ASPHYXIA, FORMULA FEEDING AND BACTERIAL COLONIZATION INCREASE PHOSPHOLIPASE A₂ ENZYME ACTIVITY AND mRNA LEVELS IN THE INTESTINE OF NEWBORN RATS† 1132

Recent experimental evidence has implicated the potent phospholipid inflammatory mediator, platelet activating factor (PAF) in the pathogenesis of necrotizing enterocolitis (NEC). We have developed a neonatal rat model of NEC using the following risk factors for human NEC; asphyxia (A), bacteria colonization (B), and formula feedings (F) in which the clinical signs and intestinal lesions in the rat model are similar to those of human NEC. PAF production is initiated by the enzyme phospholipase A₂ (PLA₂). This experiment measured PLA₂ enzyme activity and PLA₂ gene transcription in the newborn rat model of NEC. Intestinal PLA₂ enzyme activity from 26 stressed (A,B,F) and [Illegible Text] non-stressed, mother-fed control rat pups was assayed by measuring the conversion of a radiolabeled phosphatidylethanolamine substrate to free arachidonic acid. The mean PLA₂ enzyme activity for the stressed animals was 90.6 ± 10.6 vs 58.7 ± 7.7 pmol/gm protein/hr for the control pups (p=0.02). Intestinal PLA₂ mRNA was identified on Northern blots by hybridizion to a [³²P]-labeled PLA₂-II cDNA probe. Autoradiographs of the blot were analyzed by phosphoroimaging and the intensity of the hybridization bands quantified. Intestinal samples were studied from 5 stressed (A,B,F) and from 4 non-stressed, mother-fed, newborn rats. Mean PLA₂ mRNA activity was increased in the intestine of stressed newborn rats 3.5-fold over that of non-stressed pups (371 ± 107.5 vs 105 ± 52.2 units). The PLA₂ enzyme activity and mRNA levels of a given intestine did not correlate with the degree of NEC seen on gross or histologic exam. This experiment suggests that in the newborn rat model these stresses increase PLA₂ enzyme synthesis at the level of gene transcription and that the increased PLA₂ enzyme activity represents an increase in PLA₂ gene activity. The increased PLA₂ enzyme activity following exposure to risk factors for human NEC further supports the role of PAF in the pathogenesis of NEC. Supported by NIH grant HD 00999 and a grant from The March of Dimes.