Rat Model Of Necrotizing Enterocolitis Research Articles

Human \u03b2-defensin-3 reduces excessive autophagy in intestinal epithelial cells and in experimental necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of mortality in preterm newborns. Intestinal barrier dysfunction is one key event in NEC pathogenesis. Human β-defensin-3 (hBD3), one member of cationic host defence peptides, was reported to reduce the development of necrotizing enterocolitis in a neonatal rat model. And autophagy was induced in the intestine of human and animals with NEC. We hypothesized that regulation of autophagy might play a critical role in hBD3-mediated protection against NEC injury. Autophagy activity was evaluated both in intestinal epithelial cells and in NEC models. Newborn Sprague-Dawley rats were divided randomly into four groups: Control + NS, Control + rapamycin, NEC + NS, and NEC + hBD3. Body weight, histological score, survival time, enterocyte migration and mucosal barrier were recorded. Our results showed that hBD3 pretreatment could effectively inhibit autophagy activity in cultured IEC-6 and Caco2 enterocytes, and CXCR4 might be involved in hBD3-mediated autophagy suppression. Moreover, hBD3-induced inhibition of autophagy significantly promoted the intestinal epithelial cell migration by wound healing assay and transwell migration assay. In the rat model of NEC, hBD3 could noticeably reduce the expression of autophagy-activated proteins, down-regulate the expression of inflammatory mediators, and promote the mucosal integrity. Our data suggest an additional role of hBD3-mediated protection against intestinal mucosal injury: inhibition of over-activated autophagy in enterocytes.

Sumac (Rhus coriaria) for the prevention and treatment of necrotizing enterocolitis.

Leukotrienes, free oxygen radicals, tumor necrosis factor-alpha, and inflammatory mediators play major roles in the development of necrotizing enterocolitis (NEC). Rhus coriaria (RC: sumac) extracts may enhance cell viability by reinforcing defenses against free radical species in several progressive diseases as well as inflammatory diseases. The aim of the present study was to evaluate the effects of RC in a rat NEC model in terms of intestinal damage. Newborn pups were separated into three groups: control, NEC, and NEC treated with RC. Mortality and clinical sickness scores were evaluated. At the end of the study, ileum and proximal colon were obtained from all rats and histopathological and immunohistochemical studies were performed. In this study, the anti-inflammatory, antioxidant, immunomodulatory, and anti-apoptotic activities of RC were demonstrated in a rat NEC model, which suggests RC as a promising treatment option for preventing intestinal tissue damage. PRACTICAL APPLICATIONS: Free oxygen radicals, tumor necrosis factor-alpha, and inflammatory mediators play major roles in the development of NEC. Intestinal tissue damage is caused by necrosis and apoptosis as a result of intestinal inflammation and release of pro-inflammatory cytokines. Anti-inflammatory, antioxidant, immunomodulatory, and anti-apoptotic activities of RC are especially due to its phenolic compounds. In this study, the anti-inflammatory, antioxidant, immunomodulatory, and anti-apoptotic activities of RC were demonstrated in a rat NEC model. RC can suggest as a new treatment option for preventing intestinal injury.

EP2 Receptor Blockade Attenuates COX-2 Upregulation During Intestinal Inflammation.

High levels of PGE2 have been implicated in the pathogenesis of intestinal inflammatory disorders such as necrotizing enterocolitis (NEC) and peritonitis. However, PGE2 has a paradoxical effect: its low levels promote intestinal homeostasis, whereas high levels may contribute to pathology. These concentration-dependent effects are mediated by four receptors, EP1-EP4. In this study, we evaluate the effect of blockade of the low affinity pro-inflammatory receptors EP1 and EP2 on expression of COX-2, the rate-limiting enzyme in PGE2 biosynthesis, and on gut barrier permeability using cultured enterocytes and three different models of intestinal injury. PGE2 upregulated COX-2 in IEC-6 enterocytes, and this response was blocked by the EP2 antagonist PF-04418948, but not by the EP1 antagonist ONO-8711 or EP4 antagonist E7046. In the neonatal rat model of NEC, EP2 antagonist and low dose of COX-2 inhibitor Celecoxib, but not EP1 antagonist, reduced NEC pathology as well as COX-2 mRNA and protein expression. In the adult mouse endotoxemia and cecal ligation/puncture models, EP2, but not EP1 genetic deficiency decreased COX-2 expression in the intestine. Our results indicate that the EP2 receptor plays a critical role in the positive feedback regulation of intestinal COX-2 by its end-product PGE2 during inflammation and may be a novel therapeutic target in the treatment of NEC.

Recombinant human soluble thrombomodulin reduces the severity and incidence of necrotizing enterocolitis in a newborn rat model.

Necrotizing enterocolitis (NEC) remains the leading cause of death in preterm infants. Recombinant human soluble thrombomodulin (rTM) has been reported to have anti-inflammatory effects as well as antithrombogenic effects. The aim of this study was to evaluate the effect of rTM in a rat NEC model. NEC was induced by enteral feeding with hyperosmolar formula, gavage administration of lipopolysaccharide and asphyxia stress. Controls were fed by their mother ad libitum. In the treatment group, rTM was administered subcutaneously twice (once each on the first and second day). All animals surviving beyond 96h or that developed signs of distress were euthanized. The ileum was harvested for a histological evaluation and the measurement of the mRNA and protein expression. The rate of NEC-like intestinal injury in the treatment group (9/25, 36%) was significantly lower than in the NEC group (25/34, 73.5%). Tissue levels of TNF-α, IL-6 and HMGB1 were significantly elevated in the NEC group, whereas those in the treatment group were decreased to similar values as in the control group. Our experimental study showed that rTM is able to reduce the severity and incidence of NEC. It may be an alternative option for the treatment of NEC.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.

Milk Fat Globule Membrane Ameliorates Necrotizing Enterocolitis in Neonatal Rats and Suppresses Lipopolysaccharide-Induced Inflammatory Response in IEC-6 Enterocytes.

Evidence has provided support for the beneficial effects of milk fat globule membrane (MFGM) on inflammation in the intestinal tract. The objective of this study was to investigate the effects of MFGM on a rat model of necrotizing enterocolitis (NEC) and its potential mechanism of action. Sixty-two newborn Sprague Dawley rats were randomly divided into 4 experimental groups: Breast-fed normal, formula fed (FF), FF + 6 g/L MFGM, and FF + 12 g/L MFGM. The FF rats and the FF rats supplemented with MFGM were exposed to asphyxia/cold stress to induce NEC. Body weight, histological score, survival time, oxidative stress injury, enterocyte proliferation/apoptosis, and inflammatory response were assessed. Meanwhile, lipopolysaccharide (LPS)-stimulated IEC-6 enterocytes were used as a model to test the anti-inflammatory effects of MFGM. Supplementation with 12 g/L MFGM alleviated body weight loss, reduced the incidence of NEC, increased the survival rate, and attenuated the severity of bowel damage in the NEC rat model. Furthermore, 12 g/L MFGM administration inhibited the protein expression of toll-like receptor 4 (TLR4) in the animal model. In IEC-6 enterocytes, the upregulation of TLR4, myeloid differentiation primary response gene 88 (MyD88), phosphorylated nuclear factor-κB (NF-κB) p65 subunit, and the nuclear translocation of NF-κBp65 induced by LPS was partially inhibited by MFGM pretreatment. Our findings suggested that MFGM has beneficial effects on neonatal rats with NEC by suppressing inflammation via the TLR4/MyD88/NF-κB pathway.

Inhibition of miR-124 improves neonatal necrotizing enterocolitis via an MYPT1 and TLR9 signal regulation mechanism.

Necrotizing enterocolitis (NEC) was one of the main causes of morbidity and mortality in neonates. Our objective was to detect the mechanism of miR-124 in small bowel tissues of NEC. Hematoxylin and eosin (H&E) staining was used to detect the repair of the damaged tissues in rat NEC model. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the cell apoptosis level in intestinal tissue. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression level of miR-124, Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), myosin phosphatase target subunit 1 (MYPT1), and Toll-like receptor 9 (TLR9) in NEC tissues and IEC-6 cells. Luciferase reporter assay was used to verify whether ROCK1 is a direct target of miR-124. miR-124 was overexpressed in the NEC tissues, while ROCK1 and MYPT1 was downregulated in the NEC tissues. Inhibition of miR-124, suppressed the intestinal cell apoptosis and promoted the expression of ROCK1 and MYPT1. What is more, overexpression of miR-124 could inhibit the expression of ROCK1, TLR9, and MYPT1. Luciferase assay confirmedthat miR-124 can regulate the transcriptional activity of ROCK1 through binding its 3'-UTR region. miR-124 was a promoter of NEC, which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of TLR9 expression by targeting ROCK1.

Berberine reduces the occurrence of neonatal necrotizing enterocolitis by reducing the inflammatory response.

Necrotizing enterocolitis (NEC) is a life-threatening disease that occurs in premature infants. The aim of the present study was to investigate the effects of berberine, an isoquinoline alkaloid mainly used to treat digestive diseases, in a rat model of NEC. NEC models were established in newborn rats via inhalation of N2 for 90 sec every 4 h and oral administration of 4 mg/kg/day lipopolysaccharides on days 0 and 1. Berberine was administered via oral gavage. In the NEC model group, Toll-like receptor (TLR)4, nuclear factor NF-κB (NF-κB), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were upregulated. Symptoms of NEC in the berberine intervention group were significantly relieved, with a clear reduction in the incidence of NEC compared with the NEC group. TLR4, NF-κB, iNOS, TNF-α, IL-6 and IL-10 expression was decreased following berberine intervention. Furthermore, the expression of mucin-2 (MUC2) and RNA polymerase σ factor SigA (SIgA) were decreased in the NEC model group and increased following berberine intervention, when compared with the untreated group. It was also demonstrated that the incidence of NEC was reduced following berberine administration, possibly owing to changes in the inflammatory responses. The results of the current study support a potential therapeutic role of berberine for the treatment of NEC.

The protective effects of sesamol in a neonatal rat model of necrotizing enterocolitis

Background and aim: Necrotizing enterocolitis (NEC) is a severe gastrointestinal inflammatory disease associated with high rates of morbidity and mortality. Its pathophysiology includes hypoxic-ischemic injury that may be related to oxygen-derived free radical formation. Sesamol is considered to be an antioxidant and free radical scavenger with anti-inflammatory effects. The aim of this study is to investigate the effects of sesamol in a neonatal rat model of NEC.Materials and methods: The study included 1-day-old Wistar albino rat pups (n = 34) that were randomly divided into four groups: Group 1 (NEC), group 2 (NEC + intraperitoneal sesamol), group 3 (NEC + oral sesamol), and a control group. NEC was induced by exposure to hypoxia/reoxygenation, following cold stress and hyperosmolar enteral formula feeding. Sesamol 100 mg kg−1 dose−1 was administered intraperitoneally to group 2 and orally to group 3 for 3 days. On day 4 all rats were sacrificed. Histological injuries, the Bcl-2, caspase-3, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione-peroxidase (GSH-Px) activities were measured in intestinal samples.Results: The grade of intestinal damage, and Bcl-2 and caspase-3 levels in group 1 were significantly higher than in groups 2 and 3 and the control group, and intestinal damage was significantly more severe in group 1 than in groups 2 and 3. The MDA activity was significantly lower in groups 2 and 3 than in group 1 (112, 89, and 144 nmol mL−1, respectively). Groups 2 and 3 had significantly higher SOD and GSH-Px activities than group 1 (SOD: 1.75, 1.74, and 0.89 U mg−1; GSH-Px: 114, 121, and 110 nmol of NADPH min−1 mg−1, respectively).Conclusions: The present findings highlight that sesamol has beneficial effects on intestinal injury in a rat model of NEC through its antioxidant and anti-inflammatory properties.

The novel preventive effect of Daikenchuto (TJ-100), a Japanese herbal drug, against neonatal necrotizing enterocolitis in rats.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants. Daikenchuto, a Japanese herbal drug, has several effects on the digestive system, so we investigated its preventive effects in a rat model of NEC. NEC was induced in newborn rats via asphyxia (100% N2 for 90s; every 4h)+LPS (4mg/kg/day [administered orally on days 0 and 1]). The effects of Daikenchuto were evaluated in four groups (control: 0g/kg/day, I: 0.3g/kg/day, II: 0.6g/kg/day, and III: 1.0g/kg/day). Daikenchuto was administered into the stomach through a microcatheter. The incidence and severity of NEC were pathologically assessed using the NEC grade in accordance with Dovorak's previous report. Cell positivity for inflammatory cytokine (IL-6) was also evaluated. Daikenchuto reduced the incidence of NEC in control, Groups I, II, and III to 68.7, 30.0, 30.7, and 13.3%, respectively. High-dose Daikenchuto significantly improved the incidence of NEC, and the rate of IL-6 positive cells in group III was significantly lower than in the control group (p=0.04). We evaluated the effect of Daikenchuto against NEC and found that it reduced the incidence rate of NEC due to a decrease in the IL-6 production.

Probiotic Lactobacillus Species Strengthen Intestinal Barrier Function and Tight Junction Integrity in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a serious intestinal disease that occurs in newborn infants. It is associated with major morbidity and affects 5% of all infants admitted to neonatal intensive care units. Probiotics have variable efficacy in preventing necrotizing enterocolitis. Tight junctions (TJ) are protein complexes that maintain epithelial barrier integrity. We hypothesized that the probiotics Lactobacillus rhamnosus and Lactobacillus plantarum strengthen intestinal barrier function, promote TJ integrity, and protect against experimental NEC. Both an in vitro and an in vivo experimental model of NEC were studied. Cultured human intestinal Caco-2 cells were pretreated with L. rhamnosus and L. plantarum probiotics. TJ were then disrupted by EGTA calcium switch or LPS to mimic NEC in vitro. Trans-epithelial resistance (TER) and flux of fluorescein isothiocynate dextran was measured. TJ structure was evaluated by ZO-1 immunofluorescence. In vivo effects of ingested probiotics on intestinal injury and ZO-1 expression were assessed in a rat model of NEC infected with Cronobacter sakazakii (CS). Caco-2 cells treated with individual probiotics demonstrated higher TER and lower permeability compared to untreated cells (p<0.0001). ZO-1 immunofluorescence confirmed TJ stability in treated cells. Rat pups fed probiotics alone had more intestinal injury compared with controls (p=0.0106). Probiotics were protective against injury when given in combination with CS, with no difference in intestinal injury compared to controls (p=0.21). Increased permeability was observed in the probiotic and CS groups (p=0.03, p=0.05), but not in the probiotic plus CS group (p=0.79). Lactobacillus sp. strengthened intestinal barrier function and preserved TJ integrity in an in vitro experimental model of NEC. In vivo, probiotic bacteria were not beneficial when given alone, but were protective in the presence of CS in a rat model of NEC.

Unexpected Macrophage Activity Alters the Inflammatory Process in Necrotizing Enterocolitis

Objective: Macrophages play a role in clearing bacteria from the gut in the initial stages of necrotizing enterocolitis (NEC). Macrophage Inflammatory Protein-1α (MIP-1α), also known as CCL3, is a chemokine produced by macrophages that enhances the immune response. It acts as both a recruiter of immune cells and a coactivator of other macrophages. Interleukin-12 (IL-12) is a pro-inflammatory chemotactant produced by several types of phagocytic cells such as dendritic cells and neutrophils, but primarily by macrophages. We hypothesized that the pro-inflammatory state associated with NEC pathophysiology would induce increased expression of MIP-1α and IL-12 that would be detectable in serum and intestinal tissue. Methods: Timed pregnant Sprague-Dawley rats were randomized by litter. Controls were delivered vaginally and dam-fed. NEC pup groups were delivered 12 h prematurely via Cesarean section, formula fed, given a single oral dose of lipopolysaccharide, and subjected to intermittent cold and hypoxia as part of a proven NEC rat model protocol. Animals were sacrificed at 0, 12, 24, 48, 72, and 96 h of life and serum and intestinal tissue samples were collected. Samples were analysed via western blot using antibodies with affinity to MIP-1α and IL-12 respectively. Results: Serum and ileal levels of MIP-1 alpha were increased from 48 to 72 h in NEC animals when compared with controls. Serum and ileal IL-12 was downregulated in NEC groups at 12 and 24 h compared to controls. Conclusion: Macrophage function plays an important role in the first 48 h of NEC pathophysiology. Downregulation of IL-12 in the setting of increased MIP-1α expression may represent deranged or inhibited macrophage function in the context of NEC pathogenesis. Further work to elucidate the significance of these findings is warranted.

Intestinal trefoil factor increased the Bcl-2 level in a necrotizingenterocolitis neonate rat model.

The aim of this study was to investigate the therapeutic effect of intestinal trefoil factor (ITF) on necrotizing enterocolitis (NEC) by observing the pathological changes and detecting the protein level differences in Caspase-3, Bax, and Bcl-2 in an NEC neonate rat model. A Wistar rat model of NEC was established and 30 one-day-old neonate Wistar rats were randomly divided into three groups including a normal control (group A), NEC rats treated with 0.2 ml physiological saline through intraperitoneal (i.p.) injection (group B), and NEC rats treated with 0.2 mg ITF by i.p injection (group C). Compared with group B, there were statistically significant differences in Caspase-3, Bax, and Bcl-2 levels in groups A and C(P < 0.05). Moreover, there was a significant difference in the Bcl-2 level between groups A and B (P < 0.05). ITF alleviated injury of the intestinal tract in neonate rats with NEC and this mechanism was possibly related to a reduction in the expression of Caspase-3 and Bax and the increase in Bcl-2 expression.

The protective and anti-inflammatory effects of glucagon-like peptide-2 in an experimental rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease, that affects premature infants. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone and reduces the inflammation. We suspected that GLP-2 would have protective and anti-inflammatory effects in an experimental rat model of NEC. NEC was induced in newborn rats by enteral feeding with hyperosmolar formula, asphyxial stress and enteral administration of lipopolysaccharide (LPS). Rats were randomly divided into the following four groups: dam-fed, NEC, NEC+GLP-2(L) given 80μg/kg/day of GLP-2, and NEC+GLP-2(H) given 800μg/kg/day of GLP-2. GLP-2 was administered subcutaneously every 6h before stress. All animals surviving beyond 96h or any that developed signs of distress were euthanized. The clinical sickness score in the NEC+GLP-2(H) group was significantly lower than that in the NEC group. The NEC score and the survival rate in the NEC+GLP-2(H) group was significantly improved compared with those in the NEC and the NEC+GLP-2(L) groups. Villous height and crypt depth in both the GLP-2 treatment groups were significantly increased compared with those in the NEC group. There were no significant differences in the crypt cell proliferation indices among the groups. Ileal interstitial TNF-α and IL-6 level in the NEC+GLP-2(H) group was decreased to the same levels in the dam-fed group. High dose GLP-2 administration improved the incidence and survival rate for NEC. It also decreased mucosal inflammatory cytokine production. These results support a potential therapeutic role for GLP-2 in the treatment of NEC.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 μg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1β, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1β and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

Protective effects of dexpanthenol in an experimental model of necrotizing enterocolitis

Background/purposeIn pathogenesis of necrotizing enterocolitis (NEC), both oxidative stress and inflammation are considerable risk factors. The study was designed to evaluate whether administration of dexpanthenol (Dxp) is able to attenuate intestinal injury through the antioxidant and antiinflammatory mechanisms in a neonatal rat model of NEC. MethodsForty newborn pups divided into four groups were included in the study: control, control+Dxp, NEC, and NEC+Dxp. NEC was induced by hyperosmolar formula and additionally the pups were exposed to hypoxia/hyperoxia and cold stress. They were sacrificed on postnatal day four, and their intestinal tissues were analyzed biochemically and histopathologically. ResultsDxp caused a significant decrease in intestinal damage as determined by the histological score, villus height and number of goblet cells in NEC groups (p<0.0001). Tissue malondialdehyde, total oxidant status, and oxidative stress indexes levels were higher in the NEC group than in the control and control+Dxp groups (p<0.001). These values were reduced in the pups treated with Dxp (p≤0.004). Superoxide dismutase, glutathione peroxidase, and reduced glutathione activities were significantly reduced in the NEC group compared to the others (p<0.005). Treatment with Dxp significantly reduced elevations in tissue homogenate levels of tumor necrosis factor-α and interleukin-1β in the NEC+Dxp group (p=0.002 and p=0.01, respectively). ConclusionsDexpanthenol seems to have antiinflammatory and antioxidant properties. Prophylaxis with Dxp has a potential to reduce the severity of intestinal damage in NEC in the animals.

Oral relaxin maintains intestinal blood flow in a rat model of NEC

PurposeIntestinal vasoconstriction is a critical step in development of necrotizing enterocolitis (NEC). Relaxin (RLXN), a hormone found in breast milk but absent from formula, is a potent vasodilator. We hypothesized that relaxin-supplemented feeds with an NEC protocol would decrease NEC severity and increase intestinal blood flow. MethodsTimed-pregnant Sprague–Dawley rats were randomly assigned to CONTROL, NEC, NEC+1xRLXN, or NEC+All Feeds RLXN, and all but CONTROL underwent NEC protocol. NEC+1xRLXN and NEC+All Feeds RLXN groups were fed relaxin-supplemented formula with the last feed or every feed. At 48h of life, intestinal blood flow was measured at baseline and after application of 2.5% Delflex® solution. ResultsThe addition of relaxin to NEC group feeds (1x or All Feeds) improved the degree of ileal injury. Ileal blood flow was decreased in the NEC pups compared to the CONTROLS, but the addition of relaxin to one feed increased baseline ileal blood flow in the NEC group compared to NEC alone. Furthermore, the addition of relaxin to ALL feeds significantly increased baseline ileal blood flow. ConclusionPups who received relaxin with all feeds had substantially increased ileal perfusion compared to control pups. Our data suggest that relaxin supplementation maintains intestinal blood flow and results in less histologic NEC.

Recombinant Human Trefoil Factor 3 Ameliorates Bowel Injury: Its Anti-Inflammatory Effect on Experimental Necrotizing Enterocolitis

Aim. Recombinant human trefoil factor 3 (intestinal trefoil factor) has been suggested to be partially protective against necrotizing enterocolitis (NEC), but the mechanisms of this protection have not been defined. We investigated whether the protective effects of rhTFF3 are the result of an anti-inflammatory response. Methods. The rats were killed on day 4, the distal ileum was harvested for morphological studies and immunohistochemistry for NF-κB (p65), and the amounts of IL-1β, IL-6, and IL-10 in the intestinal tissue were measured using commercial ELISA assay kits. Results. In the neonatal NEC, IL-1β, IL-6, and IL-10 were significantly higher than in normal group. In normal group, IL-1β and IL-6 were significantly decreased, and the amount of IL-10 was markedly increased compared with NEC group. In the NEC model, immunohistochemical staining for NF-κB (p65) was demonstrated to be of a strong brown color and was distributed in the intestinal epithelium. Treatment with rhTFF3 significantly decreased the immunoreactivity of NF-κB (p65) in the NEC model. Conclusions. Intestinal inflammation was ameliorated after rhTFF3 was injected. rhTFF3 may protect against the intestinal injury of the neonatal rat NEC model by suppression of the inflammatory response.

L-FABP and I-FABP expression in newborn rats changes inversely in the model of necrotizing enterocolitis.

To determine the expression of hepatic L-FABP and intestinal I-FABP in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. Newborn Sprague-Dawley rats were divided into four groups: Control (C1) - exclusive breastfeeding at the first and sixth procedures (C6), NEC1 - fed formula milk and submitted to hypoxia and hypothermia at the first and sixth procedures (NEC6). The newborn pups were fed twice a day for three days, for a total of six procedures. Samples were collected for morphometric evaluation (body weight, liver weight, liver weight/body weight ratio, intestinal weight and intestinal/body weight ratio) and for immunohistochemical and Western blotting analysis. The values obtained were analyzed statistically, with the level of significance set at p<0.05. Morphometric measurements showed reduction of body and liver weights in the NEC group (p<0.05). Both immunohistochemistry and western blotting revealed that L-FABP expression in the liver was decreased and I-FABP expression in the ileum was increased in the NEC group (p<0.05). L-FABP and I-FABP expression changed inversely in the rat NEC model. These findings can contribute to a better diagnosis of NEC in human newborns.

FoxP3+ Regulatory T Cells Attenuate Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3+ regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.