Intestinal Epithelial Apoptosis Initiates Gross Bowel Necrosis in an Experimental Rat Model of Neonatal Necrotizing Enterocolitis

Abstract

The histopathology of necrotizing enterocolitis (NEC) is characterized by destruction of the mucosal layer in initial stages and by transmural necrosis of the intestinal wall in advanced stages of the disease. To test the hypothesis that enhanced epithelial apoptosis is an initial event underlying the gross histologic changes, we analyzed epithelial apoptosis and tissue morphology in an animal model of NEC and evaluated the effect of caspase inhibition on the incidence of experimental NEC in this model. Apoptosis was analyzed with terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end labeling (TUNEL) staining in intestinal sections and by measuring caspase 3 activity from intestinal lysates of neonatal rats subjected to formula feeding and cold/asphyxia stress (FFCAS) and from mother-fed (MF) controls. Morphologic evaluation was based on hematoxylin and eosin staining of intestinal sections. FFCAS resulted in histologic changes consistent with NEC, which were absent from MF animals. FFCAS was also associated with a significantly increased rate of nuclear DNA fragmentation in the small intestinal epithelium compared with MF. Elevated tissue caspase 3 activity confirmed the presence of apoptosis in samples with increased DNA fragmentation. Analysis of the coincidence of morphologic damage and apoptosis in corresponding tissue sections indicated that apoptosis precedes gross morphologic changes in this model. Furthermore, supplementation of formula with 8 boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, significantly reduced the incidences of apoptosis and experimental NEC. These findings indicate that in neonatal rats FFCAS induces epithelial apoptosis that serves as an underlying cause for subsequent gross tissue necrosis. NEC occurs in 5-15% of premature infants born weighing Ͻ1500 g and remains one of the leading causes of death in these patients (1-3). Nonetheless, the etiology of NEC remains elusive, and no specific treatment or preventive approaches have been successful. Several lines of evidence suggest that neonatal risk factors of prematurity, asphyxia, intestinal ischemia, and formula feeding are all contributing to the occurrence of the disease (4 -6). The immaturity of mucosal host defense, inappropriate bacterial colonization profile, and an imbalance of endothelin-dependent vasoconstriction and nitric oxide-dependent vasodilatation have also been implicated in the disease (7-10). Among the many inflammatory mediators that have been identified as possible culprits in the pathogenesis of NEC, PAF has been shown to play a central role. Elevated serum PAF levels in NEC patients and decreased serum PAF acetylhydrolase (i.e. the PAF degrading enzyme) in premature neonates suggests a role for this molecule in NEC pathogenesis. Initial studies in adult rats using an acute model have shown that exogenous PAF given intravenously results in ischemic bowel necrosis (11) and endotoxin, hypoxia, or tumor necrosis factor (TNF)-induced intestinal injury can be prevented by PAF receptor (PAFR...

Neonatal necrotizing enterocolitis rat model attenuated by a remote ischemic preconditioning in the pregnant

“…In the physiological process of intestinal mucosa renewal, in addition to cell proliferation, the process of apoptosis of epithelial cells also occurs 22 . Jilling et al 22 evaluated apoptosis in Neonatal necrotizing enterocolitis rat model attenuated by a remote ischemic preconditioning in the pregnant Gomes ROL et al Acta Cir Bras.…”

“…Jilling et al 22 evaluated apoptosis in Neonatal necrotizing enterocolitis rat model attenuated by a remote ischemic preconditioning in the pregnant Gomes ROL et al Acta Cir Bras. 2017;32(3):236-242 241 the gut of newborn rats, demonstrating that it was already present at a time prior to the onset of necrosis and that the frequency of apoptosis was lower in animals with higher injury degree of the intestinal mucosa.…”

“…The inability of mucosal regeneration in preterm infants may be related to the deficiency of the expression of the peptide trefoil factor, present in mucus and released together with mucin by goblet cells, contributing to the etiopathogenesis of NEC. Trefoil factor plays an important role in the intestinal epithelium, participating in its protection, its restitution and its regeneration 16 .In the physiological process of intestinal mucosa renewal, in addition to cell proliferation, the process of apoptosis of epithelial cells also occurs 22 . Jilling et al 22 evaluated apoptosis in Neonatal necrotizing enterocolitis rat model attenuated by a remote ischemic preconditioning in the pregnant Gomes ROL et al Acta Cir Bras.…”

Intestinal Epithelial Cell Apoptosis, Immunoregulatory Molecules, and Necrotizing Enterocolitisb

“…In the same study we have found that heterologous over-expression of Bcl-2, a molecular antagonist of Bax, prevented PAF-induced collapse of mitochondrial membrane potential and apoptosis in these cells, indicating that mitochondrial damage is important in the mechanism of PAF-induced apoptosis, and suggesting that understanding the expression profiles of the Bcl family of apoptosis regulators during intestinal development may be important for our understanding of NEC pathogenesis. In light of our earlier observation that wide-spread enterocyte apoptosis precedes and accompanies gross tissue necrosis in an animal model of NEC and that caspase inhibition prevents the development of experimental NEC [76], our in vitro evidence for PAF-induced enterocyte apoptosis is a significant observation to aid our mechanistic understanding of NEC pathogenesis. Additionally, we have shown that PAF does not only directly regulate apoptosis, but that signaling via the PAFR induces TLR4 expression in enterocytes (unpublished data).…”

“…Investigation of human intestinal samples that was obtained during surgery for NEC revealed that there is an abundance of apoptotic nuclei in the epithelium of intestines affected by NEC [75]. To determine whether this observation is a mere coincidence, or whether apoptosis may indeed play a pathological role in NEC, we investigated [76]. Since this early observation, several studies have shown that the development of experimental NEC is halted by various growth factors that cause enterocyte cell survival, such as EGF [77], IGF-I [78] and hb-EGF [79], or by a blocking antibody to TNFα [80] which is a proapoptotic molecule for enterocytes in vitro [81].…”