Background

Red blood cell (RBC) transfusions are necessary and essential therapeutic interventions frequently utilized in the neonatal intensive care unit and to treat critically ill infants who experience severe anemia due to physiologic and iatrogenic factors [1–4]. Nearly 80% of all infants born at <27 weeks of gestational age receive one or more RBC transfusions during their birth hospitalization [5–8]. Though repletion with packed RBC has obvious benefits (such as increased oxygen-carrying capacity), transfusions have also been implicated in the subsequent development of necrotizing enterocolitis (NEC) within 48–72 h after transfusion [7–15]. NEC is the most common acquired gastrointestinal disease of premature neonates, affecting 5%–15% of infants born with <1,500 g [5–8,16–18] and, despite technological advancement, remains a leading cause of death in neonates born between 22 and 28 weeks gestation [9–12,19,20]. NEC is also associated with multiple immediate serious complications, such as death due to sepsis, and long-term complications, including intestinal failure, growth delay, and adverse neurodevelopmental outcomes [21,22]. The causes of NEC are diverse; however, our recent findings documented that phlebotomy-induced anemia resulted in a disproportionate and persistent increase in intestinal permeability in pre-weaned mice because of the disruption of epithelial adherens junctions [23], developing a low-grade inflammatory state in the intestine with prominent macrophage precursor infiltration. Subsequent RBC transfusions activate these macrophages and thus cause NEC-like injury [24]. To examine the pathogenesis of NEC, various murine models have been established. This study will describe an improvement upon the previous version and a detailed protocol of our murine model of RBC transfusion–associated NEC.