Los genes Hox en la etiología de la hernia diafragmática congénita y la atresia de esófago / The etiology of congenital diaphragmatic hernia and esophageal atresia: the Hox genes

Introducción: La administración de adriamicina o nitrofena las ratas gestantes produce en su descencencia, respectivamente, atresiaesofágica (AE) con asociación VACTERL o hernia diafragmática congénita (HDC). La etiología de estas acciones no está clara, y han sido involucrados algunos genes como el Sonic hedgehog, la familia Gli, la cascada de los retinoides y algunos genes homeóticos. Los genes Hox son los encargados de la segmentación del embrión y de controlar aspectos fundamentales del desarrollo. Los ratones con pérdida de función para Hoxa3, Hoxb3, Hoxc3, Hoxc4 y Hoxa5 muestran fenotipos compatibles con la asociación VACTERL, malformaciones cardiacas, bronquiales y pulmonares, atresia esofágica y malformaciones diafragmáticas. El presente trabajo resume nuestros hallazgos en las alteraciones de expresión de estos genes en ambos modelos experimentales. Material y métodos: Para el modelo de AE usamos ratonas gestantes a las que se administró 4 mg/kg de adriamicina intraperitoneal en los días 7,5 y 8,5 de gestación (término=19 días). En el caso de la HDC se usó el mismo modelo, administrando nitrofen por vía intragástrica en el día 8. En ambos casos se usaron como grupo control animales alos que se dio por la misma vía el mismo volumen tan solo de excipiente. Los embriones fueron recuperados en los días 13, 14, 15 y 16 en el caso de la AE y en los días 14, 16 y 19 en el caso de la HDC. Los animales completos o sus pulmones y corazones se procesaron para técnicas morfológicas, inmunohistoquímicas y de biología molecular (RTPCR, reacción en cadena de la polimerasa a tiempo real), centrándonos en los genes Hoxa3, Hoxa5, Hoxb3, Hoxb5, Hoxc4 y Hoxd3.Resultados: AE: La inmunohistoquímica reveló grandes diferencias entre los fetos expuestos a adriaminica y los controles en la captación de los anticuerpos para Hoxa3, Hoxb3 y Hoxd3 en todos los tejidos salvo el corazón. La RT-PCR mostró una disminución en la expresión de esos genes en los pulmones pero no en el corazón en los animales tratados con el teratógeno. Lo mismo ocurrió además con Hoxc4.HDC: la expresión de Hoxa3 y Hoxb5 fue similar en los corazones delos 2 grupos. Sin embargo el nitrofen ocasionó un incremento en la expresión de Hoxa5 y Hoxb3 en el corazón de los fetos expuestos enlos días 14 y 19 y una disminución en el 16. En el análisis inmunohistoquímicono se encontraron diferencias entre los grupos. Conclusiones: En ambos modelos experimentales son evidentes las alteraciones de los genes Hox estudiados, especialmente en tejido pulmonar cardiaco. Las malformaciones de estos órganos asociadas a la AE y la HDC pueden ser debidas, entre otras causas, a alteraciones en la expresión de determinados genes Hox. Debido a su específica participación en la morfogénesis pulmonar y del intestino anterior, su estudio nos podría conducir a una mejor comprensión de la etiología de estas entidades (AU

Background: SP renatal administration of adriamycin ornitrofen to pregnant mice produce in the embryos, respectively, esophagealatresia/VACTERL association (EA) or congenital diaphragmatichernia (CDH). Various genes and signalling pathways like sonic hedgehog, Gli family, retinoic acid and homeotic genes have been pointed out in the origin of these malformations. Hox genes are master regulatory genes involved in embryo segmentation and other main development processes. Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 knock-out mice show cardiac, tracheal, lung and diaphragmatic malformations, EA and phenotypes that resemble that of VACTERL syndrome. We present herein some of our findings in the expression of these genes in both experimental models. Material and methods: Pregnant mice were exposed either to 4 mg/kg of adriamycin or vehicle on embryonic days 7,5 and 8,5; embryos were recovered at four endpoints (E13 to 16). On the other hand, nitrofen was given to pregnant mice on embryonic day 8th and embryos were recovered at E14, E16 and E19. The embryos or, separately, their lungs and hearts, were randomly processed for immunohistochemical or molecularbiology studies (RT-PCR). We used antibodies for Hoxa3, Hoxb3and Hoxd3 proteins and specific primers for Hoxa3, Hoxa5, Hoxb3,Hoxb5, Hoxc4 and Hoxd3 genes. Results: EA: Upon immunohistochemistry, adriamycin-exposed embryos showed a severe decrease in expression of Hoxa3, Hoxb3 and Hoxb3proteins in heart, skin, foregut but not in the heart. RT-PCR studies showed a statistically significant decrease of the four genes studied in the lungs of OA mice when compared to controls. CDH: Upon RT-PCRassessment the expression of Hoxa5 and Hoxb3 were higher in nitrofen-exposed mice than in controls on E14 and E19 and weaker on E16.As regards immunohistochemical localization, expression of the three genes was similar in nitrofen and control animals. Conclusions: Both experimental models exhibit an alteration in the expression of several proximal Hox genes, specially in lung and cardiac tissues. The malformations in these organs associated with CDH and EA could be in part caused by these alterations. Due to their specific participation in lung and foregut morphogenesis, their study could let us to better understand the mechanisms of CDH and EA (AU)

Expression of homeotic genes Hoxa3, Hoxb3, Hoxd3 and Hoxc4 is decreased in the lungs but not in the hearts of adriamycin-exposed mice.

Exposure of rat and mouse embryos to adriamycin (doxorubicin chlorhydrate) induces esophageal atresia (EA) and VACTERL association. Sonic hedgehog (Shh) and Gli2/Gli3 pathways are involved in these conditions and knockout mice for homeotic Hox genes Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 show phenotypes with some of the associated VACTERL features. This study aims at evaluating the possible influence of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 as upstream regulators of this complex signalling. Pregnant mice were exposed either to 4 mg/kg of adriamycin (EA group) or vehicle (controls) on embryonic days 7.5 and 8.5. Embryos were recovered at four endpoints (E12.5-E15.5) and randomly assigned for immunohistochemical or molecular biology studies. Lungs and hearts were separately harvested and processed for Hoxa3, Hoxb3, Hoxd3 and Hoxc4 quantitative RT-PCR measurements. Antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins were used for immunohistochemical studies. RT-PCR studies showed a drastic and statistically significant decrease of the four genes in the lungs of EA mice when compared to controls, with a slight recovery from E15.5. Hearts of both groups showed a similar expression of all the genes throughout gestation. Control embryos expressed the hox3 paralogous genes in heart, skin, foregut derivatives and their surrounding mesoderm through E12.5-E15.5 whereas adriamycin-exposed embryos showed a severe decrease in expression of these three proteins in the same tissues but not in the heart. Adriamycin drastically reduced the expression of Hoxa3, Hoxb3, Hoxd3 and Hoxc4 in mice embryonic lungs. Their expression in the heart did not seem to be influenced by adriamycin in this experimental setting.

[The etiology of congenital diaphragmatic hernia and esophageal atresia: the Hox genes]. / Los genes Hox en la etiología de la hernia diafragmática congénita y la atresia de esófago.

BACKGROUND: Prenatal administration of adriamycin or nitrofen to pregnant mice produce in the embryos, respectively, esophageal atresia/VACTERL association (EA) or congenital diaphragmatic hernia (CDH). Various genes and signalling pathways like sonic hedgehog, Gli family, retinoic acid and homeotic genes have been pointed out in the origin of these malformations. Hox genes are master regulatory genes involved in embryo segmentation and other main development processes. Hoxa3, Hoxb3, Hoxc3, Hoxc4 and Hoxa5 knock-out mice show cardiac, tracheal, lung and diaphragmatic malformations, EA and phenotypes that resemble that of VACTERL syndrome. We present herein some of our findings in the expression of these genes in both experimental models. MATERIAL AND METHODS: Pregnant mice were exposed either to 4 mg/kg of adriamycin or vehicle on embryonic days 7,5 and 8,5; embryos were recovered at four endpoints (E13 to 16). On the other hand, nitrofen was given to pregnant mice on embryonic day 8th and embryos were recovered at E14, E16 and E19. The embryos or, separately, their lungs and hearts, were randomly processed for immunohistochemical or molecular biology studies (RT-PCR). We used antibodies for Hoxa3, Hoxb3 and Hoxd3 proteins and specific primers for Hoxa3, Hoxa5, Hoxb3, Hoxb5, Hoxc4 and Hoxd3 genes. RESULTS: EA: Upon immunohistochemistry, adriamycin-exposed embryos showed a severe decrease in expression of Hoxa3, Hoxb3 and Hoxb3 proteins in heart, skin, foregut but not in the heart. RT-PCR studies showed a statistically significant decrease of the four genes studied in the lungs of OA mice when compared to controls. CDH: Upon RT-PCR assessment the expression of Hoxa5 and Hoxb3 were higher in nitrofen-exposed mice than in controls on E14 and E19 and weaker on E16. As regards immunohistochemical localization, expression of the three genes was similar in nitrofen and control animals. CONCLUSIONS: Both experimental models exhibit an alteration in the expression of several proximal Hox genes, specially in lung and car- diac tissues. The malformations in these organs associated with CDH and EA could be in part caused by these alterations. Due to their specific participation in lung and foregut morphogenesis, their study could let us to better understand the mechanisms of CDH and EA.

[Thyroid C cells are decreased in experimental CDH]. / Las células C tiroideas están disminuidas en la hernia diafragmática experimental.

BACKGROUND/AIM: Experimental CDH is often associated with malformations of neural crest origin. Several of these features are present in human CDH and therefore likely similar pathogenic mechanisms should be explored. The aim of the present study is to examine whether thyroid C-cells, another neural crest derivative, are abnormal in this rat model. METHODS: Pregnant rats were exposed either to 100 mg of 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofén) or vehicle (controls) on 9.5 day of gestation. Fetuses were recovered on day 21st and the thyroids of those with CDH (68%) were immuno-histochemically stained with anti-calcitonin antibody. The number of positively stained cells per high power field were counted using a computer-assisted image analysis method in at least 5 sections per thyroid. The distribution of the cells within the gland was assessed as well. Comparisons between CDH and control rats were made by non-parametric tests with a significance threshold of p<0.05. RESULTS: The number of c-cells was dramatically reduced in CDH animals in comparison with controls (101.2 +/- 61.3 vs 23.1 +/- 37, p<0.0001). Histology of the thyroid was similar in both groups, but the distribution of positive C-cells within the gland followed an abnormal pattern in CDH rats with the cells tending to be located at the periphery rather than at the core of the lobes. CONCLUSIONS: Nitrofén induces a severe decrease in thyroid C cells accompanied by abnormal distribution patterns. These results add further evidence of the involvement of a neural crest dysregulation as a component of the pathogenesis of experimental CDH. Whether there is or not a clinical counterpart to these findings is still unknown, but the nature of the cardiovascular and craneo-facial malformations in some babies with CDH strongly support further research in this field.

Organ changes and bacterial translocation in a rat model of chronic rejection after small bowel transplantation.

UNLABELLED: Rejection after small bowel transplantation (SBTx) may allow bacterial translocation damaging the liver and lungs. This study investigated these issues in a rat model of chronic rejection. MATERIALS AND METHODS: Orthotopic SBTx was performed in syngeneic (SYN) (ACI-ACI, n=8) and allogeneic (ALLO) (ACI-Lewis, n=8) rat strain combinations. Cyclosporine was given to ALLO rats for 28 days. Animals were sacrified between 55 and 65 days. Lymph nodes and venous samples were cultured; Escherichia coli DNA was assessed by polymerase chain reaction. We measured intestine, liver, spleen, and lung protein and DNA contents. Chronic rejection was histologically confirmed. RESULTS: Two of eight and four of eight rats died in the first week after SYN and ALLO SBTx, respectively. There were no differences in organ weights or DNA and protein contents compared with the controls. Gram-negative enteric bacteria were found in two of four ALLO and two of six SYN rats (ns), and aerobic gram-positive were found in two of four and two of six (ns), respectively. Anaerobic growth occurred in mesenteric lymph nodes in only one ALLO rat. E. coli DNA was negative in all animals. Lungs were severely emphysematous in ALLO rats with no histologic changes observed in the other phagocytic organs. Mild rejection was found in the intestine of ALLO rats. CONCLUSIONS: Bowel lesions in ALLO rats might be consistent with chronic rejection and lung lesions could be related to bacterial translocation after SBTx. However, contrary to our expectations, no significant bacterial translocation was demonstrated in either group at the end of the experiments.

Las células C tiroideas están disminuidas en la hernia diafragmática experimental / Thyroid C cells are decreased in experimental CDH

Introducción/objetivo. La hernia diafragmática congénita (HDC) experimental a menudo se asocia con malformaciones de origen neurocristal. Algunas de estas alteraciones están también presentes en la entidad humana, por lo que el mecanismo patogénico pudiera ser similar. El objetivo de este trabajo es estudiar si las células parafoliculares tiroideas (células C), también directamente derivadas de la cresta neural, son anormales en este modelo animal. Metodos. Ratas gestantes fueron expuestas a 100 mg de nitrofén por vía intragástrica (grupo nitrofén) o 1 mL de excipiente (grupo control) en el día 9,5 de gestación. Los fetos fueron recuperados en el día 21 de gestación y los tiroides de aquellos que presentaban HDC (68%) fueron procesados para su estudio inmunohistoquímico con anticuerpo anticalcitonina. El número de células teñidas por campo microscópico de gran aumento fue establecido usando un método informático automatizado en al menos 5 cortes de cada tiroides. Se estudió igualmente la distribución de las células dentro de la glándula. Las comparaciones entre grupos se hicieron con métodos no paramétricos, estableciendo un nivel de significación estadística de p < 0,05. Resultados. El número total de células estaba reducido drásticamente en los animales del grupo nitrofén comparados con los controles (23,1±37 vs 101,2±61,3; p < 0,0001). La histología del tiroides fue similar en los 2 grupos, pero la distribución de las células parafoliculares dentro de la glándula seguía un patrón anormal en el grupo nitrofén. Conclusiones. El herbicida nitrofén induce una disminución severa del número total de células parafoliculares así como una distribución anormal de las mismas dentro del tiroides. Estos resultados refuerzan la evidencia del papel patogénico de una disregulación de la cresta neural como mecanismo de esta malformación en roedores. La semejanza del modelo experimental y la HDC en humanos invita a realizar investigaciones paralelas en las 2 especies (AU)

Background/aim. Experimental CDH is often associated with malformations of neural crest origin. Several of these features are present in human CDH and therefore likely similar pathogenic mechanisms should be explored. The aim of the present study is to examine whether thyroid C-cells, another neural crest derivative, are abnormal in this rat model. Methods. Pregnant rats were exposed either to 100 mg of 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofén) or vehicle (controls) on 9.5 day of gestation. Fetuses were recovered on day 21st and the thyroids of those with CDH (68%) were immuno-histochemically stained with anti-calcitonin antibody. The number of positively stained cells per high power field were counted using a computer-assisted image analysis method in at least 5 sections per thyroid. The distribution of the cells within the gland was assessed as well. Comparisons between CDH and control rats were made by non-parametric tests with a significance threshold of p<0.05. Results. The number of c-cells was dramatically reduced in CDH animals in comparison with controls (101.2±61.3 vs 23.1±37, p<0.0001). Histology of the thyroid was similar in both groups, but the distribution of positive C-cells within the gland followed an abnormal pattern in CDH rats with the cells tending to be located at the periphery rather than at the core of the lobes. Conclusions. Nitrofén induces a severe decrease in thyroid C cells accompanied by abnormal distribution patterns. These results add further evidence of the involvement of a neural crest dysregulation as a component of the pathogenesis of experimental CDH. Whether there is or not a clinical counterpart to these findings is still unknown, but the nature of the cardiovascular and craneo-facial malformations in some babies with CDH strongly support further research in this field

Reconstrucción digital tridimensional para el análisis de las malformaciones intratorácicas congénitas inducidas con nitrofen / Aplication of a ·D reconstruction model for the analysis of nitrofeninduced intrathoracic malformations

Introducción. Los distintos modelos informáticos de reconstrucción tridimensional ofrecen múltiples ventajas a la hora de analizar y comparar diferentes procesos biológicos y estructuras anatómicas. El modelo experimental en roedores de hernia diafragmática congénita (HDC) inducida por nitrofen ocasiona múltiples anomalías en estructuras derivadas de la cresta neural. El objetivo de este trabajo es analizar mediante un método de reconstrucción tridimensional las posibles alteraciones de los nervios vago y recurrente laríngeo en esta malformación. Material y métodos. Se usó el modelo experimental de HDC en ratas. Se estudiaron 9 fetos control y otros 9 con HDC. En cada uno se obtuvo un bloque torácico (desde laringe a la bifurcación traqueal) que fue seccionado seriadamente. Se tiñeron uno de cada 10 cortes y las imágenes resultantes (de 90 a 120 en cada feto) se digitalizaron usando un software biológico específico que permitió la reconstrucción tridimensional (TDR-3dbase®) de las estructuras señaladas (nervios vago y recurrente laríngeo, tráquea, esófago y grandes vasos). Resultados. Se encontraron diferencias en la anatomía nerviosa de los fetos con HDC comparados con los controles: 1) Ausencia de uno de los nervios vagos (4/9). 2) Ausencia de uno de los recurrentes laríngeos (6/9). 3) Hipoplasia evidente de los nervios vagos (2/9). 4) Desviaciones anatómicas groseras del recorrido de los nervios. Conclusiones. La reconstrucción tridimensional permitió un análisis detallado y proporcionó una aproximación real a la anatomía de las malformaciones. Los fetos con HDC presentaron anomalías de los nervios vago y recurrente laríngeo que apoyan la teoría de una alteración en la organogénesis mediada por la cresta neural en el origen de esta enfermedad (AU)

Background. Three dimensional computer-assisted reconstruction offers some adventages for analysis and comparison of biological phenomena and anatomical structures. The CDH nitrofen-induced animal model associates multiple anomalies in neural-crest derived tissues. The goal of this study is to analyse by a 3-D reconstruction software the malformations in the extrinsic innervation of the esophagus in this model. Methods. Nine control fetuses from 4 dams and 9 fetuses with CDH from 7 dams were studied. A thoracic block from the larynx to tracheal bifurcation was serially sectioned in the horizontal plane in every embryo. One in every 10 sections was stained with HE. The image was digitalized using biological software (TDR-3dbase®). Vagus and recurrent laryngeal nerves, trachea, esophagus and the great vessels were examined. In order to obtain the 3-D reconstructions, 90 to 120 consecutive images were used. Results. In comparison with controls there were striking abnormalities of these nerves in fetuses with CDH: 1) Absence of the left (2/9) or right (2/9) vagus nerves. 2) Absence of the left (3/9) or right (3/9) recurrent laryngeal nerves. 3) Marked hypoplasia of the trunk of the vagus (2/9). 4) Deviations of their normal course and change of normal anatomical relationships into the mediastinum (2/9). Conclusions. To fullfill our goals 3-D reconstructions allow a detailed analysis and provide a precise insight into the real anatomy. Rat fetuses with CDH have anomalies of the vagus and recurrent laryngeal nerves that support the concept of a neural crest involvement in the origin of this malformation. These observations may explain esophageal motility disorders in CDH (AU)

Bacterial translocation in acute rejection after small bowel transplantation in rats.

Acute rejection after small bowel transplantation (SBTx) may facilitate bacterial translocation (BT) and subsequent changes in the liver, spleen, and lungs. This study investigated whether BT occurs after acute rejection and whether this is followed by changes in the structure of the intestine and the phagocytic organs interposed between the gut and the general circulation. Orthotopic SBTx was performed in allogeneic (ALLO) rat-strain combinations (BN-Wistar, n=5). For comparison we used syngeneic SBTx (SYN) (BN-BN, n=6) controls. Animals were sacrificed on postoperative day 7. Mesenteric lymph nodes and portal and caval blood were cultured for aerobes and anaerobes. Escherichia coli beta-galactosidase DNA was assessed by polymerase chain reaction in the blood samples. Intestine, liver, spleen, and lung protein and DNA contents were measured. Histologic changes were graded according to standard criteria of acute rejection. For comparisons we used chi(2) and nonparametric Mann-Whitney test with a threshold of significance of p<0.05. ALLO rats lost more weight after SBTx than SYN rats (-13.02+/-4.39% vs. -8.04+/-5.08% of preoperative weight), although the difference was not significant (ns). A variable degree of graft rejection was histologically demonstrated in all ALLO rats, and DNA/protein content in the graft was significantly higher in this group (0.245+/-0.85 vs. 0.134+/-0.21, p<0.05). Gram-negative enteric bacteria were found in 4/5 ALLO and 4/6 SYN rats (ns), and aerobic Gram-positive bacteria in 2/5 and 3/6 (ns), respectively. Anaerobic growth occurred in mesenteric lymph nodes in one ALLO rat and in the bloodstream in another one. E. coli DNA was isolated in none of the ALLO but in two SYN rats (ns). BT was frequent after SBTx in both syngeneic and allogeneic strain combinations. Contrary to our expectations, BT after SBTx was not higher in ALLO group rats. However, anaerobic germs were isolated only in this group.

Pax3 mRNA is decreased in the hearts of rats with experimental diaphragmatic hernia.

Rats with nitrofen-induced congenital diaphragmatic hernia (CDH) have heart hypoplasia and cardiovascular malformations. The mechanism of action of nitrofen involves changes in neural crest signaling. Pax3 function is required for cardiac neural crest cells to complete their migration to the developing heart. The aim of this study was to examine whether Pa x 3 expression is changed at two gestational endpoints in rat embryos or fetuses exposed to nitrofen. On day E9.5 of gestation, pregnant rats received either 100 mg of nitrofen (n=10) or vehicle alone (control, n=10). The fetuses were recovered on E15 or E21. Their hearts were dissected out and weighed. Pax3 mRNA expression was determined by real-time polymerase chain reaction. We used two-tailed Student's t-tests to compare groups, with a threshold of significance of p<0.05. Compared with controls, nitrofen-exposed fetuses had heart hypoplasia in terms of heart/body weight ratio (0.62+/-0.10% vs. 0.77+/-0.17%, p<0.05). Pax3 mRNA expression in the heart was significantly decreased on E15 in nitrofen-treated embryos (32.94+/-17.11 U vs. 55.09+/-11.56 U, p<0.05), and it was still decreased, although not significantly, in the hearts of nitrofen-exposed fetuses recovered on E21 (15.67+/-5.56 U vs. 20.51+/-5.92 U, not significant). In conclusion, Pax3 is underexpressed in the hearts of nitrofen-exposed embryonal rats before the end of gestation. The mechanism of action of Pax3 should be further investigated because it could be one of the targets for future prenatal transplacental intervention.

[Aplication of a 3D reconstruction model for the analysis of nitrofen induced intrathoracic malformations]. / Reconstrucción digital tridimensional para el análisis de las malformaciones intratorácicas congénitas inducidas con nitrofen.

BACKGROUND: Three dimensional computer-assisted reconstruction offers some adventages for analysis and comparison of biological phenomena and anatomical structures. The CDH nitrofen-induced animal model associates multiple anomalies in neural-crest derived tissues. The goal of this study is to analyse by a 3-D reconstruction software the malformations in the extrinsic innervation of the esophagus in this model. METHODS: Nine control fetuses from 4 dams and 9 fetuses with CDH from 7 dams were studied. A thoracic block from the larynx to tracheal bifurcation was serially sectioned in the horizontal plane in every embryo. One in every 10 sections was stained with HE. The image was digitalized using biological software (TDR-3dbase). Vagus and recurrent laryngeal nerves, trachea, esophagus and the great vessels were examined. In order to obtain the 3-D reconstructions, 90 to 120 consecutive images were used. RESULTS: In comparison with controls there were striking abnormalities of these nerves in fetuses with CDH: 1) Absence of the left (2/9) or right (2/9) vagus nerves. 2) Absence of the left (3/9) or right (3/9) recurrent laryngeal nerves. 3) Marked hypoplasia of the trunk of the vagus (2/9). 4) Deviations of their normal course and change of normal anatomical relationships into the mediastinum (2/9). CONCLUSIONS: To fullfill our goals 3-D reconstructions allow a detailed analysis and provide a precise insight into the real anatomy. Rat fetuses with CDH have anomalies of the vagus and recurrent laryngeal nerves that support the concept of a neural crest involvement in the origin of this malformation. These observations may explain esophageal motility disorders in CDH.

Decrease of parafollicular thyroid C-cells in experimental esophageal atresia: further evidence of a neural crest pathogenic pathway.

Adriamycin-induced experimental esophageal atresia (EA) is often associated with malformations of neural crest (NC) origin, such as abnormal pharyngeal pouch derivatives like the thymus and the parathyroids. The aim of the present study was to examine whether NC-derived thyroid C-cells were abnormal in a rat model. Pregnant rats received intraperitoneally either 2 mg/kg Adriamycin (EA) or vehicle (controls) on days 8 and 9 of gestation. Fetuses were recovered on day 21, and blocks including the trachea and thyroid were fixed in formalin, coronally sectioned at 3-mum widths, and stained with standard hematoxylin and eosin until the largest area of thyroid was reached. From this point on, the 1st, 10th, and 20th slices were immunohistochemically stained with anti-calcitonin antibody. Positively-stained cells in each section of the gland were counted using a computer-assisted image analysis method, and the results were averaged. The distribution of the cells within the gland was assessed as well. Comparisons between EA and control rats were made by nonparametric tests with a significance threshold of p<0.05. The number of C-cells was dramatically reduced in EA animals compared with controls (32.4+/-36 vs. 92.3+/-60.5, p<0.001). Histology of the thyroid was similar in both groups, but the distribution of positive C-cells within the gland followed an abnormal pattern in EA rats. Adriamycin causes a pattern of NC-derived malformations, including a severe decrease in thyroid C-cells accompanied by abnormal distribution or migration patterns. These results represent further evidence of the involvement of NC organogenic control dysregulation in the pathogenesis of EA and its associated malformations. The similarities between the rat model and the clinical picture strongly support investigating other subclinical NC-derived anomalies in patients with EA.