Adriamycin produces a reproducible teratogenic model of gastrointestinal atresia in the mouse.

Gastrointestinal atresia is a major cause of bowel obstruction in the newborn. Experimental models and clinical observations have demonstrated the heterogeneous nature of its pathogenesis. A proportion is due to late intra-uterine vascular insults and some are genetic in nature. Epidemiological studies have found gastrointestinal atresia to occur with other birth defects, in particular VACTERL anomalies, suggesting that a subset of cases may result from an early disturbance to intestinal morphogenesis. Adriamycin is teratogenic in rats, producing gastrointestinal atresia and VACTERL anomalies. The mouse is the foremost mammal studied by developmental biologists, offering an expanding wealth of knowledge and scientific research techniques. The aim of this study was to create an Adriamycin mouse model for investigating the development of gastrointestinal atresia. CBA/Ca mice were accurately time-mated (n = 30). Four different doses of Adriamycin (0-saline control, 4, 5 and 6 mg/kg) at three different timings of injections were compared (12 groups). Dams received two intraperitoneal injections, 24 h apart, commencing on day 7, 7.5 or 8. Foetuses were harvested on day 18. Gastrointestinal atresia and VACTERL anomalies were examined using a dissecting microscope. Adriamycin produced type IIIa gastrointestinal atresia in six treatment groups. The effect of Adriamycin depended on the timing and dose of the injections. VACTERL anomalies were only found in four treatment groups, proposing overlapping critical embryological windows for these malformations. Gastrointestinal atresia can be induced by the teratogen Adriamycin, occurring with and without VACTERL anomalies. This produces a reproducible mouse model in which the molecular pathogenesis of gastrointestinal atresia may be studied.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography.

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9-12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Adriamycin produces a reproducible teratogenic model of vertebral, anal, cardiovascular, tracheal, esophageal, renal, and limb anomalies in the mouse.

BACKGROUND/PURPOSE: The Adriamycin rat model is an established model for vertebral, anal, cardiac, tracheal, esophageal, renal, and limb (VACTERL) anomalies and gastrointestinal atresias. Mice are the foremost mammal studied by developmental biologists, providing greater availability of molecular probes, antibodies, and transferable knowledge with transgenic studies. Only tracheoesophageal malformations have been previously described in the Adriamycin mouse model. The aim of this study was to carry out a dose-response analysis of the teratogenicity of Adriamycin in the mouse to determine the effect of the dose and timing of exposure in producing tracheoesophageal malformations and show if it causes other VACTERL anomalies. METHODS: CBA/Ca mice were accurately time mated (n = 30). Four different doses (0 [saline], 4, 5, and 6 mg/kg) of Adriamycin (EBEWE Pharma Ges.m.b.H. Nfg.KG, A-4866 Unterach, Austria) at 3 different timings of injections were compared. Dams received 2 intraperitoneal injections, 24 hours apart, commencing on day 7, 7.5, or 8. Fetuses were harvested on day 18. Anomalies were examined using a dissecting microscope and serial transverse sections. RESULTS: Administering Adriamycin at 6 mg/kg on days 7 and 8 had the most teratogenic effect, with 80% of fetuses having 3 or more VACTERL anomalies: anorectal malformation, 100%; tracheoesophageal malformation, 50%; right-sided aortic arch, 58.3%; bladder agenesis/bilateral hydronephrosis, 100%. CONCLUSION: This study establishes a mouse model that should provide insights into the cellular and molecular mechanisms underlying VACTERL anomalies.

Abnormal separation of the respiratory primordium in the adriamycin mouse model of tracheoesophageal malformations.

BACKGROUND/PURPOSE: Organogenesis relies on temperospatially coordinated signaling systems. The adriamycin rat model provided insights into the dysmorphogenesis of tracheoesophageal malformations. An adriamycin mouse model (AMM) would facilitate the investigation of their molecular pathogenesis. To transfer the knowledge gained from the rat, we describe a histological account of the critical period of organogenesis of these malformations in the AMM. METHOD: CBA/Ca mice were accurately time-mated (n = 18). Dams received intraperitoneal injections of adriamycin (6 mg/kg) (n = 12) or saline control (n = 6) on days 7 and 8. Fetuses were harvested on days 9, 9.5, 10, 11, 12, and 13, resin embedded, and 1-mum sections of the developing foregut were examined. RESULTS: Day 11 control fetuses showed normal separation of the respiratory primordium, with apoptotic bodies at the point of separation. A more caudal point of separation of the distal foregut without apoptotic bodies was found in 4 of 10 AMM fetuses. Day 13 AMM fetuses had dorsal or ventral outpouchings of the foregut, indicating which malformation they would develop. Abnormal branching of the notochord was seen from day 9.5 in AMM fetuses. This was not always associated with abnormal tracheoesophageal development. CONCLUSION: This study confirms that the abnormal observations made in the rat model apply to the mouse.

Adriamycin mouse model: a variable but reproducible model of tracheo-oesophageal malformations.

A spectrum of tracheo-oesophageal malformations is seen in humans: oesophageal atresia, tracheal agenesis and laryngotracheo-oesophageal clefts. They are thought to share a common but unknown aetiology. These birth defects are frequently associated with other VACTERL anomalies. The adriamycin rat model (ARM) has proved to be a valuable model of the VACTERL anomalies, illustrating the dysmorphogenesis of oesophageal atresia and tracheal agenesis. As organogenesis relies on temporaspatially co-ordinated signalling systems, the next step would be to study the molecular pathogenesis of tracheo-oesophageal malformations. However, the mouse is the foremost mammal studied by developmental biologists, offering an expanding wealth of knowledge and scientific research techniques with which to investigate these anomalies. A limited dose response analysis of the teratogenicity of adriamycin in the mouse has identified a dose and timing of injections that produced tracheo-oesophageal malformations and other VACTERL anomalies. A clear account of the types and variability of the tracheo-oesophageal malformations produced by this dose is essential in order to be able to plan and interpret any future investigations of early gestation fetuses. CBA/Ca mice were accurately time-mated (n = 10). Nine dams received intraperitoneal injections of adriamycin (6 mg/kg) and one control dam received saline injections, on days 7 and 8. Fetuses were harvested on day 18, near term. Tracheo-oesophageal malformations were examined by dissecting microscope and serial transverse sections. Results are reported in the standard teratological manner as mean percentage per litter (+/-SEM). The resorption rate of the adriamycin treated fetuses was 50.4%. There were 29 adriamycin treated fetuses for inspection. Tracheo-oesophageal malformations were found in 29.2% (+/-10.3), affecting five out of nine litters. Oesophageal atresia occurred in 15.6% (+/-8.1), laryngotracheo-oesophageal cleft in 10.4% (+/-7) and tracheal agenesis in 3.1% (+/-3.1). All of these malformations occurred with a tracheo-oesophageal fistula. Unlike the ARM, the AMM can produce fetuses with complete laryngotracheo-oesophageal cleft as well as oesophageal atresia or tracheal agenesis. Their occurrence was found to be reproducible but variable. These are important considerations when planning and interpreting experiments using this model.

Notable sequential alterations in notochord volume during development in the Adriamycin rat model.

BACKGROUND/PURPOSE: The Adriamycin rat model (ARM) is a well-established model of the vertebral, anorectal, cardiac, tracheoesophageal, renal, and limb association. An important finding in the ARM is that Adriamycin induces abnormal notochord morphology in the region of the foregut. Having recently demonstrated notochord hypertrophy in ARM embryos, the authors designed this study to assess notochord volume sequentially from gestational days 10 to 14 (E10-E14) to test the hypothesis that notochord hypertrophy occurs maximally soon after Adriamycin administration. METHODS: Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on E7, E8, and E9. Control animals were given saline. Embryos were recovered at E10, E11, E12, E13, and E14 and embedded in paraffin. Quantitative morphology using the Cavalieri technique was performed on hematoxylin and eosin-stained transverse serial sections to determine total embryo and total notochord volume. RESULTS: The percentage volume of notochord per embryo was significantly increased (P < .05) in Adriamycin-treated embryos at all gestational time frames from E10 to E14 when compared with equivalent controls. This increased volume of notochord was found to be maximal at E11. CONCLUSIONS: These data support the authors' previous finding that Adriamycin induces notochord hypertrophy and suggest that notochord volume is increased relative to embryo volume soon after Adriamycin administration and is maximal on E11. The abnormal increase in notochord volume during the critical phase of development may interfere with organogenesis, resulting in the vertebral, anorectal, cardiac, tracheoesophageal, renal, and limb association.

Type 2 nitric oxide synthase and protein nitration in chronic lung infection.

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen-free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS.