Fetal Rabbit Lung Research Articles

Fetal Tracheal Occlusion Increases Lung Basal Cells via Increased Yap Signaling.

Fetal endoscopic tracheal occlusion (FETO) is an emerging surgical therapy for congenital diaphragmatic hernia (CDH). Ovine and rabbit data suggested altered lung epithelial cell populations after tracheal occlusion (TO) with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA sequencing (mRNA-seq) data and used quantitative image analysis in fetal rabbit lung TO, which had increased basal cells and reduced ciliated cells after TO. In a fetal mouse TO model, flow cytometry showed increased basal cells, and immunohistochemistry demonstrated basal cell extension to subpleural airways. Nuclear Yap, a known regulator of basal cell fate, was increased in TO lung, and Yap ablation on the lung epithelium abrogated TO-mediated basal cell expansion. mRNA-seq of TO lung showed increased activity of downstream Yap genes. Human lung specimens with congenital and fetal tracheal occlusion had clusters of subpleural basal cells that were not present in the control. TO increases lung epithelial cell nuclear Yap, leading to basal cell expansion.

Complementary Effect of Maternal Sildenafil and Fetal Tracheal Occlusion Improves Lung Development in the Rabbit Model of Congenital Diaphragmatic Hernia.

To evaluate the effect of combining antenatal sildenafil with fetal tracheal occlusion (TO) in fetal rabbits with surgically induced congenital diaphragmatic hernia (CDH). Although antenatal sildenafil administration rescues vascular abnormalities in lungs of fetal rabbits with CDH, it only partially improves airway morphometry. We hypothesized that we could additionally stimulate lung growth by combining this medical treatment with fetal TO. CDH was created on gestational day (GD)23 (n=54). Does were randomized to receive either sildenafil 10 mg/kg/d or placebo by subcutaneous injection from GD24 to GD30. On GD28, fetuses were randomly assigned to TO or sham neck dissection. At term (GD30) fetuses were delivered, ventilated, and finally harvested for histological and molecular analyses. Unoperated littermates served as controls. The lung-to-body-weight ratio was significantly reduced in sham-CDH fetuses either (1.2 ± 0.3% vs 2.3 ± 0.3% in controls, P=0.0003). Sildenafil had no effect on this parameter, while CDH fetuses undergoing TO had a lung-to-body-weight ratio comparable to that of controls (2.5 ± 0.8%, P<0.0001). Sildenafil alone induced an improvement in the mean terminal bronchiolar density (2.5 ± 0.8 br/mm2 vs 3.5 ± 0.9 br/mm2, P=0.043) and lung mechanics (static elastance 61 ± 36 cmH2O /mL vs 113 ± 40 cmH2O/mL, P=0.008), but both effects were more pronounced in fetuses undergoing additional TO (2.1 ± 0.8 br/mm2, P=0.001 and 31 ± 9 cmH2O/mL, P<0.0001 respectively). Both CDH-sham and CDH-TO fetuses treated with placebo had an increased medial wall thickness of peripheral pulmonary vessels (41.9 ± 2.9% and 41.8 ± 3.2%, vs 24.0 ± 2.9% in controls, P<0.0001). CDH fetuses treated with sildenafil, either with or without TO, had a medial thickness in the normal range (29.4% ± 2.6%). Finally, TO reduced gene expression of vascular endothelial growth factor and surfactant protein A and B, but this effect was counteracted by sildenafil. In the rabbit model for CDH, the combination of maternal sildenafil and TO has a complementary effect on vascular and parenchymal lung development.

Heterogeneous Response in Rabbit Fetal Diaphragmatic Hernia Lungs After Tracheal Occlusion

BackgroundFetal tracheal occlusion (TO) is an experimental therapeutic approach to stimulate lung growth in the most severe congenital diaphragmatic hernia (CDH) cases. We have previously demonstrated a heterogeneous response of normal fetal rabbit lungs after TO with the appearance of at least two distinct zones. The aim of this study was to examine the fetal lung response after TO in a left CDH fetal rabbit model. MethodsFetal rabbits at 25 d gestation underwent surgical creation of CDH followed by TO at 27 d and harvest on day 30. Morphometric analysis, global metabolomics, and fluorescence lifetime imaging microscopy (FLIM) were performed to evaluate structural and metabolic changes in control, CDH, and CDH + TO lungs. ResultsRight and left lungs were different at the baseline and had a heterogeneous pulmonary growth response in CDH and after TO. The relative percent growth of the right lungs in CDH + TO was higher than the left lungs. Morphometric analyses revealed heterogeneous tissue-to-airspace ratios, in addition to size and number of airspaces within and between the lungs in the different groups. Global metabolomics demonstrated a slower rate of metabolism in the CDH group with the left lungs being less metabolically active. TO stimulated metabolic activity in both lungs to different degrees. FLIM analysis demonstrated local heterogeneity in glycolysis, oxidative phosphorylation (OXPHOS), and FLIM “lipid-surfactant” signal within and between the right and left lungs in all groups. ConclusionsWe demonstrate that TO leads to a heterogeneous morphologic and metabolic response within and between the right and left lungs in a left CDH rabbit model.

MicroRNA 200b is upregulated in the lungs of fetal rabbits with surgically induced diaphragmatic hernia.

Profiling of miR-200b expression and its targets (transforming growth factor [TGF]-β2 and ZEB2) in the surgical rabbit congenital diaphragmatic hernia (DH) model before and after tracheal occlusion (TO). Thirty-eight timed-pregnant rabbits had left DH creation on gestational day (GD) 23. On GD28, 17 randomly selected fetuses had TO. We harvested fetuses at GD23, GD28, or GD30. We calculated lung-to-body weight ratios, processed lungs for miR-200b in situ hybridization and real-time quantitative polymerase chain reaction, and evaluated effects on downstream targets TGF-β2 or ZEB2. We obtained 16 DH fetuses (n=7 GD28 and n=9 GD30), 13 TO fetuses (GD30), and 38 control fetuses (n=15 GD23, n=11 GD28, and n=12 GD30). Diaphragmatic hernia lungs were hypoplastic, and TO resulted in control lung-to-body weight ratio levels. Term miR-200b-3p levels were significantly upregulated in the hypoplastic compared with control ipsilateral lung (1.906±0.90 vs 0.7429±0.44) (P<.01). Fetal TO ipsilateral lungs displayed a variable miR-200b response on in situ hybridization and polymerase chain reaction, with levels similar to control and congenital DH lungs. The TGF-β2 was unchanged in hypoplastic and TO lungs, and ZEB2 tended to be reduced in TO compared with DH lungs (1.79 [0.4-2.9] vs 0.73 [0.5-1.4]). Hypoplastic fetal rabbit lungs display upregulation of miR-200b expression although downstream targets are not different from controls. Following TO, fetal rabbit lungs display a variable miR-200b response.

Unique Heterogeneous Topological Pattern of the Metabolic Landscape in Rabbit Fetal Lungs following Tracheal Occlusion

Introduction: Fetal tracheal occlusion (TO) is currently an experimental approach to drive accelerated lung growth. It is stimulated by mechanotransduction that results in increased cellular proliferation and growth. However, it is currently unknown how TO affects the metabolic landscape of fetal lungs. Materials and Methods: TO or sham was performed on fetal rabbits at 26 days followed by lung harvest on day 30. Mass spectrometry was performed to evaluate global metabolic changes. Fluorescence lifetime intensity microscopy (FLIM) was performed to estimate local free/bound NADH relative ratio as an indicator of aerobic glycolysis versus oxidative phosphorylation (glycolysis/OXPHOS). Results: TO results in a metabolic shift from tricarboxylic acid cycle towards glycolysis. FLIM reveals uniform structures in control lungs characterized by similar ratios of free/bound NADH indicating a homogenous topological pattern. Similar uniform structures are observed in shams with some variability in the glycolysis/OXPHOS ratio. In contrast, lungs following TO demonstrate different types of unique distinct topological zones: one with enlarged alveoli and a shift towards glycolysis; the other maintains balance between glycolysis/OXPHOS similar to control lungs. Conclusion: We demonstrate for the first time a unique variable topological pattern of metabolism in fetal lungs following TO with a wide variation of metabolism between zones.

The amniotic fluid as a source of mesenchymal stem cells with lung-specific characteristics.

The amniotic fluid is a clinically accessible source of mesenchymal stem cells (AF-MSC) during gestation, which enables autologous cellular therapy for perinatal disorders. The origin of AF-MSC remains elusive: renal and neuronal progenitors have been isolated from the AF-MSC pool, yet no cells with pulmonary characteristics. We analyzed gene expression of pulmonary and renal markers of 212 clonal lines of AF-MSC isolated from amniocentesis samples. AF-MSC were cultured on dishes coated with extracellular matrix (ECM) proteins from decellularized fetal rabbit lungs. In vivo differentiation potential of AF-MSC that expressed markers suggestive of lung fate was tested by renal subcapsular injections in immunodeficient mice. Of all the isolated AF-MSC lines, 26% were positive for lung endodermal markers FOXA2 and NKX2.1 and lacked expression of renal markers (KSP). This AF-MSC subpopulation expressed other lung-specific factors, including IRX1, P63, FOXP2, LGR6, SFTC, and PDPN. Pulmonary marker expression decreased over passages when AF-MSC were cultured under conventional conditions, yet remained more stable when culturing the cells on lung ECM-coated dishes. Renal subcapsular injection of AF-MSC expressing lung-specific markers resulted in engrafted cells that were SPTB positive. These data suggest that FOXA2+/NKX2.1+/KSP- AF-MSC lines have lung characteristics which are supported by culture on lung ECM-coated dishes.

Association of Surfactant Protein with Expression of Hoxa5 and Hoxb5 in Rabbit Fetal Lung

Hox genes regulate organ formation and identity of the embryo, and expressed in specific temporo-spatial patterns in the developing embryo. We compared the expression levels of the Hoxa5, Hoxb5, surfactant protein (SP)-A, and SP-B genes in immature and mature rabbit fetal lung tissues, and to uncover roles for Hoxa5, Hoxb5, SP-A, and SP-B. Cesarean sections were performed after rabbits were divided into two groups of 30-31 days of gestation (term group, n = 24) and 26-27 days of gestation (preterm group, n = 24). mRNA levels of Hoxa5, Hoxb5, SP-A, and SP-B were compared by quantitative reverse transcriptase polymerase chain reaction, and protein expression of Hoxa5 and Hoxb5 was compared by western blot analysis. Fetal lung tissue histology was observed by hematoxylin and eosin (H&E) staining. The relative expression ratios of SP-A and SP-B mRNA in the term to preterm groups were 2.45:1 and 2.94:1, respectively. Hoxb5 mRNA and protein levels decreased in the term group, with a relative expression ratio of 0.48:1 and 0.50:1, however, Hoxa5 mRNA and protein levels increased in the term group with a relative expression ration of 2.99:1 and 2.33:1, respectively, for the term to preterm groups. Moreover, a significant positive correlation was found between the expression of Hoxa5 and SP-A, SP-B in the term group. Hoxa5 gene may be essential for the expression of SP-A and SP-B in term rabbits. The Hoxb5 gene may be an important factor for lung maturation in preterm rabbits.

Excessive Reversal of Epidermal Growth Factor Receptor and Ephrin Signaling Following Tracheal Occlusion in Rabbit Model of Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) causes severe pulmonary hypoplasia from herniation of abdominal contents into the thorax. Tracheal occlusion (TO) for human CDH improves survival, but morbidity and mortality remain high, and we do not fully understand the cellular pathways and processes most severely impacted by CDH and TO. We created a left diaphragmatic hernia (DH) in rabbit fetuses with subsequent TO and collected left lung sections for NextGen mRNA sequencing. DH, TO, and DHTO fetuses had comparable body and organ growth to control except for lower lung weights in DH (p<0.05). Of 13,687 expressed genes, DHTO had 687 differentially expressed genes compared to DH, but no other group-group comparison had more than 10. Considering genes in combination, many of the genes reduced in DH were more highly expressed in DHTO than in control. Benchmarking fetal rabbit lung gene expression to published lung development data, both DH and DHTO lungs were more highly correlated with the gene expression of immature lung. DNA synthesis was upregulated in DHTO compared to DH and ribosome and protein synthesis pathways were downregulated. DH reduced total and epithelial cell proliferation by half and two-thirds respectively, and DHTO increased proliferation by 2.5 and 3.4-fold respectively. Signaling pathways downregulated by DH and upregulated in DHTO were epidermal growth factor receptor signaling, ephrin signaling, and cell migration; however, levels of ephrin and EGFR signaling in DHTO exceeded that of control. Identification and inhibition of the ligands responsible for this dysregulated signaling could improve lung development in CDH.

317: Intratracheal delivery of amniotic fluid derived stem cells (AFSc) in perfluorcarbon as compared to tracheal occlusion (TO)

Perfluoro-octyl-bromide (PFOB) and amniotic fluid derived stem cells (AFSc) can be used to modulate lung development. We assessed the compatibility of AFSc with PFOB and their effects on fetal rabbit lungs in vivo as compared to tracheal occlusion (TO).

Signaling molecules in the fetal rabbit model for congenital diaphragmatic hernia

Little is known about molecular changes in lungs of fetal rabbits with surgically induced diaphragmatic hernia (DH). Therefore, we examined in this model gene expressions of pivotal molecules for the developing lung. At day 23 of gestation, DH was created in 12 fetuses from 4 does. Both lungs from six live DH fetuses and from six unoperated controls were harvested and weighed at term. Transcription of 15 genes involved in alveolarization, angiogenesis, regulation of vascular tone, or epithelial maturation was investigated by real-time quantitative polymerase chain reaction. DH decreased lung-to-body weight ratio (P < 0.001). A bilateral downregulation was seen for genes encoding for tropoelastin (P < 0.01), lysyl oxidase (P < 0.05), fibulin 5 (P < 0.05), and cGMP specific phosphodiesterase 5 (P < 0.05). Lower mRNA levels for endothelial nitric oxide synthase occurred in the ipsilateral lung (P < 0.05). Experimental DH in fetal rabbits disrupted transcription of genes implicated in lung growth and function. Similarities with the human disease make this model appropriate for investigation of new prenatal therapies.

Ultrastructural evaluation of type II pneumocytes in the hypoplastic lung of rabbit fetuses induced by oligohydramnios

The present study was carried out using electron microscopy to evaluate the expression of lamellar bodies in type II pneumocytes of fetal rabbit lungs with hypoplasia induced by oligohydramnios. Using an amniotic shunting rabbit model, the lungs obtained from 32 fetuses, including 16 experimental fetuses (shunting group) and 16 control fetuses (control group), were used for electron microscopic studies. At least 250 type II pneumocytes per fetus were photographed. The number of lamellar bodies per type II pneumocyte was counted. The number of lamellar bodies per type II pneumocyte of the shunting group was consistently and significantly less than that of the control group (5.49 +/- 2.07 vs 7.58 +/- 2.08; P < 0.01). These results suggest that the occurrence of hypoplastic lungs induced by oligohydramnios is associated with an immaturity of the surfactant system, especially the expression of the lamellar bodies of type II pneumocytes.

Effect of Maternal Administration of Betamethasone on Peripheral Arterial Development in Fetal Rabbit Lungs

Objectives: 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. Study Design: 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 α-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. Results: 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 µm), to an increase in the percentage of non-muscularized peripheral vessels (ED <60 µm), 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. Conclusions: Maternal administration of BM caused thinning of the arterial wall of pulmonary vessels (ED <100 µm) and a decrease in muscularization in peripheral vessels (ED <60 µm). 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.

Effect of maternal dexamethasone treatment on the type II pneumocytes in hypoplastic lung by oligohydramnios: an ultrastructural study

A previous study documented the effects of maternal corticosteroid treatment on structural growth and functional development in fetal lungs associated with pathogenic conditions such as oligohydramnios using immunohistochemical and morphometric analyses. The purpose of the present study was to examine the effect of maternal dexamethasone treatment the expression of lamellar body in type II pneumocytes of the fetal rabbit lungs with hypoplasia induced by oligohydramnios using electron microscopy. Using an amniotic shunting rabbit model, pregnant rabbits were injected intravenously with either 0.1 ml of saline or 0.25 mg/kg/day of dexamethasone in 0.1 ml of saline 48 and 24 h before the delivery of fetuses, at day 30 of gestation. The number of lamellar bodies per type II pneumocyte was counted in each group using electron micrographs. The number of lamellar bodies per type II pneumocyte in the lungs of the shunted group that received saline was consistently and significantly less than that of the other three groups (5.49 +/- 2.07 vs. 7.34 +/- 2.27: shunted group that received dexamethasone, 7.58 +/- 2.08: non-shunted group that received saline, 7.79 +/- 1.90: non-shunted group that received dexamethasone; P < 0.01). These results suggest that maternal dexamethasone treatment accelerates the maturation of the surfactant system, especially the expression of lamellar bodies in type II pneumocytes, even in hypoplastic lungs induced by oligohydramnios.

Effect of maternal betamethasone administrated at different stages of fetal rabbit lung development

Effect of maternal betamethasone administrated at different stages of fetal rabbit lung development

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.

Mechanical stretch-induced serotonin release from pulmonary neuroendocrine cells: implications for lung development

Pulmonary neuroendocrine cells (PNEC) produce amine (serotonin, 5-HT) and peptides (e.g., bombesin, calcitonin) with growth factor-like properties and are thought to play an important role in lung development. Because physical forces are essential for lung growth and development, we investigated the effects of mechanical strain on 5-HT release in PNEC freshly isolated from rabbit fetal lung and in the PNEC-related tumor H727 cell line. Cultures exposed to sinusoidal cyclic stretch showed a significant 5-HT release inhibitable with gadolinium chloride (10 nM), a blocker of mechanosensitive channels. In contrast to hypoxia (Po2 approximately 20 mmHg), stretch-induced 5-HT release was not affected by Ca2+-free medium or nifedipine (50 microM), excluding the exocytic pathway. In H727 cells, stretch failed to release calcitonin, a peptide stored within dense core vesicles (DCV), whereas hypoxia caused massive calcitonin release. 5-HT released by mechanical stretch is derived predominantly from the cytoplasmic pool, because it is rapid ( approximately 5 min) and is releasable from early (20 days of gestation) fetal PNEC containing few DCV. Both mechanical stretch and hypoxia upregulated expression of tryptophan hydroxylase, the rate-limiting enzyme of 5-HT synthesis. We conclude that mechanical strain is an important physiological stimulus for the release of 5-HT from PNEC via mechanosensitive channels with potential effects on lung development and resorption of lung fluid at the time of birth.

Transcription Factors NF-κB and C/EBPδ and IL-1-Induced Expression of Surfactant Protein A in Lung Explants during the Perinatal Period

Interleukin-1 (IL-1) increases the expression of surfactant protein A (SP-A) in rabbit, lamb and human fetal lung. The upregulation disappears towards term. Among the transcription factors, IL-1 activates nuclear factor ĸB (NF-ĸB) and CCAAT/enhancer-binding protein δ (C/EBPδ). NF-ĸB presumably has a role in IL-1-induced upregulation of SP-A. Also, C/EBPδ may regulate SP-A expression. The aim was to study the role of these transcription factors in the induced effect of IL-1 on SP-A expression. Explants from fetal and neonatal rabbit lung were cultured in vitro followed by studies using immunohistochemistry, electrophoretic mobility shift assay and Northern analysis. We found gestation-dependent changes in IL-1-induced immunoreactivities of NF-ĸB and C/EBPδ in the nuclei of alveolar cells. This increase in nuclear transcription factors correlated with IL-1-induced SP-A expression levels. As studied in the explants from fetal and newborn lung, the SP-A mRNA expression additionally associated with C/EBPδ mRNA and with the binding of nuclear extracts from the lung explants to the C/EBP consensus probe. On the basis of the present and previous studies, we propose that NF-ĸB and C/EBPδ have potential mediator roles in IL-1-induced upregulation of SP-A in immature lung.

Postnatal lung mechanics, lung composition, and surfactant synthesis after tracheal occlusion vs prenatal intrapulmonary instillation of perfluorocarbon in fetal rabbits

Background/Purpose Fetal tracheal occlusion (TO) accelerates lung growth but decreases surfactant production. We have previously shown that instillation of perfluorooctylbromide (PFOB) into fetal rabbit lungs leads to lung growth similar to TO. This study compares neonatal lung mechanics and surfactant production after prenatal intrapulmonary PFOB instillation vs TO. Methods In each of 18 pregnant rabbits on gestational day 27, sets of 4 fetuses underwent either (1) intrapulmonary instillation of 1 mL PFOB, (2) TO, (3) instillation of 1 mL 0.9% NaCl (saline), and (4) hysteroamniotomy without fetal manipulation (control). Fetuses were born by cesarean delivery after 48 hours. Fetuses of 12 rabbits were mechanically ventilated for 15 minutes to evaluate lung compliance and airway resistance. Pulmonary surfactant protein B (SP-B) was quantified by immunohistochemistry in fetuses of the remaining 6 rabbits. Results Compliance was decreased in the TO group after cesarean delivery (0.33 ± 0.13 mL/cm H 2O) compared with PFOB (0.59 ± 0.12 mL/cm H 2O), saline (0.50 ± 0.12 mL/cm H 2O), and control (0.52 ± 0.10 mL/cm H 2O) fetuses. Mean fetal lung to body weight ratio was higher in TO and PFOB fetuses compared with saline and control. Higher water content and lower numbers of surfactant protein B–positive cells were found in the TO-treated fetuses. Conclusions Both prenatal intrapulmonary instillation of PFOB and TO accelerate lung growth, but TO is associated with decreased postnatal lung compliance, possibly influenced by decreased surfactant production and increased fluid retention. Conversely, instillation of PFOB preserved lung compliance and surfactant synthesis.

Effects of betamethasone and thyroid releasing hormone on fetal lung maturation: an experimental and morphometric study

Objective: To determine the effects of betamethasone and thyroid releasing hormone (TRH) on morphologic fetal rabbit lung maturation when administered separately and in combination. Material and Methods: Twenty-five pregnant rabbits were divided into five groups. Study groups were as follows: TRH or betamethasone alone (between 24 and 27th days of gestation), TRH+betamethasone or betamethasone+TRH (second dose of drugs were added in the last 2 days), and the control. The pregnancies were terminated on the 27th day of gestation. Weights and volumes of the fetal lungs were determined and the results were compared. Results: Mean lung weights in all treatment groups were significantly heavier than controls ( P<0.05). The differences between mean lung weights of TRH alone, TRH+betamethasone, and betamethasone alone groups were not statistically significant ( P>0.05), but the mean lung weight of betamethasone+TRH group was significantly lower than the other treatment groups ( P<0.05). Mean lung volume of TRH alone group was significantly higher than control group; however, there were no significant differences between other treatment groups and the control. No correlation was found between the lung weights and volumes in all groups ( r=0.1, P>0.05). Conclusion: Only TRH alone treatment produced a significant increase in both fetal lung volume and weight compared to the control group. Administering TRH alone can lead to increased lung maturation. Combining TRH to corticosteroid treatment significantly decreased mean fetal lung weight, instead of an increase. According to these results combined used of these two substances probably counteract each others effect, instead of having a synergism.

Innervation of pulmonary neuroendocrine cells and neuroepithelial bodies in developing rabbit lung.

We investigated the development of innervation of the pulmonary neuroendocrine cell (PNEC) system composed of single cells and organoid cell clusters, neuroepithelial bodies (NEB) in rabbit fetal and neonatal lungs. To visualize the nerve fibers and their contacts with PNECs/NEBs, we used confocal microscopy and multilabel immunohistochemistry (IHC) with pan-neural marker, synaptic vesicle protein 2 (SV2), and serotonin (5-HT) as markers for PNECs/NEBs, and smooth muscle actin or cytokeratin to identify airway landmarks. The numbers and distribution of PNEC/NEB at different stages of lung development (E16, 18, 21, 26, and P2) and the density of innervation were quantified. First PNECs immunoreactive for 5-HT were identified in primitive airway epithelium at E18 as single cells or as small cell clusters with or without early nerve contacts. At E21 a significant increase in the number of PNECs with formation of early innervated NEB corpuscules was observed. The overall numbers of PNECs/NEBs and the density of mucosal, submucosal, and intercorpuscular innervation increased with progressing gestation and peaked postnatally (P2). At term, the majority of NEBs and single PNECs within airway mucosa possessed neural contacts. Such an extensive and complex innervation of the PNEC system indicates a multifunctional role in developing lung and during neonatal adaptation.