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Surgical experimental jejunoileal atresia in rat embryo
Journal of Pediatric Surgery (2009) 44, 1725–1729
www.elsevier.com/locate/jpedsurg
Surgical experimental jejunoileal atresia in rat embryo Naziha Khen-Dunlop a,b,⁎, Laurent Fourcade a , Frédérique Sauvat a , Guénolée de Lambert b , Anais Victor b , Nadine Cerf-Bensussan b , Sabine Sarnacki a,b a
Department of Pediatric Surgery, Necker-Enfants Malades Hospital, 75015 Paris, France Laboratory INSERM U913, Paris Descartes University, Necker Faculty, 75015 Paris, France
b
Received 15 October 2008; revised 26 November 2008; accepted 12 December 2008
Key words: Rat; Fetal surgery; Intestinal atresia; Dysmotility
Abstract Purpose: Jejunoileal atresia represents about 40% of intestinal atresia. After surgical repair, intestinal motility disorders are burdened with the postoperative outcome, and the origin of these troubles remains unclear. To specify the physiopathologic feature of jejunoileal atresia, we developed an experimental surgical model in fetal rat. Methods: Time-dated pregnant rats were operated on at 18 days of gestational age. Hysterotomy was performed, followed by fetal wall incision. The exteriorization of the bowel loop was obtained by saline injection; the intestine was ligated and returned to the abdominal cavity before incisions were closed. Fetal intestine was excised at day 21, after cesarean delivery. Results: Twenty-one pregnant rats underwent operation with 90% maternal survival rate. Among the 56 fetuses successfully operated on, 49 survived (87%). In fetuses with atresia, the mean birth weight (4.5 ± 0.6 g) and the mean intestinal length (12.8 ± 1.3 cm) were significantly lower compared to sham fetuses and controls. Conclusion: The rat model offers the advantage of a low-expense mammal model with a wide panel of probes and reagents available for the study of the gut. This model of jejunoileal atresia could be used to study the consequences of prenatal intestinal obstruction on fetal gut. © 2009 Elsevier Inc. All rights reserved.
The prevalence of intestinal atresia is about 3 for 10,000 births [1], and the diagnosis is currently made during the second or the third trimester of gestation on a dilated bowel during an ultrasound examination. Three types are recognized depending on the level of the obstruction. Duodenal atresia is observed in 50% of cases and is considered to be the result of a lack of revacuolization of the solid cord stage of intestinal development [2,3]. Associated anomalies are ⁎ Corresponding author. Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France. Tel.: +33 1 44 49 41 94; fax: +33 1 44 49 41 60. E-mail address: [email protected] (N. Khen-Dunlop). 0022-3468/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2008.12.017
frequent, especially cardiac malformations (40%) and Down's syndrome (25%) [4,5]. At the distal part of the gut, colonic atresia is a rare entity found in 10% of cases whose origin is still unclear [6]. Jejunoileal atresia represents about 40% of cases and is most often isolated [3,5]. According to the first classic description of Louw and Barnard [7] in 1955, in this form, the obstruction seems to be the consequence of a fetal vascular accident. Experimental models of ligation or electrocoagulation of the mesenteric vessels in fetal dogs, lambs, or chicks reproduced the macroscopic lesion observed in human jejunoileal atresia and provided strong elements for this hypothesis [8,9]. Atresia
1726
N. Khen-Dunlop et al.
could also occur secondary to intestinal volvulus, intussusception, or strangulation in a tight parietal defect [10-12]. The treatment performed at birth is based on the resection of the atretic segment, followed either by anastomosis or intestinal derivation. Despite a usual favorable prognosis after the surgical repair, serious complications are still observed in the postoperative issue, especially because of impaired intestinal function [13,14]. The origin of these postoperative troubles remains unclear, and thus, further studies are required to improve the management of patients. To allow more accurate insight into the physiopathologic feature of jejunoileal atresia and particularly the direct impact of the obstruction on gut development, we created an experimental surgical model in fetal rat.
1. Material and methods
Fig. 2 Introduction of the catheter intraperitonally for the injection of the saline solution (original magnification ×16).
This experimental protocol was reviewed and approved by the regional animal care and use committee (N° P2. SS.025.07). Time-dated pregnant Wistar rats were obtained from a commercial breeder (Janvier SA, Le Genest St Isle, France). The mothers were kept in individual cages and maintained under 12-hour light/12-hour dark cycling until the surgical procedure that was realized on day 18 of the gestation (term, days 22 ± 1 day). Maternal general anesthesia was obtained by an intramuscular injection of 12 mg/100 g of ketamine (50 mg/mL) and 0.07 mg/100 g of chlorpromazine (0.25 mg/mL). It maintained adequate depth anesthesia for 90 minutes. Under aseptic conditions, a midline laparotomy was performed, and one of the uterine horns was exposed. The fetuses were mobilized to expose their abdomen, avoiding the umbilical vessels. All the fetal manipulations were performed using microsurgical instruments, under a stereo dissection microscope at
a magnification ×16 (Zeiss, OPMI 1FC, Micro Mecanique, Evry, France). The fetuses were maintained into the amniotic space during the all procedure. A hysterotomy was performed after the placement of 8-0 polypropylene purse-string suture, incorporating the amniotic membranes (Fig. 1). The fetal wall incision was performed, after pulling the abdominal wall by 9-0 polypropylene suture on the lower right or left abdominal quadrant, depending on the fetal position, just above the limb. Such a low incision was essential to avoid liver injury. The fetal abdominal wall opening was realized by a 24-gauge needle where a catheter was introduced intraperitonally (Fig. 2). A sterile saline solution was then injected as long as needed to exteriorize a bowel loop. The hole has to be large enough to allow the exteriorization of one intestinal loop but not too large, to avoid the exteriorization of the liver or a complete evisceration that would be impossible to reintegrate. The intestine was ligated by 11-0 polypropylene and pushed back into the fetal abdomen using a smooth cotton patty (Fig. 3). The laparotomy and the hysterotomy were then
Fig. 1 Purse-string suture face to the right lower abdominal quadrant, easily located through the uterine wall (original magnification ×16).
Fig. 3 Ligation of the exteriorized intestinal loop (original magnification ×16).
Jejunoileal atresia in rat embryo
1727
closed (with, respectively, 9-0 and 8-0 sutures initially placed). The amniotic fluid was restored by an injection of 1 mL of sterile saline solution. During the 90 minutes of the surgical procedure, tocolysis was unnecessary. The maternal laparotomy was closed with 2 layers of 4-0 polypropylene running sutures. Shams procedures were realized at the same time consisting in hysterotomy and fetal laparotomy without intestinal ligation. The other littermates served as controls. On day 21.5 of gestation, fetuses were harvested by cesarean delivery, weighted, and killed after maternal general anesthesia, according to the same protocol described for the first procedure. The fetal intestinal tract was removed through a large midline incision, from the stomach to the rectum. The atresia was located by measuring the whole intestinal length and its distance from the duodenum and the cecum.
2. Operative results The surgical procedure involved a significant learning curve, which was not reported. At the beginning of our experience, we observed most deaths among operated fetuses because of hemorrhage (after liver injury or liver exteriorization) or failure of intestinal reintegration. In most of cases, death could be diagnosed immediately by the complete fading of fetal body. Despite the gentle manipulation of the fetuses, the mobilization of the horn could lead to a mild intrauterine hemorrhage. After improvement of the surgical technique, we limited the operative time to 90 minutes. Criteria of success were a living fetus at the cesarean delivery, a clear visible stricture, a discrepancy in gut diameter above and below the ligation, and a difference in the color of the 2 segments with a green bilious content in the proximal segment contrasting with an uncolored distal segment. The fetus had to meet all these criteria to be included.
Fig. 4 Fetuses of rat delivered by cesarean delivery. The fetus with intestinal atresia (left side) shows an abdominal distension compared to the littermate (right side) (original magnification ×16).
Fig. 5 Small intestine after intestinal atresia. The distension of the bowel above the occlusion can be important (A) or absent (B) depending on the level of the atresia. Both intestinal segments behind the atresia do not contain meconium.
Twenty-one pregnant rats underwent operation, 2 died preoperatively of anesthetic complications (90% survival rate). There were no infectious complications. A total of 56 fetuses were successfully operated on (from 2 to 4 per liter) and 49 survived (87%). On gross examination, a discreet scar was visible in the operated lower abdominal quadrant, and the abdominal distension was moderate (Fig. 4). At laparotomy, there were no signs of peritoneal inflammation, perforation or intestinal adhesions, and no obvious malformations. The mean weight of fetuses with atresia was 4.5 g (±0.6 g), and the mean intestinal length was 12.8 cm (±1.3 cm). The mean weight was 5.3 g (±0.6 g) in sham fetuses and 5.6 g (±0.7 g) in controls. The mean intestinal length was 15.4 cm (±1.2 cm) in sham fetuses and 15.7 cm (±1.5 cm) in controls. Intestinal length and birth weight were significantly lower in fetuses with atresia compared to sham fetuses (P = .007 and P b .001, respectively) and compared to controls (P = .006 and P b .001, respectively). The atresia was located in the proximal part of the gut in 8 cases (16.5%), in the middle part in 16 cases (32.5%), and in the distal part in 25 cases (51%). In all cases, no meconium was seen in the rectum. The distension of the bowel above
1728 the occlusion was variable, depending on the length of the intestinal segment above the occlusion, and on the presence or not of ingested blood (Fig. 5).
3. Discussion The current study describes the successful development of a new model of jejunoileal atresia in rat, with a high maternal and fetal survival rate (90% and 87%, respectively). The umbilical hernia withdraws at embryonic day 16 (E16) to E17 in rats; thus, this fetal surgery at E18 appears to be the earliest term for this experimental model. Moreover, despite a short length of occlusion (3 days), the features of the small bowel at the cesarean delivery reproduce the macroscopic aspect of the intestine in neonates born with intestinal atresia. Compared with shams and controls, the intestinal length and the birth weight are lower in the fetuses with atresia, arguing for direct consequences of the atresia not only on the intestinal development but also on the prenatal growth. Previous studies have shown that prenatal interruption of the amniotic fluid transit in intestinal atresia contributes to fetal undergrowth in chick embryo model [9]. It was also analyzed in human intestinal atresia where growth retardation seems to be linked to the length of the unused segment, that is, more pronounced in jejunal than in ileal atresia [15]. Two other experimental animal studies established the nutritive role of amniotic fluid on fetus by showing an increase in growth after amniotic fluid infusion [16,17]. The differences we observed between fetuses with atresia and controls confirmed that functional exclusion of a part of the fetal intestine leads to fetal growth impairment. Experimental animal models of jejunoileal atresia have been described in fetal dogs and lambs [8,18]. Although the observations were very similar to what is observed in humans, such animals cannot be extensively used because of animal expense, husbandry requirements, small number of fetuses per ewe, and long gestation. Experimental jejunoileal atresia has also been described in chicks. A large number of eggs can be studied, and numerous atresias can be obtained. Although this avian model gives important data concerning the alteration of the enteric nervous system, the extrapolation of the results to humans is however restrained [19]. The rat model offers the advantage of a mammal model, a lowexpense, numerous littermate controls, short length of gestation, and high resistance to infection. At last, a wide panel of probes and reagents is available in rats that allow an accurate study of all the components of the gut system. Based on these advantages, a rat model of intestinal atresia relying on the administration of adriamycin to the gestational mother have been successfully developed [20]. Multiple gastrointestinal atresias were obtained associated with tail and genital anomalies, all observed in 70% to 90% of the treated rats [21]. Regarding the associated malformations, this model is far from the classical human jejunoileal atresia. Although useful to elucidate the origin of syndromic
N. Khen-Dunlop et al. atresias, it results from disturbed embryogenesis and changes observed in the intestine could overtake the consequences of the only atresia [22,23]. In our model, atresia was obtained by intestinal ligation and not by focal mesenteric ischemia, but both techniques had previously shown to lead to the same morphological aspect in the lamb model [24]. Thus, this model appears appropriate for the study of the consequences of intestinal obstruction that was our aim. Recent embryology studies have shown that antenatal mechanical events can interfere with the normal development of organs and our model allows investigating this hypothesis in intestinal atresia. Numerous examples suggest the influence of mechanosensitive genes as follows: in rats, fetal tracheal occlusion accelerates lung growth [25]; in Drosophila, in vivo laser-pulse stimulation creates tissue deformation during gastrulation and can modulate the morphogenetic movements [26]; and in chick embryo, alteration of venous return to the heart after venous clip can cause congenital cardiac malformations, linked to modifications in the expression of genes involved in cardiovascular development [27]. We developed an in vivo model of experimental fetal jejunoileal atresia in the rat that could be used to study the consequences of a prenatal intestinal obstruction in fetal guts. Precise description of gut lesions, especially in the enteric nervous system, may help the understanding of the pathogenesis of motility disorders observed after the surgical repair of intestinal atresia. Such studies are the first step toward the development of new therapeutic strategies.
References [1] Cragan JD, Martin ML, Moore CA, et al. Descriptive epidemiology of small intestinal atresia, Atlanta, Georgia. Teratology 1993;48:441-50. [2] Lynn HB, Espinas EE. Intestinal atresia. Arch Surg 1959;79:357-61. [3] Walker K, Badawi N, Hamid CH, et al. A population-based study of the outcome after small bowel atresia/stenosis in New South Wales and the Australian Capital Territory, Australia, 1992-2003. J Pediatr Surg 2008;43:484-8. [4] Hemming V, Rankin J. Small intestinal atresia in a defined population: occurrence, prenatal diagnosis and survival. Prenat Diagn 2007;27: 1205-11. [5] Dalla Vecchia LK, Grosfeld JL, West KW, et al. Intestinal atresia and stenosis: a 25-year experience with 277 cases. Arch Surg 1998;133: 490-6. [6] Fairbanks TJ, Kanard RC, Del Moral PM, et al. Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway. J Pediatr Surg 2005;40:390-6. [7] Louw JH, Barnard CN. Congenital intestinal atresia; observations on its origin. Lancet 1955;269:1065-7. [8] Pawlik NA, Hardy Jr FE, Hill JL. Myoelectric activity differences in acute and chronic models of lamb intestinal atresia. J Pediatr Surg 1987;22:1203-6. [9] Lopez de Torre B, Tovar JA, et al. The nutrition of the fetus with intestinal atresia: studies in the chick embryo model. J Pediatr Surg 1992;27:1325-8. [10] Komuro H, Hori T, Amagai T, et al. The etiologic role of intrauterine volvulus and intussusception in jejunoileal atresia. J Pediatr Surg 2004; 39:1812-4.
Jejunoileal atresia in rat embryo [11] Huang WC, Wang CH, Yuh YS, et al. A rare type of ileal atresia due to intrauterine intussusception. Eur J Pediatr 2007;166:1177-8. [12] Ogunyemi D. Prenatal ultrasonographic diagnosis of ileal atresia and volvulus in a twin pregnancy. J Ultrasound Med 2000;19:723-6. [13] Piper HG, Alesbury J, Waterford SD, et al. Intestinal atresias: factors affecting clinical outcomes. J Pediatr Surg 2008;43:1244-8. [14] Khen N, Jaubert F, Sauvat F, et al. Fetal intestinal obstruction induces alteration of enteric nervous system development in human intestinal atresia. Pediatr Res 2004;56:975-80. [15] Surana R, Puri P. Small intestinal atresia: effect on fetal nutrition. J Pediatr Surg 1994;29:1250-2. [16] Buchmiller TL, Kim CS, Chopourian HL, et al. Transamniotic fetal feeding: enhancement of growth in a rabbit model of intrauterine growth retardation. Surgery 1994;116:36-41. [17] Trahair JF, Sangild PT. Fetal organ growth in response to oesophageal infusion of amniotic fluid, colostrum, milk or gastrin-releasing peptide: a study in fetal sheep. Reprod Fertil Dev 2000;12:87-95. [18] Koga Y, Hayashida Y, Ikeda K, et al. Intestinal atresia in fetal dogs produced by localized ligation of mesenteric vessels. J Pediatr Surg 1975;10:949-53. [19] Fiegel HC, Schönberg RA, Roth B, et al. Submucosal plexus of dilated gut disappears after ligation in chicken embryos: preliminary results. Eur J Pediatr Surg 2006;16:407-10.
1729 [20] Gillick J, Giles S, Bannigan S, et al. Midgut atresias result from abnormal development of the notochord in an adriamycin rat model. J Pediatr Surg 2002;37:719-22. [21] Fourcade L, Shima H, Miyazaki E, et al. Multiple gastrointestinal atresias result from disturbed morphogenesis. Pediatr Surg Int 2001; 17:361-4. [22] Dawrant MJ, Giles S, Bannigan J, et al. Adriamycin produces a reproducible teratogenic model of gastrointestinal atresia in the mouse. Pediatr Surg Int 2008;24:731-5. [23] Merei JM. Embryogenesis of adriamycin-induced hindgut atresia in rats. Pediatr Surg Int 2002;18:36-9. [24] Patricolo M, Noia G, Rossi L, et al. An experimental animal model of intestinal obstruction to simulate in utero therapy for jejunoileal atresia. Fetal Diagn Ther 1998;13:298-301. [25] Kitano Y, Yang EY, von Allmen D, et al. Tracheal occlusion in the fetal rat: a new experimental model for the study of accelerated lung growth. J Pediatr Surg 1998;33:1741-4. [26] Supatto W, Débarre D, Moulia B, et al. In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses. Proc Natl Acad Sci U S A 2005;102:1047-52. [27] Groenendijk BC, Hierck BP, Vrolijk J, et al. Changes in shear stressrelated gene expression after experimentally altered venous return in the chicken embryo. Circ Res 2005;96:1291-8.
www.elsevier.com/locate/jpedsurg
Surgical experimental jejunoileal atresia in rat embryo Naziha Khen-Dunlop a,b,⁎, Laurent Fourcade a , Frédérique Sauvat a , Guénolée de Lambert b , Anais Victor b , Nadine Cerf-Bensussan b , Sabine Sarnacki a,b a
Department of Pediatric Surgery, Necker-Enfants Malades Hospital, 75015 Paris, France Laboratory INSERM U913, Paris Descartes University, Necker Faculty, 75015 Paris, France
b
Received 15 October 2008; revised 26 November 2008; accepted 12 December 2008
Key words: Rat; Fetal surgery; Intestinal atresia; Dysmotility
Abstract Purpose: Jejunoileal atresia represents about 40% of intestinal atresia. After surgical repair, intestinal motility disorders are burdened with the postoperative outcome, and the origin of these troubles remains unclear. To specify the physiopathologic feature of jejunoileal atresia, we developed an experimental surgical model in fetal rat. Methods: Time-dated pregnant rats were operated on at 18 days of gestational age. Hysterotomy was performed, followed by fetal wall incision. The exteriorization of the bowel loop was obtained by saline injection; the intestine was ligated and returned to the abdominal cavity before incisions were closed. Fetal intestine was excised at day 21, after cesarean delivery. Results: Twenty-one pregnant rats underwent operation with 90% maternal survival rate. Among the 56 fetuses successfully operated on, 49 survived (87%). In fetuses with atresia, the mean birth weight (4.5 ± 0.6 g) and the mean intestinal length (12.8 ± 1.3 cm) were significantly lower compared to sham fetuses and controls. Conclusion: The rat model offers the advantage of a low-expense mammal model with a wide panel of probes and reagents available for the study of the gut. This model of jejunoileal atresia could be used to study the consequences of prenatal intestinal obstruction on fetal gut. © 2009 Elsevier Inc. All rights reserved.
The prevalence of intestinal atresia is about 3 for 10,000 births [1], and the diagnosis is currently made during the second or the third trimester of gestation on a dilated bowel during an ultrasound examination. Three types are recognized depending on the level of the obstruction. Duodenal atresia is observed in 50% of cases and is considered to be the result of a lack of revacuolization of the solid cord stage of intestinal development [2,3]. Associated anomalies are ⁎ Corresponding author. Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France. Tel.: +33 1 44 49 41 94; fax: +33 1 44 49 41 60. E-mail address: [email protected] (N. Khen-Dunlop). 0022-3468/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2008.12.017
frequent, especially cardiac malformations (40%) and Down's syndrome (25%) [4,5]. At the distal part of the gut, colonic atresia is a rare entity found in 10% of cases whose origin is still unclear [6]. Jejunoileal atresia represents about 40% of cases and is most often isolated [3,5]. According to the first classic description of Louw and Barnard [7] in 1955, in this form, the obstruction seems to be the consequence of a fetal vascular accident. Experimental models of ligation or electrocoagulation of the mesenteric vessels in fetal dogs, lambs, or chicks reproduced the macroscopic lesion observed in human jejunoileal atresia and provided strong elements for this hypothesis [8,9]. Atresia
1726
N. Khen-Dunlop et al.
could also occur secondary to intestinal volvulus, intussusception, or strangulation in a tight parietal defect [10-12]. The treatment performed at birth is based on the resection of the atretic segment, followed either by anastomosis or intestinal derivation. Despite a usual favorable prognosis after the surgical repair, serious complications are still observed in the postoperative issue, especially because of impaired intestinal function [13,14]. The origin of these postoperative troubles remains unclear, and thus, further studies are required to improve the management of patients. To allow more accurate insight into the physiopathologic feature of jejunoileal atresia and particularly the direct impact of the obstruction on gut development, we created an experimental surgical model in fetal rat.
1. Material and methods
Fig. 2 Introduction of the catheter intraperitonally for the injection of the saline solution (original magnification ×16).
This experimental protocol was reviewed and approved by the regional animal care and use committee (N° P2. SS.025.07). Time-dated pregnant Wistar rats were obtained from a commercial breeder (Janvier SA, Le Genest St Isle, France). The mothers were kept in individual cages and maintained under 12-hour light/12-hour dark cycling until the surgical procedure that was realized on day 18 of the gestation (term, days 22 ± 1 day). Maternal general anesthesia was obtained by an intramuscular injection of 12 mg/100 g of ketamine (50 mg/mL) and 0.07 mg/100 g of chlorpromazine (0.25 mg/mL). It maintained adequate depth anesthesia for 90 minutes. Under aseptic conditions, a midline laparotomy was performed, and one of the uterine horns was exposed. The fetuses were mobilized to expose their abdomen, avoiding the umbilical vessels. All the fetal manipulations were performed using microsurgical instruments, under a stereo dissection microscope at
a magnification ×16 (Zeiss, OPMI 1FC, Micro Mecanique, Evry, France). The fetuses were maintained into the amniotic space during the all procedure. A hysterotomy was performed after the placement of 8-0 polypropylene purse-string suture, incorporating the amniotic membranes (Fig. 1). The fetal wall incision was performed, after pulling the abdominal wall by 9-0 polypropylene suture on the lower right or left abdominal quadrant, depending on the fetal position, just above the limb. Such a low incision was essential to avoid liver injury. The fetal abdominal wall opening was realized by a 24-gauge needle where a catheter was introduced intraperitonally (Fig. 2). A sterile saline solution was then injected as long as needed to exteriorize a bowel loop. The hole has to be large enough to allow the exteriorization of one intestinal loop but not too large, to avoid the exteriorization of the liver or a complete evisceration that would be impossible to reintegrate. The intestine was ligated by 11-0 polypropylene and pushed back into the fetal abdomen using a smooth cotton patty (Fig. 3). The laparotomy and the hysterotomy were then
Fig. 1 Purse-string suture face to the right lower abdominal quadrant, easily located through the uterine wall (original magnification ×16).
Fig. 3 Ligation of the exteriorized intestinal loop (original magnification ×16).
Jejunoileal atresia in rat embryo
1727
closed (with, respectively, 9-0 and 8-0 sutures initially placed). The amniotic fluid was restored by an injection of 1 mL of sterile saline solution. During the 90 minutes of the surgical procedure, tocolysis was unnecessary. The maternal laparotomy was closed with 2 layers of 4-0 polypropylene running sutures. Shams procedures were realized at the same time consisting in hysterotomy and fetal laparotomy without intestinal ligation. The other littermates served as controls. On day 21.5 of gestation, fetuses were harvested by cesarean delivery, weighted, and killed after maternal general anesthesia, according to the same protocol described for the first procedure. The fetal intestinal tract was removed through a large midline incision, from the stomach to the rectum. The atresia was located by measuring the whole intestinal length and its distance from the duodenum and the cecum.
2. Operative results The surgical procedure involved a significant learning curve, which was not reported. At the beginning of our experience, we observed most deaths among operated fetuses because of hemorrhage (after liver injury or liver exteriorization) or failure of intestinal reintegration. In most of cases, death could be diagnosed immediately by the complete fading of fetal body. Despite the gentle manipulation of the fetuses, the mobilization of the horn could lead to a mild intrauterine hemorrhage. After improvement of the surgical technique, we limited the operative time to 90 minutes. Criteria of success were a living fetus at the cesarean delivery, a clear visible stricture, a discrepancy in gut diameter above and below the ligation, and a difference in the color of the 2 segments with a green bilious content in the proximal segment contrasting with an uncolored distal segment. The fetus had to meet all these criteria to be included.
Fig. 4 Fetuses of rat delivered by cesarean delivery. The fetus with intestinal atresia (left side) shows an abdominal distension compared to the littermate (right side) (original magnification ×16).
Fig. 5 Small intestine after intestinal atresia. The distension of the bowel above the occlusion can be important (A) or absent (B) depending on the level of the atresia. Both intestinal segments behind the atresia do not contain meconium.
Twenty-one pregnant rats underwent operation, 2 died preoperatively of anesthetic complications (90% survival rate). There were no infectious complications. A total of 56 fetuses were successfully operated on (from 2 to 4 per liter) and 49 survived (87%). On gross examination, a discreet scar was visible in the operated lower abdominal quadrant, and the abdominal distension was moderate (Fig. 4). At laparotomy, there were no signs of peritoneal inflammation, perforation or intestinal adhesions, and no obvious malformations. The mean weight of fetuses with atresia was 4.5 g (±0.6 g), and the mean intestinal length was 12.8 cm (±1.3 cm). The mean weight was 5.3 g (±0.6 g) in sham fetuses and 5.6 g (±0.7 g) in controls. The mean intestinal length was 15.4 cm (±1.2 cm) in sham fetuses and 15.7 cm (±1.5 cm) in controls. Intestinal length and birth weight were significantly lower in fetuses with atresia compared to sham fetuses (P = .007 and P b .001, respectively) and compared to controls (P = .006 and P b .001, respectively). The atresia was located in the proximal part of the gut in 8 cases (16.5%), in the middle part in 16 cases (32.5%), and in the distal part in 25 cases (51%). In all cases, no meconium was seen in the rectum. The distension of the bowel above
1728 the occlusion was variable, depending on the length of the intestinal segment above the occlusion, and on the presence or not of ingested blood (Fig. 5).
3. Discussion The current study describes the successful development of a new model of jejunoileal atresia in rat, with a high maternal and fetal survival rate (90% and 87%, respectively). The umbilical hernia withdraws at embryonic day 16 (E16) to E17 in rats; thus, this fetal surgery at E18 appears to be the earliest term for this experimental model. Moreover, despite a short length of occlusion (3 days), the features of the small bowel at the cesarean delivery reproduce the macroscopic aspect of the intestine in neonates born with intestinal atresia. Compared with shams and controls, the intestinal length and the birth weight are lower in the fetuses with atresia, arguing for direct consequences of the atresia not only on the intestinal development but also on the prenatal growth. Previous studies have shown that prenatal interruption of the amniotic fluid transit in intestinal atresia contributes to fetal undergrowth in chick embryo model [9]. It was also analyzed in human intestinal atresia where growth retardation seems to be linked to the length of the unused segment, that is, more pronounced in jejunal than in ileal atresia [15]. Two other experimental animal studies established the nutritive role of amniotic fluid on fetus by showing an increase in growth after amniotic fluid infusion [16,17]. The differences we observed between fetuses with atresia and controls confirmed that functional exclusion of a part of the fetal intestine leads to fetal growth impairment. Experimental animal models of jejunoileal atresia have been described in fetal dogs and lambs [8,18]. Although the observations were very similar to what is observed in humans, such animals cannot be extensively used because of animal expense, husbandry requirements, small number of fetuses per ewe, and long gestation. Experimental jejunoileal atresia has also been described in chicks. A large number of eggs can be studied, and numerous atresias can be obtained. Although this avian model gives important data concerning the alteration of the enteric nervous system, the extrapolation of the results to humans is however restrained [19]. The rat model offers the advantage of a mammal model, a lowexpense, numerous littermate controls, short length of gestation, and high resistance to infection. At last, a wide panel of probes and reagents is available in rats that allow an accurate study of all the components of the gut system. Based on these advantages, a rat model of intestinal atresia relying on the administration of adriamycin to the gestational mother have been successfully developed [20]. Multiple gastrointestinal atresias were obtained associated with tail and genital anomalies, all observed in 70% to 90% of the treated rats [21]. Regarding the associated malformations, this model is far from the classical human jejunoileal atresia. Although useful to elucidate the origin of syndromic
N. Khen-Dunlop et al. atresias, it results from disturbed embryogenesis and changes observed in the intestine could overtake the consequences of the only atresia [22,23]. In our model, atresia was obtained by intestinal ligation and not by focal mesenteric ischemia, but both techniques had previously shown to lead to the same morphological aspect in the lamb model [24]. Thus, this model appears appropriate for the study of the consequences of intestinal obstruction that was our aim. Recent embryology studies have shown that antenatal mechanical events can interfere with the normal development of organs and our model allows investigating this hypothesis in intestinal atresia. Numerous examples suggest the influence of mechanosensitive genes as follows: in rats, fetal tracheal occlusion accelerates lung growth [25]; in Drosophila, in vivo laser-pulse stimulation creates tissue deformation during gastrulation and can modulate the morphogenetic movements [26]; and in chick embryo, alteration of venous return to the heart after venous clip can cause congenital cardiac malformations, linked to modifications in the expression of genes involved in cardiovascular development [27]. We developed an in vivo model of experimental fetal jejunoileal atresia in the rat that could be used to study the consequences of a prenatal intestinal obstruction in fetal guts. Precise description of gut lesions, especially in the enteric nervous system, may help the understanding of the pathogenesis of motility disorders observed after the surgical repair of intestinal atresia. Such studies are the first step toward the development of new therapeutic strategies.
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