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In utero repair of gastroschisis in the sheep (Ovis aries) model
Journal of Pediatric Surgery (2010) 45, 65–69
www.elsevier.com/locate/jpedsurg
In utero repair of gastroschisis in the sheep (Ovis aries) model Jacob T. Stephenson a,b,c , Kullada O. Pichakron b , Lan Vu a , Tim Jancelewicz a , Ramin Jamshidi a , J. Kevin Grayson c , Kerilyn K. Nobuhara a,⁎ a
Fetal Treatment Center, Pediatric Surgery Department University of California, San Francisco, San Francisco, CA 94143-0570, USA b Department of Surgery David Grant Medical Center, Travis AFB, CA 94535, USA c Clinical Investigations Facility David Grant Medical Center, Travis AFB, CA 94535, USA Received 26 September 2009; accepted 6 October 2009
Key words: Gastroschisis; Amniotic fluid; Fetal surgery
Abstract Objective: Gastroschisis is associated with inflammatory changes in the exposed bowel which leads to intestinal dysmotility after postnatal repair. The insult is a combined effect of amniotic fluid exposure and mechanical constriction. We hypothesized that in utero anatomic repair is possible in a sheep model, and that it may halt the inflammatory damage caused by both mechanisms. Methods: Gastroschisis was surgically created in mid-gestation (day 75) in 8 sheep fetuses. On gestational day 100, 2 fetuses underwent open fetal gastroschisis repair, where the eviscerated bowel was returned to the peritoneal cavity, and the abdominal wall was primarily closed. All fetuses were harvested at 135 days of gestation. Results: Six fetuses survived the initial operation, and both fetuses that underwent gastroschisis repair survived to term. At 100 and 135 days of gestation, the eviscerated bowel showed progressive signs of inflammation and peel development. The gross and microscopic inflammatory changes in the gastroschisis bowel at 100 days of gestation were completely resolved at term following in utero repair. Conclusion: In utero anatomic repair of gastroschisis is possible in mid-gestation in the fetal lamb model, and it appears to ameliorate the inflammatory process. © 2010 Published by Elsevier Inc.
Gastroschisis is a congenital abdominal wall defect that causes intestinal evisceration early in development. The herniation of the bowel allows constant exposure to amniotic
Presented at the 40th Annual Meeting of the American Pediatric Surgical Association, Fajardo, Puerto Rico, May 28-June 1, 2009. ⁎ Corresponding author. Department of Surgery, University of California, San Francisco, Box 0570, San Francisco, CA 94143-0570, USA. Tel.: +1 415 476 0449; fax: +1 415 476 2314. E-mail address: [email protected] (K.K. Nobuhara). 0022-3468/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2009.10.012
fluid throughout gestation. Additionally, the defect causes a mechanical constriction of the bowel and its mesentery which is especially profound as the fetus grows more than the abdominal cavity. Both of these mechanisms are felt to be independent insults of varying degrees to the developing intestine. The combination leads to a thickened and inflamed appearance of the bowel at term, with matting and a fibrous peel over the serosa [1]. The gross and microscopic inflammatory changes in the bowel of fetuses with gastroschisis are well described in
66 humans and in animal models to include foreshortened mesentery, fibrous peel, and thickened bowel wall. The biochemical analysis of amniotic fluid has also revealed an increase in inflammatory markers such as total protein, ferritin, and the cytokines IL-6 and IL-8 [2]. This has led to an interest in amnioinfusion or amnioexchange in an effort to halt the continued inflammatory bowel damage prenatally [3]. This approach succeeds in reducing the amniotic content of waste products [4] and may blunt the inflammatory process, but it does not directly deal with the underlying inflammatory insult. We hypothesized that complete reduction and abdominal wall closure is possible in the mid-gestation fetal lamb by open fetal surgery, and that this anatomic repair would halt the ongoing inflammatory damage to the fetal bowel.
1. Methods 1.1. Animals and experimental design Timed-pregnant Q-fever negative sheep (Ovis aries) with ultrasound confirmed twin gestation were individually housed in climate-controlled runs, with ad libitum access to chow and water. Animals were allowed to acclimate to the housing facility for 7 to 10 days before first intervention. All procedures were approved and in accordance with the Institutional Animal Care and Use Committee at David Grant Medical Center, Travis AFB, Calif.
1.2. Initial operation—creation of gastroschisis— 75 days Ewes were fasted the night before surgery, and then were premedicated with ketamine (10 mg/kg IM) and brought to the operative suite on gestational day 75. Intravenous access was obtained in the external jugular vein, and preoxygenation achieved prior to induction with 5% inhaled isofluorane. Orotracheal intubation was then performed and anesthesia was provided with 1.5% to 2.5% isofluorane. Isotonic fluid was administered intravenously, and a single perioperative dose of cephazolin (25 mg/kg IV) was given. Intraoperative monitoring included pulse oximetry and cardiac Doppler of each fetus. Maternal laparotomy was performed to access the bifid uterus with twin gestation. Ultrasound was used to assess viability and position of the two lambs. A full thickness pursestring suture was placed through one uterine wall between cotyledons. Electrocautery and sharp dissection were then used to gain access using an amniotic bubble technique. Amniotic fluid was sampled prior to any manipulation. The fetal hindquarters were then gently brought into the wound, and a 10-mm full-thickness abdominal wall defect was created to the left of the umbilicus in order to avoid injury to the liver and umbilical vein. Lateral pressure was applied to achieve evisceration of the
J.T. Stephenson et al. small bowel. The fetus was replaced, and amniotic fluid volume was restored with warm saline with 100 mg of cephazolin. The uterus was closed with a 3.5-mm transverse stapling device, and the maternal abdominal wall was closed in layers. The nonoperated twin sibling served as control. Fetal well-being was confirmed with ultrasound before the cessation of anesthesia. Postoperative analgesia was accomplished with buprenorphine (0.01 mg/kg IM) every 12 hours for 48 hours after the procedure.
1.3. Gastroschisis repair group (n = 2)—100 days of gestation At gestational day 100, the ewes were brought back to the operating suite with identical pre-operative preparation. Endotracheal anesthesia was again obtained, and the maternal laparotomy was re-opened. The experimental fetus was identified, and a new hysterotomy site was chosen and opened in the same fashion. Amniotic fluid was again sampled prior to intervention. The fetal hindquarters and abdomen were gently brought out of the uterus with care taken to avoid handling of the eviscerated bowel and to ensure unimpeded umbilical flow at all times. The gastroschisis defect was then extended 1-cm laterally with electrocautery. The abdominal wall was manually stretched to create additional abdominal domain for intestinal reduction. The intestine was then reduced to the peritoneal cavity, and closure of the defect was accomplished with interrupted full-thickness horizontal mattress sutures of 3-0 polypropelene suture. Amniotic fluid volume was replaced with warm saline, and the uterus and maternal abdominal wall were closed in identical fashion to the first operation. Routine postoperative care was again used.
1.4. Terminal delivery – 135 days At gestational day 135 (term, 145 days), the ewes were once again returned to the operating suite after standard preparation. Endotracheal anesthesia was again obtained, and the maternal laparotomy was re-opened. Amniotic fluid was sampled before any other manipulation. The experimental and control lambs were individually removed, and euthanasia was accomplished with overdose of sodium pentobarbital (80 mg/kg IV, B-euthanasia solution) while on placental blood support. The ewe was then euthanized in like manner. Stomach, small intestine, colon, and liver were then harvested from experimental and control animals.
1.5. Histology Tissues were submerged in 4% paraformaldehyde preservative overnight, then transferred to a solution containing 30% sucrose in 4% paraformaldehyde until saturated. Tissue was embedded in paraffin, sectioned (5 μm), mounted onto Superfrost Plus (Fisher, Pittsburgh,
In utero repair of gastroschisis
67 25 days of evisceration was edematous and thickened, with fibrinous debris in the amniotic fluid and the beginning of fibrous peel formation. The abdominal cavity was small, but accepted the reduction of the intestine. In one fetal lamb, transient bradycardia was noted on echocardiography after reduction, presumably from diminished umbilical flow related to the increase in abdominal pressure. The bradycardia resolved after full anesthesia recovery and this lamb
Fig. 1 Appearance of the intestine at 135 days of gestation. A, Lamb with gastroschisis. B, after gastroschisis repair—note the normal appearance of the small bowel and the meconium in the colon, with few adhesions to the anterior abdominal wall.
Pa) slides, and stained with hematoxylin/eosin for light microscopy. Standard light microscopy was employed, and measurements were taken of the small intestinal serosa, outer longitudinal smooth muscle, and the inner circular smooth muscle at 3 sites, with results averaged for each subject.
2. Results All 8 ewes had viable twin pregnancies at the time of initial operation at 75 days of gestation. Gastroschisis was surgically created in 8 fetuses, and 8 nonoperated siblings served as controls. Six experimental fetuses (75%) survived the initial operation, one fetus had early intrauterine fetal demise, and one spontaneously aborted. All control animals survived, but the twin siblings of the fetuses who died were not used for evaluation, leaving 6 animals in each group for comparison. At 100 days of gestation, 2 gastroschisis fetuses underwent repair in the operating room with 100% survival of experimental animals and control siblings. The bowel after
Fig. 2 Hematoxylin and eosin of small intestine at 135 days of gestation (original magnification ×40). A, Control. B, Gastroschisis. C, Gastroschisis repaired.
68 survived to term but did show mild evidence of growth restriction at delivery. At the time of terminal harvest at 135 days, the small bowel of the animals with unrepaired gastroschisis was severely foreshortened, matted, and covered with a thick fibrous peel (Fig. 1A). The outer longitudinal layer of the muscularis of eviscerated bowel measured 0.115 ± 0.069 mm. This was 7-fold higher than in control animals, with an outer muscle layer measured at 0.0159 ± 0.003 mm. The inner muscular layer showed a 4-fold difference, 0.102 ± 0.053 mm in the gastroschisis bowel and 0.0246 ± 0.008 mm in the control animals. The serosa with associated peel revealed thick connective tissue which was rich in macrophages (Fig. 2). Those animals whose gastroschisis defect was repaired exhibited grossly normal appearing bowel with a few adhesions underlying a nearly scarless abdominal wall. Meconium was present throughout the colon (Fig. 1B). The outer layer of muscularis was 0.0540 ± 0.006 mm and the inner layer of muscularis was 0.0560 ± 0.018 mm.
3. Discussion Despite optimal postnatal treatment of gastroschisis, intestinal motility can be slow to return. This leads to poor feeding, prolonged intensive care nursery admissions, and complications of protracted parenteral nutrition [5]. This intestinal dysfunction may continue well into childhood even in the absence of surgical complications or concomitant disease processes [6]. There is growing evidence that the poor intestinal motility may result from damage to the enteric nervous system and the interstitial cells of Cajal during their development in mid-gestation [7-9]. Fetal intervention, therefore, is being investigated in an effort to remove or control the ongoing insult to the prenatal bowel to allow for resumption of normal development of this complex process. Luton and others have documented the inflammatory milieu in the amniotic fluid of gastroschisis animals and humans [2], and have begun using amnioexchange as a therapeutic option to blunt the ongoing damage [10-12]. Continuous amnioinfusion has been shown to improve the inflammatory bowel changes in the mid-gestation sheep model [13,14], and a recent study using fetal chicks documented increased count of c-Kit bearing interstitial cells of Cajal in those treated with amnioallantoic exchange [15]. While these results are encouraging, amnioexchange seems to function in this setting in a fashion similar to dialysis. While this may temper the inflammatory environment, it does not correct the underlying problem. A recent study by Midrio et al [16] found no improvement in inflammatory markers or in time to feeding in a small cohort of gastroschisis patients treated with prenatal amnioexchange. We believe from prior studies that the insult is 2-fold: exposure of the bowel to harmful amniotic fluid and
J.T. Stephenson et al. ischemic constriction at the level of the abdominal wall [1]. Our goal in performing an anatomic abdominal wall repair in this gastroschisis model was to prevent ongoing insult by both mechanisms of injury to the developing bowel. Although we do not yet have postnatal functional data, the gross and microscopic appearance of the bowel of the repaired animals at term was completely normal, with biletinged meconium throughout a decompressed intestinal tract, suggesting effective prenatal motility. While both animals who underwent in utero repair survived to term with normal appearing bowel, we obviously cannot draw conclusions on the safety of this procedure with such small numbers. Of specific interest are the post-repair abdominal compartment pressures which need to be more completely investigated. The use of bioengineered scaffolds may serve to provide definitive closure of the abdominal wall defect, while recreating the lost domain. We are, of course, not advocating open fetal surgery for human patients with gastroschisis at this point. As we further define the pathophysiology of bowel damage in gastroschisis and continued improvements are made in techniques of fetal interventions, this may be an option with reasonable risk profile in the future.
4. Conclusions With this study, we have shown that the anatomic repair of a full thickness abdominal wall defect is possible in the mid-gestation sheep model of gastroschisis. This repair appears to ameliorate the inflammatory cycle by eliminating the constrictive insult to the developing bowel and preventing ongoing exposure of the bowel to amniotic fluid. Larger functional studies are necessary to thoroughly investigate the effect that this has on the motility of the bowel at term gestation, especially as compared to amnioexchange.
References [1] Langer JC, Longaker MT, Crombleholme TM, et al. Etiology of intestinal damage in gastroschisis. I: Effects of amniotic fluid exposure and bowel constriction in a fetal lamb model. J Pediatr Surg 1989;24 (10):992-7. [2] Guibourdenche J, Berrebi D, Vuillard E, et al. Biochemical investigations of bowel inflammation in gastroschisis. Pediatr Res 2006;60(5):565-8. [3] Akgur FM, Olguner M. Amniotic fluid exchange vs amniofusion in gastroschisis. Surg Endosc 2003;17(12):2032-3 [author reply 2034]. [4] Marder AL, Moise K, Chuang A, et al. Amnioexchange for the treatment of gastroschisis- an in vitro study to determine the volume and number of exchanges needed. Fetal Diagn Ther 2008;23(2):95-9. [5] Sydorak RM, Nijagal A, Sbragia L, et al. Gastroschisis: small hole, big cost. J Pediatr Surg 2002;37(12):1669-72. [6] Berseth CL, Malachowski N, Cohn RB, et al. Longitudinal growth and late morbidity of survivors of gastroschisis and omphalocele. J Pediatr Gastroenterol Nutr 1982;1(3):375-9.
In utero repair of gastroschisis [7] Midrio P, Faussone-Pellegrini MS, Vannucchi MG, et al. Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells. J Pediatr Surg 2004;39(10): 1541-7. [8] Vannucchi MG, Midrio P, Flake AW, et al. Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: an immunohistochemical study in a rat model. Neurosci Lett 2003;339 (1):77-81. [9] Santos MM, Tannuri U, Maksoud JG. Alterations of enteric nerve plexus in experimental gastroschisis: is there a delay in the maturation? J Pediatr Surg 2003;38(10):1506-11. [10] Luton D, Guibourdenche J, Vuillard E, et al. Prenatal management of gastroschisis: the place of the amnioexchange procedure. Clin Perinatol 2003;30(3):551-72, viii. [11] Sapin E, Mahieu D, Borgnon J, et al. Transabdominal amnioinfusion to avoid fetal demise and intestinal damage in fetuses with
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[12]
[13]
[14]
[15]
[16]
gastroschisis and severe oligohydramnios. J Pediatr Surg 2000;35 (4):598-600. Luton D, de Lagausie P, Guibourdenche J, et al. Effect of amnioinfusion on the outcome of prenatally diagnosed gastroschisis. Fetal Diagn Ther 1999;14(3):152-5. Guys JM, Esposito C, Simeoni J, et al. An experimental model of gastroschisis using fetoendoscopy: preliminary results and technical considerations. Surg Endosc 2002;16(2):317-9. Luton D, de Lagausie P, Guibourdenche J, et al. Influence of amnioinfusion in a model of in utero created gastroschisis in the pregnant ewe. Fetal Diagn Ther 2000;15(4):224-8. Vargun R, Aktug T, Heper A, et al. Effects of intrauterine treatment on interstitial cells of Cajal in gastroschisis. J Pediatr Surg 2007;42(5): 783-7. Midrio P, Stefanutti G, Mussap M, et al. Amnioexchange for fetuses with gastroschisis: is it effective? J Pediatr Surg 2007;42(5):777-82.
www.elsevier.com/locate/jpedsurg
In utero repair of gastroschisis in the sheep (Ovis aries) model Jacob T. Stephenson a,b,c , Kullada O. Pichakron b , Lan Vu a , Tim Jancelewicz a , Ramin Jamshidi a , J. Kevin Grayson c , Kerilyn K. Nobuhara a,⁎ a
Fetal Treatment Center, Pediatric Surgery Department University of California, San Francisco, San Francisco, CA 94143-0570, USA b Department of Surgery David Grant Medical Center, Travis AFB, CA 94535, USA c Clinical Investigations Facility David Grant Medical Center, Travis AFB, CA 94535, USA Received 26 September 2009; accepted 6 October 2009
Key words: Gastroschisis; Amniotic fluid; Fetal surgery
Abstract Objective: Gastroschisis is associated with inflammatory changes in the exposed bowel which leads to intestinal dysmotility after postnatal repair. The insult is a combined effect of amniotic fluid exposure and mechanical constriction. We hypothesized that in utero anatomic repair is possible in a sheep model, and that it may halt the inflammatory damage caused by both mechanisms. Methods: Gastroschisis was surgically created in mid-gestation (day 75) in 8 sheep fetuses. On gestational day 100, 2 fetuses underwent open fetal gastroschisis repair, where the eviscerated bowel was returned to the peritoneal cavity, and the abdominal wall was primarily closed. All fetuses were harvested at 135 days of gestation. Results: Six fetuses survived the initial operation, and both fetuses that underwent gastroschisis repair survived to term. At 100 and 135 days of gestation, the eviscerated bowel showed progressive signs of inflammation and peel development. The gross and microscopic inflammatory changes in the gastroschisis bowel at 100 days of gestation were completely resolved at term following in utero repair. Conclusion: In utero anatomic repair of gastroschisis is possible in mid-gestation in the fetal lamb model, and it appears to ameliorate the inflammatory process. © 2010 Published by Elsevier Inc.
Gastroschisis is a congenital abdominal wall defect that causes intestinal evisceration early in development. The herniation of the bowel allows constant exposure to amniotic
Presented at the 40th Annual Meeting of the American Pediatric Surgical Association, Fajardo, Puerto Rico, May 28-June 1, 2009. ⁎ Corresponding author. Department of Surgery, University of California, San Francisco, Box 0570, San Francisco, CA 94143-0570, USA. Tel.: +1 415 476 0449; fax: +1 415 476 2314. E-mail address: [email protected] (K.K. Nobuhara). 0022-3468/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2009.10.012
fluid throughout gestation. Additionally, the defect causes a mechanical constriction of the bowel and its mesentery which is especially profound as the fetus grows more than the abdominal cavity. Both of these mechanisms are felt to be independent insults of varying degrees to the developing intestine. The combination leads to a thickened and inflamed appearance of the bowel at term, with matting and a fibrous peel over the serosa [1]. The gross and microscopic inflammatory changes in the bowel of fetuses with gastroschisis are well described in
66 humans and in animal models to include foreshortened mesentery, fibrous peel, and thickened bowel wall. The biochemical analysis of amniotic fluid has also revealed an increase in inflammatory markers such as total protein, ferritin, and the cytokines IL-6 and IL-8 [2]. This has led to an interest in amnioinfusion or amnioexchange in an effort to halt the continued inflammatory bowel damage prenatally [3]. This approach succeeds in reducing the amniotic content of waste products [4] and may blunt the inflammatory process, but it does not directly deal with the underlying inflammatory insult. We hypothesized that complete reduction and abdominal wall closure is possible in the mid-gestation fetal lamb by open fetal surgery, and that this anatomic repair would halt the ongoing inflammatory damage to the fetal bowel.
1. Methods 1.1. Animals and experimental design Timed-pregnant Q-fever negative sheep (Ovis aries) with ultrasound confirmed twin gestation were individually housed in climate-controlled runs, with ad libitum access to chow and water. Animals were allowed to acclimate to the housing facility for 7 to 10 days before first intervention. All procedures were approved and in accordance with the Institutional Animal Care and Use Committee at David Grant Medical Center, Travis AFB, Calif.
1.2. Initial operation—creation of gastroschisis— 75 days Ewes were fasted the night before surgery, and then were premedicated with ketamine (10 mg/kg IM) and brought to the operative suite on gestational day 75. Intravenous access was obtained in the external jugular vein, and preoxygenation achieved prior to induction with 5% inhaled isofluorane. Orotracheal intubation was then performed and anesthesia was provided with 1.5% to 2.5% isofluorane. Isotonic fluid was administered intravenously, and a single perioperative dose of cephazolin (25 mg/kg IV) was given. Intraoperative monitoring included pulse oximetry and cardiac Doppler of each fetus. Maternal laparotomy was performed to access the bifid uterus with twin gestation. Ultrasound was used to assess viability and position of the two lambs. A full thickness pursestring suture was placed through one uterine wall between cotyledons. Electrocautery and sharp dissection were then used to gain access using an amniotic bubble technique. Amniotic fluid was sampled prior to any manipulation. The fetal hindquarters were then gently brought into the wound, and a 10-mm full-thickness abdominal wall defect was created to the left of the umbilicus in order to avoid injury to the liver and umbilical vein. Lateral pressure was applied to achieve evisceration of the
J.T. Stephenson et al. small bowel. The fetus was replaced, and amniotic fluid volume was restored with warm saline with 100 mg of cephazolin. The uterus was closed with a 3.5-mm transverse stapling device, and the maternal abdominal wall was closed in layers. The nonoperated twin sibling served as control. Fetal well-being was confirmed with ultrasound before the cessation of anesthesia. Postoperative analgesia was accomplished with buprenorphine (0.01 mg/kg IM) every 12 hours for 48 hours after the procedure.
1.3. Gastroschisis repair group (n = 2)—100 days of gestation At gestational day 100, the ewes were brought back to the operating suite with identical pre-operative preparation. Endotracheal anesthesia was again obtained, and the maternal laparotomy was re-opened. The experimental fetus was identified, and a new hysterotomy site was chosen and opened in the same fashion. Amniotic fluid was again sampled prior to intervention. The fetal hindquarters and abdomen were gently brought out of the uterus with care taken to avoid handling of the eviscerated bowel and to ensure unimpeded umbilical flow at all times. The gastroschisis defect was then extended 1-cm laterally with electrocautery. The abdominal wall was manually stretched to create additional abdominal domain for intestinal reduction. The intestine was then reduced to the peritoneal cavity, and closure of the defect was accomplished with interrupted full-thickness horizontal mattress sutures of 3-0 polypropelene suture. Amniotic fluid volume was replaced with warm saline, and the uterus and maternal abdominal wall were closed in identical fashion to the first operation. Routine postoperative care was again used.
1.4. Terminal delivery – 135 days At gestational day 135 (term, 145 days), the ewes were once again returned to the operating suite after standard preparation. Endotracheal anesthesia was again obtained, and the maternal laparotomy was re-opened. Amniotic fluid was sampled before any other manipulation. The experimental and control lambs were individually removed, and euthanasia was accomplished with overdose of sodium pentobarbital (80 mg/kg IV, B-euthanasia solution) while on placental blood support. The ewe was then euthanized in like manner. Stomach, small intestine, colon, and liver were then harvested from experimental and control animals.
1.5. Histology Tissues were submerged in 4% paraformaldehyde preservative overnight, then transferred to a solution containing 30% sucrose in 4% paraformaldehyde until saturated. Tissue was embedded in paraffin, sectioned (5 μm), mounted onto Superfrost Plus (Fisher, Pittsburgh,
In utero repair of gastroschisis
67 25 days of evisceration was edematous and thickened, with fibrinous debris in the amniotic fluid and the beginning of fibrous peel formation. The abdominal cavity was small, but accepted the reduction of the intestine. In one fetal lamb, transient bradycardia was noted on echocardiography after reduction, presumably from diminished umbilical flow related to the increase in abdominal pressure. The bradycardia resolved after full anesthesia recovery and this lamb
Fig. 1 Appearance of the intestine at 135 days of gestation. A, Lamb with gastroschisis. B, after gastroschisis repair—note the normal appearance of the small bowel and the meconium in the colon, with few adhesions to the anterior abdominal wall.
Pa) slides, and stained with hematoxylin/eosin for light microscopy. Standard light microscopy was employed, and measurements were taken of the small intestinal serosa, outer longitudinal smooth muscle, and the inner circular smooth muscle at 3 sites, with results averaged for each subject.
2. Results All 8 ewes had viable twin pregnancies at the time of initial operation at 75 days of gestation. Gastroschisis was surgically created in 8 fetuses, and 8 nonoperated siblings served as controls. Six experimental fetuses (75%) survived the initial operation, one fetus had early intrauterine fetal demise, and one spontaneously aborted. All control animals survived, but the twin siblings of the fetuses who died were not used for evaluation, leaving 6 animals in each group for comparison. At 100 days of gestation, 2 gastroschisis fetuses underwent repair in the operating room with 100% survival of experimental animals and control siblings. The bowel after
Fig. 2 Hematoxylin and eosin of small intestine at 135 days of gestation (original magnification ×40). A, Control. B, Gastroschisis. C, Gastroschisis repaired.
68 survived to term but did show mild evidence of growth restriction at delivery. At the time of terminal harvest at 135 days, the small bowel of the animals with unrepaired gastroschisis was severely foreshortened, matted, and covered with a thick fibrous peel (Fig. 1A). The outer longitudinal layer of the muscularis of eviscerated bowel measured 0.115 ± 0.069 mm. This was 7-fold higher than in control animals, with an outer muscle layer measured at 0.0159 ± 0.003 mm. The inner muscular layer showed a 4-fold difference, 0.102 ± 0.053 mm in the gastroschisis bowel and 0.0246 ± 0.008 mm in the control animals. The serosa with associated peel revealed thick connective tissue which was rich in macrophages (Fig. 2). Those animals whose gastroschisis defect was repaired exhibited grossly normal appearing bowel with a few adhesions underlying a nearly scarless abdominal wall. Meconium was present throughout the colon (Fig. 1B). The outer layer of muscularis was 0.0540 ± 0.006 mm and the inner layer of muscularis was 0.0560 ± 0.018 mm.
3. Discussion Despite optimal postnatal treatment of gastroschisis, intestinal motility can be slow to return. This leads to poor feeding, prolonged intensive care nursery admissions, and complications of protracted parenteral nutrition [5]. This intestinal dysfunction may continue well into childhood even in the absence of surgical complications or concomitant disease processes [6]. There is growing evidence that the poor intestinal motility may result from damage to the enteric nervous system and the interstitial cells of Cajal during their development in mid-gestation [7-9]. Fetal intervention, therefore, is being investigated in an effort to remove or control the ongoing insult to the prenatal bowel to allow for resumption of normal development of this complex process. Luton and others have documented the inflammatory milieu in the amniotic fluid of gastroschisis animals and humans [2], and have begun using amnioexchange as a therapeutic option to blunt the ongoing damage [10-12]. Continuous amnioinfusion has been shown to improve the inflammatory bowel changes in the mid-gestation sheep model [13,14], and a recent study using fetal chicks documented increased count of c-Kit bearing interstitial cells of Cajal in those treated with amnioallantoic exchange [15]. While these results are encouraging, amnioexchange seems to function in this setting in a fashion similar to dialysis. While this may temper the inflammatory environment, it does not correct the underlying problem. A recent study by Midrio et al [16] found no improvement in inflammatory markers or in time to feeding in a small cohort of gastroschisis patients treated with prenatal amnioexchange. We believe from prior studies that the insult is 2-fold: exposure of the bowel to harmful amniotic fluid and
J.T. Stephenson et al. ischemic constriction at the level of the abdominal wall [1]. Our goal in performing an anatomic abdominal wall repair in this gastroschisis model was to prevent ongoing insult by both mechanisms of injury to the developing bowel. Although we do not yet have postnatal functional data, the gross and microscopic appearance of the bowel of the repaired animals at term was completely normal, with biletinged meconium throughout a decompressed intestinal tract, suggesting effective prenatal motility. While both animals who underwent in utero repair survived to term with normal appearing bowel, we obviously cannot draw conclusions on the safety of this procedure with such small numbers. Of specific interest are the post-repair abdominal compartment pressures which need to be more completely investigated. The use of bioengineered scaffolds may serve to provide definitive closure of the abdominal wall defect, while recreating the lost domain. We are, of course, not advocating open fetal surgery for human patients with gastroschisis at this point. As we further define the pathophysiology of bowel damage in gastroschisis and continued improvements are made in techniques of fetal interventions, this may be an option with reasonable risk profile in the future.
4. Conclusions With this study, we have shown that the anatomic repair of a full thickness abdominal wall defect is possible in the mid-gestation sheep model of gastroschisis. This repair appears to ameliorate the inflammatory cycle by eliminating the constrictive insult to the developing bowel and preventing ongoing exposure of the bowel to amniotic fluid. Larger functional studies are necessary to thoroughly investigate the effect that this has on the motility of the bowel at term gestation, especially as compared to amnioexchange.
References [1] Langer JC, Longaker MT, Crombleholme TM, et al. Etiology of intestinal damage in gastroschisis. I: Effects of amniotic fluid exposure and bowel constriction in a fetal lamb model. J Pediatr Surg 1989;24 (10):992-7. [2] Guibourdenche J, Berrebi D, Vuillard E, et al. Biochemical investigations of bowel inflammation in gastroschisis. Pediatr Res 2006;60(5):565-8. [3] Akgur FM, Olguner M. Amniotic fluid exchange vs amniofusion in gastroschisis. Surg Endosc 2003;17(12):2032-3 [author reply 2034]. [4] Marder AL, Moise K, Chuang A, et al. Amnioexchange for the treatment of gastroschisis- an in vitro study to determine the volume and number of exchanges needed. Fetal Diagn Ther 2008;23(2):95-9. [5] Sydorak RM, Nijagal A, Sbragia L, et al. Gastroschisis: small hole, big cost. J Pediatr Surg 2002;37(12):1669-72. [6] Berseth CL, Malachowski N, Cohn RB, et al. Longitudinal growth and late morbidity of survivors of gastroschisis and omphalocele. J Pediatr Gastroenterol Nutr 1982;1(3):375-9.
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