Experimental Model Of Necrotizing Enterocolitis Research Articles

Modification of Experimental Model of Necrotizing Enterocolitis (NEC) in Rat Pups by Single Exposure to Hypothermia and Hypoxia and Impact of Mother's Milk on Incidence of Disease.

BACKGROUND Necrotizing enterocolitis (NEC) is a potentially life-threatening disease that affects the intestine of the neonate, causing necrosis and general inflammation. Treatment consists of feeding cessation and antibiotic therapy. In more severe cases, surgical intervention is necessary. Recently, different NEC models have been used to study the development of novel diagnostic and therapeutic methods. This work modified an experimental NEC model in rat pups by a single exposure of animals to NEC-causing factors and testing the impact of mother's milk on prevalence of the disease. MATERIAL AND METHODS Fifty rat pups were subjected to the NEC protocol, in which they were exposed to 100% nitrogen atmosphere and cold stress for set periods of time and formula feeding with exposure to mother's milk and artificial milk. Twenty-nine pups were used for control. After a set time of 72 h, bowel fragments were obtained and examined histologically by hematoxylin-eosin staining with a modified 3-grade scale. RESULTS Histological features of NEC were present in most of the samples (10/14) in the group exposed to 1 min of hypoxia (P=0.016), 10 min of cold stress (P=0.4) and formula feeding every 3 h with no mother's milk (P=0.001). In the group of 11 animals with the same stress conditions but fed mother's milk right after birth, only 1 sample of NEC was present. CONCLUSIONS The modified experimental NEC model based on formula feeding and single exposure to hypothermia and hypoxia was assessed statistically and histologically. In this model, mother's milk had a protective effect against necrotizing enterocolitis.

Recombinant IGF-1/BP3 protects against intestinal injury in a neonatal mouse NEC model.

Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.

Experimental necrotizing enterocolitis using oral lipopolysaccharide and protective role of breastmilk.

Necrotizing enterocolitis (NEC) is a life-threatening condition that afflicts neonates. Breastfeeding has demonstrated to play a protective role against it. By administering lipopolysaccharides (LPS) orally in newborn rats (NBR), we have developed an experimental model to induce NEC-like gut damage. Our aim was to assess the macroscopic and microscopic appearance of the gut, to evaluate the presence of NEC and study the role of breast milk (BM). NBR were divided into 3 groups: Group A (control, n=10) remained with the mother, group B (LPS, n=25) was isolated after birth, gavage-fed with special rat formula and oral LPS, then submitted to stress (hypoxia after gavage) and group c (BM, n=12) was breastfed once after birth, then isolated, and submitted to stress like group B. On day 4, NBR were sacrificed, and intestine was harvested and assessed. In the control group NEC was not present either macroscopically or histologically. Both groups submitted to stress (B and C) presented a global incidence of NEC of 73%. Most of group B developed histologic signs of NEC (85%) and group C showed a statistically lower incidence of NEC (50%, p=0.04), playing the BM a protective role against NEC (OR=0.19; 95% CI: 0.40-0.904). Our model showed a significant incidence of NEC in NBR (73%) with the same protective role of BM as in newborn humans, achieving a reliable and reproducible experimental NEC model. This will allow us to investigate new potential therapeutic targets for a devastating disease that currently lacks treatment.

Effect of Oral Chondroitin Sulfate Supplementation on Acute Brain Injury in a Murine Necrotizing Enterocolitis Model.

Necrotizing enterocolitis (NEC) is a devastating condition where inflammatory changes and necrosis in the gut results in activation of brain microglia and subsequent neurodevelopmental impairment. Chondroitin sulfate (CS) is a glycosaminoglycan in human breast milk that is absent in conventional formulas. We hypothesized that oral formula supplementation with CS during a murine model of experimental NEC would not only attenuate intestinal injury, but also brain injury. NEC was induced in mouse pups on postnatal days (PNDs) 5 to 8. Three conditions were studied: (1) breastfed controls, (2) NEC, and (3) NEC+enteral CS (formula+200 mg/kg/d of CS). Pups were euthanized on PND 9 or reunited with dams by the evening of PND 8. Intestinal segments were H&E stained, and immunohistochemistry was performed on brain tissue for Iba-1 to assess for microglial morphology and cortical changes. Neurodevelopmental assays were performed on mice reunited with foster dams on PND 9. Single-cell RNA-sequencing analysis was performed on human intestinal epithelial cells exposed to (1) nothing, (2) hydrogen peroxide (H 2 O 2 ) alone, or (3) H 2 O 2 + CS to look at the differential gene expression between groups. Groups were compared with ANOVA or Kruskal-Wallis tests as appropriate with p < 0.05 considered significant. Compared with NEC, mice treated with oral CS showed improved clinical outcomes, decreased intestinal injury, and attenuated microglial activation and deleterious cortical change. Mice with CS performed better on early neurodevelopmental assays when compared with NEC alone. Single-cell analysis of HIEC-6 cells demonstrated that CS treatment down regulated several inflammatory pathways including nuclear factor κB-suggesting an explanation for the improved Th17 intestinal cytokine profile. Oral CS supplementation improved both physiological, clinical, and developmental outcomes. These data suggest that CS is a safe compound for formula supplementation for the prevention of NEC.

Chondroitin sulfate supplementation improves clinical outcomes in a murine model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5-day-old pups. Six groups were studied (n = 9-15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well-established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p < 0.05 considered significant. CS supplementation in formula improved experimental NEC outcomes when compared to NEC alone. CS supplementation resulted in similar improvement in NEC in both the WT and eNOS KO mice. CS supplementation did not result in microbial changes when compared to NEC alone. Our data suggest that although CS supplementation improved outcomes in NEC, this protection is not conferred via the eNOS pathway or alteration of microbial diversity. CS therapy in NEC does improve the intestinal cytokine profile and further experiments will explore the mechanistic role of CS in altering immune pathways in this disease.

The role of human milk nutrients in preventing necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an intestinal disease that primarily impacts preterm infants. The pathophysiology of NEC involves a complex interplay of factors that result in a deleterious immune response, injury to the intestinal mucosa, and in its most severe form, irreversible intestinal necrosis. Treatments for NEC remain limited, but one of the most effective preventative strategies for NEC is the provision of breast milk feeds. In this review, we discuss mechanisms by which bioactive nutrients in breast milk impact neonatal intestinal physiology and the development of NEC. We also review experimental models of NEC that have been used to study the role of breast milk components in disease pathophysiology. These models are necessary to accelerate mechanistic research and improve outcomes for neonates with NEC.

Prenatal Heparin-Binding Epidermal Growth Factor–Like Growth Factor Administration Decreases Lung Inflammation in the Experimental Model of Necrotizing Enterocolitis

INTRODUCTION: Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death in premature infants. Postnatal enteral and prenatal intraperitoneal Heparin-Binding EGF-like Growth Factor (HB-EGF) administration has decreased the incidence and severity of intestinal injury in a neonatal rat model of NEC. The aim was to determine the effect of prenatally administered HB-EGF on lung injury in neonatal rats exposed to the experimental model of NEC. METHODS: Pregnant rats and pups were separated into 3 groups: control (natural birth, no NEC protocol, no HB-EGF), NEC (cesarean section, NEC protocol), and HB-EGF (prenatal HB-EGF administration, cesarean section, NEC protocol). Pregnant rats were given HB-EGF (800 μg/kg/dose) via intraperitoneal injection 2 hours before timed cesarean section. After delivery, the premature rat pups were exposed to the NEC model (hypercaloric feds, hypothermia, and hypoxia). Pups were sacrificed upon clinical signs of NEC; the intestines and lungs were hematoxylin and eosin stained for grading. RESULTS: The median alveolar wall thicknesses were significantly different between control (71,646 μm2) vs NEC (75,398 μm2; p < 0.0001) and HB-EGF (71,646 μm2) vs NEC (p < 0.0001); however, the control and HB-EGF groups were similar (p = 0.307; Fig. 1A). The median alveolar areas were significantly different between control (85.23 μm2) vs NEC (66.40 μm2; p < 0.0001) and HB-EGF (64.14 μm2) vs NEC (p < 0.0001); however, again, the control and HB-EGF were similar (p = 0.215; Fig. 1B).CONCLUSION: Our results support the prenatal HB-EGF role in preventing lung injury by means of preserving alveolar wall thickness and alveolar surface area in this animal model of NEC; therefore, maternal administration of HB-EGF before delivery in prematurity could protect lungs from NEC-related injury.

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 NECin 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- / - derivedIMwere 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.

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

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. 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 5μm 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. In vivo imaging required 60-100s 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. Multicontrast imaging coupled with IVU-based in vivo vascular phenotyping is a powerful new tool for elucidating NEC pathogenesis.

FOXM1 Promotes Intestinal Vascular Endothelial Cell Proliferation and Protects Neonatal Mice against Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a life‐threatening gastrointestinal illness affecting mostly premature infants characterized by intestinal inflammation and necrosis. While several factors such as abnormal intestinal colonization and an immature immune response have been shown to play a role in NEC, we previously found that defective intestinal microvascular development promotes NEC development. FOXM1 is a protein expressed in cells undergoing proliferation and a member of the Forkhead box family, a master regulator of cell cycle progression. FoxM1 has been shown to mediate endothelial cell proliferation under stress conditions. Here we hypothesize that promoting FOXM1 expression protects neonatal mice against intestinal injury in an experimental NEC model by promoting endothelial cell proliferation. To test our hypothesis, we first examined FOXM1 mRNA expression during NEC development in a neonatal mouse model. Second, we investigated the role of FOXM1 in NEC development using FOXM1 transgenic and knockout animals. Finally, we examined in vitro whether FOXM1 can protect endothelial cell proliferation under stress condition. We found that FOXM1 expression was significantly decreased in the intestine of pups exposed to the NEC model for 24 and 48 hours compared to dam‐fed littermate controls. Neonatal mice that lack of FOXM1 in endothelial cells showed higher mortality rate and more severe intestinal injury when exposed to the experimental NEC model. While their 3‐day survival remained unchanged, pups with endothelial‐cell specific overexpression of FOXM1 had decreased incidence of severe intestinal injury. Furthermore, when cultured in vitro, intestinal endothelial cells obtained from pups overexpressing FOXM1 showed increased proliferation, which was unaffected by VEGFR2 inhibition using VEGFR2 kinase inhibitor Ki‐8751. In conclusion, FOXM1 protects against neonatal NEC by promoting endothelial cell proliferation.

Nicotinamide riboside relieves the severity of experimental necrotizing enterocolitis by regulating endothelial function via eNOS deacetylation

BackgroundNicotinamide adenine dinucleotide (NAD+) is involved in regulating oxidative stress. Although NAD+ is associated with various health issues, its role in the intestinal microcirculation in necrotizing enterocolitis (NEC) remains to be confirmed. In the current study, we explored whether nicotinamide riboside (NR), a natural NAD + precursor, ameliorates the severity of NEC through endothelial nitric oxide synthase(eNOS) signaling. MethodsA mouse experimental NEC model was induced by formula gavage and hypoxia in full-term mouse pups. Intestinal endothelial cells (MIMECs) were isolated and subjected to stress using tumor necrosis factor (TNF)-α. NR was administered to assess the intestinal microcirculation and lipid peroxidation levels and to explore the involved signaling pathways. ResultsNAD + levels were reduced after induction of NEC stress, which was associated with intestinal injury. NR administration promoted NAD + levels, attenuated oxidative stress and relieved the symptoms of experimental NEC, which were relevant to increased intestinal microcirculatory perfusion through the sirtuin (SIRT) 1 pathway in experimental NEC mice. However, this improvement was not found in eNOS-knockout mice. Consistently, MIMECs exposed to TNFα showed decreased SIRT1 activity associated with increased eNOS acetylation, which could bring about endothelial dysfunction due to limited nitric oxide production. NR administration increased the NAD + content and repressed the production of reactive oxygen species (ROS) in MIMECs under TNFα stress. NR also promoted SIRT1 activity and accordingly suppressed the eNOS acetylation levels under TNFα stress. ConclusionThe current data indicate that NR administration improves the survival of experimental NEC mice via SIRT1-associated eNOS acetylation/deacetylation modulation, which is implicated in endothelial dysfunction. Although NR is commonly found in the human diet, it may also be a promising strategy for NEC treatment because of its pathogenic association with NEC.

Cytokine therapy in necrotizing enterocolitis: A promising treatment for preterm infants

Necrotizing enterocolitis (NEC) is an intestinal disorder that disproportionately affects premature infants and lacks in effective therapeutics. Mihi and colleagues1 demonstrated that the cytokine interleukin-22 promotes intestinal epithelial regeneration and reduces disease severity in an experimental model of NEC.

Astaxanthin Reduces the Severity of Intestinal Damage in a Neonatal Rat Model of Necrotizing Enterocolitis.

This study aimed to ascertain the effects of astaxanthin (ASX) in an experimental necrotizing enterocolitis (NEC) model using rat pups. Forty-two pups born from five Wistar albino rats were randomly divided into three groups as the control group, NEC + placebo (saline), and NEC + ASX. Pups in the NEC + ASX group were given 100 mg/kg/day oral ASX from day 1 to day 4 of the study. Saline of 2 mL/kg was given to the NEC + placebo group. Histopathological, immunohistochemical (caspase-3), and biochemical evaluations including the total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), glutathione (GSH), lipid hydroperoxide (LPO), 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products (AOPP), myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nuclear factor erythroid 2-related factor 2 (Nfr-2) activities were all performed. A better survival rate and weight gain were demonstrated in the NEC + ASX group (p < 0.05). In the histopathological evaluation, the severity of intestinal damage was significantly reduced in the NEC + ASX group, as well as decreased apoptosis (enzyme-linked immunosorbent assay [ELISA] for caspase-3; p = 0.001). The biochemical analyses of intestinal tissue TOS, oxidative stress index (OSI; TOS/TAS), IL-1β, LPO, 8-OHdG, AOPP, caspase-3 (p < 0.001 for all), and TNF-α and MPO (p = 0.001 for both parameters) levels were lower in the NEC + ASX group than in the NEC + placebo group. Nrf-2, TAS, GSH, and SOD levels were higher in the NEC + ASX group than in the NEC + placebo group (p = 0.001, 0.001, <0.001, and 0.01, respectively). ASX treatment has been shown to effectively reduce the severity of intestinal damage in NEC due to its antioxidant, anti-inflammatory, and antiapoptotic properties. · NEC causes extremely high morbidity and mortality, as well as many complications.. · We investigated the effectiveness of ASX in the experimental NEC model created in rat pups.. · First study examining the effect of ASX on the experimental NEC rat model..

Human placental-derived stem cell therapy ameliorates experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), a life-threatening intestinal disease, is becoming a larger proportionate cause of morbidity and mortality in premature infants. To date, therapeutic options remain elusive. Based on recent cell therapy studies, we investigated the effect of a human placental-derived stem cell (hPSC) therapy on intestinal damage in an experimental NEC rat pup model. NEC was induced in newborn Sprague-Dawley rat pups for 4 days via formula feeding, hypoxia, and LPS. NEC pups received intraperitoneal (ip) injections of either saline or hPSC (NEC-hPSC) at 32 and 56 h into NEC induction. At 4 days, intestinal macroscopic and histological damage, epithelial cell composition, and inflammatory marker expression of the ileum were assessed. Breastfed (BF) littermates were used as controls. NEC pups developed significant bowel dilation and fragility in the ileum. Further, NEC induced loss of normal villi-crypt morphology, disruption of epithelial proliferation and apoptosis, and loss of critical progenitor/stem cell and Paneth cell populations in the crypt. hPSC treatment improved macroscopic intestinal health with reduced ileal dilation and fragility. Histologically, hPSC administration had a significant reparative effect on the villi-crypt morphology and epithelium. In addition to a trend of decreased inflammatory marker expression, hPSC-NEC pups had increased epithelial proliferation and decreased apoptosis when compared with NEC littermates. Further, the intestinal stem cell and crypt niche that include Paneth cells, SOX9+ cells, and LGR5+ stem cells were restored with hPSC therapy. Together, these data demonstrate hPSC can promote epithelial healing of NEC intestinal damage.NEW & NOTEWORTHY These studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can be utilized to elucidate critical neonatal repair mechanisms to overcome NEC.

Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

Levels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Beneficial effects of butyrate in intestinal injury

PurposeButyrate is a short-chain fatty acid produced in the intestine. It is controversial whether butyrate is protective or destructive for the intestinal epithelium in the development of diseases like necrotizing enterocolitis (NEC), and its mechanism of action remains unclear. We aimed to determine the effect of butyrate on the intestinal epithelium by studying its effects on intestinal epithelial cells (IEC-18) exposed to injury and in vivo by investigating the effects on the intestine in an experimental model of NEC. MethodsA) In vitro study: Butyrate was given to normal IEC-18 to determine the dose triggering injury. Based on above results, low dose butyrate (1 mM) was given to H2O2-injured cells to determine its effect against inflammation. B) In vivo study: NEC was induced by hypoxia and gavage feeding between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Butyrate (150 mM) was administered by enema on P6 in NEC (n = 6). Distal ileum was harvested on P9. ResultsHigh dose (16 mM) butyrate upregulated inflammatory marker IL-6, while low dose butyrate protected cells from injury by reducing IL-6 expression. Similarly, compared with NEC alone, NEC mice who received butyrate had reduced intestinal damage, reduced IL-6 and NF-ĸB expression, and increased intestinal tight junction marker Claudin-7. ConclusionButyrate has opposite effects depending on the dose administered. Butyrate can protect cells from H2O2-induced injury and can in vivo protect the intestine from NEC. This beneficial effect is because of downregulation of inflammation and enhancement of intestinal barrier.

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Caveolin 1 is Associated with Upregulated Claudin 2 in Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

Therapeutic and preventative effects of ankaferd blood stopper in an experimental necrotizing enterocolitis model

Necrotizing enterocolitis (NEC) is a major neonatal health problem that especially affects preterm infants and causes severe morbidity and mortality. Although its pathogenesis is not fully understood, important risk factors include prematurity, oxidative stress, inflammation, and apoptosis. Ankaferd Blood Stopper® (ABS) has antioxidant, antiinflammatory, antimicrobial, antiapoptotic, and wound healing accelerant properties. In this study, we aimed to investigate whether treatment with ABS reduced the severity of NEC in rat pups in an experimental NEC model. Thirty-six newborn Wistar albino rat pups were randomly assigned to the control, NEC + saline, or NEC + ABS groups. NEC was induced by intraperitoneal injection of lipopolysaccharide, feeding with hyperosmolar enteral formula, and exposure to hypoxia/hyperoxia and cold stress. ABS was administered intraperitoneally to the pups in the NEC + ABS group daily starting on day 1 of the study at a dose of 2 ml/kg by diluting 2 ml with saline at a ratio of 1:3. All pups were sacrificed on day 4. The terminal ileum including the proximal colon was removed for histopathological and immunohistochemical examination and biochemical analysis. Macroscopic assessment and intestinal injury scores were lower in NEC + ABS group compared to the NEC + saline group (p < 0.05). Immunohistochemical evaluations of caspase-3, -8, and -9 revealed significantly reduced apoptosis in the NEC + ABS group compared to the NEC + saline group (p = 0.001). Total oxidant status, oxidative stress index, tumor necrosis factor α and interleukin-1β levels, and lipid, protein, and deoxyribonucleic acid oxidation products were significantly lower in the NEC + ABS group compared to NEC + saline group (p < 0.001 for all), while total antioxidant status, glutathione, and superoxide dismutase levels were higher in the NEC + ABS group (p < 0.001, p < 0.001, p = 0.01, respectively). ABS treatment has the potential to effectively reduce the severity of intestinal damage in NEC due to its antioxidant, antiinflammatory, and antiapoptotic properties. Therefore, NEC may be an alternative option for treatment.

Targeting the PXR-TLR4 signaling pathway to reduce intestinal inflammation in an experimental model of necrotizing enterocolitis.

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR−/− mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR−/− mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.