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...