Evaluation of the protective and therapeutic effects of extra virgin olive oil rich in phenol in experimental model of neonatal necrotizing enterocolitis by clinical disease score, inflammation, apoptosis, and oxidative stress markers.

BACKGROUND AND AIM: Necrotizing Enterocolitis (NEC) is an inflammation-associated ischemic necrosis of the intestine. To investigate the effects of extra virgin olive oil (EVOO) on inflammation, oxidative stress, apoptosis, and histological changes in NEC-induced newborn rats. MATERIALS AND METHODS: 24 rats were randomly divided into three groups: control, NEC and NEC + EVOO. NEC induction was performed using hypoxia-hyperoxia, formula feeding, and cold stress. The NEC + EVOO group received 2 ml/kg EVOO with high phenolic content by gavage twice a day for 3 days. 3 cm of bowel including terminal ileum, cecum, and proximal colon was excised. RESULTS: Weight gain and clinical disease scores were significantly higher in the NEC + EVOO group than in the NEC group (p < 0.001). EVOO treatment caused significant decreases in IL1ß, IL6 levels (p = 0.016, p = 0.029 respectively) and EGF, MDA levels (p = 0.032, p = 0.013 respectively) compared to NEC group. Significant decreases were observed in IL6 gene expression in the NEC + EVOO group compared to the NEC group (p = 0.002). In the group NEC + EVOO, the number of Caspase-3 positive cells was found to be significantly reduced (p < 0.001) and histopathological examination revealed minimal changes and significantly lower histopathological scores (p < 0.001). CONCLUSION: Phenol-rich EVOO prevents intestinal damage caused by NEC by inhibiting inflammation, oxidative stress, apoptosis.

Comparison of preoperative and intraoperative surgeon diagnosis and pathologic findings in spontaneous intestinal perforation vs necrotizing enterocolitis.

OBJECTIVE: To investigate the accuracy of preoperative and intraoperative diagnosis via comparison to pathologic diagnosis in spontaneous intestinal perforation (SIP) vs. necrotizing enterocolitis (NEC). STUDY DESIGN: A retrospective review of neonates <1500 g treated for pneumoperitoneum between 07/2004-09/2022 was conducted. Patients treated for NEC medically prior to diagnosis and those treated with drain only were excluded. Fleiss' Kappa analysis assessed agreement between all three diagnoses: preoperative, intraoperative, and pathologic. RESULT: Overall, 125 patients were included with mean birthweight 834.2 g (SD:259.2) and mean gestational age 25.8 weeks (SD:2.2). Preoperative and intraoperative diagnoses agreed in 90.3%, intraoperative and pathologic agreed in 71.1%, and preoperative and pathologic agreed in 75.2% of patients. Fleiss' Kappa was 0.55 (95% CI:0.43,0.68), indicating moderate agreement between the three diagnoses. CONCLUSION: Our study shows moderate agreement between preoperative, intraoperative, and pathologic diagnoses. Further studies investigating the clinical characteristics of SIP and NEC are needed to improve diagnostic accuracy and management.

Potential role of bile acids in the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most common acute gastrointestinal diseases in preterm infants. Recent studies have found that NEC is not only caused by changes in the intestinal environment but also by the failure of multiple systems and organs, including the liver. The accumulation of bile acids (BAs) in the ileum and the disorder of ileal BA transporters are related to the ileum injury of NEC. Inflammatory factors such as tumor necrosis factor (TNF)-α and interleukin (IL)-18 secreted by NEC also play an important role in regulating intrahepatic BA transporters. As an important link connecting the liver and intestinal circulation, the bile acid metabolic pathway plays an important role in the regulation of intestinal microbiota, cell proliferation, and barrier protection. In this review, we focus on how bile acids explore the dynamic changes of bile acid metabolism in necrotizing enterocolitis and the potential therapeutic value of targeting the bile acid signaling pathways.

Óbitos neonatais por enterocolite necrosante: estudo de coorte retrospectiva / Neonatal deaths from necrotizing enterocolitis: a retrospective cohort study / Muertes neonatales por enterocolitis necrozante: estudio de cohorte retrospectivo

Introdução:A enterocolite necrosante é uma doença que pode afetar o trato gastrointestinal de recém-nascidos,cujas manifestações clínicas podem ser caracterizadas por vômitos biliosos, sangue nas fezes, distensão abdominal, além de alterações nos parâmetros hemodinâmicos e instabilidade térmica. As populações mais vulneráveis a essa enfermidade são recém-nascidos de baixo peso,expostos ao ambiente de terapia intensiva neonatal. Objetivos: Identificar o perfil de recém-nascidos e os fatores maternos e neonatais associados à ocorrência de óbitos por enterocolite necrosante, em maternidade de referência do Ceará-Brasil. Metodologia: Trata-se de coorte retrospectiva, estudo que objetiva a descrição da incidência de determinado evento ao longo do tempo, além do estabelecimento de relações causais entre os fatores associados ao acontecimento. Incluíram-serecém-nascidos que tiveram óbitos por enterocolite necrosante entre 2019 e 2021, comficha de investigação de óbitos neonatais preenchida corretamente, não sendo excluído nenhum recém-nascido, totalizando amostra de 29 óbitos.Resultados: Identificou-se que o perfil dos recém-nascidos foi,em maioria, deprematuros e com baixo peso e fatores de risco para outras doenças associadas,como a sepse, o que acarretourealização de procedimentos invasivos e internação em ambiente de terapia intensiva neonatal.Conclusões: A prematuridade e o baixo peso ao nascer foram as variáveis relevantes no estudo e podem estar associadas à piora das condições clínicas do recém-nascido e ao desenvolvimento de enterocolite necrosante (AU).

Introduction: Necrotizing Enterocolitis is a disease that can affect the gastrointestinal tract of newborns, whose clinical manifestations can be characterized by bilious vomiting, blood in stool, abdominal distension, in addition to changes in hemodynamic parameters and thermal instability. The populations most vulnerable to this disease are low birth weight newborns exposed to the neonatal intensive care environment. Objectives: This study aimed to identify the profile of newborns and maternal and neonatal factors associated with the occurrence of deaths from necrotizing enterocolitis in a reference maternity hospital in Ceará, Brazil. Methodology: This is a retrospective cohort study seeking to describe the incidence ofa particular event over time, as well as establish causal relationships between the factors associated with the event. The study population comprised newborns who died from necrotizing enterocolitis between 2019 and 2021, who had neonatal death investigation forms filled out correctly, with no newborns being excluded, totaling a sample of 29 deaths. Results: It was identified that the profile of newborns was mostly premature, of low birth weight and with risk factors for other associated diseases such as sepsis, leading to invasive procedures and hospitalization in a neonatal intensive care environment. Conclusions: Prematurity and low birth weight were relevant variables in the study and may be associated with worsening of the newborn's clinical conditionsand development of necrotizing enterocolitis (AU).

ntroducción:La Enterocolitis Necrotizante es enfermedad que puede afectar el tracto gastrointestinal del recién nacido, cuyas manifestaciones clínicas pueden caracterizarse por vómitos biliosos, sangre en las heces, distensión abdominal, además de cambios en los parámetros hemodinámicos e inestabilidad térmica.Las poblaciones más vulnerables a esta enfermedad son recién nacidos con bajo peso expuestos al entorno de cuidados intensivos neonatales.Objetivos: Identificar el perfil de recién nacidos y los factores maternos y neonatales asociados a la ocurrencia de muertes por enterocolitis necrotizante, en maternidad de referencia en el Ceará-Brasil.Metodología: Estudio de cohorte retrospectivo, para describir la incidencia de determinado evento a lo largo del tiempo, además de establecer relaciones causales entre los factores asociados al evento.Se incluyeron recién nacidos fallecidos por enterocolitis necrotizante entre 2019 y 2021, quienes tuvieron formulario de investigación de muerte neonatal correctamente diligenciado, no excluyéndose ningún recién nacido, totalizando muestra de 29 defunciones.Resultados:El perfil de los recién nacidos fue mayoritariamente prematuro y de bajo peso al nacer y con factores de riesgo para otras enfermedades asociadas, como sepsis, con procedimientos invasivos y hospitalización en ambiente de cuidados intensivosneonatales.Conclusiones:La prematuridad y el bajo peso al nacer fueron variables relevantes en el estudio y pueden estar asociados con empeoramiento de las condiciones clínicas de recién nacidos y desarrollo de enterocolitis necrotizante (AU).

Active polypeptide MDANP protect against necrotizing enterocolitis (NEC) by regulating the PERK-eIF2ɑ-QRICH1 axis.

The effect of MDANP effects on ER stress signalling not well known or elucidated. Endoplasmic reticulum (ER) stress plays a critical role in necrotizing enterocolitis (NEC) pathogenesis through the PERK-eIF2ɑ-QRICH1 axis. The present study aimed to explore the protective effects of MDANP in NEC development. Firstly, a function screening was designed to identify the candidate peptides in human milk, and then the identified peptides were validated in NEC patients. In vivo, NEC was induced in mice pups and divided into four groups: (1) control group, (2) NEC group, (3) MDANP + NEC group, and (4) NS + NEC group. In vitro, lentivirus-mediated QRICH1 silencing, was used to transfect NCM460 cell lines, then stimulated with LPS. After LPS stimulation, cells were treated with chemically synthesized MDANP, and the essential proteins in the QRICH1 signalling pathway in cells were tested and compared. After the small-scale screening, a peptide (SKSKKFRRPDIQYPDATDED) named MDANP was determined as the principal peptide. Its protective effect against NEC through inhibiting the expression of ERS key proteins and impeding the intestinal cells' apoptosis was observed in the animal models. Furthermore, the inhibitive effect of MDANP on apoptosis of intestinal epithelial cells through modulating the PERK-eIF2ɑ-QRICH1 ERS pathway was also confirmed in vitro. Taken together, our data suggest that MDANP effectively ameliorates apoptosis in NEC through attenuating PERK-eIF2ɑ-QRICH1.

Persistent Proclivity to a Proinflammatory State in a Human Enteroid Model of Necrotizing Enterocolitis.

Background: Necrotizing enterocolitis (NEC) is a devastating disease of premature neonates with substantial morbidity and mortality. Necrotizing enterocolitis is associated with prematurity, a hyperinflammatory response, and dysregulation of intestinal barrier function. We hypothesize that patients with NEC will have an increased hyperinflammatory intestinal response compared with those without NEC. Patients and Methods: Enteroids were generated from intestinal tissue from neonates undergoing resection. They were treated with 100 mcg/mL lipopolysaccharide (LPS), subjected to 24 hours of hypoxia inducing experimental NEC, then compared with untreated controls. Expression of tumor necrosis factor (TNF-α) and interleukin 8 (IL-8) were evaluated via reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) to measure inflammatory response. Analysis of variance (ANOVA) determined statistical significance (p < 0.05). Results: Treated NEC-derived enteroids expressed significantly higher levels of IL-8 (RT-qPCR, p = 0.003; ELISA, p = 0.0002) compared with untreated NEC-derived enteroids with an increase in inflammatory marker concentration in those with a greater degree of prematurity (ELISA, p = 0.0015). A higher level of IL-8 was seen in NEC-derived enteroids compared with control after treatment (RT-qPCR, p = 0.024). Tumor necrosis factor-α levels were elevated in treated NEC-derived enteroids compared with untreated NEC-derived enteroids (RT-qPCR, p = 0.006; ELISA, p = 0.002) and compared with treated non-NEC-derived enteroids (RT-qPCR, p = 0.025; ELISA, p < 0.0001). Conclusions: Enteroids generated from neonates with NEC have an elevated hyperinflammatory response in response to NEC-inducing stimuli compared with controls. Enteroids generated from neonates with NEC with a greater degree of prematurity have a larger increase in inflammatory markers. This tendency toward a hyperinflammatory state may be correlated with an infant's proclivity to develop NEC and further demonstrates the hyperinflammatory state of prematurity.

Astragaloside IV regulates TL1A and NF-κB signal pathway to affect inflammation in necrotizing enterocolitis.

AIM: This study aims to explore the possible effect of Astragaloside IV (AS-IV) on necrotizing enterocolitis (NEC) neonatal rat models and verify the possible implication of TNF-like ligand 1 A (TL1A) and NF-κB signal pathway. METHODS: NEC neonatal rat models were established through formula feeding, cold/asphyxia stress and Lipopolysaccharide (LPS) gavage method. The appearance, activity and skin as well as the pathological status of rats subjected to NEC modeling were assessed. The intestinal tissues were observed after H&E staining. The expression of oxidative stress biomarkers (SOD, MDA and GSH-Px) and inflammatory cytokines (TNF-α, IL-1ß and IL-6) were detected by ELISA and qRT-PCR. Western blotting and immunohistochemistry were applied to detect expressions of TL1A and NF-κB signal pathway-related proteins. Cell apoptosis was assessed by TUNEL. RESULTS: NEC neonatal rat models were established successfully, in which TL1A was highly expressed and NF-κB signal pathway was activated, while TL1A and NF-κB signal pathway can be suppressed by AS-IV treatment in NEC rats. Meanwhile, inflammatory response in intestinal tissues was increased in NEC rat models and AS-IV can attenuate inflammatory response in NEC rats through inhibiting TL1A and NF-κb signal pathway. CONCLUSION: AS-IV can inhibit TL1A expression and NF-κb signal pathway to attenuate the inflammatory response in NEC neonatal rat models.

Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. METHODS: Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines. RESULTS: In all four cases, major immune cells, such as T cells (15.1-47.7%), B cells (3.1-19.0%), monocytes (16.5-31.2%), macrophages (1.6-17.4%), dendritic cells (2.4-12.2%), and natural killer cells (7.5-12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells. CONCLUSION: Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

ß-glucan protects against necrotizing enterocolitis in mice by inhibiting intestinal inflammation, improving the gut barrier, and modulating gut microbiota.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease with high morbidity and mortality, affecting preterm infants especially those with very low and extremely low birth weight. ß-glucan has manifested multiple biological effects including anti-inflammatory, regulation of gut microbiota, and immunomodulatory activities. This study aimed to investigate the effects of ß-glucan on NEC. METHODS: Neonatal C57BL/6 mice were randomly divided into three groups: Control group, NEC group and ß-glucan group. Newborn 3-day-old mice were gavaged with either 1 mg/ml ß-glucan or phosphate buffer saline at 0.03 ml/g for 7 consecutive days before NEC induction and a NEC model was established with hypoxia combined with cold exposure and formula feeding. All the pups were killed after 72-h modeling. Hematoxylin-eosin staining was performed to assess the pathological injury to the intestines. The mRNA expression levels of inflammatory factors in intestinal tissues were determined using quantitative real-time PCR. The protein levels of TLR4, NF-κB and tight junction proteins in intestinal tissues were evaluated using western blotting and immunohistochemistry. 16S rRNA sequencing was performed to determine the structure of the gut microbiota. RESULTS: ß-glucan administration ameliorated intestinal injury of NEC mice; reduced the intestinal expression of TLR4, NF-κB, IL-1ß, IL-6, and TNF-α; increased the intestinal expression of IL-10; and improved the expression of ZO-1, Occludin and Claudin-1 within the intestinal barrier. Pre-treatment with ß-glucan also increased the proportion of Actinobacteria, Clostridium butyricum, Lactobacillus johnsonii, Lactobacillus murinus, and Lachnospiraceae bacterium mt14 and reduced the proportion of Klebsiella oxytoca g Klebsiella in the NEC model. CONCLUSION: ß-glucan intervention prevents against NEC in neonatal mice, possibly by suppressing the TLR4-NF-κB signaling pathway, improving intestinal barrier function, and partially regulating intestinal microbiota.

Association of Placental Pathologic Findings with the Severity of Necrotizing Enterocolitis in Preterm infants - A Matched Case-Control Study.

OBJECTIVE: To determine the association of placental pathology with the severity of necrotizing enterocolitis (NEC) in preterm infants. METHODS: This single-center matched case-control study included infants with NEC (n = 107) and gestational age and birth weight-matched controls (n = 130), born between 2013 and 2020. Placentas were evaluated according to the Amsterdam Placental Workshop Group Consensus Statement. RESULTS: Acute histologic chorioamnionitis with the fetal response was significantly more common in infants with surgical NEC vs. medical NEC (35.4% vs. 15.3%; p = 0.02). On regression model, infants with multiple placental pathologies (OR 2.16; 95% CI 1.01 - 4.73; p = 0.04) and maternal vascular malperfusion (OR 2.2; 95% CI 1.12 - 4.51; p = 0.02) had higher odds of either medical or surgical NEC than controls. CONCLUSION: Infants with multiple placental lesions, including placental inflammatory and vascular lesions, were at higher risk of medical or surgical NEC in the postnatal period.

Knockdown of PHLDA1 Alleviates Necrotizing Enterocolitis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling.

OBJECTIVE: Pleckstrin homology-like domain family A member 1 (PHLDA1) is involved in the progression of intestine-related diseases, but its role and related mechanisms in Necrotizing enterocolitis (NEC) are unclear. The aim of this study was to better understand the function of PHLDA1 in NEC and the underlying mechanisms. METHODS: A neonatal mouse model of NEC was established by hypoxic hypothermia, and sh-PHLDA1 was transfected into mice to observe the mortality of each group within 4 days. The levels of IL-1ß, IL-6, IL-18 and TNF-α were measured by PCR and ELISA. ROS, MDA, SOD, and GSH-Px levels were detected by Dihydroethidium (DHE) method and kit; expression of pyroptosis-related factors including NLRP3, ASC, cleaved-caspase1, GSDMD-N, IL-1ß, IL-18, and Nrf2 were detected by western-blot; mechanistically, the effects of transfection of sh-PHLDA1 and ML385 (Nrf2 inhibitor) were investigated, and the expression of pyroptosis-related factors was detected again. RESULTS: PHLDA1 was highly expressed in the intestinal tissues of NEC mice, and transfection of sh-PHLDA1 improved the survival rate, alleviated intestinal lesions, improved intestinal inflammation, oxidative stress and cellular scorching in NEC. In addition, sh-PHLDA1 was able to inhibit NLRP3 activation and pyroptosis by activating Nrf2. CONCLUSION: Knockdown of PHLDA1 attenuated necrotizing small intestinal colitis by enhancing Nrf2 expression to inhibit NLRP3 inflammasome activation and pyroptosis.

Intestinal epithelium in early life.

Rapid development of the fetal and neonatal intestine is required to meet the growth requirements of early life and form a protective barrier against external insults encountered by the intestinal mucosa. The fetus receives nutrition via the placenta and is protected from harmful pathogens in utero, which leads to intestinal development in a relatively quiescent environment. Upon delivery, the intestinal mucosa is suddenly tasked with providing host defense and meeting nutritional demands. To serve these functions, an array of specialized epithelial cells develop from intestinal stem cells starting in utero and continuing postnatally. Intestinal disease results when these homeostatic processes are interrupted. For preterm neonates, the most common pathology resulting from epithelial barrier dysfunction is necrotizing enterocolitis (NEC). In this review, we discuss the normal development and function of the intestinal epithelium in early life as well as how disruption of these processes can lead to NEC.

Bioinformatics analysis of potential key genes and pathways in neonatal necrotizing enterocolitis.

OBJECTIVE: To detect differentially expressed genes in patients with neonatal necrotizing enterocolitis (NEC) by bioinformatics methods and to provide new ideas and research directions for the prevention, early diagnosis and treatment of NEC. METHODS: Gene chip data were downloaded from the Gene Expression Omnibus database. The genes that were differentially expressed in NEC compared with normal intestinal tissues were screened with GEO2R. The functions, pathway enrichment and protein interactions of these genes were analyzed with DAVID and STRING. Then, the core network genes and significant protein interaction modules were detected using Cytoscape software. RESULTS: Overall, a total of 236 differentially expressed genes were detected, including 225 upregulated genes and 11 downregulated genes, and GO and KEGG enrichment analyses were performed. The results indicated that the upregulated differentially expressed genes were related to the dimerization activity of proteins, while the downregulated differentially expressed genes were related to the activity of cholesterol transporters. KEGG enrichment analysis revealed that the differentially expressed genes were significantly concentrated in metabolism, fat digestion and absorption pathways. Through STRING analysis, 9 key genes in the protein network interaction map were identified: EPCAM, CDH1, CFTR, IL-6, APOB, APOC3, APOA4, SLC2A and NR1H4. CONCLUSION: Metabolic pathways and biological processes may play important roles in the development of NEC. The screening of possible core targets by bioinformatics is helpful in clarifying the pathogenesis of NEC at the gene level and in providing references for further research.

A potential pathogenic hypoxia-related gene HK2 in necrotizing enterocolitis (NEC) of newborns.

BACKGROUND: Necrotizing enterocolitis (NEC) is a disastrous gastrointestinal disease of newborns, and the mortality rate of infants with NEC is approximately 20%-30%. The exploration of pathogenic targets of NEC will be conducive to timely diagnosis of NEC. METHODS: The whole transcriptome RNA sequencing was performed on NEC samples to reveal the expression of lncRNAs, circRNAs, miRNAs and mRNAs. Using differential expression analysis, cross analysis, target prediction, enrichment analysis, the pathogenic ceRNA network and target was found. RESULTS: Preliminarily, 281 DEmRNAs, 21 DEmiRNAs, 253 DElncRNAs and 207 DEcircRNAs were identified in NEC samples compared with controls. After target prediction and cross analyses, a key ceRNA regulatory network was built including 2 lncRNAs, 4 circRNAs, 2 miRNAs and 20 mRNAs. These 20 mRNAs were significantly enriched in many carbohydrate metabolism related pathways. After cross analysis of hypoxia-, carbohydrate metabolism-related genes, and 20 core genes, one gene HK2 was finally obtained. Dendritic cells activated were significantly differentially infiltrated and negatively correlated with HK2 expression in NEC samples. CONCLUSIONS: The promising pathogenic hypoxia-related gene HK2 has been firstly identified in NEC, which might also involve in the carbohydrate metabolism in NEC.

NF-κB inactivation attenuates the M1 macrophage polarization in experimental necrotizing enterocolitis by glutaredoxin-1 deficiency.

The pathogenesis of necrotizing enterocolitis (NEC) is severe inflammatory injury in preterm infants, which resulted from macrophage polarization. Nuclear factor-κB (NF-κB) is implicated to be involved in macrophage polarization. We here evaluated the essential role of NF-κB in macrophage polarization in NEC in human samples from neonates with NEC and the mouse experimental NEC model. Enhanced intestinal macrophage (IM) infiltration was presented in human neonates with NEC, the majority of which were M1 macrophages. Meanwhile, NF-κB was activated in the IMs in human NEC samples. NF-κB inhibition by BAY promoted the M1 to M2 macrophage polarization. Furthermore, glutaredoxin-1 (Grx1) deficiency promoted M2 polarization via NF-κB inactivation from the lipopolysaccharide-induced proinflammatory macrophages. The IMs isolated from Grx1- / - mice presented with decreases in total numbers and less macrophage differentiation. Grx1- / - derived IM were effective in the increased survival in experimental NEC through inflammation blocking. Our study provides evidence that NF-κB inactivation by Grx1 depletion contributed to the alleviation of NEC via inhibiting M1 macrophage polarization. The modulation to alternative macrophages in the intestines may provide a promising benefits for NEC treatment.

Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease that impacts the intestine of premature infants. Sildenafil has shown benefit in colitis and ischemia/reperfusion models but has not been adequately studied in NEC. Sildenafil's best studied mechanism involves augmenting nitric oxide induced vasodilation. We hypothesized that sildenafil would improve outcomes during experimental NEC in an eNOS dependent manner. MATERIALS: NEC was induced in five-day old mouse pups with gavage formula feeds plus intermittent hypoxia and hypothermia. Using wild type (WT) mice, the route of sildenafil administration was studied in the following groups: (1) breastfed controls, (2) NEC + oral (PO) sildenafil, (3) NEC + PO vehicle, (4) NEC + intraperitoneal (IP) sildenafil, (5) NEC + IP vehicle. The eNOS KO groups studied included: (1) breastfed controls, (2) NEC + PO sildenafil, (3) NEC + PO vehicle. Data were tested for normality and compared using t-tests or Mann-Whitney with a p-value <0.05 considered significant. RESULTS: In WT mice, oral and IP sildenafil resulted in improved clinical outcomes compared to their respective vehicle group. Only orally administered sildenafil significantly improved perfusion to the intestine and protected it from macroscopic and histologic injury. When repeated in eNOS KO mice, oral sildenafil improved clinical scores and attenuated intestinal injury scores, despite no effect on intestinal perfusion. CONCLUSIONS: Sildenafil, when administered orally, improves clinical outcomes and protects the intestine in a murine model of experimental necrotizing enterocolitis. While sildenafil requires eNOS to impact mesenteric perfusion, it does not appear to be dependent on eNOS to attenuate intestinal injury.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-α for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-α, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

[Expression and Role of PD-L1 in a Mouse Model of Necrotizing Enterocolitis].

Objective: To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC). Methods: A total of 20 wild-type C57 BL/6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L1 (+/+) mice and 14 PD-L1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors. Results: The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P<0.05). According to the results of the PD-L1 gene mouse experiment, the control groups of PD-L1 (+/+) mice and PD-L1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L1 (+/+) NEC mouse ( P<0.05). In addition, serum interleukin (IL)-10 in PD-L1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1ß all increased by more than 25% (all P<0.05). Conclusion: PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes.

In vivo phenotyping of the microvasculature in necrotizing enterocolitis with multicontrast optical imaging.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal emergency in premature infants and is characterized by a dysfunctional gut microcirculation. Therefore, there is a dire need for in vivo methods to characterize NEC-induced changes in the structure and function of the gut microcirculation, that is, its vascular phenotype. Since in vivo gut imaging methods are often slow and employ a single-contrast mechanism, we developed a rapid multicontrast imaging technique and a novel analyses pipeline for phenotyping the gut microcirculation. METHODS: Using an experimental NEC model, we acquired in vivo images of the gut microvasculature and blood flow over a 5000 × 7000 µm2 field of view at 5 µm resolution via the following two endogenous contrast mechanisms: intrinsic optical signals and laser speckles. Next, we transformed intestinal images into rectilinear "flat maps," and delineated 1A/V gut microvessels and their perfusion territories as "intestinal vascular units" (IVUs). Employing IVUs, we quantified and visualized NEC-induced changes to the gut vascular phenotype. RESULTS: In vivo imaging required 60-100 s per animal. Relative to the healthy gut, NEC intestines showed a significant overall decrease (i.e. 64-72%) in perfusion, accompanied by vasoconstriction (i.e. 9-12%) and a reduction in perfusion entropy (19%)within sections of the vascular bed. CONCLUSIONS: Multicontrast imaging coupled with IVU-based in vivo vascular phenotyping is a powerful new tool for elucidating NEC pathogenesis.