Pediatr Surg Int DOI 10.1007/s00383-012-3195-2
ORIGINAL ARTICLE
Abnormal control of lung branching in experimental esophageal atresia Ana Catarina Fragoso • Rosa Aras-Lopez Leopoldo Martinez • Jose´ Esteva˜o-Costa • Juan A. Tovar
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Ó Springer-Verlag Berlin Heidelberg 2012
Abstract Purpose Esophageal atresia and tracheo-esophageal fistula (EA-TEF) result from abnormal division of the foregut into esophagus and trachea thus, it may influence airway branching and lung development. The present study examined lung morphogenesis in fetuses with EA-TEF focusing in the expression of FGF10 and its receptor FGFR2 IIIb. Methods Pregnant rats received either 1.75 mg/kg i.p. adriamycin or vehicle on E7, E8 and E9. Embryos were recovered at E15, E18 and E21 and lungs processed for immunohistochemistry and RT-PCR. Three groups were studied: control, adriamycin-exposed with EA-TEF, and adriamycin-exposed without EA-TEF. Comparisons were performed with Mann–Whitney or t tests (significance level, 5 %). Results Lung weight at E15 and E18 were significantly lower in adriaEA fetuses in which the relative mRNA levels of FGF10 were significantly higher. These differences disappeared near term. The receptor FGFR2 IIIb
A. C. Fragoso L. Martinez J. A. Tovar (&) Department of Pediatric Surgery, INGEMM and Idipaz Research Laboratory, Hospital Universitario La Paz, Paseo de La Castellana, 261, 28046 Madrid, Spain e-mail:
[email protected] A. C. Fragoso L. Martinez J. A. Tovar Department of Pediatrics, Universidad Autonoma de Madrid, Madrid, Spain A. C. Fragoso J. Esteva˜o-Costa Faculty of Medicine, University of Porto, Porto, Portugal R. Aras-Lopez Universidad Autonoma de Madrid, Madrid, Spain
messenger was only significantly increased in adria noEA fetuses at E15. Immunohistochemical study was consistent with these findings. Conclusions Abnormal expression of FGF10 during earlier stages of development, when the lungs are smaller than controls, suggests a compensatory response aimed at ‘‘catching up’’ delayed tracheobronchial branching. Whether similar changes take place in the human condition and influence respiratory physiology remain to be determined. Keywords Esophageal atresia Tracheo-esophageal fistula Lung morphogenesis FGF10 FGFR2 IIIb Adriamycin rat model Lung branching
Introduction Most patients with esophageal atresia and tracheo-esophageal fistula (EA-TEF) survive and the scarcity of specimens available for structural and molecular studies results in a limited knowledge of the embryogenesis of the respiratory tract. The adriamycin rat model that mimics in a precise manner EA-TEF and its associated malformations [1] provides a unique opportunity to unravel its pathogenesis. Similar to humans, EA-TEF induced by adriamycin in rats results from abnormal division of the foregut into esophagus and trachea. If emergence of the lung bud from the foregut is abnormal, it is likely for further branching to be also abnormal and hence, for lung development to be defective. Lung hypoplasia and defective airway branching were recently reported in the adriamycin EA-TEF rat model [2]. We hypothesized that signalling by one of the key regulators of branching, FGF10 and its receptor FGFR2 IIIb, would be changed during lung growth in this model.
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Materials and methods
Reverse transcriptase polimerase chain reaction (RT-PCR)
EA-TEF rat model After IdiPaz Animal Care Committee approval, time-dated pregnant Sprague–Dawley rats (gestational day 0, E0 = sperm in vaginal smear) were treated either with 1.75 mg/kg i.p. adriamycin (Farmiblastina. Pharmacia, Madrid, Spain) or vehicle on E7, E8 and E9.
Embryo harvesting and dissection Cesarean section was performed on E15, E18 and E21, after euthanizing the dams with intracardiac injection of potassium chloride. At the elected time points, embryos were recovered, weighed and dissected under a dissecting microscope to document the presence of EA-TEF. Lungs were harvested, weighed, photographed and processed for immunohistochemistry and RNA extraction. Three groups of offsprings were compared: control (C, n = 26), adriamycin-treated with EA-TEF (adria EA, n = 32) and adriamycin-treated without EA-TEF (adria noEA, n = 28).
Immunohistochemistry Lungs were fixed overnight in 4 % paraformaldehyde. After inclusion in paraffin, 5 lm sections were stained. Immunohistochemical staining was performed using standard techniques with anti-FGF10 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) after a dilution of 1:100. Briefly, antigen recovery was performed with sodium citrate (10 mM, pH 6) in microwave for 10 min and quenching of endogenous peroxidase activity using 3 % H2O2. Immunostaining was detected using avidin–biotinhorseradish peroxidase complexes (Vector Laboratories, Burlington, CA). Images were obtained from 6 different slides from each group.
FGF10 and FGFR2 IIIb messenger expressions were quantified in a Light Cycler 480 with LightCycler 480 probes master (Roche Applied Science, Mannheim, Germany) for FGF10 (Rn00564115_m1; Applied Biosystems) and LightCycler 480 SYBR Green I Master (Roche Applied Science, Mannheim, Germany) for FGFR2 IIIb (04887352001) that was amplified using the following primer: 50 -GAGCACCGTACTGGACCAACAC and 30 -TG GTAGGTGTGGTTGATGGACC. All RT-PCR reactions were run in duplicate in a total reaction volume of 5 ng of cDNA. For FGFR2 IIIb, the RT-PCR conditions were: 95 8C for 5 min, followed by 50 cycles at 95 8C for 10 s, 60 8C for 10 s and 72 8C for 10 s; for the FGF10 probe, RTPCR conditions were: 95 8C for 10 min, followed by 45 cycles at 95 8C for 10 s, 60 8C for 30 s and at 72 8C for 1 s. Results were normalized to the expression of the 18S. The relative mRNA levels were determined by calculating the threshold cycle for each gene using the threshold cycle method. Statistical methods Data are presented as mean ± SD (standard deviation). Comparison between groups was performed with Mann– Whitney U test or unpaired t test where appropriate. The statistical significance level was set at 5 %.
Results Lung macroscopy The macroscopic appearance of the studied groups at the elected end points are shown in Fig. 1. On day 15, the lungs of adriamycin-exposed embryos were less branched; at day 18, the gross morphology still showed a smaller lung in the adria EA group, but at day 21 the lungs looked similar. Lung weight and lung weight/body weight ratio
mRNA extraction and cDNA synthesis Data is shown in Fig. 2. Total mRNA was isolated from snap-frozen lungs using High Pure RNA Tissue Kit (Roche Applied Science, Mannheim, Germany). Concentration and purity of RNAs were determined spectrophotometrically. RNA integrity was analyzed by 1 % agarose gel electrophoresis; 250 ng of total RNAs was retrotranscripted to complementary DNAs (cDNA) by reverse transcription reactions using a high capacity RNA-to-cDNA Kit (Applied Biosystems, Carlsbad, CA). All cDNAs were stored at -80 8C, until further use.
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Intra-group analysis Comparing the progress of the absolute lung weight and the lung weight/body weight ratio (LW/BW %) it could be observed that the former increased throughout gestation in all groups (p \ 0.0001), but the latter only increased significantly between E15 and E18. From E18 to E21 the LW/ BW % decreased significantly in the control and in the adria noEA groups.
Pediatr Surg Int
Fig. 1 Gross lung morphology of Control, adria noEA (adriamycinexposed without esophageal atresia) and adria EA (adriamycinexposed with esophageal atresia) groups, at E15, E18 and E21. Lungs
of adriamycin-exposed embryos are smaller and less branched on E15; on E18, the adria EA lungs remain smaller, and on day 21 all lungs looked similar
Inter-group analysis
Intra-group analysis
On E15, both absolute lung weights and LW/BW % were significantly decreased in adriamycin-treated groups (all p \ 0.0001). On E18, both absolute lung weight and LW/ BW % were significantly lower only in the adria EA group (p = 0.0166 and p = 0.0004, respectively). At E21 the adria noEA group showed a significant higher absolute lung weight when compared with control and adria EA groups (p = 0.0295 and p = 0.0037, respectively), as well as a higher LW/BW % when compared with controls.
The expression in controls demonstrated a profile of gradual increase of mRNA FGF10 levels that was significant from E15 to E18 and from E18 to E21. In the adriamycin-treated groups there were no significant changes although an increasing trend was observed in the adria EA group. The comparison between E15 and E21 revealed that controls exhibited a significant increase in the expression levels of FGF10; adria EA group showed a lower increase and adria noEA had a stable expression throughout the studied time points.
Immunohistochemistry
Inter-group analysis
FGF10 immunoreactivity was detected at all stages of development of all the studied groups. With some variations within individual sections, immunoreactivity was intense in all groups and time points, with a peak of expression at day 18. The location of the protein expression was more focal, with mesenchymal predominance, at the pseudoglandular and saccular stages of development than in the canalicular stage, when an intense ubiquitous expression was observed (Fig. 3).
The comparison revealed a significantly higher level of FGF10 messenger in the adriamycin-treated groups on E15 that was maintained in adria EA fetuses at E18. At day 21, adria noEA fetuses had significantly lower levels.
RT-PCR analysis of expression of FGF10
Intra-group analysis
Results obtained from real time RT-PCR analysis of expression of mRNA FGF10 are shown in Fig. 4.
All groups showed a peak of expression on E18. Regarding the intra-group profile, we observed a
RT-PCR analysis of expression of FGFR2 IIIb isoform The real time RT-PCR results of the expression of mRNA FGFR2 IIIb in the lung are shown in Fig. 5.
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LW/BW ratio % Control
E15 1.612±0.250
E15 vs E18
E18
p
