Amniotic Fluid Stem Cells Prevent Development of Ascites in a Neonatal Rat Model of Necrotizing Enterocolitis

Abstract

Aim It has been demonstrated that in a neonatal rat model of necrotizing enterocolitis (NEC), amniotic fluid stem (AFS) cells decrease intestinal damage and improve survival via modulation of stromal cells expressing cyclooxygenase 2 in the lamina propria. Herein, we aimed to evaluate the effect of AFS cells on body weight and fluid retention in this NEC model.

Methods AFS cells were obtained from green fluorescent protein (GFP) + pregnant rats at E16 and expanded in culture. A total of 185 neonatal rats had NEC induced by gavage feeding of hypertonic formula + hypoxia + oral lipopolysaccharide (4 mg/kg/d) and were randomized to intraperitoneal phosphate buffered saline (PBS, n = 93) or AFS cells (n = 92). A total of 36 breastfed (BF) rats were used as controls. All rats were being killed at 96 hours of life. Groups were compared for body weight and presence of free intraperitoneal fluid using nonparametric and contingency tests. Data are expressed as mean ± standard deviation.

Results There were no differences in birth weight among the groups (PBS = 5.6 ± 0. 3 g; AFS cells = 5.6 ± 0. 3 g; BF = 5.6 ± 0. 3 g; p = 1). The body weight at randomization was not different between PBS (5.61 ± 0. 5 g) and AFS cells (5.60 ± 0. 5; p = 1) rats. After the rats were killed, BF rats were significantly heavier (12.5 ± 0.1 g) than PBS (5.12 ± 0.4 g) and AFS cell rats (4.95 ± 0.3; p < 0.0001). From randomization to being killed, PBS rats had 9% of weight loss in comparison with 12% in AFS cell rats (p = 0.08). After the rats were killed, 42 (45%) PBS rats developed ascites with evident abdominal distension in comparison with 19 (21%) AFS cells (p = 0.0005). None of BF animals had ascites.

Conclusion Gavage feeding and undernutrition severely affect growth in this model of NEC. Administration of AFS cells result in lower incidence of ascites than in PBS rats. This could explain the differences in body weight between the two groups of rats that were killed. Furthermore, studies on liver function and fluid composition are needed to investigate our speculation.

Keywords

stem cell - COX-2 - fetal cells - cell therapy

• References

• 1 Castle S, Grikscheit T, Ford HR. Necrotizing enterocolitis. In: Puri P, , ed. Newborn Surgery. 3rd ed. London: Hodder Arnold; 2011: 547-553
• 2 Wu SF, Caplan M, Lin HC. Necrotizing enterocolitis: old problem with new hope. Pediatr Neonatol 2012; 53 (3) 158-163
• 3 Zani A, Cananzi M, Fascetti-Leon F , et al. Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism. Gut 2013;
• 4 Good M, Siggers RH, Sodhi CP , et al. Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium. Proc Natl Acad Sci U S A 2012; 109 (28) 11330-11335
• 5 Siggers JL, Ostergaard MV, Siggers RH , et al. Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates. Am J Physiol Gastrointest Liver Physiol 2013; 304 (10) G864-G875
• 6 Barlow B, Santulli TV, Heird WC, Pitt J, Blanc WA, Schullinger JN. An experimental study of acute neonatal enterocolitis—the importance of breast milk. J Pediatr Surg 1974; 9 (5) 587-595
• 7 Zani A, Cordischi L, Cananzi M , et al. Assessment of a neonatal rat model of necrotizing enterocolitis. Eur J Pediatr Surg 2008; 18 (6) 423-426
• 8 De Coppi P, Bartsch Jr G, Siddiqui MM , et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007; 25 (1) 100-106
• 9 Zani A, Eaton S, Leon FF , et al. Captopril reduces the severity of bowel damage in a neonatal rat model of necrotizing enterocolitis. J Pediatr Surg 2008; 43 (2) 308-314
• 10 Nadler EP, Dickinson E, Knisely A , et al. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. J Surg Res 2000; 92 (1) 71-77
• 11 Walsh MC, Kliegman RM. Necrotizing enterocolitis: treatment based on staging criteria. Pediatr Clin North Am 1986; 33 (1) 179-201
• 12 Kim SS, Albanese CT. Necrotizing enterocolitis. In: Grosfeld JL, O'Neill JA, Fonkalsrud EW, , eds. Pediatric Surgery. 6th ed. Philadelphia, PA: Mosby Elsevier; 2006: 1427-1452
• 13 McBride WJ, Roy S, Brudnicki A, Stringel G. Correlation of complex ascites with intestinal gangrene and perforation in neonates with necrotizing enterocolitis. J Pediatr Surg 2010; 45 (5) 887-889
• 14 Zani A, Ghionzoli M, Lauriti G , et al. Does intestinal permeability lead to organ failure in experimental necrotizing enterocolitis?. Pediatr Surg Int 2010; 26 (1) 85-89
• 15 Zhang Z, Lin H, Shi M , et al. Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients. J Gastroenterol Hepatol 2012; 27 (Suppl. 02) 112-120
• 16 Zheng YB, Zhang XH, Huang ZL , et al. Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure. PLoS ONE 2012; 7 (7) e41392
• 17 Liu H, Liu DQ, Li BW , et al. Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo. In Vitro Cell Dev Biol Anim 2011; 47 (9) 601-608
• 18 Moschidou D, Mukherjee S, Blundell MP , et al. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach. Mol Ther 2012; 20 (10) 1953-1967