Necrotizing enterocolitis is one disease with many origins and potential means of prevention.

BACKGROUND: The field of necrotizing enterocolitis (NEC) research has been in existence for over 60 years. During the first five decades little progress in NEC prevention and no definitive progress in treatment was achieved. One of the major determinants of this ineffectiveness may have been a global propensity to lump NEC into a single disease entity rather than a spectrum of diseases with a common outcome. The driver of this philosophy was most likely statistical, in that researchers desired large cohorts to optimize statistical power. Additionally, in the past quarter century, our preterm NEC cohorts were (and in some cases still are) contaminated with spontaneous intestinal perforations (SIP). This completely different acquired neonatal intestinal disease (ANID) markedly alters clinical characteristics and outcomes in NEC cohorts and subsets if not addressed. Unfortunately, cohort size has been proven to be less important than data quality when it comes to NEC over this last decade of research. Emerging progress in NEC prevention has been greatly enhanced as a result of dividing well-defined NEC into subsets of disease origin and investigating these entities individually. REVIEW OBJECTIVES: The purpose of this review is to offer the bedside clinician a concise, up-to-date review of recent advances in NEC reductionism. The reader should understand the history and basic theory behind NEC subsets, their application to NEC prevention, and comprehend that prevention of NEC requires a comprehensive quality improvement strategy that is likely best realized with a zero tolerance approach. CONCLUSIONS: We are entering a new era of NEC prevention. NICUs that embrace and achieve effective NEC prevention strategies will rapidly outpace their contemporaries. Because NEC is still the major driver of morbidity and mortality in most NICUs today, those who reject or fail in this pursuit will likely face increasingly severe consequences due to growing requirements for outcomes transparency.

Spontaneous intestinal perforation (SIP) will soon become the most common form of surgical bowel disease in the extremely low birth weight (ELBW) infant.

Recent data have revealed declines in the prevalence rates of NEC over the last decade in premature infants. In contrast, SIP has either remained steady or risen during the same epoch. These trends are consistent with our knowledge of the clinical arena. The ability to discern SIP contamination within NEC datasets has slowly improved. Additionally, quality improvement efforts are being utilized to reduce NEC through stewardship of antibiotics, acid inhibitors, central lines and blood products, as well as optimization of human milk diets. These forces are moving us to a new era, where NEC will no longer be the dominant surgical intestinal disease of the extremely preterm neonate. Indeed, in the extremely low birth weight (ELBW) population, SIP may already be the most prevalent reason for abdominal surgery. In this perspective, the reader will find supporting data and references for these assertions as well as predictions for the future.

Understanding intestinal vulnerability to perforation in the extremely low birth weight infant.

Spontaneous intestinal perforation (SIP) occurs commonly in extremely low birth weight (ELBW) infants. Our understanding of its etiologies has improved dramatically over the last decade. Included in this comprehension is an ongoing reconciliation of the iatrogenic risk factors, the microbiology, and the histopathology. The latter shows focal perforations with necrosis of the muscularis externa and no sign of ischemic damage (typically characterized by mucosal necrosis in the preterm bowel). Associations include extreme prematurity, early postnatal steroids (EPS), early use of indomethacin (EUI), and two common pathogens (Candida and Staphylococcus epidermis). Animal models of SIP suggest that all risk factors converge on a common collection of signaling pathways: those of nitric oxide synthases (NOS), insulin-like growth factors (IGFs), and epidermal growth factors (EGFs). Many of these factors skew trophism of the ileum (defined as thinning of the submucosa concomitant with hyperplasia of the muscosa). Global depletion of NOS is associated with disturbed intestinal motility and diminished transforming growth factor-alpha (TGF-alpha) in the muscularis externa. This constellation of insults seems to make the distal intestine vulnerable to perforation during recovery of motility.

Understanding clinical literature relevant to spontaneous intestinal perforations.

Spontaneous intestinal perforation (SIP) has emerged as a disease of extremely low-birth-weight (ELBW) infants over the last two decades. Several risk factors have been associated with this disease including early postnatal steroids (EPS; use within the first week of life), early use of indomethacin (EUI; use within the first 3 postnatal days), and the synergistic combination of the two. These two risk factors are thought to play a causal role in the etiology of SIP through their effects on ileal trophism and motility. Two infectious agents ( Candida and Staphylococcus epidermidis) are commonly grown from peritoneal cultures of patients with SIP. It is less clear whether these infections play a causal role or if they represent comorbidities of perforation. Chorioamnionitis is thought to be a risk factor for SIP, as is the stress and elevated cortisol that accompanies it. Recent analyses suggest that antenatal indomethacin may also be a risk factor for SIP, particularly when given close to birth. These latter variables are more challenging to rank in importance compared with EPS and EUI, which have been repeatedly associated with SIP in both retrospective cohorts and randomized controlled trials. Because neonatal care of the ELBW infant is commonly standardized, the habitual combination of any of these risk factors potentially amplifies the risk of SIP. Many of these factors are medicines, thus SIP risk is exacerbated by select forms of polypharmacy. Our challenge lies in understanding how these drug interactions lead to harm.

A role for plasmin in platelet aggregation: differential regulation of IGF release from IGF-IGFBP complexes?

OBJECTIVES: To determine if plasmin differentially augments platelet aggregation through variable efficiencies of IGF-IGFBP complex cleavage. METHODS: We utilized ADP-triggered platelet aggregation assays to test the effects of IGF-I versus IGF-II in complex with IGFBP-2 or IGFBP-3 upon the efficiency of plasmin (a known IGFBP protease) as a pro-aggregatory stimulus. In vitro proteolysis assays were performed as controls. RESULTS: We found that IGF-I complexes augmented platelet-mediated aggregation whereas IGF-II either had no effect (IGFBP-2) or inhibited platelet-mediated aggregation (IGFBP-3). In vitro proteolysis assays of IGFBP-2 and IGFBP-3 using plasmin revealed that three of the four aggregation findings were explained by the disparate efficiencies of IGFBP proteolysis associated with each IGF. Only IGF-II-IGFBP-2 complex resulted in a finding that could not be explained by the concept of differential regulation of plasmin's proteolysis efficiency by the two IGF ligands. CONCLUSIONS: Our findings demonstrate that the plasmin can differentially modulate platelet aggregation in response to intrinsic heterogeneities within the IGF axis.

An analysis of IGFBP evolution.

OBJECTIVES: To perform a synonymous, non-synonymous codon mutational analysis of the IGFBP gene family and identify mechanisms by which the IGFBP subfamilies diverged. METHODS: We identified 78 intact nucleotide sequences from 6 IGFBP subfamilies and 12 different species and used them for phylogenetic and synonymous, non-synonymous codon mutational analysis. Deletion and insertion comparisons were performed across subfamilies to determine if this might play a unique role in subfamily genesis. RESULTS: IGFBP-2 was identified by phylogenetic analysis to be the most related subfamily of the IGFBP progenitor, followed by IGFBP-4. Insertions and deletions within the variable domains were associated with divergence of each subfamily from its progenitor, suggesting a common motif for IGFBP evolution. Insertions unique to mammals were also found within the amino terminus of IGFBP-2. CONCLUSION: IGFBP subfamily divergence is associated with variable domain insertion or deletion and vigorous non-synonymous codon mutation. Our findings suggest strong selective pressure for IGFBP divergence in terrestrial vertebrates.

Changing the paradigm of defining, detecting, and diagnosing NEC: Perspectives on Bell's stages and biomarkers for NEC.

Better means to diagnose and define necrotizing enterocolitis are needed to guide clinical practice and research. Adequacy of Bell's staging system for clinical practice and clarity of cases used in NEC clinical datasets has been a topic of controversy for some time. This article provides reasons why a better global definition for NEC is needed and offers a simple alternative bedside definition for preterm NEC called the "Two out of Three" rule. Some argue that biomarkers may fill knowledge gaps and provide greater precision in defining relevant features of a clinical disease like NEC. NEC biomarkers include markers of inflammation, intestinal dysfunction, hematologic changes, and clinical features. Development and reporting of NEC biomarkers should be guided by the FDA's BEST Consensus resource, "Biomarkers, EndpointS, & other Tools" and consistently report metrics so that studies can be compared and results pooled. Current practice in the NICU would be enhanced by clinical tools that effectively inform the clinical team that a baby is at increasing risk of NEC. Ideally, these tools will incorporate both clinical information about the baby as well as molecular signals that are indicative of NEC. While meaningful biomarkers for NEC and clinical tools exist, translation into practice is mediocre.

A neonatal mouse model of intestinal perforation: investigating the harmful synergism between glucocorticoids and indomethacin.

BACKGROUND: Early postnatal steroids and indomethacin in combination have been shown to increase the risk of spontaneous intestinal perforation (SIP) in infants with extremely low birth weight (ELBW), but the mechanism behind this synergistic effect is unknown. MATERIALS AND METHODS: Based on literature in a variety of models suggesting that glucocorticoids and nonsteroidal antiinflammatory agents diminish complementary isoforms of nitric oxide synthase (NOS), we hypothesized that perturbations in NO metabolism contribute to SIP. RESULTS: Our results using newborn wild-type (WT) and endothelial NOS-knockout (eNOS KO) mice treated with dexamethasone and/or indomethacin indicate that indomethacin treatment diminishes ileal eNOS abundance; dexamethasone treatment diminishes ileal inducible NOS and neuronal NOS (nNOS); 100% of dexamethasone-treated eNOS KO mice die after 3 days; eNOS KO mice treated for 2 days with dexamethasone develop acute pyloric stenosis in association with reduced expression of pyloric nNOS; and isolated ileum from eNOS KO mice treated for 2 days with dexamethasone exhibit a significant decrease in spontaneous peristalsis, decreased circumference, and decreased capacitance for forced volume before ileal perforation compared with ileum from untreated controls. CONCLUSIONS: Our findings suggest that eNOS and nNOS display functional overlap in the newborn mouse gastrointestinal tract and that simultaneous reduction in the activity of both NOS isoforms may be a risk factor for neonatal ileal perforation. If this holds true in human infants, then it provides a plausible etiologic explanation for the strong temporal association between SIP and the simultaneous treatment of ELBW infants with glucocorticoids and indomethacin.

A critical question for NEC researchers: Can we create a consensus definition of NEC that facilitates research progress?

In the last decades the reported incidence of preterm necrotizing enterocolitis (NEC) has been declining in large part due to implementing comprehensive NEC prevention initiatives, including breast milk feeding, standardized feeding protocols, transfusion guidelines, and antibiotic stewardship and improving the rigor with which non-NEC cases are excluded from NEC data. However, after more than 60 years of NEC research in animal models, the promise of a "magic bullet" to prevent NEC has yet to materialize. There are also serious issues involving clinical NEC research. There is a lack of a common, comprehensive definition of NEC. National datasets have their own unique definition and staging definitions. Even within academia, randomized trials and single center studies have widely disparate definitions. This makes NEC metadata of very limited value. The world of neonatology needs a comprehensive, universal, consensus definition of NEC. It also needs a de-identified, international data warehouse.

A methodology for distinguishing divergent cell fates within a common progenitor population: adenoma- and neuroendocrine-like cells are confounders of rat ileal epithelial cell (IEC-18) culture.

BACKGROUND: IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation/transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage. RESULTS: IEC-18 cells were cultured for 72 hours in serum free media (SFM), with and without various insulin-like growth factor agonists to differentially boost the basal rate of proliferation. A strategy was employed to identify constitutive genes as markers of divergent fates through gene array analysis by cross-referencing fold-change trends for individual genes against crypt cell abundance in each treatment. We then confirmed the cell-specific phenotype by immunolocalization of proteins corresponding to those genes. The majority of IEC-18 cells in SFM alone had a loss in expression of the adenomatous polyposis coli (APC) gene at the mRNA and protein levels, consistent with adenoma-like transformation. In addition, a small subset of cells expressed the serotonin receptor 2A gene and had neuroendocrine-like morphology. CONCLUSIONS: IEC-18 cells commonly undergo a change in cell fate prior to reaching confluence. The most common fate switch that we were able to detect correlates with a down regulation of the APC gene and transformation into an adenoma-like phenotype.

A randomized-controlled trial of prophylactic hydrocortisone supplementation for the prevention of hypotension in extremely low birth weight infants.

OBJECTIVE: Extremely low birth weight (ELBW) infants are at risk for hypotension. Abnormal adrenal function may play a role in the pathogenesis of hypotension, and therefore, the administration of hydrocortisone (HC) may be an effective treatment for hypotension in some infants. However, the efficacy of prophylactic HC to prevent the use of vasopressors for a defined hypotensive state has not been studied. We conducted a randomized-controlled trial to determine the potential role on adrenal insufficiency in early neonatal hypotension and to determine the effectiveness of prophylactic HC in reducing treatment of hypotension in ELBW infants. STUDY DESIGN: Infants were assigned to receive either HC or placebo within the first 3 hours of life. Therapy was continued for 5 days. The presence of hypotension was based on an operational definition and treatment with vasopressors (VP) was standardized based on an a priori protocol. RESULTS: A total of 34 patients were enrolled. Baseline characteristics were similar between groups. Of the HC group 25% received VP at 24 hours of age compared to 44% of the placebo group. On day of life 2, only 7% of the HC group received VP compared to 39% of the placebo group (p<0.05). CONCLUSION: Prophylactic treatment with HC reduces the incidence of hypotension, defined by treatment with VP, among ELBW infants during the first 2 days of life. However, the mounting evidence that prophylactic administration of glucocorticoids in the first days of life is harmful to ELBW infants makes HC prophylaxis unwise until the efficacy of treatment relative to safety can be clearly established.

Necrotizing enterocolitis among neonates in the United States.

BACKGROUND: Prior studies have identified individual risk factors that are associated with necrotizing enterocolitis (NEC); however, the small sample sizes of these previous studies have not allowed the analysis of potential interaction between multiple variables and NEC. Our purpose was to describe the incidence and risk factors for NEC in premature neonates admitted for intensive care. METHODS: We identified neonates as having NEC if they met accepted diagnostic criterion for necrotizing enterocolitis. Using a national database, we assessed the association between NEC and a battery of risk factors previously reported in peer-reviewed literature. RESULTS: There were 15,072 neonates that met inclusion criteria; 14,682 did not have NEC, while 390 (2.6%) met criterion for NEC. Multivariate analysis showed that low birth weight was the most important risk factor for NEC. Other factors that were associated with an increased risk of NEC were exposure to antenatal glucocorticoids, vaginal delivery, need for mechanical ventilator support, exposure to both glucocorticoids and indomethacin during the first week of life, absence of an umbilical arterial catheter, and low Apgar score at 5 minutes. Length of hospital stay and mortality were higher in neonates with NEC than in neonates without NEC. CONCLUSIONS: NEC remains an important cause of morbidity and mortality in prematurely born neonates. In contrast to previous studies, we found that exposure to antenatal glucocorticoids was associated with an increased risk for NEC independent of birth weight.

Necrotizing enterocolitis in term infants.

This article is an overview of NEC in term neonates and also summarizes data from 52 cases within Intermountain Healthcare during the last 11 years. In all 52, NEC occurred among neonates already admitted to a neonatal intensive care unit for some other reason; thus, NEC invariably developed as a complication of treatment, not as a primary diagnosis. The authors speculate that the incidence of term NEC can be reduced by identifying neonatal intensive care unit patients at risk for NEC and applying appropriate-volume human milk feeding programs for these patients.

Ileal Immunoglobulin Binding by the Neonatal Fc Receptor: A Previously Unrecognized Mechanism of Protection in the Neonatal Rat Model of Necrotizing Enterocolitis?

BACKGROUND: Mucosal apoptosis is the initiating event in models of necrotizing enterocolitis (NEC) within rodents. It is possible there are species-specific differences that make apoptosis a more prominent feature of NEC in rodents than in humans. HYPOTHESIS: A lower threshold for mucosal apoptosis in the rodent distal intestine might have evolutionary advantages (via enhanced opsonization with the neonatal Fc receptor [FcRn]), since many short-gestation mammals are comparatively premature (histomorphologically) but are protected from NEC by breast milk. METHODS: We utilized a rat intestinal epithelial cell (IEC-18) model to determine if cell death alters FcRn - IgG binding, and rodent models of NEC to determine if cell death results in increased opsonization of IgG. Cultured IEC-18 cells were treated with H2O2 and analyzed. Neonatal Sprague-Dawley rats were cold and hypoxia stressed and intestinal sections were frozen for analysis. RESULTS: IgG binding was increased in H2O2-treated cells. Co-incubation of treated cells with either insulin-like growth factor or tunicamycin decreased IgG binding. Sprague-Dawley rats formula fed with exogenous bacteria showed a significant decrease in intestinal FcRn mRNA but increased ileal IgG binding. CONCLUSIONS: We speculate that FcRn plays a role in passive opsonization and subsequent bacterial pathogen clearance, making rodents resistant to NEC.

A 14-kDa cathepsin L-derived carboxyl IGFBP-2 fragment is sequestered by cultured rat ileal crypt cells.

IGF-II gut drives mucosal growth during gestation. IGF binding protein-2 (IGFBP-2) has a high affinity for IGF-II and tightly regulates IGF-II availability during fetal and early neonatal growth. We have previously demonstrated that glucocorticoids alter IGF homeostasis in the neonatal ileum, but the mechanism(s) by which this occurs is poorly understood. We hypothesized that dexamethasone alters proteolytic regulation of IGFBP-2 in ileal crypt cells. To test this, ileal crypt [ileal epithelial (IEC)-18] cells were cultured in serum-free media and used to study IGFBP-2 catabolism by immunochemistry, gene array analysis, and pharmacological perturbation with dexamethasone. In addition, isolated human IGFBP-2, IGF-II, and cathepsins B, D, and L were utilized for in vitro protease assays. We found IGFBP-2 to be highly abundant in IEC-18 culture, and sequestration of carboxyl IGFBP-2 antigen was seen within vesicular bodies of some cells. Dexamethasone significantly decreased the number of these cells and decreased IGFBP-2 in the media. On gene array analysis, cathepsin L's message abundance was significantly increased by dexamethasone, and, by in vitro assay, cathepsin L created a 14-kDa carboxyl fragment that corresponded to the sole antigen detected in IEC-18 cell lysates as well as a 16.5-kDa fragment found in the media. The sequestered fragment size was formed preferentially when IGF-II was present, whereas the larger fragment size was formed preferentially when IGF-II was absent. Cathepsins B and D did not produce these fragments in vitro and were not detected in IEC-18 media. We conclude that dexamethasone alters IGFBP-2 catabolism through its effects on cathepsin L.