Adriamycin Rat Model Research Articles

The optimal dose of Adriamycin to create a viable rat model potentially applicable to congenital obstructive uropathy

Purpose The Adriamycin rat model is an established model for different organ anomalies including congenital obstructive uropathy. In the current study, we carried out a dose-response analysis to find out the optimal dose of Adriamycin to create a viable rat model of obstructive uropathy. Methods Thirty time-mated Sprague-Dawley rats were divided into 5 groups including 1 control group and 4 different treatment groups. The 4 Adriamycin dosage regimens investigated in this study were 1.25, 1.5, 1.75, and 2 mg/(kg d). Experimental rats (n = 24) were injected intraperitoneally with different doses of Adriamycin on gestational days 7 to 9 (6 rats in each group). Control rats (n = 6) were injected with an equivalent volume of saline on the same days. Viable term fetuses were harvested on gestational day 21 by cesarean delivery and dissected under a dissecting microscope. Serial transverse sections from urinary tract system were obtained for histological examination. Results One hundred thirty-three viable fetuses were recovered from Adriamycin-treated rats, and 50 were from rats in the control group. There were no resorptions in the control group; however, 52 resorptions were recorded in Adriamycin groups. The rates of hydronephrosis and resorptions were 60% and 0%, 80.5% and 5.8%, 100% and 17.3%, and 100% and 76.9% at doses of 1.25, 1.50, 1.75, and 2 mg/(kg d), respectively. Histologic examination of the kidneys in the treated groups showed a significant decrease in renal parenchyma compared with the control group. Conclusions The dosage of 1.5 mg/(kg d) of Adriamycin yielded the highest number of viable hydronephrotic fetuses. At this dose, urinary abnormalities are milder; but the highest number of viable fetuses is provided, which is necessary to create a reproducible and viable animal model.

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- μm 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.

Bilateral megaureters in the Adriamycin rat model

Congenital obstructive uropathy is associated with significant morbidity and mortality in the human neonate. The pathophysiology of congenital obstructive uropathy is poorly understood. There are very few experimental models of prenatal obstruction of the urinary tract, except in the fetal lamb or inbred rats. Prenatal exposure to Adriamycin in a rat model leads to a spectrum of malformations including urinary tract anomalies. We hypothesized that Adriamycin administration during a particular time frame could yield a high incidence of urinary tract anomalies and therefore designed this study to investigate the rates of urinary tract anomalies at different windows of Adriamycin injection in rat embryos. Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats at different times from days 6 to 10 of gestation. Control animals were given saline. Embryos recovered on gestational day 21 by cesarean section were examined for urinary tract anomalies, and malformations were noted. Sections were then processed for paraffin embedding, sectioned at 5 mum, and stained with hematoxylin and eosin for histological examination. Anomalies of the urinary tract occurred maximally following Adriamycin administration on days 7, 8, and 9 of gestation (91.6%) compared with 16% of controls. The most common urinary tract anomaly in the Adriamycin group was bilateral megaureters with a hypoplastic bladder (81%). Other anomalies included unilateral or bilateral ureterohydronephrosis with a normal-sized bladder, duplex kidney, and unilateral or bilateral renal agenesis. In conclusion, the critical embryologic window for the development of bilateral megaureters with a small bladder in the Adriamycin rat model occurs following Adriamycin administration on gestational days 7-9. This simple experimental model of bilateral megaureter may allow further research into the pathophysiology of this condition.

Esophageal atresia and other visceral anomalies in a modified Adriamycin rat model and their correlations with amniotic fluid volume variations.

The Adriamycin rat model (ARM) has been used to produce visceral malformations in fetuses to explain the mechanisms of foregut division. The models vary in the dosage of Adriamycin (ADR) and in the number of applications. Our study of a modified ARM using 2.2 mg/kg of ADR for 2 days only, intraperitoneally in pregnant rats, is presented. A total of 81 fetuses were obtained with this model from the ADR group, 74 (91%) alive. Uretero-hydronephrosis (UHN) was observed in 70 fetuses (95%), esophageal atresia (EA) in 68 (92%), duodenal atresia (DA) in 68 (92%), bladder hypoplasia (BH) in 67 (90%), plus other malformations. In evaluating amniotic fluid (AF) volume of the fetuses with EA with tracheo-esophageal fistula (TEF) (group I) and EA without TEF (group II), both associated with bilateral UHN when compared with the control group (group III), groups I and II showed higher AF volume in groups I and II than the control group (group III) did ( p=0.0001). In conclusion, ARM was adequate to produce EA and other visceral malformations. The use of ADR in a higher dosage for a shorter period of time produced better results than those presented in previous literature. The increase of AF volume obtained in fetuses presenting EA plus bilateral UHN strongly suggests, despite ureteral dilatation (urinary obstruction), that a malformed communication may exist between the urinary system and the amniotic cavity, permitting the existence of polyhydramnios that is due to digestive obstruction such as EA and DA.

The development of the proximal oesophageal pouch in the adriamycin rat model of oesophageal atresia with tracheo-oesophageal fistula.

This study examined the morphological development of the proximal oesophagus in the Adriamycin-induced rat model of oesophageal atresia. The proximal oesophageal segment in oesophageal atresia with tracheo-oesophageal fistula (OA\TOF) has been assumed to be of similar embryological origin to the distal oesophagus. However, recent research using the Adriamycin model of OA\TOF has indicated that these structures may have a different origin. Time-mated Sprague-Dawley rats were administered either Adriamycin intraperitoneally or saline of an equivalent volume between days 6-9 of gestation. The rats were sacrificed between days 11-19 of gestation, their embryos removed and histologically sectioned. These sections were analysed to observe the morphological changes occurring in the proximal foregut. The proximal oesophageal pouch first appeared on day 15.25 as a dorsal outpouching of the proximal foregut immediately cranial to an area of apoptosis in the dorsal epithelium of the distal pharynx. It elongated through a process of cellular proliferation until it was clearly formed on day 16. Relatively little growth occurred from days 17-19. In the rat developing oesophageal atresia, the proximal oesophageal pouch has an origin different to that of the distal oesophagus. This study may explain the difference in immunohistological properties and intrinsic nervous supply between the proximal and distal oesophageal segments in oesophageal atresia.

Adriamycin induces notochord hypertrophy with conservation of sonic hedgehog expression in abnormal ectopic notochord in the adriamycin rat model

Background/purpose The Adriamycin rat model (ARM) is a well-established model of the Vertebral, Anorectal, Cardiac, Tracheoesophageal, Renal, Limb (VACTERL) association. The notochord, which expresses Sonic Hedgehog ( Shh), has been found to be grossly malformed with ventral ectopic branches in the foregut region of embryos in the ARM. The authors designed this study to test the hypothesis that Shh-expressing ectopic notochord could contribute to an increased volume of notochord relative to total embryo volume, resulting in an increased concentration of Shh in the notochord of affected embryos. Methods Adriamycin was administered intraperitoneally to rats on days 7 (E7), E8, and E9 of gestation and saline to control animals. Embryos recovered at E12 and E14 were examined immunohistochemically for Shh expression. Quantitative morphology using the Cavalieri technique was performed to determine embryo and notochord volume. Results Embryos in both Adriamycin and control groups at E12 and E14 showed comparable levels of Shh expression in notochord at all locations. The percentage of notochord per embryo was significantly increased in Adriamycin embryos at E12 and E14 compared with equivalent controls. Conclusions These data suggest that Adriamycin induces notochord hypertrophy. With all regions of the notochord secreting Shh, this could result in a higher concentration gradient of Shh in close abnormal proximity to the foregut, possibly contributing to the malformations found in the VACTERL association.

Increased fibronectin expression in developing embryos is associated with abnormal notochord in the Adriamycin rat model.

The VACTERL association is a spectrum of clinical conditions, including esophageal atresia (EA) and tracheoesophageal fistula (TEF), which affects approximately 1 in 5,000 live human births. The administration of intraperitoneal Adriamycin to pregnant rats reliably induces anomalies, such as EA and TEF, in their offspring, in what is known as the Adriamycin rat model (ARM). In affected embryos the presence of gross notochord abnormalities is commonly found, with typical features being ectopic ventral branches and adherence of the notochord to the foregut. Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein present on most cell surfaces, in extracellular fluids and in plasma. FN is involved in various functions, including cell adhesion, cell motility and wound healing. Previous studies in rats have shown that a single dose of Adriamycin can produce an appreciable rise in FN levels in various organs such as kidney and heart. We hypothesised that Adriamycin administration could promote upregulation of FN expression contributing to increased gut-notochord adherence and the development of abnormal ventral notochordal branching in the ARM. This study was designed to investigate FN expression in ARM embryos. Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on days 7,8 and 9 of gestation (E7, E8 and E9 respectively). Control animals were given saline. Embryos recovered on E10-E14 were fixed, embedded in paraffin and sectioned. Immunohistochemistry using an anti-FN rabbit polyclonal antibody was performed. FN expression in both Adriamycin and control embryos on E10, E11 and E12 was comparable. However, the levels of FN expression in Adriamycin embryos on E13 and E14 were significantly greater in embryos with abnormal notochords than in equivalent control embryos. Adriamycin-induced increased expression of FN, in the ARM, may contribute to abnormal notochord development leading to the VACTERL association.

Adriamycin effects on the chick embryo.

Adriamycin is an anthracycline, anti-neoplastic drug with known teratogenic effects on foetal rats in what is known as the Adriamycin rat model (ARM). This includes conditions similar to those in newborn humans, known collectively as the VACTERL association. This comprises vertebral (V), anorectal (A), cardiac (C), tracheoesophageal (TE), renal (R) and limb (L) anomalies. We designed this study to test the hypothesis that the administration of Adriamycin to chick embryos would cause similar anomalies to those in the VACTERL association seen in the ARM. Fertilized Ross eggs received Adriamycin doses from 2-50 microg into the air sac and from 0.9-6 microg into the albumin. Administration varied from day 0-3 (D(0-3)) with D(0) being the first day of incubation. Control eggs received saline. Embryos were incubated at 38 degrees C and a relative humidity of 70%. Embryos were recovered on D(14), paraffin-embedded and transverse sections studied for morphological abnormalities. In the air sac group ( n=142), 71% of Adriamycin embryos survived versus 86% of controls (n=29). In the albumin group (n=121), 42% of Adriamycin embryos survived versus 55% of controls (n=69). No embryos demonstrated anomalies consistent with the VACTERL association. Ventral defects affected 1% of surviving Adriamycin embryos versus 4% of controls in the air sac group. In the albumin group, 19.8% of surviving Adriamycin embryos had ventral defects compared to 15.7% of surviving controls. Anophthalmia affected 1% of the surviving embryos in the Adriamycin air sac group and 2% of the Adriamycin albumin group. No controls developed anophthalmia. Exencephaly affected 2% of the survivors in the Adriamycin air sac group but none of the albumin group or controls. The administration of Adriamycin to chick embryos in comparable doses and times to those used in the ARM does not appear to produce comparable effects in relation to developmental anomalies, such as the VACTERL association. Despite examining different administration routes and mimicking the ARM, by giving Adriamycin to embryos at gastrulation, we were unable to re-create the anomalies seen in the ARM.

Notochord anomalies in the adriamycin rat model: A morphologic and molecular basis for the VACTERL association

Background/Purpose: The Adriamycin rat model (ARM) is a reliable model of the VACTERL association. The notochord is structurally abnormal in the region of the foregut, midgut, and hindgut in the ARM. The authors hypothesised that notochord anomalies allow ectopic expression of molecular signals in the developing embryo and thus lead to VACTERL malformations. This study was designed to investigate this hypothesis. Methods: Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on days 7, 8, and 9 of gestation. Control animals were given saline. Embryos were recovered on gestational days 10.5 to 14 at½-day intervals and at full term. The first group of embryos were embedded in resin, and sagittal sections stained with Toluidine blue were studied for morphologic abnormalities. The second group of embryos were examined using in situ hybridization for the expression of Sonic Hedgehog (Shh), a patterning gene implicated in the etiology of the VACTERL association. Results: Twenty-seven of the 28 (96.4%) full-term embryos showed VACTERL anomalies. Forty-five of the 50 (90%) experimental embryos (gestational days 10.5 to 14) showed notochord abnormalities. Abnormal ventral branches from the notochord toward the gut were a commonly observed abnormality. These were seen to impinge on the developing foregut, midgut, dorsal aorta, and kidney. In situ hybridization for Shh showed that these branches from the notochord expressed Shh in 66.6% of experimental embryos. This abnormal Shh expression was not seen in the control embryos. Conclusions: Adriamycin diffusely induces altered notochord morphology in the rat embryo. The abnormal notochord morphology may allow ectopic expression of Sonic Hedgehog, and, thus, contribute to the malformations found in the VACTERL association. J Pediatr Surg 38:469-473. Copyright 2003, Elsevier Science (USA). All rights reserved.

Cell death in the early adriamycin rat model.

The adriamycin rat model (ARM) exhibits many features of the VACTERL association. Adriamycin is a cytotoxic drug used in cancer chemotherapy. Although its exact mode of action is not clear, it is presumed to have a similar cytotoxic role in the developing embryo. Lysotracker red (LT) is a dye that stains phagolysosomes and apoptotic bodies and allows entire rodent embryos to be stained for apoptosis. We hypothesised that there was increased cell death in adriamycin-exposed embryos. To investigate this hypothesis, adriamycin (1.75 mg/kg) was given intraperitoneally to rats on days 7, 8, and 9 of pregnancy. A control group was given saline on the same schedule. Embryos were recovered at 3, 12, 24, and 48 h following the last dose and also at term (21 days) to confirm that the usual incidence of congenital anomalies found in the ARM was obtained in our animal model. Embryos were embedded in resin, sectioned, and studied by light microscopy. Embryos from the 3-h and 24-h groups were studied using LT and confocal microscopy to search for evidence of apoptosis. All term newborns (100%) from the adriamycin-treated group demonstrated the typical abnormalities found in the ARM, i.e., oesophageal atresia, multiple gastrointestinal atresias, vertebral malformations, absent tails, ureterohydronephrosis, etc. In the 9.5-day adriamycin group there was no difference in appearance between the experimental and control embryos. Specifically, no cellular debris or increased cell turnover indicative of adriamycin cytotoxicity was observed in the experimental group. At day 10.5, 90% of embryos from two separate litters had evidence of notochordal distortion and tethering to the gut or gut-tube abnormalities. These findings were not observed in the control embryos. Confocal microscopy and LT examination of the embryos from litters killed at 3 and 24 h following the last dose of adriamycin demonstrated no evidence of increased cell death in adriamycin-exposed embryos compared to control embryos. The absence of significant apoptosis in the developing embryos in the immediate period following administration of adriamycin suggests that the teratogenic effect of adriamycin is not caused by cell death.

Midgut atresias result from abnormal development of the notochord in an Adriamycin rat model

Background/Purpose: Prenatal exposure to Adriamycin in a rat model (ARM) has been reported to lead to a spectrum of tracheoesophageal and associated malformations of the gastrointestinal tract, including multiple intestinal atresias. An abnormal relationship of the notochord with the foregut has been implicated in the formation of esophageal atresias. The authors hypothesised that midgut atresias arise from abnormal notochord development in the region of the midgut. This study was designed to examine the gut-notochord relationship during early embryonic development. Methods: Timed pregnant Wistar rats were given 1.75 mg/kg of Adriamycin intraperitoneally on days 7, 8, and 9 of gestation. Embryos were recovered at 12-hour intervals from days 9.5 to 14, and at term. A control group was given saline instead of Adriamycin. Embryos were embedded in resin or wax, sectioned, and studied using light microscopy, paying particular attention to the notochord and surrounding structures. Results: The notochord appeared identical in controls and experimental embryos on day 9.5. However, on day 10.5 the notochord was diffusely abnormal in ARM, distorted, and tethered to foregut as well as midgut compared with controls. This abnormality was not seen in control embryos. On day 12 the notochord abnormalities were more exaggerated in the region of the midgut in ARM embryos. Full-term ARM animals had esophageal and multiple intestinal atresias. Conclusions: The notochord is abnormal in the region of the developing midgut, and this may account for the occurrence of atresias found in this region. J Pediatr Surg 37:719-722. Copyright 2002, Elsevier Science (USA). All rights reserved.

Dorsoventral patterning in oesophageal atresia with tracheo-oesophageal fistula: Evidence from a new mouse model

Background/Purpose: The well-established Adriamycin rat model of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) complements recently described mouse genetic models in which loss of function mutations in foregut patterning genes, such as Nkx2.1 (Ttf 1), lead to OA/TOF. The authors aimed to integrate the 2 systems by adapting the Adriamycin model to the mouse to study molecular aspects of tracheo-oesophageal development. Methods: Pregnant CBA/Ca mice were injected intraperitoneally with 4 mg/kg of Adriamycin on embryonic days 7.5 and 8.5. Embryos and fetuses of various gestational ages were subjected to morphologic or histologic examination. Sections were stained with H & E or processed for immunohistochemistry using an antibody specific for Nkx2.1. Results: Tracheo-oesophageal malformations were observed in 47% of Adriamycin-treated embryos. Early foregut development was similar in Adriamycin-exposed and control embryos but, by E11.5, many treated embryos had an undivided oesophago-trachea, which gave rise to the lung buds and a fistula to the stomach. The fistula originated from the dorsal aspect of the undivided tube and was negative for Nkx2.1, or showed only transient Nkx2.1 expression, compared to the strongly positive bronchi ventrally. Conclusions: The Adriamycin model of OA is adaptable to the mouse. In the absence of tracheo-oesophageal separation, the dorsal fistula retains its nonrespiratory commitment suggesting that dorsoventral patterning of foregut development is undisturbed by Adriamycin exposure. J Pediatr Surg 37:185-191. Copyright © 2002 by W.B. Saunders Company.

Multiple gastrointestinal atresias result from disturbed morphogenesis.

Multiple gastrointestinal atresias (MGA) have been reported to account for 6% to 32% of all intestinal atresias. Controversy exists regarding the pathogenesis. Many investigators believe MGA to be the result of multiple ischemic infarctions of the intestinal tract. However, some have suggested that MGA results from a malformative process early in fetal life. Prenatal exposure to adriamycin in a rat model has been reported to lead to a spectrum of tracheoesophageal and associated malformations of the gastrointestinal tract, including intestinal atresias, identical to these observed in humans. The aim of this study was to determine the incidence and histopathologic findings of MGA in order to understand the pathogenesis. Timed-pregnant Sprague-Dawley rats were injected with adriamycin (1.75 mg/kg) in nine different gestational-day protocols. MGA was only seen in those rats who received adriamycin on gestational days 7, 8, and 9. The litters were recovered on day 21 by cesarean section. The digestive tracts (DT) of the fetuses were harvested for macroscopic and microscopic examination. Ten rats who received adriamycin on gestational days 7, 8, and 9 produced 87 newborns; 1 was damaged during dissection. DT anomalies occurred in 80 (93%) of the 86 newborns; 94% of these demonstrated MGA. There was a very high incidence of associated anomalies in newborns with MGA. Histologically, the blind-ending atresias showed different degrees of villous hyperplasia with or without intraluminal material. This is the first report demonstrating a high rate of occurrence of MGA in the adriamycin rat model. The injection of adriamycin early in gestation, the high incidence of associated malformations, and the anatomic and histologic findings in MGA indicate that MGA is a result of a malformative rather than an ischemic process.

Notochord involvement in experimental esophageal atresia.

Esophageal atresia (EA) is often accompanied by vertebral defects and other anomalies. The adriamycin rat model of EA has disclosed the embryology of the malformation and shown that the vertebrae and notochord are also abnormal. This study describes the nature of notochord malformations in rat embryos exposed to adriamycin. Time-mated rats received either 1.75 mg/kg adriamycin or vehicle i.p. on gestational days (E) 6 to 9; E-12, E-12.5, and E-13 embryos were harvested, embedded in paraffin, and serially sectioned at 3 microm in transverse plane from the head to the stomach for subsequent PAS staining. The findings in both groups were compared at the three endpoints. Control embryos had neither tracheoesophageal nor notochord malformations. On day 12, only 11/36 adriamycin embryos were normal; 7/36 had abnormal notochords, 11/36 had EA, and 7/36 had both. The corresponding figures for 12.5 days were 12/27, 0/27, 7/27, and 8/27 and those for the day 13 7/23, 5/23, 3/23, and 8/23. The malformed notochords were thickened, bifurcated, or trifurcated in the sagittal plane. The simultaneous presence of notochord and esophageal malformations suggests a direct link between both defects, but our observation of isolated occurrence of both shows that they reflect two expressions of the profound disturbance of embryonic para-axial organization responsible for the cluster of malformations rather than a cause-effect association.

Embryology of esophageal atresia in the adriamycin rat model

Purpose: The aim of this study was to describe the dysmorphogenetic process leading to esophageal atresia and tracheoesophageal fistula (EA + TEF) in the recently developed Adriamycin model of the malformation. Methods: Time-mated pregnant rats were given either Adriamycin (1.75 mg/kg intraperitoneally) or saline on days 6 to 9 of gestation, and their embryos recovered on days 12, 12.5, and 13 were serially sectioned in the transversal plane and studied microscopically after H&E and PAS staining. The findings were compared with those of age-matched untreated embryos. Results: All untreated and saline embryos were normal, whereas 49% of Adriamycin embryos had foregut malformations. Tracheoesophageal separation was complete on day 12 in control embryos, whereas 9 of 10 Adriamycin-exposed embryos had a common esophagotrachea with low emergence of the bronchi at that stage. This pattern had evolved into that of a regular EA + TEF in all nine malformed embryos by day 13. On day 12.5, esophagotrachea was found in 6 of 13 and EA + TEF in 5 of 13 embryos. Two had less well-defined malformations. Conclusions: Esophagotrachea equivalent to complete tracheoesophageal cleft is the first step leading to EA + TEF in this model. The full-blown malformation is finally acquired by partial loss of the posterior wall of the foregut, which tapers-off in the mediastinal mesenchyme and respiratory differentiation of the anterior wall down to the level of bronchial bifurcation, where it constitutes the fistula and the distal esophagus.