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Multiple studies have documented the safety of intestinal anastomosis after resection for necrotizing enterocolitis (NEC). We sought to evaluate a large population of infants with surgical NEC and assess outcomes after primary anastomosis versus enterostomy. The Pediatric Health Information System database was used to identify infants with Bell Stage 3 NEC who underwent an intestinal resection for acute disease between 2016 and 2021. Demographics and preoperative physiology were assessed, and nutritional, infectious, and surgical outcomes were analyzed. Two hundred twenty-two infants at 38 children's hospitals were included. Thirty-five (15.8%) were managed with a primary anastomosis. Among infants who underwent a resection within 10 d of their first operative intervention and survived for at least 3 d, a primary anastomosis was used in 26 (13.7%). These patients were older but had similar weight and physiological status at the time of resection as those managed with an enterostomy. The incidence of wound and infectious complications, duration of parenteral nutrition and length of stay were similar after anastomosis or enterostomy. In a large, geographically heterogenous population of infants with NEC, only 15.8% were managed with a primary anastomosis after intestinal resection. Survivors who underwent resection within 10 d were demographically and physiologically comparable to those who underwent enterostomy and had similar surgical outcomes. While there are clearly indications for enterostomy in some infants with NEC, these data confirm the conclusions of smaller, single-center studies that a primary anastomosis should be considered more frequently. [ABSTRACT FROM AUTHOR]

Certain congenital cardiac lesions are at increased risk for the development of necrotizing enterocolitis (NEC). These patients are often reliant on pulmonary and systemic vasomodulators to maintain adequate perfusion and oxygenation. This study sought to determine whether pulmonary or systemic vasodilator treatment is protective against the development of NEC in this population. We utilized International Classification of Diseases (ICD) codes to identify high risk congenital cardiac disease patients ≤6 mo of age, cared for at a tertiary children's hospital between January 2011 and January 2021. Cardiac anomalies were stratified into ductal dependent (pulmonary DD-P or systemic DD-S) or independent lesions. The rate of NEC development in those who received vasodilators (inhaled nitric oxide [iNO], pulmonary vasodilators, systemic vasodilators) was compared to controls in a multivariate analysis. Of the 352 patients, who met inclusion criteria, 77.6% had ductal dependent lesions (DD-S 41.9%, DD-P 35.7%), 19.5% received iNO, and 37.5% received other vasodilatory drugs. The overall NEC rate was 15.1%. On univariate analysis, DD-S, iNO use, and systemic vasodilators was associated with a significantly higher risk of NEC, while DD-P was associated with lower NEC risk. On multivariate analysis, only iNO (odds ratio 2.725, confidence interval [1.36-5.44]) and DD-S (odds ratio 2.279, confidence interval [1.02-5.11]) were independent risk factors for NEC. In patients with at-risk congenital cardiac disease lesions, a ductus dependent systemic circulation or iNO treatment is associated with an increased risk of developing NEC. The presence of iNO or DD-S should be utilized as markers of increased risk both in the prevention and workup of suspected NEC. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a gastrointestinal disease of premature neonates. We previously validated a NEC enteroid model derived from human infant intestinal tissue. Typical enteroid configuration is basolateral-out (BO) without direct access to the luminal (apical) surface. Apical access is necessary to allow physiologic comparison of pathogen interaction with the intestinal epithelial barrier. We hypothesize that apical-out (AO) enteroids will provide a relevant NEC model to study this relationship. Following the institutional review board approval (#11610-11611), neonatal intestinal tissue was collected from surgical specimens. Stem cells were collected; enteroids were generated and grown to maturity in BO conformation then everted to AO. Enteroids were untreated or treated for 24 h with 100 μg/mL lipopolysaccharide and hypoxia. Protein and gene expression were analyzed for inflammatory markers, tight junction (TJ) proteins and permeability characteristic of NEC. Apical TJ protein zonula occludens-1 and basolateral protein β-catenin immunofluorescence confirmed AO configuration. Treated AO enteroids had significantly increased messenger RNA (P = 0.001) and protein levels (P < 0.0001) of tumor necrosis factor-α compared to controls. Corrected total cell fluorescence of toll-like receptor 4 was significantly increased in treated AO enteroids compared to control (P = 0.002). Occludin was found to have significantly decreased messenger RNA in treated AO enteroids (P = 0.003). Expression of other TJ proteins claudins-1, -4 and zonula occludens-1 was significantly decreased in treated AO enteroids (P < 0.05). AO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC. • Apical-out (AO) enteroids subjected to lipopolysaccharide and hypoxia show necrotizing enterocolitis characteristics. • Chemically everted enteroids (AO) allow ready access to their luminal surface. • AO enteroids allow study of pathogen-intestinal epithelium interaction. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor (HIF) 1α is essential for the pathogenesis of necrotizing enterocolitis (NEC). HIF-1α is stabilized by glutaredoxin-1 (Grx1) deletion. The precise role of HIF-1α in the intestinal microcirculation in NEC is not well defined. We aimed to determine the role of HIF-1α in the regulation of the intestinal microcirculation during the development of NEC. Experimental NEC was induced in full-term C57BL/6 mice and Grx1−/− pups through the formula gavage and hypoxia technique. HIF-1α signaling was blocked using the HIF-1α inhibitor, YC-1 [3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole]. Intestinal tissues were collected at predetermined time points for the assessment of the intestinal microcirculation and HIF-1α activity and signaling. We found that NEC induction impaired the intestinal microcirculation, but the impairment of the intestinal blood flow and capillary density was ameliorated in Grx1−/− mice. This amelioration was associated with tripeptide glutathione–protein adducts in the intestinal tissue. Grx1 ablation also promoted vascular endothelial growth factor A production in the intestinal tissue. This intestinal microvascular improvement was not found in HIF-1α-inhibited mice, suggesting that HIF-1α was involved in the intestinal microcirculatory perfusion. The current data demonstrated that HIF-1α signaling is involved in the intestinal microvascular modification during the pathogenesis of NEC, suggesting that targeting HIF-1α might be a promising strategy for NEC treatment. [ABSTRACT FROM AUTHOR]

Fecal microbiota transplantation (FMT) is a promising therapy, but it has not been used to treat neonatal necrotizing enterocolitis (NEC) due to reports of adverse side effects. Probiotics are considered relatively safe with practicable administrative procedures; however, no systematic research has compared the results of FMT and probiotic consortium transplantation (PCT) on oxidative stress in the intestines of patients with NEC. We conducted this study to provide a basis for optimizing NEC therapy. Eight-day-old newborn C57BL/6 mice were randomly divided into the following four groups: the dam-fed group (control group); the NEC induction group (NEC group); the NEC induction and transplantation of Lactobacillus reuteri and Bifidobacterium infantis consortium group (NEC + PCT group); and the NEC induction and the FMT group (NEC + FMT). Intestinal injury, oxidative stress indexes, intestinal barrier function, and inflammatory cytokines were assessed in the terminal ileum. FMT more effectively modulates oxidative stress in the intestine than does PCT; however, the difference between the effects of PCT and FMT was not significant. The protective effect was associated with enhanced antioxidant capacity, regulation of the main components of the mucus layer, reduced inflammatory reactions, and improved intestinal integrity. Intestinal dysbiosis affects oxidative stress, inflammatory response, and mucosal integrity. Although FMT is more effective than PCT in regulating oxidative stress, PCT may be preferred in pediatrics because the proportion and dose of transplanted bacteria can be standardized and individualized according to individual conditions. [ABSTRACT FROM AUTHOR]

Introduction: Although pneumoperitoneum from necrotizing enterocolitis or spontaneous intestinal perforation is a surgical emergency, risk stratification to determine which neonates benefit from initial peritoneal drainage (PD) is lacking., Methods: Using a single-center retrospective review of very low birth weight neonates under 1500 g who underwent PD for pneumoperitoneum (January 2015 to December 2023) from necrotizing enterocolitis or spontaneous intestinal perforation, two cohorts were created: drain "responders" (patients managed definitively with PD; includes placement of a second drain) and "nonresponders" (patients who underwent subsequent laparotomy or died after PD). Antenatal/postnatal characteristics, periprocedural clinical data, and hospital outcomes were compared between responders and nonresponders using Student's t-test, chi-squared test, or Kruskal-Wallis test as appropriate, with P < 0.05 considered significant., Results: Fifty-six neonates were included: 31 (55%) drain responders and 25 (45%) nonresponders. Birth weight, gestational age, sex, ethnicity, use of postnatal steroids, and enteral feeds were similar between the cohorts. Nonresponders had higher base deficits (-3.4 versus -5.0, P = 0.032) and FiO 2 (0.25 versus 0.52, P = 0.001) after drain placement. Drain responders had significantly shorter lengths of stay (89 versus 148 days, P = 0.014) and lower mortality (6.4% versus 56%, P < 0.001). A subgroup analysis of the nonresponders showed no differences in birth weight, vasopressor requirement, FiO 2 , or postdrain base deficit between nonresponders who had a drain alone versus laparotomy following drain placement., Conclusions: PD remains a viable initial therapy for pneumoperitoneum in premature very low birth weight neonates (< 1500 g), demonstrating clinical response in more than half. Ongoing clinical assessment and judgment is imperative after drain placement to ensure continued clinical improvement., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Immune factors are important antecedents in the pathophysiology of necrotizing enterocolitis (NEC). However, studies on the peripheral blood lymphocyte subsets changes in NEC patients among different Bell stages and in patients requiring surgery are scarce., Methods: 34 infants with NEC and 33 age-matched controls were included. Peripheral blood was collected within 48 h after NEC diagnosis. Peripheral blood B and T lymphocytes subsets were detected by 12-color flow cytometry. Cell ratios were calculated, and their relationship to disease severity and their roles as indicators for surgery were assessed., Results: NEC patients showed elevated percentages of unSwB cells (CD27+IgD+ unswitched memory/activated B cells)/B cells, SwB cells (CD27+IgD-switched memory B cells)/B cells, CD8+ T (CD3+CD8+ T cells)/T cells, Tem (CD45RA-CCR7-effector memory T cells)/CD4+ T cells, Tem/CD8+ T cells and decreased Bn (CD27-IgD+ naïve B cells)/B cells, CD4+T (CD3+CD4+ T cells)/T cells, CD45RA+ CCR7+ naïve T cells (CD45RA+CCR7+ naïve T cells)/CD8+T cells. Compared to NEC patients at BELL stage I + II, patients at BELL stage III showed increased percentages of SwB cells/B cells, antibody secreting cell (ASC, CD3-CD20-CD27high CD38high ASCs)/B cells and Tem/CD4+ T cells, and decreased percentages of CD45RA+CCR7+ naïve T cells/CD4+ T cells. The Receiver Operating Characteristic Curve analysis showed that the sensitivity of ASC/B cells ratio (0.52%) is 86.67% and the specificity of Tem/CD4+T ratio (5.22%) is 100%, indicating that NEC patients required surgery., Conclusions: The severity of NEC exhibits codirectional changes with the maturation of B and T lymphocytes, especially CD4+ T cells. The increased ASC/B and Tem/CD4+ T cells could serve as potential indicators for NEC patients requiring surgery., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Necrotizing Enterocolitis (NEC) remains a significant cause of morbidity and mortality. Recently, there has been an increased recognition of the importance of intestinal immunity and the associations with antibiotics and enteral feeds in the pathophysiology of NEC. The primary purpose of this study is to examine the association of enteral feeds on the survival of premature neonates with NEC. A retrospective review using the Vermont Oxford Network for a Level IV NICU from January 1, 2013 through December 31, 2019 was performed. All neonates had a gestational age between 22 to 29 weeks, weighed at least 300 grams (n = 653), had a reported enteral feed status and were treated for NEC (n = 43). Data analysis utilized two-tailed t -tests for NEC and infection rates then Fisher's exact tests for survival status. The incidence of NEC in the population was 6.6% (43/653). Of the 43 neonates treated for NEC, 27 were enterally fed, while the other 16 were not. All 27 neonates with NEC that were able to achieve enteral feeds survived and had an infection rate of 22.2%. Meanwhile, all 16 neonates with NEC that were unable to achieve enteral feeds died and had an infection rate of 62.5%. There is a significant association between enteral feeds and NEC, survival, and infection rates in premature neonates. These findings support the importance of intestinal immunity and the microbiota in NEC. Given the limitations of the retrospective review, the profound survival advantage with enteral feeds reinforces the need for further study. [ABSTRACT FROM AUTHOR]

Surgical simulation-based training (SBT) can increase resident confidence and improve performance. SBT in pediatric surgery is in its infancy and often geared toward training pediatric surgery fellows. Since case volume for various pediatric surgery–specific procedures can be low based on the rarity of the pathology involved and the level of care provided by the institution, our aim was to create a pediatric surgery simulation-based curriculum for general surgery residents to address this need. We performed an institutional needs assessment consisting of 4 pediatric surgeons' and 28 general surgery residents' confidence in resident ability to independently perform pediatric surgery–specific tasks and procedures using a Likert-scaled survey. These included the placement of a silastic silo for gastroschisis, a percutaneous drain for perforated necrotizing enterocolitis, and completion of a laparoscopic pyloromyotomy for pyloric stenosis. Models simulating these pathologies and curriculum for performing each procedure were generated. We successfully created a model and SBT curriculum to teach general surgery residents how to place a silastic silo for patients with gastroschisis, a percutaneous drain for patients with perforated necrotizing enterocolitis, and how to complete a laparoscopic pyloromyotomy for patients with pyloric stenosis. These were deemed high fidelity models based on a survey of our pediatric surgeons. We created a pediatric surgery SBT curriculum for general surgery residents, which can be used to supplement learning of various high-acuity, low-occurrence procedures. Assessment of residents and validation of scores is underway. [ABSTRACT FROM AUTHOR]

There is no clear consensus on the optimal operative management of premature infants with surgical necrotizing enterocolitis (sNEC) or spontaneous intestinal perforation (SIP); thus, a protocol was developed to guide surgical decision making regarding initial peritoneal drainage (PD) versus initial laparotomy (LAP). We sought to evaluate outcomes after implementation of the protocol. Pre-post study including multiple urban hospitals. Premature infants with sNEC/SIP were accrued after implementation of surgical protocol-directed care (June 2014-June 2019). Patients with a birth weight of <750 g and less than 2 wk of age without pneumatosis or portal venous gas were treated with PD on perforation. PD patients received subsequent LAP for clinical deterioration or continued meconium/bilious drainage. Postprotocol characteristics and outcomes were compared with institutional historical controls. Significance set at P < 0.05. Preprotocol and postprotocol cohorts comprise 35 and 73 patients, respectively. There was a statistically significant difference in age at intervention between historical control PD (14 ± 13 d) and postprotocol PD (9 ± 4 d) groups (P = 0.01), PD patient's birth weight (716 ± 212 g versus 610 ± 141 g, P = 0.02) and estimated gestational age of LAP patients (27 ± 1.7 wk versus 31 ± 4 wk, P = 0.002). PD was definitive surgery in 27% (12 of 44) of postprotocol patients compared with 13% (3 of 23) historical controls. A trend in improved survival postprotocol occurred in all PD infants (73% versus 65%), all LAP (75% versus 70%), and for initial PD and subsequent LAP (82% versus 67%). Utilization of a surgical protocol in sNEC/SIP is associated with improved success of PD as definitive surgery and improved survival. [ABSTRACT FROM AUTHOR]

The assessment of fecal volatile organic compounds (VOCs) has emerged as a noninvasive biomarker in many different pathologies. Before assessing whether VOCs can be used to diagnose intestinal diseases, including necrotizing enterocolitis (NEC), it is necessary to measure the impact of variable infant demographic factors on VOC signals. Stool samples were collected from term infants at four hospitals in a large metropolitan area. Samples were heated, and fecal VOCs assessed by the Cyranose 320 Electronic Nose. Twenty-eight sensors were combined into an overall smellprint and were also assessed individually. 16s rRNA gene sequencing was used to categorize infant microbiomes. Smellprints were correlated to feeding type (formula versus breastmilk), sex, hospital of birth, and microbial enterotype. Overall smellprints were assessed by PERMANOVA with Euclidean distances, and individual sensors from each smellprint were assessed by Mann–Whitney U -tests. P < 0.05 was significant. Overall smellprints were significantly different according to diet. Individual sensors were significantly different according to sex and hospital of birth, but overall smellprints were not significantly different. Using a decision tree model, two individual sensors could reliably predict microbial enterotype. Assessment of fecal VOCs with an electronic nose is impacted by several demographic characteristics of infants and can be used to predict microbiome composition. Further studies are needed to design appropriate algorithms that are able to predict NEC based on fecal VOC profiles. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotizing enterocolitis (NEC) is one of the most devastating diseases in neonates and is characterized by high morbidity and mortality. It has been suggested that neutrophils play a crucial role in NEC pathogenesis and contribute to the hyperinflammatory reaction after bacterial colonization, which ultimately induces NEC. The aim of this study was to investigate whether dissolution of neutrophil extracellular traps (NETs) by systemic DNase1 therapy reduces NEC manifestation and morbidity. Methods NEC was induced in neonatal mice by gavage feeding of lipopolysaccharide mixed in Neocate, followed by hypoxia q12 h for 5d. Inactivated DNase1 and DNase1 were administered intraperitoneally twice daily in the control and treatment groups, respectively, starting on day 5 for 72 h. Survival, NEC score, intestinal damage (Chiu score, malondialdehyde [MDA], glutathione peroxidase [GPx]), inflammation (neutrophil elastase [NE], myeloperoxidase [MPO], toll-like receptor 4 [TLR4]), and NETs markers (SYTOX orange, cell-free DNA [cfDNA], DNase, citrullinated Histone 3 [H3cit]) were then assessed. Results In total, 44 neonatal mice were used in the experiment. Mice in the treatment group demonstrated significantly reduced NEC rates (44 versus 86%, P = 0.029) and improved survival in comparison to controls (65 versus 35%, P = 0.01). Furthermore, mice treated with DNase1 showed significantly less tissue damage (cfDNA, Chiu score), oxidative stress (MDA, GPx), and inflammation (NE, MPO, H3cit, TLR4), which ultimately lead to a significant reduction in mortality. Conclusions The results of the study indicate that systemic DNase1 treatment leads to a significant reduction in tissue damage, NEC severity, and mortality. Therefore, after validation of our findings in human subjects, DNase1 treatment should be considered as a therapeutic option in neonates diagnosed with NEC. [ABSTRACT FROM AUTHOR]

Abstract Background Psychological stress during pregnancy has been shown to cause subsequent harm to the fetus and newborn. Many studies focus on neurodevelopmental outcomes, but little is known about the effect of gestational stress on intestinal immunity and development. The purpose of this study was to determine the effect of psychological stress during pregnancy on intestinal architecture and growth in newborns. Methods Eight-week-old C57BL6 littermates underwent timed breeding. Pregnant dams were subjected to 1 h of daily psychological stress by using a well-established restraint model during days E7-E14. The distal ileum of 2-wk-old offspring of stressed mothers and nonstressed controls was harvested for histologic analysis. Slides were blinded to measure villus height and crypt depth and surface area. Serum was obtained to measure serum corticosterone levels. An explant model was used to measure corticosterone on the intestinal stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and growth factors epidermal growth factor receptor and insulin-like growth factor-1. Results The villus height, crypt depth, and surface area were significantly decreased in newborn exposed to stress during gestation. In addition, corticosterone levels were elevated in 2-wk-old mice exposed to stress. Real-time polymerase chain reaction revealed that explants exposed to corticosterone had a decrease in LGR5 compared with controls and an increase in epidermal growth factor receptor. Conclusions Here, we establish that neonatal mice from mothers that were subjected to psychological stress during pregnancy have significantly shorter villi and crypts compared with controls. In addition, pups from stressed mothers have decreased expression levels of the intestinal stem cell marker LGR5. These findings will aid in determining the effect of gestational psychological stress on intestinal development and stem cell plasticity. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotizing enterocolitis (NEC) in premature infants is often a devastating surgical condition with poor outcomes. GYY4137 is a long-acting donor of hydrogen sulfide, a gasotransmitter that is protective against intestinal injury in experimental NEC, likely through protection against injury secondary to ischemia. We hypothesized that administration of GYY4137 would improve mesenteric perfusion, reduce intestinal injury, and reduce inflammatory responses in experimental NEC and ischemia-reperfusion injury, and that these benefits would be mediated through endothelial nitric oxide synthase–dependent pathways. Methods NEC was induced in C57BL/6 wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (eNOSKO) pups via maternal separation, formula feeding, enteral lipopolysaccharide, and intermittent hypoxic and hypothermic stress. Pups received daily intraperitoneal injections of 50 mg/kg GYY4137 or phosphate buffered saline vehicle. In separate groups, adult male WT and eNOSKO mice underwent superior mesenteric artery occlusion for 60 min. Before abdominal closure, 50 mg/kg GYY4137 or phosphate buffered saline vehicle was administered into the peritoneal cavity. Laser doppler imaging was used to assess mesenteric perfusion of pups at baseline and on postnatal day 9, and the adult mice at baseline and 24 h after ischemic insult. After euthanasia, the terminal ileum of each animal was fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Sections were blindly graded using published injury scores. Intestinal tissue was homogenized and cytokines measured by ELISA. Data were compared using Mann–Whitney U test, and P -values <0.05 were significant. Results After NEC and ischemia reperfusion (I/R) injury, GYY4137 improved perfusion in WT mice compared to vehicle, but this effect was lost in the eNOSKO animals. Histologic injury followed a similar pattern with reduced intestinal injury in WT mice treated with GYY4137, and no significant improvement in the eNOSKO group. Cytokine expression after GYY4137 administration was altered by the ablation of eNOS in both NEC and I/R injury groups, with significant differences noted in Interleukin 6 and vascular endothelial growth factor. Conclusions GYY4137, a long-acting donor of hydrogen sulfide, has potential as a therapeutic compound for NEC. It improves mesenteric perfusion and intestinal injury in experimental NEC and intestinal I/R injury, and these benefits appear to be mediated through eNOS-dependent pathways. [ABSTRACT FROM AUTHOR]

Background To evaluate peritoneal drainage (PD) and laparotomy ± resection/ostomy (LAP) as initial approaches to the surgical management of necrotizing enterocolitis (NEC) in premature, extremely low birth weight (ELBW) infants. Methods Kids' Inpatient Database (2003-2012) was searched for cases of NEC ( International Classification of Diseases, ninth revision, Clinical Modification [ICD-9-CM] 777.5x) in premature (<37 weeks), extremely low birth weight (<1000 g) infants. Infants were admitted at <28 days of life. Propensity score (PS)-matched analyses were performed, using end points of hospital mortality, length of stay (LOS), and cost of hospitalization. Cases were matched 1:1 on 48 confounding variables (demographic, clinical, and hospital characteristics and 39 comorbidities). Results On PS-matched comparison, PD had higher survival versus LAP, P = 0.0009. LOS and cost were higher for PD versus LAP, P < 0.003. Survival rates did not differ between PD + LAP and PD-only treatments. LOS and cost were higher for PD + LAP versus PD-only, P < 0.02. PD + LAP infants had higher survival versus LAP, P = 0.0193. LOS and cost were higher for PD + LAP, P < 0.005. Conclusions A risk-adjusted PS-matched analysis of operative management in premature, ELBW infants with NEC found higher survival rates associated with PD placement versus LAP, whether PD was used as definitive treatment or with subsequent LAP even after controlling for potential contributors to selection bias (i.e., stability influencing management preference). [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. Increased intestinal permeability is central to NEC development. We have shown that stem cells (SCs) can reduce the incidence and severity of NEC. Our current goal was to investigate the efficacy of four different types of SC in preservation of gut barrier function during NEC. Materials and methods We compared (1) amniotic fluid–derived mesenchymal SC, (2) bone marrow–derived mesenchymal SC, (3) amniotic fluid–derived neural SC, and (4) enteric neural SC. Premature rat pups received an intraperitoneal injection of 2 × 10 6 SC or phosphate-buffered saline only and were then subjected to experimental NEC. Control pups were breastfed and not subjected to NEC. After 48 h, animals received a single enteral dose of fluorescein isothiocyanate -labeled dextran (FD70), were sacrificed 4 h later, and serum FD70 concentrations determined. Results Compared to breastfed, unstressed pups with intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 μg/mL), untreated pups exposed to NEC had impaired barrier function with significantly increased permeability (18.6 ± 4.25 μg/mL, P = 0.047). Pups exposed to NEC but treated with SC had significantly reduced intestinal permeability: Amniotic fluid–derived mesenchymal SC (9.45 ± 1.36 μg/mL, P = 0.017), bone marrow–derived mesenchymal SC (6.73 ± 2.74 μg/mL, P = 0.049), amniotic fluid–derived neural SC (8.052 ± 1.31 μg/mL, P = 0.0496), and enteric neural SC (6.60 ± 1.46 μg/mL, P = 0.033). Conclusions SCs improve gut barrier function in experimental NEC. Although all four types of SC reduce permeability equivalently, SC derived from amniotic fluid may be preferable due to availability at delivery and ease of culture, potentially enhancing clinical translation. [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) is a severe intestinal disease of premature infants with high mortality. Studies suggest a causative relationship between red blood cell (RBC) transfusion and NEC; however, whether RBC transfusion leads to worse outcomes in NEC is unknown. We sought to determine whether RBC transfusion was associated with an increased risk of surgical NEC and mortality. Methods In this retrospective study, 115 patients were enrolled with NEC Bell's stage 2A or greater from 2010-2015. Patients were classified based on the timing of RBC transfusion before NEC: ≤72 h, >72 h, and no transfusion. Variables including gestational age (GA), birth weight (BW), feedings, and hematocrit levels were analyzed. Outcomes were surgical intervention for NEC following RBC transfusion and mortality. Results Twenty-three (20%) infants developed NEC ≤ 72 h after RBC transfusion, 16 (69.6%) required surgery with a mortality rate of 21.7% ( n = 5). Seventeen (15%) infants developed NEC > 72 h after RBC transfusion, 12 (70.6%) required surgery with a mortality rate of 23.5% ( n = 4). 75 (65%) patients developed NEC without RBC transfusion, 17 (22.7%) required surgery with a mortality rate of 4% ( n = 3). Lower GA and BW were significantly associated with RBC transfusion and the need for surgical intervention. RBC transfusion ≤72 h before NEC was associated with surgical NEC (pairwise adjusted P < 0.001) and mortality (pairwise adjusted P = 0.048). However, multivariable logistic regression analysis revealed RBC transfusion is not an independent risk factor for surgical NEC. Conclusions Infants of lower GA and BW were more likely to receive an RBC transfusion before NEC, which was significantly associated with surgical intervention and an increasing risk of mortality. Judicious use of transfusions in premature infants may improve NEC outcomes. [ABSTRACT FROM AUTHOR]

Background Direct peritoneal resuscitation (DPR) has previously been shown to alter blood flow in the small bowel mesenteric vessels in models of intestinal ischemia. However, a survival advantage or its effects on local tissue inflammation have not been previously demonstrated. We hypothesized that DPR would increase survival and decrease intestinal tissue inflammation after intestinal ischemia and reperfusion (I/R) injury. Methods Eight-week-old male C57Bl6J mice were anesthetized and underwent midline laparotomy. I/R and DPR groups were exposed to superior mesenteric artery occlusion for 60 min with a nontraumatic clamp. Immediately after removal of the clamp, 1 mL of phosphate-buffered saline, 1 mL of minimal essential media, or 1 mL of minimal essential media supplemented with fetal bovine serum, penicillin and/or streptomycin, and glutamine were placed into the abdominal cavity of DPR groups. Animals were then closed in two layers and allowed to reperfuse for 6 h (cytokine analysis, n = 6 per group) or 7 d (survival analysis, n = 10 per group). After 6 h of reperfusion, animals were euthanized. Intestines were harvested and homogenized. Extracts were quantified for total protein content (Bradford assay), myeloperoxidase activity, tissue inflammatory cytokine, and growth factor production. P < 0.05 was significant. Results I/R caused marked intestinal ischemia, significant mortality, and a significant increase in tissue cytokine and growth factor levels ( P < 0.05). Seven-day survival was 30% for I/R without treatment and rose to 60% with DPR therapy using phosphate-buffered saline as the dialysate. DPR using plain MEM or MEM with supplements after ischemia increased 7-d survival to 90% ( P < 0.05). DPR also significantly decreased intestinal tissue levels of myeloperoxidase, as well as intestinal tissue levels of multiple growth factors and inflammatory cytokines. Conclusions DPR increases survival and decreases intestinal inflammation after intestinal I/R injury. Translational applications are readily achievable and should be considered for patients with intestinal ischemic pathology. [ABSTRACT FROM AUTHOR]

Background Immunoglobulin A (IgA) plays a key role in coating luminal antigens and preventing translocation of harmful bacteria. The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor that when stimulated activates factors important for barrier function and intestinal homeostasis. We hypothesize that AhR signaling is critical for establishment of intestinal homeostasis in neonates. Material and methods Mice: C57BL/6 (B6) AhR+/+ wild type (WT), B6.AhR−/− Aryl-hydrocarbon receptor knockout (KO), and B6.AhR+/+ raised on an AhR ligand-free diet (AhR LF). Enzyme-linked immunosorbent assay was used to measure fecal and serum IgA levels. Bacterial translocation was measured by culturing the mesenteric lymph nodes. Results Two week old KO mice had significantly less fecal IgA compared with WT (and AhR LF, P value = 0.0393. The amount of IgA from the gastric contents of 2-wk-old mice was not significantly different. At age 8 wk, AhR LF mice had significantly less fecal IgA than WT and KO P value = 0.0077. At 2 wk, KO mice had significantly higher levels of bacterial translocation and at 8 wk AhR LF had significantly higher levels of bacterial translocation compared with WT. Conclusions In neonatal mice, the lack of AhR signaling is associated with loss of intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation. In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity. [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) involves impaired ileal blood flow due to alterations in vascular tone control and intestinal angiogenesis. Platelet-derived growth factor (PDGF) is a mediator of normal angiogenesis in intestinal epithelium. We hypothesized that gene dysregulation during experimental NEC results in altered PDGF expression. Methods Sprague–Dawley rats were randomized to groups by litter. Controls were delivered vaginally and dam-fed. NEC groups were delivered prematurely by cesarean section and subjected to an established NEC protocol. Ileum was obtained at 0, 12, 24, 48, 72, and 96 h of life from all animals ( N = 108 animals). Western blot analysis was carried out for every time point, and samples were evaluated by immunohistochemistry. Antibodies against PDGF-A, PDGF-B, and their receptors, PDGFR-α and PDGFR-β, were used. Statistical analysis was performed using two-way analysis of variance with a priori P < 0.05. Results Ileal PDGF-A concentration was higher in controls versus NEC from 24–96 h of life. Its receptor, PDGFR-α, was low in concentration in both groups at all time points. PDGF-B concentration was increased in controls at 24 and 72 h of life but decreased at the 48-h mark. Its receptor, PDGFR-β, was also low in both groups at 12 and 24 h but increased in controls at 48 and 72 h. Conclusions These data support our hypothesis that PDGF and PDGF receptor expression are altered in experimental NEC. Dysregulation of PDGF during intestinal maturation could contribute to the development of NEC. Further investigation into this pathway could yield new therapeutic targets for this devastating disease. [ABSTRACT FROM AUTHOR]

Background Macrophages can be polarized into proinflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. Materials and methods Macrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/propidium iodide flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/M2 markers and real time polymerase chain reaction. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia, and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. Results In vitro, FHs-74 cell apoptosis was increased after coculture with macrophages and lipopolysaccharide (LPS). This apoptosis was increased by exposure to M1-conditioned medium and suppressed by exposure to M2-conditioned medium. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the proapoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, most of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization and protected the intestines from NEC. Conclusions M1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization. [ABSTRACT FROM AUTHOR]

Background Intestinal alkaline phosphatase (IAP) activity is decreased in necrotizing enterocolitis (NEC), and IAP supplementation prevents NEC development. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after NEC induction would not reverse intestinal injury. Materials and methods NEC was induced in Sprague-Dawley pups by delivery preterm followed by formula feedings with lipopolysaccharide (LPS) and hypoxia exposure and continued up to 4 d. IAP was added tofeeds on day 2 until being sacrificed on day 4. NEC severity was scored based on hematoxylin and eosin-stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and interleukin-6 expression were measured using real time polymerase chain reaction. Results: NEC pups' alkaline phosphatase (AP) activity was decreased to 0.18 U/mg compared with controls of 0.57 U/mg (P < 0.01). Discontinuation of LPS and hypoxia after 2 d increased AP activity to 0.36 U/mg (P < 0.01). IAP supplementation in matched groups did not impact total AP activity or expression. Discontinuing LPS and hypoxia after NEC onset improved intestinal injury scores to 1.14 compared with continued stressors, score 2.25 (P < 0.01). IAP supplementation decreased interleukin-6 expression two-fold (P < 0.05), though did not reverse NEC intestinal damage (P = 0.5). Conclusions This is the first work to demonstrate that removing the source of NEC improves intestinal damage and increases AP activity. When used as a rescue treatment, IAP decreased intestinal inflammation though did not impact injury making it likely that IAP is best used preventatively to those neonates at risk. [ABSTRACT FROM AUTHOR]

Background Despite emergence of markers of intestinal mucosal damage such as intestinal fatty-acid binding protein (i-FABP), there are no specific markers of damage extending into the muscle layers. We hypothesized that smooth muscle actin (SMA) released from the intestinal muscularis would be detectable in plasma after severe intestinal injury. Materials and methods Serial blood samples were collected from rats (n = 10) undergoing intestinal ischemia-reperfusion injury (IRI) and controls (n = 5). Additionally, admission and/or preoperative plasma samples were collected from twelve neonates with necrotizing enterocolitis (NEC), and five age- and weight-matched controls. Plasma ileal fatty-acid binding protein (rat) or i-FABP (human) were measured by enzyme-linked immunosorbent assay, and plasma SMA was detected by western blotting. Results Plasma ileal fatty-acid binding protein was low in both the control group and IRI at baseline, but became rapidly elevated in the IRI group even during ischemia. SMA was detected in reperfusion plasma samples of all IRI rats, but in none of the control samples. Plasma i-FABP was higher in infants with NEC than age- and weight-matched controls. Although i-FABP was higher in infants with severe surgical disease compared with focal disease, there was no difference between the operative and nonoperative groups. SMA was detected in the plasma of all four neonates with severe surgical NEC, but not in those with focal disease or those who were successfully conservatively managed. Conclusions SMA is detectable in plasma after severe intestinal injury and maybe a clinically useful maker of intestinal muscle damage. [ABSTRACT FROM AUTHOR]

Abstract: Background: Necrotizing enterocolitis (NEC) alters intestinal microvascular control mechanisms causing significant vasoconstriction. Our prior work with intraperitoneal 2.5% dextrose solution demonstrated increased intestinal perfusion in experimentally induced NEC. In the current study, we examine whether a buffered solution with lower glucose and osmolar loads similarly increases intestinal blood flow. We hypothesized that buffered 1.5% dextrose solution would increase ileal blood flow compared with baseline in NEC. Methods: We randomly assigned pregnant Sprague-Dawley rats to control (n = 103) or NEC (n = 123) groups, by litter. We induced NEC by previously published methods. Control pups were vaginally delivered and dam-fed. We used laser Doppler flowmetry to evaluate perfusion in the terminal ileum at 12, 24, 48, 72, or 96 h after delivery at baseline and after application of topical 1.5% dextrose solution. We evaluated differences between groups and time points by analysis of variance and Tukey post hoc test. Results: Baseline blood flow in the terminal ileum increased with gestational age in both groups (P < 0.05). Control groups had significantly greater baseline blood flow than NEC groups (P < 0.05), and topical application of buffered 1.5% dextrose solution increased blood flow compared with baseline in both groups at all time points (P < 0.05). Conclusions: Topical 1.5% dextrose solution significantly enhanced blood flow in the terminal ileum to the same degree as 2.5% dextrose solution. Thus, the use of buffered 1.5% dextrose solution might be more beneficial in treating clinical NEC, because it places a lower glucose and osmotic load on NEC-injured intestine. [Copyright &y& Elsevier]

Abstract: Background: The pathophysiology of necrotizing enterocolitis (NEC) includes the massive production of endogenous cytokines with exaggerated activation of inflammatory pathways. Colchicine has been used as an anti-inflammatory agent. The aim of this study was to investigate the possible beneficial effects of colchicine in a neonatal rat model of NEC. Materials and methods: We randomly divided rat pups into three groups: a control group, a saline-treated NEC group, and a colchicine-treated NEC group. We induced NEC by hyperosmolar enteral formula feeding and exposure to hypoxia/reoxygenation after cold stress. Intestinal samples were harvested for biochemical and histopathologic analyses. Results: The grade of intestinal injury of pups in the saline-treated NEC group was significantly higher than in the control and colchicine-treated groups (P < 0.001 and 0.003, respectively). The median level of intestinal malondialdehyde was significantly higher in the saline-treated NEC group compared with the control group (P = 0.006) or the colchicine-treated NEC group (P = 0.015). We observed significantly higher activity levels of intestinal superoxide dismutase and glutathione peroxidase in the colchicine-treated NEC group compared with the saline-treated NEC group (P = 0.033 and 0.030, respectively). The tissue levels of tumor necrosis factor-α and interleukin-1β were significantly higher in the saline-treated NEC group compared with the colchicine-treated NEC group (P < 0.001 and 0.003, respectively). Conclusions: We observed that in this model of NEC, colchicine had favorable effects on intestinal histologic and biochemical changes. [Copyright &y& Elsevier]

Abstract: Background: Cytidine 5′-diphosphocholine (CDP-choline) is an endogenous intermediate in the biosynthesis of phosphatidylcholine, a contributor to the mucosal defense of the intestine. The aim of this study was to evaluate the possible cytoprotective effect of CDP-choline treatment on intestinal cell damage, membrane phospholipid content, inflammation, and apoptosis in a neonatal rat model of necrotizing enterocolitis (NEC). Methods: We divided a total of 30 newborn pups into three groups: control, NEC, and NEC + CDP-choline. We induced NEC by enteral formula feeding, exposure to hypoxia-hyperoxia, and cold stress. We administered CDP-choline intraperitoneally at 300 mg/kg/d for 3 d starting from the first day of life. We evaluated apoptosis macroscopically and histopathologically in combination with proinflammatory cytokines in the gut samples. Moreover, we determined membrane phospholipid levels as well as activities of xanthine oxidase, superoxide dismutase, glutathione peroxidase, and myeloperoxidase enzymes and the malondialdehyde content of intestinal tissue. Results: Mean clinical sickness score, macroscopic gut assessment score, and intestinal injury score were significantly improved, whereas mean apoptosis score and caspase-3 levels were significantly reduced in pups in the NEC + CDP-choline group compared with the NEC group. Tissue proinflammatory cytokine (interleukin-1β, interleukin-6, and tumor necrosis factor-α) levels as well as tissue malondialdehyde content and myeloperoxidase activities were reduced, whereas glutathione peroxidase and superoxide dismutase activities were preserved in the NEC + CDP-choline group. In addition, NEC damage reduced intestinal tissue membrane phospholipids, whereas CDP-choline significantly enhanced total phospholipid and phosphatidylcholine levels. Long-term follow-up in additional experiments revealed increased body weight, decreased clinical sickness scores, and enhanced survival in CDP-choline–receiving versus saline-receiving pups with NEC lesions. Conclusions: Our study reports, for the first time, beneficial effects of CDP-choline treatment on intestinal injury in a neonatal rat model of NEC. Our data suggest that CDP-choline may be used as an effective therapeutic agent to prevent NEC. [Copyright &y& Elsevier]

Abstract: Background: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with an incidence of 0.5–2.4 cases per 1000 live births and a mortality rate between 10% and 50%. Neonates affected by NEC develop a septic injury that is associated with increased risk of neurological impairment due to intraventricular bleeding and chronic lung disease. Intestinal alkaline phosphatase (IAP) is an endogenous protein that has been shown to inactivate the endotoxin lipopolysaccharide (LPS), and has recently been used successfully as an adjunct to treat sepsis in adult patients. We tested the hypothesis that systemic, exogenous IAP will mitigate the inflammatory response as measured by serum levels of proinflammatory cytokines in a rat model of NEC. Methods: Newborn Sprague-Dawley rats were divided into groups. Control pups were dam fed. NEC was induced by feeding formula containing LPS and exposure to intermittent hypoxia. NEC pups were given intraperitoneal injections of 4 or 40 glycine units (U) of IAP or placebo twice daily. Intestine and serum was collected for cytokine analysis as well as measurement of alkaline phosphatase activity. Results: Systemic IAP administration significantly increased serum alkaline phosphatase activity in a dose- and time-dependent fashion. The proinflammatory cytokines tumor necrosis factor α, interleukin 6, and interleukin 1β were significantly increased in NEC rats versus controls on days 2 and 3. Importantly, treatment with 40 U systemic IAP decreased these proinflammatory cytokines back to near-control levels. Conclusions: Systemic IAP administration appears effective in mitigating the systemic inflammatory response associated with NEC, and may prove to be a valuable adjunctive treatment for NEC. [Copyright &y& Elsevier]

Abstract: Background: Supplementation of intestinal alkaline phosphatase (IAP), an endogenous protein expressed in the intestines, decreases the severity of necrotizing enterocolitis (NEC)–associated intestinal injury and permeability. We hypothesized that IAP administration is protective in a dose-dependent manner of the inflammatory response in a neonatal rat model. Materials and methods: Pre- and full-term newborn Sprague–Dawley rat pups were sacrificed on day of life 3. Control pups were vaginally delivered and dam fed. Preterm pups were delivered via cesarean section and exposed to intermittent hypoxia and formula feeds containing lipopolysaccharide (NEC) with and without IAP. Three different standardized doses were administered to a group of pups treated with 40, 4, and 0.4U/kg of bovine IAP (NEC+IAP40, IAP4, or IAP0.4U). Reverse transcription-real-time polymerase chain reaction (RT-PCR) for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α on liver and lung tissues and serum cytokine analysis for interleukin (IL)-1β, IL-6, IL-10, and TNF-α were performed. Data were analyzed by Kruskal–Wallis and Mann–Whitney tests, expressed as mean±standard error of the mean and P ≤0.05 considered significant. Results: Levels of cytokines IL-1β, IL-6, and TNF-α increased significantly in NEC versus control, returning to control levels with increasing doses of supplemental enteral IAP. Hepatic and pulmonary TNF-α and iNOS messenger ribonucleic acid expressions increased in NEC, and the remaining elevated despite IAP supplementation. Conclusions: Proinflammatory cytokine expression is increased systemically with intestinal NEC injury. Administration of IAP significantly reduces systemic proinflammatory cytokine expression in a dose-dependent manner. Early supplemental enteral IAP may reduce NEC-related injury and be useful for reducing effects caused by a proinflammatory cascade. [Copyright &y& Elsevier]

Background: Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis. Materials and Methods: CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay. Results: Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells. Conclusion: These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains. [ABSTRACT FROM AUTHOR]

Background: Cronobacter sakazakii (CS) is a highly virulent gram-negative opportunistic pathogen that has been implicated in clinical outbreaks of necrotizing enterocolitis (NEC). The role of mucosal immune cells in CS infection is not well understood. In this study, we sought to elucidate the role of neutrophils (polymorphonuclear leukocytes; PMNs) and macrophages in the pathogenesis of NEC induced by CS using a novel newborn mouse model. Materials and Methods: PMNs and macrophages were depleted in newborn mice using Gr-1 antibody and carrageenan, respectively, and then infected with 103 CFU of CS. The development of NEC in these mice was assessed by a pathologist based on the morphologic changes in the intestine. Cytokine production was determined in the serum and intestinal homogenates of infected mice by enzyme-linked immunosorbent assay (ELISA). Inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was determined by flow cytometry and Greiss method, respectively. Results: Depletion of PMNs and macrophages in newborn mice led to increased recruitment of dendritic cells (DCs) in the intestine compared with wild-type mice upon infection with CS. PMN- and macrophage-depleted mice showed increased bacterial load, production of pro-inflammatory cytokines, iNOS expression, and NO production in the intestines in comparison to wild-type mice fed with CS. In addition, depletion of PMNs and macrophages prior to infection in mice resulted in severe inflammation, villus destruction, and enhanced enterocyte apoptosis in the intestines compared with CS-infected wild-type mice. Conclusions: Our data suggest that depletion of PMNs and macrophages from the lamina propria (LP) exacerbates experimental NEC, indicating that both of these immunocytes play an important role in the clearance of CS during the initial stages of infection. The increased mucosal cytokine response and NO production in the absence of these immunocytes may be responsible for the observed increase in mucosal injury. Understanding how CS manipulates these cells, employing novel mouse model of NEC reported in this study, will provide significant insights for the development of novel therapeutic and preventive strategies to combat NEC. [Copyright &y& Elsevier]

The ability of the host to respond to intestinal injury requires the regeneration of native tissue through a highly orchestrated response from the intestinal stem cells, a population of cells located within the intestinal crypts that have the capability to repopulate the entire villous. The field of intestinal stem cell biology is thus of great interest to surgeons and non-surgeons alike, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel disease, trauma, and necrotizing enterocolitis. The field of intestinal stem cell research has been advanced recently by the identification of the putative marker, Lgr5, which has allowed for the isolation and further characterization of the intestinal stem cell. Under the control of the WNT signaling pathway, Lgr5 marks the rapidly dividing cells of the intestinal crypt, and identifies a population of cells that is capable of regenerating the entire villous. We now review the identification of Lgr5 as an intestinal stem cell marker, identify controversies in the intestinal stem cell field, and highlight the response of the intestinal stem cell to injury within the intestinal mucosa that may occur clinically. [ABSTRACT FROM AUTHOR]

Background: Enterocytes produce intestinal alkaline phosphatase (iAP), which detoxifies lipopolysaccharide (LPS), a mediator in necrotizing enterocolitis (NEC) pathogenesis. We hypothesize that aberrant expression or function of iAP contributes to the pathogenesis of NEC. Materials and Methods: Newborn Sprague Dawley rat pups were divided into three main groups. Control pups were breast fed, while two groups were exposed to intermittent hypoxia, LPS, and formula feeding for 4 d to induce NEC. Bovine iAP, with and without the presence of LPS, was administered orally to one of the NEC groups. The intestine was harvested and used to detect alkaline phosphatase (AP) activity and protein expression. Terminal ileum sections were used to grade intestinal injury and stained for AP. Comparisons were made with adult rat duodenum. Results: Compared with adult rats, control pups expressed significantly less AP protein but had 2-fold higher AP activity. NEC pup AP activity was significantly decreased compared to controls (P ≤ 0.05), which paralleled both the AP protein expression and immunofluorescence assay results. Following iAP administration, immunofluorescence, protein expression, and activity of AP were significantly increased compared with NEC pups without iAP supplementation. All NEC pups had intestinal injury grades ≥ 2 on a 4-point scale, while control and iAP-treated pups had grades < 0.25 (P < 0.001). Conclusions: Enteral administration of iAP to rat pups with experimental NEC increased AP activity levels to that of controls, and appears to protect the intestine. This opens up a new area of study in NEC pathophysiology as well as a potential novel treatment strategy to prevent the development of NEC. [ABSTRACT FROM AUTHOR]

Background: To determine whether peritoneal drain (PD) or laparotomy (LAP) is the most effective intervention in premature neonates with necrotizing enterocolitis (NEC) or intestinal perforation (IP). Methods: A systematic review of the published literature between January 2000 and December 2008 was undertaken. Prospective studies with at least 25 patients in each of the PD and LAP arms were selected. Gestational age, birth weight, operation, and mortality data were extracted. Results: Five prospective studies (two level I, three level II) with 523 (273 PD, 250 LAP) participants followed for mortality met selection criteria. Using a fixed effect model, the combined estimate indicates an increased mortality of 55% with PD (OR 1.55, 95% CI: 1.08–2.22, P =0.02) without statistical heterogeneity (χ2 =5.88, P =0.21). PD patients were 0.78 wk younger (P =0.0002) and 67g smaller (P =0.0006). Analysis of the three level II trials yielded a combined estimate indicating an excess mortality of 89% with PD patients (95% CI: 1.20–2.98, P =0.006) without statistical heterogeneity (χ2 =3.74, P =0.15). Conclusions: PD is associated with 55% excess mortality compared with LAP. Pediatric surgeons must individually assess and select patients with NEC and IP for optimal surgical therapy. [Copyright &y& Elsevier]

Background: A proposed mechanism of intestinal injury in necrotizing enterocolitis (NEC) involves vascular dysfunction through altered nitric oxide synthase (NOS) activity. We hypothesize that this dysfunction results in an imbalance in nitric oxide (•NO) and superoxide (O2 •–) production by the intestinal vascular endothelium, which contributes to the intestinal injury seen in NEC. Materials and Methods: Neonatal rat pups were divided into two groups. Control pups were breast fed and housed with their mother. Experimental NEC pups were housed separately and either exposed to formula feeding and 5% to 10% hypoxia alone (FF/H) or with the addition of lipopolysaccharide (FF/H/LPS). Mesenteries from each group were analyzed for •NO and O2 •– production with and without NOS inhibition by NG-monomethyl-L-arginine (L-NMMA). Western blot analysis for eNOS, phosphorylated eNOS (phospho-eNOS), and inducible NOS (iNOS) was performed, and each terminal ileum was graded for intestinal injury by histology. Results: Histology revealed mild intestinal injury (grade 1–2 on a 4-point scale) in the FF/H group and severe injury (grade 3–4) in the FF/H/LPS group. The FF/H cohort had significantly increased •NO and lower O2 •– production, while the FF/H/LPS group shifted to significantly decreased •NO and increased O2 •– production. L-NMMA inhibited >50% of O2 •– production in all three groups but only inhibited •NO production in control and FF/H pups. Western blot analysis revealed increased levels of phospho-eNOS in FF/H pups and increased iNOS in FF/H/LPS pups. Conclusions: This study demonstrates in the progression of NEC, intestinal ischemia is associated with a shift from •NO to O2 •– production, which is NOS-dependent. Potentially greater injury results from impaired vasodilatation and over-production of reactive oxygen species. [Copyright &y& Elsevier]

Background: Infants who develop necrotizing enterocolitis (NEC) are usually managed with fecal diversion. The integrity of the bowel being diverted is often suboptimal. Our clinical impression is that stomas created in this circumstance are fraught with complications. The purpose of this study is to quantify the rate of these complications and identify risk factors. Methods: A retrospective data collection from May 1999 to May 2008 on infants undergoing laparotomy for NEC was conducted. Data collected included gestational age, birth weight, age, and weight at operation, indications for surgical therapy, procedure performed, time to stoma output, time to takedown of stoma, complication directly related to the ostomy, and mortality. Data comparisons were analyzed statistically using χ2, Pearson''s correlation, Fisher''s exact test, or a 2-tailed Student''s t-test with significance reported for P <0.05. Results: A total of 73 patients were identified. Mean gestational age was 28 (±4) wk, mean birth weight was 1247 (±713) g. Mean age at the time of surgery was 23 (±27) d, and mean weight at operation was 1513 (±1306) g. The most common indication for surgical intervention was pneumoperitoneum (n =43, 58%). The most common level of intestinal diversion was the ileum (n =63, 85%). In-house mortality was 13%. There were 31 patients (42%) who developed 32 stoma-related complications. Demographic or preoperative variables that were a significant predictor of stoma-related complications were gestational age (P =0.003) and preoperative weight (P =0.024). Conclusion: Premature infants carry a risk for developing stoma-related complications. Within that cohort, there is significantly increased risk of stoma-related complications in patients who are younger in gestational age and who have low preoperative weight. Future prospective studies may allow insight into preventative practices. [Copyright &y& Elsevier]

Background: Reactive oxygen species (ROS) are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. We have recently found that activation of multiple cellular signaling transduction pathways occurs during ROS-induced intestinal cell apoptosis; the phosphatidylinositol 3-kinase (PI3-K) pathway plays an anti-apoptotic role during this process. Insulin-like growth factor (IGF)-1 activates PI3-K pathway to promote cell survival; however, the effects of IGF-1 treatment during gut injury are not clearly defined. The purpose of this study was to determine whether IGF-1 protects intestinal cells from ROS-induced apoptosis. Materials and methods: Rat intestinal epithelial (RIE)-1 cells were treated with either IGF-1 (100 nm), hydrogen peroxide (H2O2; 500 μm), or combination. Western blotting was performed to assess phosphorylation of Akt, a downstream effector of PI3-K. Cell Death Detection ELISA, DCHF, and JC-1 assays were performed to demonstrate protective effects of IGF-1. Wortmannin, an inhibitor of PI3-K, was used to show PI3-K-dependent mechanism of action for IGF-1. Results: H2O2 treatment resulted in increased intestinal epithelial cell apoptosis with intracellular ROS generation and mitochondrial membrane depolarization; IGF-1 pre-treatment attenuated this response without affecting ROS production. H2O2-induced phosphorylation of Akt was further increased with IGF-1 treatment; wortmannin abolished these effects in RIE-1 cells. Conclusions: PI3-K pathway is activated during ROS-induced intestinal epithelial cell injury; IGF-1 exerted an anti-apoptotic effect during this response by PI3-K activation. A better understanding of the exact role of IGF-1-mediated activation of PI3-K may allow us to facilitate the development of novel therapy against NEC. [Copyright &y& Elsevier]