nine sheep carrying 14 fetuses underwent fetMRI under general anesthesia at a mean of 118 days' gestational age (term = 145 days). A total of 61 organs were measured in nine normal fetal sheep and five that underwent surgical creation of diaphragmatic hernia (DH), so as to induce pulmonary hypoplasia. Lungs were measured on T2-WI (weighted images) in three different planes, while liver and kidneys were measured in the axial (T1-WI) and sagittal (T2-WI) planes, respectively. Necropsy was done within 24 h after fetMRI to determine the volume postmortem by the water displacement method. Values were linearly correlated and a Bland and Altman analysis was done for volume measurement comparison, calculating means +/- SD, bias (mean of the difference of volume measurements), precision (SD of the difference) and absolute and proportionate limits of agreement for both methods. The accuracy of fetMRI volume measurement was determined for different organ groups by calculating the median relative error and precision index, both being measures of error in proportion to the magnitude of the volume measured, as a clinically relevant proxy of potential errors. RESULTS: The fetMRI volume measurements were on average larger than postmortem volumes, except for the kidneys. Kidney volume determination had a relative error of 29%, while measurements of larger organs had larger relative errors (42% for liver). Normal lungs were less accurately measured in the coronal or sagittal than in the axial plane (relative error 53%, 73% and 38%, respectively; P < 0.05 for sagittal vs. axial). Axially-measured lung volumes were more accurate for lungs of normal sheep compared to DH lungs (relative error 38% vs. 73%, respectively; P < 0.05). CONCLUSION: FetMRI measured systematically higher volumes for organs such as fetal liver or lung. This may be related to fluid loss or lack of perfusion at the time of necropsy. Measurement of lung volume by fetMRI was most accurate in the axial plane. Measurements of lung and liver volumes by fetMRI in normal sheep were both in agreement with volumes measured at necropsy. Loss of accuracy for DH-lungs in comparison with the accuracy when measuring other similarly small organs, such as kidneys, suggests that fetMRI measurements can be less accurate for hypoplastic lungs related to CDH. With improving hardware, it might become easier to render the fetal lung and determine its volume reliably.

Pulmonary vascular effects of sildenafil on the development of chronic pulmonary hypertension in the ovine fetus.

We investigated the pulmonary vascular effects of prophylactic use of sildenafil, a specific phosphodiesterase-5 inhibitor, in late-gestation fetal lambs with chronic pulmonary hypertension. Fetal lambs were operated on at 129 +/- 1 days gestation (term = 147 days). Ductus arteriosus (DA) was compressed for 8 days to cause chronic pulmonary hypertension. Fetuses were treated with sildenafil (24 mg/day) or saline. Pulmonary vascular responses to increase in shear stress and in fetal PaO2 were studied at, respectively, day 4 and 6. Percent wall thickness of small pulmonary arteries (%WT) and the right ventricle-to-left ventricle plus septum ratio (RVH) were measured after completion of the study. In the control group, DA compression increased PA pressure (48 +/- 5 to 72 +/- 8 mmHg, P < 0.01) and pulmonary vascular resistance (PVR) (0.62 +/- 0.08 to 1.15 +/- 0.11 mmHg x ml(-1) x min(-1), P < 0.05). Similar increase in PAP was observed in the sildenafil group, but PVR did not change significantly (0.54 +/- 0.06 to 0.64 +/- 0.09 mmHg x ml(-1) x min(-1)). Acute DA compression, after brief decompression, elevated PVR 25% in controls and decreased PVR 35% in the sildenafil group. Increased fetal PaO2 did not change PVR in controls but decreased PVR 60% in the sildenafil group. %WT and RVH were not different between groups. Prophylactic sildenafil treatment prevents the rise in pulmonary vascular tone and altered vasoreactivity caused by DA compression in fetal lambs. These results support the hypothesis that elevated PDE5 activity is involved in the consequences of chronic pulmonary hypertension in the perinatal lung.

Pulmonary circulation of fetal sheep with lung hyperplasia following tracheal occlusion.

OBJECTIVES: To determine the alteration of blood flow velocity profile in the pulmonary arteries (PAs) of fetal sheep after tracheal occlusion (TO). METHODS: Doppler ultrasound investigations of the PAs, the pulmonary trunk and the ductus arteriosus were performed weekly in nine ewes (gestational age 92-98 days, term 145 days) with singleton pregnancies after TO (n = 5) and in control fetuses (n = 4). Histological examinations with morphometry of pulmonary arterial vessels and of airways were performed in both groups. RESULTS: In the control group the experiments lasted 38 +/- 6 days (mean +/- SD), and in the TO group 25 +/- 7 days. Relative lung weight was significantly higher in the TO group compared with the control group (14.5 +/- 3.4% and 4.0 +/- 0.5% of body weight). There were no significant differences in the Doppler parameters (pulsatility index, resistance index, the systolic peak, the diastolic minimum, time averaged maximum velocity) between groups. When the Doppler values of PAs in TO and control fetuses were combined, no significant differences between the left and right PA could be detected. The external diameter of peripheral PAs was significantly higher in the TO group as compared to control group (105.7 +/- 2.5 microm vs. 96.9 +/- 1.3 microm). CONCLUSIONS: We found a threefold increase of fetal lung volume after TO without significant changes of blood flow velocity profiles in the PAs of fetal sheep.

Pulmonary vascular morphology in a fetal rabbit model for congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Although the pulmonary vascular abnormalities in congenital diaphragmatic hernia (CDH) are described from a morphologic point of view, the treatment of pulmonary hypertension (PH) remains one of the main unsolved problems in clinical daily practice. For this reason, detailed studies in well-validated animal models could still be of significance in our understanding of the pathogenesis of CDH. METHODS: In does of 23 days' gestational age (GA), 39 fetuses underwent creation of diaphragmatic hernia (DH) and 15 fetuses a sham thoracotomy (SHAM). Thirty-nine nonoperated littermates served as internal controls (CTR). Fetuses were harvested by cesarean section on days 25, 27, 29, and 30 of gestation. Lung specimens were obtained and formalin fixed for further vascular morphometry studies. RESULTS: Lung vessels from DH fetuses started to show significantly smaller internal diameter (ID), external diameter (ED), larger arterial wall thickness (WT), and, in particular, a proportionally higher medial thickness (%MT) and adventitial thickness (%AT) from day 27 onward when compared with control fetuses. SHAM fetuses, which were harvested at term, showed no differences with CTR. CONCLUSIONS: This is the first report documenting changes over time in the vascular system in a rabbit fetal model of surgically induced DH. These changes mimic pathologic findings observed in human fetuses and are also concordant with earlier observations in the surgical ovine model and the toxic nitrofen rat model.