[show abstract][hide abstract] ABSTRACT: The mechanisms by which tracheal occlusion (TO) improves alveolarization in congenital diaphragmatic hernia (CDH) are incompletely understood. Therefore transcriptional and histological effects of TO on alveolarization were studied in the rabbit model for CDH. The question of the best normalization strategy for gene expression analysis was also addressed.
Fetal rabbits were randomized for CDH or sham operation on gestational day 23/31 and for TO or sham operation on day 28/31 resulting in four study groups. Untouched littermates were added. At term and before lung harvest, fetuses were subjected to mechanical ventilation or not. Quantitative real-time PCR was performed on lungs from 4-5 fetuses of each group with and without previous ventilation. Stability of ten housekeeping genes (HKGs) and optimal number of HKGs for normalization were determined, followed by assessment of HKG expression levels. Expression levels of eleven target genes were studied in ventilated lungs, including genes regulating elastogenesis, cell-environment interactions, and thinning of alveolar walls. Elastic staining, immunohistochemistry and Western blotting completed gene analysis.
Regarding HKG expression, TO increased β-actin and β-subunit of ATP synthase. Mechanical ventilation increased β-actin and β2-microglobulin. Flavoprotein subunit of succinate dehydrogenase and DNA topoisomerase were the most stable HKGs. CDH lungs showed disorganized elastin deposition with lower levels for tropoelastin, fibulin-5, tenascin-C, and α6-integrin. After TO, CDH lungs displayed a normal pattern of elastin distribution with increased levels for tropoelastin, fibulin-5, tenascin-C, α6-integrin, ß1-integrin, lysyl oxidase, and drebrin. TO increased transcription and immunoreactivity of tissue inhibitor of metalloproteinase-1.
Experimental TO might improve alveolarization through the mechanoregulation of crucial genes for late lung development. However part of the transcriptional changes involved genes that were not affected in CDH, raising the question of TO-induced disturbances of alveolar remodeling. Attention should also be paid to selection of HKGs for studies on mechanotransduction-mediated gene expressions.
PLoS ONE 01/2013; 8(7):e69210. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with severe congenital diaphragmatic hernia. Limited data are available regarding the effect of duration of TO on lung development. Our objective was to evaluate the effects of long (2 and 2.5 days) versus short (1 day) TO on lung development in rats with nitrofen-induced diaphragmatic hernia.
Nitrofen was administered on embryonic day (ED) 9 and fetal TO performed either on ED18.5, 19 or 20 (term = 22 days). Sham-operated and untouched littermates served as controls. On ED21, lungs were harvested and only fetuses with a left-sided diaphragmatic defect were included in further analyses.
Lung-body-weight ratio incrementally increased with the duration of TO. Increased proliferation following long TO was confirmed by immunohistochemistry and qRT-PCR for the proliferation marker Ki-67. Irrespective of duration, TO induced more complex airway architecture. Medial wall thickness of pulmonary arteries was thinner after long rather than short TO.
In the nitrofen rat model of congenital diaphragmatic hernia, a longer period of TO leads to enhanced lung growth and less muscularized pulmonary arteries.
[show abstract][hide abstract] ABSTRACT: Fetal lung development may be impaired by some congenital anomalies or in utero events. Animal models are used to understand the pathophysiology of these diseases and explore therapeutic strategies. Our group has an interest in the prenatal management of congenital diaphragmatic hernia (CDH). Isolated CDH remains associated with a 30% mortality because of lung hypoplasia and pulmonary hypertension. On day 23 of gestation (pseudoglandular stage) CDH was created in both ovarian-end fetuses (n= 28) in 14 time-mated pregnant white rabbits (hybrid of Dendermonde and New-Zealand White). At term (day 30) all survived operated fetuses and size-matched controls were harvested. Fetuses/lungs were assigned randomly to formalin fixation either under pressure of 25 cm H2O (CDH25 n=5; CTR25 n=5) or without (0 cm H2O (CDH0 n=7; CTR0 n=7). Fetuses and lungs were first weighed, and then the lungs were processed for morphometry. Pulmonary development was evaluated by lung-to-body weight ratio (LBWR) and airway and vascular morphometry. Surgical induction of CDH does reduce the LBWR to hypoplastic levels. The contralateral lung weight is 81% of what is expected, whereas the ipsilateral lung is only 46% of the normal. This was accompagnied by a loss of conducting airway generations, precisely, terminal bronchioles (TB), which were surrounded by less alveoli. The ipsilateral CDH lung demonstrated a thickened media in the peripheral arteries as well. As a result, in the severely hypoplastic ipsilateral lung, an airway fixation pressure of 25 H2O has no significant effect on the morphometric indices. The contralateral lung has a normal amount of alveoli around a single TB, which also behave like alveoli of the normal lung, i.e. expand under pressure fixation. The present study on severely hypoplastic lungs that never respirated, shows that in contrast to normal lungs, the morphometric indices are not significantly influenced by a difference in fixation pressure. Increasing fixation pressure seems to expand the lung only when sufficient alveolated parenchyma is present.
Histology and histopathology 09/2010; 25(9):1105-16. · 2.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: To assess the effects of fetal tracheal administration of VEGF on pulmonary maturation in a preterm rabbit model.
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.
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.
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.
Early human development 02/2010; 86(2):99-105. · 2.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: We sought to investigate effects of intratracheal albumin injection prior to tracheal occlusion (TO) on lung proliferation in fetal rats with nitrofen-induced congenital diaphragmatic hernia.
On embryonic day 19, nitrofen-exposed fetuses underwent TO, TO and 50 microL of either intratracheal albumin 20% or saline, or remained untouched. Main outcome at embryonic day 21.5 was expression of the proliferation marker Ki-67. Secondary outcomes were lung-to-bodyweight ratio (LBWR), tropoelastin expression, density and spatial distribution of elastin, pulmonary/alveolar morphometry, and fetal survival.
TO increased Ki-67 messenger RNA and LBWR. Albumin further increased LBWR and density of Ki-67-positive cells but also fetal mortality. TO with or without adjuncts induced elastin deposits at the tips of arising secondary crests, increased air space size, and decreased septal thickness.
TO had effects on lung proliferation and advanced the morphologic appearance. Addition of albumin increased density of proliferating cells and LBWR, yet at the expense of additional fetal loss.
American journal of obstetrics and gynecology 02/2010; 202(2):198.e1-9. · 3.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: We studied the vascular effects of betamethasone (BM) and/or tracheal occlusion (TO) in fetal rabbits with surgically induced congenital diaphragmatic hernia (CDH).
At day 23 (pseudoglandular phase; term = 31 d), 54 ovarian-end fetuses from 27 does underwent induction of CDH. Thirteen did receive either 0.05 mg/kg BM, on days 28 and 29 with a 24-h interval, or 14 saline [controls (CTR)]. At day 28, one ovarian-end fetus underwent TO and harvesting was at term. In total, we compared (ANOVA) lung-to-body weight ratio (LBWR) and vascular morphometric indices in survivors from the following groups (n - number alive at delivery): CDH (9); CDH + TO (10); unoperated controls (14); CDH + BM (10); CDH + TO (9); controls CTR + BM (13).
Maternal BM had no effect on LBWR. LBWR was comparable to normal in CDH fetuses undergoing TO. Both TO and BM have an effect on medial thickening due to CDH which is larger when both interventions are combined.
Both TO and BM lessen peripheric muscularization present in CDH lungs and their effect is cumulative.
[show abstract][hide abstract] ABSTRACT: Fetal tracheal occlusion (TO) is currently used to treat severe cases of congenital diaphragmatic hernia (DH). Clinical and experimental studies suggest an improved postnatal outcome, but lung tissue mechanics after TO have not been studied. We determined the effect of TO on mechanical impedance and lung tissue components in a rabbit model for DH. At 23 days of gestation (term = 31 days) either a sham thoracotomy or a diaphragmatic defect was induced. DH fetuses were randomly assigned to undergo 5 days later TO. Fetuses were delivered by term cesarean section to determine lung to body weight ratio (LBWR), dynamic lung mechanics and lung impedance. Airway resistance (R(aw)), elastance (H(L)), tissue damping (G(L)) and hysteresivity (G(L)/H(L)) were calculated from impedance data. Collagen I and III and elastin were quantified histologically. LBWR was significantly increased by TO compared to DH (P < 0.001) and resistance and compliance of the respiratory system (R(rs), C(rs)) were improved as well. TO resulted in a significant decrease of R(aw) comparable to observations in sham-fetuses, without effect on lung tissue mechanics H(L), G(L) and hysteresivity. This coincides with a significant decrease of collagen I, III and elastin in comparison to DH fetuses. In this first report on lung tissue mechanics in a rabbit model of DH, TO had a substantial effect on tissue morphology yet this was not mirrored in lung mechanics. We conclude that the effect of TO on lung mechanics without in utero reversal of occlusion, is dominated by airway remodeling.
[show abstract][hide abstract] ABSTRACT: Glucocorticoids are given antenatally to promote pulmonary epithelial maturation and prevent respiratory distress syndrome in premature newborns. In contrast to airway changes, effects on vessels are less documented. We hypothesized that antenatal betamethasone (BM) administration promotes vascular development. Does received either a course of BM = 0.05 mg/kg/day (18 does, 70 fetuses), BM = 0.1 mg/kg/day (20 does, 75 fetuses), or saline (11 does, 92 fetuses) starting on d25, 26 (canalicular stage), d27, d28 (saccular stage), and d29 (alveolar stage) of gestation. In total 236 fetuses from 49 does were examined at term (d31) in terms of vascular development. Lung specimens were weighed and formalin fixed for morphometry. We determined differences in fetal body, liver and lung weight, proportionate medial thickness, muscularization of intra-acinar vessels, number of vessels under 100 microm, as well as immunoreactivity to Flk-1 in vascular smooth muscle and endothelial cells. A dose-dependent reduction in neonatal body and organ weight was observed in fetuses exposed to BM at d25. In contrast, term liver weight increased after late administration of BM (d28, 29). There was a dose- and time-dependent thinning of the pulmonary arterial media, which coincided with a decreased proportion of intra- and pre-acinar muscularized arteries (ED <or= 60 microm) and increased microvascularization (ED <or= 30), as well as increased endothelial immunoreactivity for Flk-1. Analyzing our data using a purpose designed geometrical model, the results suggest that maternal administration of BM promotes changes in vascular morphology, which may be compatible with remodeling and vessel formation.
[show abstract][hide abstract] ABSTRACT: Maternal retinoid administration has beneficial effects on lung development in the nitrofen rodent toxic model of congenital diaphragmatic hernia (DH). We wanted to investigate the effects in a surgical model, where the retinoid signaling pathway is not primarily disrupted by the toxic agent. We created DH in fetal rabbits at day 23 of gestation, administrated to the does all trans-retinoic acid (ATRA) or vehicle (VHC) intramuscularly for 8 consecutive days and harvested normal and operated (DH) fetuses at 31 d (n = 7 in each group). Normal lungs exposed to ATRA had increased surfactant protein mRNA levels without change in type II pneumocyte density. There was no measurable effect on lung-to-body weight ratio and airway morphometry by ATRA. In DH lungs (DH/VHC) surfactant protein mRNA levels were increased, as well as the density of type II pneumocytes. When supplemented with ATRA (DH/ATRA) these parameters returned to normal (VHC). Cell proliferation or apoptosis were not influenced by ATRA supplementation. In conclusion, maternal ATRA supplementation does not affect gross anatomic, morphologic or proliferation indices in hypoplastic lungs related to surgically induced DH in rabbit. However, ATRA lowers surfactant protein expression and normalizes type I/II pneumocyte ratio to what is observed in normal lungs.
[show abstract][hide abstract] ABSTRACT: Glucocorticoids promote lung maturation and reduce the incidence of respiratory distress syndrome in premature newborns. We hypothesized that betamethasone (BM), which is known to induce thinning of the alveolar walls, would also thin the arterial media and adventitia of intra-parenchymatic vessels in developing rabbit lungs.
112 fetuses from 21 time-mated, pregnant, giant white rabbits received maternal injections of BM at either 0.05 or 0.1 mg/kg/day on days 25-26 of gestational age. Controls received either saline (10 does, 56 fetuses) or no injection (10 does, 59 fetuses). Fetuses were harvested from day 27 onwards until term (day 31). 44 additional fetuses (8 does) were harvested between days 23 and 26. Endpoints were wet lung-to-body weight ratio, vascular morphometric indices and immunohistochemistry staining for alpha-smooth muscle actin, Flk-1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). ANOVA (Tukey's test) and independent t test (p < 0.05) were used for comparison between BM and saline groups.
Maternal BM injected on days 25-26 to pregnant rabbits induced a significant decrease in fetal body and lung weight and the lung-to-body weight ratio in the preterm pups shortly after injection. BM led to a dose-dependent thinning of the arterial media and adventitia (pulmonary arteries with an external diameter (ED) of <100 microm), to an increase in the percentage of non-muscularized peripheral vessels (ED <60 microm), in eNOS and VEGF immunoreactivity of the endothelial and smooth muscle cells in the pulmonary vessels and to an increase in Flk-1-positive pulmonary epithelial cell density.
Maternal administration of BM caused thinning of the arterial wall of pulmonary vessels (ED <100 microm) and a decrease in muscularization in peripheral vessels (ED <60 microm). This coincided with increased expression of Flk-1 in the endothelium and smooth muscle cells of the pulmonary arteries. All the effects studied were dose-dependent.
[show abstract][hide abstract] ABSTRACT: As an aid to evaluate lung hypoplasia, we investigated the difference between T2 value in fetal lungs of lambs with surgically-induced left-sided diaphragmatic hernia (DH) and gestational age (GA)-matched control littermates (normal [NL]).
Lungs were divided into two groups: DH (n = 4) and NL (n = 6). DH was induced at 65-75 days GA (term = 145 days). Fetal MRI was performed (mean GA: 120 days) with half-Fourier acquisition single-shot turbo spin-echo (TE: 60 ms) and rapid acquisition with relaxation enhancement (TE: 350 ms) in the same location (1.5-T Philips, Gyroscan, Best, The Netherlands). T2 of each lung was calculated for multiple regions of interest by taking natural logarithm of signal-to-noise ratio. Mean T2 was compared between DH and NL (unpaired analysis for entire group). Paired comparison between left/right lung was made within DH and NL.
Unpaired analysis showed significantly lower T2 of left respectively right lungs in DH (p = 0.02 [respectively] 0.05]) compared to NL (n = 6), as well as between the T2 of all DH versus NL (p = 0.001). In DH, calculated T2 appeared to be lower in left than in right lungs (difference ranged from -2 to +49%). In NL, left and right lungs showed comparable T2.
Measurement of T2 signal intensity in DH lungs is feasible and show lower T2 in comparison to NL lungs. Left lungs from lambs with DH show lower T2 than right lungs.
Fetal Diagnosis and Therapy 02/2007; 22(2):143-8. · 1.90 Impact Factor