Experimental Necrotizing Enterocolitis Research Articles

Mucosal loss with increased expression of IL-6, IL-8, and COX-2 in a formula-feeding only neonatal rat model of necrotizing enterocolitis

IntroductionThe aim of our study is to establish a reliable neonatal rat model by formula feeding only for evaluation of early surgical intervention on the course of experimental necrotizing enterocolitis (NEC). Material and methodsNewborn Sprague–Dawley rats were divided into 50 breast-fed (group 1) and 38 formula fed (Similac/Esbilac, group 2) animals. The pups were sacrificed on the 4th, 5th, and 6th day of life and the terminal intestine examined for macroscopic and histologic changes as well as cytokine expression. ResultsThe histological mucosal damage was significantly higher of group 2 compared to group 1. The area of the vital mucosa of group 2 was significantly (58.57%, p<0.001) lower compared to group 1 (75.12%). The mRNA expression of the inflammatory cytokines IL-6, IL-8 and COX-2 was significantly 2-, 5- and 10-fold increased in group 2 compared to group 1. DiscussionFormula fed newborn rats displayed an inflammatory enterocolitis similar to human NEC. Our study demonstrates a significant loss of mucosa in animals with NEC having increased expression levels of IL-6, IL-8 and COX-2. Mucosal loss appears to be a distinct feature of experimental NEC and has to be correlated with the human disease.

Beneficial effects of Etanercept on experimental necrotizing enterocolitis

Tissue damage in necrotizing enterocolitis (NEC) of infants occurs as a result of an uncontrolled inflammatory response. The aim of this study was to investigate any potential anti-inflammatory effects that Etanercept may have on the inflammatory response in an experimental NEC model in newborn rats. Newborn pups were randomized into three groups immediately after birth (Control, NEC + Placebo and NEC + Etanercept). Pups in the NEC + Placebo and NEC + Etanercept groups were subjected to an NEC-inducing protocol (hypercarbia, hypothermia and hyperoxia) twice a day for 3 days. Pups in the NEC + Etanercept group were given an intraperitoneal injection of Etanercept. Rats were harvested for biochemical and histopathological examinations. The histopathological injury score of rats in the NEC + Placebo group was significantly higher compared to the NEC + Etanercept and Control groups (p < 0.05 for both comparisons). Tissue levels of tumor necrosis factor-α, interleukin-1β, and malondialdehyde were higher in the placebo group compared to the Etanercept group. Our results suggest that Etanercept attenuates intestinal tissue damage in NEC by reducing inflammation and blocking the production of free-oxygen radicals, while also reducing tissue levels of tumor necrosis factor-α and interleukin-1β.

Helicobacter hepaticusincreases intestinal injury in a rat model of necrotizing enterocolitis

Enterohepatic helicobacter species (EHS) infect the intestinal tract and biliary tree, triggering intestinal and hepatic disorders. Helicobacter hepaticus, the prototypic murine EHS, is also associated with inflammation. Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. The cause of NEC is not fully understood, but anomalies of bacterial colonization (dysbiosis) are thought to play an important role in disease onset. To evaluate the effect of H. hepaticus infection on the development of NEC, premature formula-fed rats were kept either in H. hepaticus-free conditions or colonized with H. hepaticus; both groups were exposed to asphyxia and cold stress. The incidence of NEC, expression of Toll-like receptors (TLRs), production of cytokines and mucins, and presence of autophagy regulators were evaluated at the site of injury. H. hepaticus infection increased the incidence of NEC from 39 to 71% and significantly increased levels of TLR4 receptor, expression of proinflammatory cytokines CXCL1, IL-1β, IL-12, and IL-23, and altered activation of autophagy. H. hepaticus induces inflammation and increases the incidence and severity of experimental NEC; this is consistent with observations in neonates of blooms of proinflammatory microbes just before the onset of NEC. Future studies using rodent NEC models should include testing for H. hepaticus infection. Further studies in neonates of early identification and/or diminution of proinflammatory microbes may be beneficial in decreasing the incidence of NEC.

Sa1824 Amniotic Fluid Attenuates Experimental Murine and Piglet Necrotizing Enterocolitis via Reduced TLR4 Signaling

G A A b st ra ct s Hypothesis: We hypothesized that LPS increases the intestinal Treg population in mice. Methods: Total spleen cells or purified splenic CD3+ T cells from C57BL/6 mice were stimulated in vitro with anti-CD3/28 and TGFβ to promote Treg generation. Parallel cultures were performed with additional LPS. Four days later, cells were stained for CD3, FoxP3 and helios and analyzed by flow cytometry. To analyze the effect of intestinal LPS on intestinal Treg population in vivo, germ-free C57BL/6 mice were administered ultrapure LPS by daily gastric gavage for 3 or 4 weeks. Mesenteric lymph node cells from LPS treated or untreated germ-free mice were analyzed for CD3, FoxP3 and helios expression. Last, peripheral T cells from conventional flora C57BL/6 WT, C57BL/10 WT and C57BL/10ScN (TLR4 deficient) mice kept under pathogen-free conditions were analyzed for Treg subpopulations. Results: The addition of LPS to anti-CD3/28 and TGF β stimulated, purified CD3 T cell cultures increased the ratio of helios negative Tregs to total Tregs from 43±19% to 89±6 % (N=2; p=0.02). Addition of LPS to total spleen cells stimulated with anti-CD3/28 in the presence of TGFβ increased the percentage of helios negative Tregs among total Tregs from 20±5 % to 40±2 % (N=3; p,0.01). In vivo, in a germ-free environment devoid of gut microbiota, orally administered LPS increased the total number of Treg cells 3.4-fold (p ,0.01; at least 2 experiments for each group). The percentage of helios negative Tregs (iTregs) also increased from 15±2% to 32±3% after 3 or 4 week of LPS treatment (p,0.001). Conversely, in animals populated with intestinal microbiota and maintained under pathogen-free conditions, the percentage of helios negative iTregs was decreased from 27±4% in C57BL/6 WT and 24±3% in C57BL/10 WT to 17±3% in C57BL/10 ScN TLR4 deficient mice (at least two experiments for each animal group; p,0.05 between C57BL/6 WT and TLR4 deficient as well as between C57BL/10 WT and TLR4 deficient). Conclusions: These findings support an important role for LPS-triggered TLR4 signaling in intestinal mucosal and peripheral Treg development.

The role of the intestinal microbiota in the pathogenesis of necrotizing enterocolitis

Development of necrotizing enterocolitis (NEC) requires a susceptible host, typically a premature infant or an infant with congenital heart disease, enteral feedings and bacterial colonization. Although there is little doubt that microbes are critically involved in the pathogenesis of NEC, the identity of specific causative pathogens remains elusive. Unlike established normal adult gut microbiota, which is quite complex, uniform, and stable, early postnatal bacterial populations are simple, diverse, and fluid. These properties complicate studies aimed at elucidating characteristics of the gut microbiome that may play a role in the pathogenesis of NEC. A broad variety of bacterial, viral, and fungal species have been implicated in both clinical and experimental NEC. Frequently, however, the same species have also been found in physiologically matched healthy individuals. Clustered outbreaks of NEC, in which the same strain of a suspected pathogen is detected in several patients suggest, but do not prove, a causative relationship between the specific pathogen and the disease. Studies in Cronobacter sakazakii, the best characterized NEC pathogen, have demonstrated that virulence is not a property of a bacterial species as a whole, but rather a characteristic of certain strains, which may explain why the same species can be pathogenic or non-pathogenic. The fact that a given microbe may be innocuous in a full-term, yet pathogenic in a pre-term infant has led to the idea of opportunistic pathogens in NEC. Progress in understanding the infectious nature of NEC may require identifying specific pathogenic strains and unambiguously establishing their virulence in animal models.

Protective Function of FoxP3+ Regulatory T Cells in 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 Tregs are significantly reduced in the ileum of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigates whether Treg development is delayed in newborn rat pups and whether Tregs play a protective role in experimental NEC induction. Utilizing a 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. There were few Tregs in the mesenteric lymph nodes (MLN) and ileums of newborn rats, which were further reduced after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to over 85%, and intestinal damage based on preservation of cellular composition and reduction of NEC score from maximum of 3 to 1 in severity. The Tregs suppressed the upregulation of costimulatory molecule CD80 on dendritic cells (DCs) and regulated the trafficking of effector cells in the MLN. There is a developmental delay in Tregs which contributes to the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the induction of NEC by regulating DCs and modulating effector cell trafficking. Treg immunotherapy has an enormous therapeutic potential in the prevention and treatment of NEC.

The hepatic bile acid transporters Ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.

Heat Shock Pretreatment Reduces Intestinal Injury in a Neonatal Rat Model of Early Necrotizing Enterocolitis

Background: Increased pro-inflammatory cytokines are suggested in the pathogenesis of necrotizing enterocolitis (NEC). The transcription factor, nuclear factor-ĸB (NF-ĸB), is a central regulator of inflammatory and immune responses, and recent rodent NEC models have shown that NF-ĸB may have a critical role in the disease processes that underlie NEC. Heat shock proteins have important functions in response to stress-related events in a variety of systems, including digestive organs. Objectives: We investigated whether heat shock pretreatment protects intestinal epithelial damage in the early NEC rat model. Methods: We generated human NEC-like lesions in neonatal rat ileum by administering oral endotoxin (10 mg/kg), intermittent 8% hypoxia, and hypertonic formula. Heat shock was administered by raising the chamber temperature to 42°C for 20 min, 3 h prior to endotoxin ingestion. Results: Heat shock pretreatment increased the expression of HSP70 in the ileal tissue and attenuated histological severity of early experimental NEC. NF-ĸB was activated in the ileal tissue of the NEC group and this activation was attenuated by heat shock pretreatment, which was determined by electrophoretic mobility shift assay and Western blot analysis of p50 in subcellular fractionated samples. Conclusions: Heat shock pretreatment reduced the incidence and severity of early experimental NEC in rats. A possible mechanism underlying this protective effect includes inhibition of NF-ĸB activation through increased HSP70 expression.

Heparin-Binding Epidermal Growth Factor-Like Growth Factor and Mesenchymal Stem Cells Act Synergistically to Prevent Experimental Necrotizing Enterocolitis

We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo. In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined. Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05). Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.

Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)--a devastating inflammatory disease of the premature intestine--upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ-deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.

Direct peritoneal resuscitation augments ileal blood flow in necrotizing enterocolitis via a novel mechanism

PurposeEndothelin-1, prostaglandins (PGs), and nitric oxide (NO) have been implicated in the intestinal microvascular dysfunction of necrotizing enterocolitis (NEC). We hypothesized that direct peritoneal resuscitation (DPR) dilates the intestinal microvasculature and improves blood flow independent of these mechanisms. MethodsRat pups were assigned by litter to experimental NEC or CONTROL groups. Laser Doppler flowmetry evaluation of intestinal microvascular blood flow was studied at baseline, with mediator blockade (endothelin-A receptor, endothelin-B receptor, PG synthesis, or NO synthase) and with DPR. Repeated-measures analysis of variance test was applied with Tukey-Kramer honestly significant difference test (P < .05). ResultsAt baseline, NEC animals demonstrated significantly decreased ileal blood flow as compared with CONTROLs (P < .05). Endothelin-A receptor and PG inhibition increased flow in the intestinal microvasculature, but this was significantly augmented by the addition of DPR (P < .05). Blockade of NO synthase decreased intestinal blood flow, which was overcome with addition of DPR (P < .05). ConclusionIleal blood flow was significantly reduced in NEC animals as compared with CONTROLs. The addition of DPR to the peritoneum increased ileal blood flow significantly in all groups in spite of blockade of these known vasoactive mechanisms. Direct peritoneal resuscitation may be a novel strategy to improve intestinal blood flow in NEC.

Transactivation of EGFR by LPS Induces COX-2 Expression in Enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.

Bifidobacterium bifidum in a rat model of necrotizing enterocolitis: antimicrobial peptide and protein responses

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear. We report that like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF+BIF). All groups were exposed to asphyxia and cold stress. The expression of lysozyme, secretory phospholipase A2, pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF (NEC) group, compared to the DF and FF+BIF groups where disease was attenuated. We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.

Active Transport of Bile Acids Decreases Mucin 2 in Neonatal Ileum: Implications for Development of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants, but its etiology remains unclear. We have previously shown that mucin 2 (Muc2) positive goblet cells are significantly decreased in NEC. We have also shown that ileal bile acids (BAs) are significantly increased during the development of this disease. Because BAs can affect mucins, we hypothesized that elevated ileal BAs contribute to decreased Muc2 in experimental NEC. The role of Muc2 in NEC was evaluated in Winnie +/+ mice, a strain that produces aberrant Muc2. Muc2 and trefoil factor 3 (Tff3) were assessed in neonatal rats subjected to the NEC protocol when bile acids were removed, and in ileal explants from newborn and older rats cultured with and without BAs. Further, the role of active transport of BAs was determined using neonatal rats given the apical sodium dependent bile acid transporter (Asbt) inhibitor SC-435 and in neonatal Asbt knockout mice subjected to the NEC protocol. Mice with aberrant Muc2 had significantly greater incidence and severity of NEC. Using both in vivo and ex vivo techniques, we determined that BAs decrease Muc2 positive cells in neonatal but not older ileum. However, Tff3 positive cells are not decreased by BAs. In addition, active transport of BAs is required for BAs to decrease Muc2 in immature ileum. These data show that functional Muc2 plays a critical role in the prevention of NEC and BAs can potentiate the decreased Muc2 in disease development. Further, BAs have a more profound effect on Muc2 in immature versus older ileum, which may explain at least in part why NEC occurs almost exclusively in premature infants.

Role of Neutrophils and Macrophages in the Pathogenesis of Necrotizing Enterocolitis Caused by Cronobacter sakazakii

Cronobacter sakazakii (CS) is a highly virulent gram-negative opportunistic pathogen that has been implicated in clinical outbreaks of necrotizing enterocolitis (NEC). The role of mucosal immune cells in CS infection is not well understood. In this study, we sought to elucidate the role of neutrophils (polymorphonuclear leukocytes; PMNs) and macrophages in the pathogenesis of NEC induced by CS using a novel newborn mouse model. PMNs and macrophages were depleted in newborn mice using Gr-1 antibody and carrageenan, respectively, and then infected with 10(3) CFU of CS. The development of NEC in these mice was assessed by a pathologist based on the morphologic changes in the intestine. Cytokine production was determined in the serum and intestinal homogenates of infected mice by enzyme-linked immunosorbent assay (ELISA). Inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was determined by flow cytometry and Greiss method, respectively. Depletion of PMNs and macrophages in newborn mice led to increased recruitment of dendritic cells (DCs) in the intestine compared with wild-type mice upon infection with CS. PMN- and macrophage-depleted mice showed increased bacterial load, production of pro-inflammatory cytokines, iNOS expression, and NO production in the intestines in comparison to wild-type mice fed with CS. In addition, depletion of PMNs and macrophages prior to infection in mice resulted in severe inflammation, villus destruction, and enhanced enterocyte apoptosis in the intestines compared with CS-infected wild-type mice. Our data suggest that depletion of PMNs and macrophages from the lamina propria (LP) exacerbates experimental NEC, indicating that both of these immunocytes play an important role in the clearance of CS during the initial stages of infection. The increased mucosal cytokine response and NO production in the absence of these immunocytes may be responsible for the observed increase in mucosal injury. Understanding how CS manipulates these cells, employing novel mouse model of NEC reported in this study, will provide significant insights for the development of novel therapeutic and preventive strategies to combat NEC.

Efectos de la administración de glucocorticoides prenatales en el desarrollo de la enterocolitis necrosante en neonatos de rata wistar

Necrotizing enterocolitis (NEC) is a frequent problem in preterm infants. Prenatal treatment with steroids proved to be effective for lung maturation and it is thought to have a protective effect on the immature bowel. To study the effects of prenatal treatment with steroids at the onset, clinical course and histological pattern of NEC in an animal model. Pregnant rats received treatment with intraperitoneal hydrocortisone (5 mg/kg) 24 and 48 hrs prior to the expected date of delivery (group S). Control pregnant rats were injected with normal saline, at the same timing (group P). After term delivery by cesarean section, both groups were kept in identical conditions in a neonatal incubator at 35ºC, away from their mothers to prevent any exposure to breast milk. Pups were fed every three hours with neonatal formula via an orogastric tube. To further increase the susceptibility to NEC, pups were exposed to hypoxia followed by hypothermia three times a day for 72 hrs (H-H) or until development of clinical signs of NEC. At that point, each animal was anesthetized and euthanized. The intestine was fixed for histological analysis. Those animals which died before 72 hours were excluded to prevent false positive results in the histopathological exam. The clinical signs of NEC include oral intolerance, gastric residuals, respiratory distress, abdominal distension, wall erythema and hematochezia; 60% of animals in group P (n= 16) presented with at least one clinical sign, vs. 40% of pups in group S (n= 15). The onset of clinical signs and clinical course in group S was more benign than in group P. Mortality rate was 40% for pups in group P vs. 20% for group S (NS). Histological analysis indicated that 80% of the animals from group P showed signs of NEC, of which 50% reached grade 3-4 (maximum score of damage), whereas only 40% of the animals in group S presented with signs of NEC (p<0.05), all were of grade 0-1 (minimum histological damage). Prenatal treatment with steroids was effective for amelioration of the onset and clinical presentation in this model of experimental NEC.

Effects of intestinal trefoil factor on PI3K and caspase-3/9 in newborn rats with necrotizing enterocolitis

PI3K/Akt signal pathway was blocked by Wortmannin, a specific inhibitor of phosphoinositide-3 kinase (PI3K), and the effects of intestinal trefoil factor (ITF) on PI3K and caspase-3/9 in newborn rats with necrotizing enterocolitis (NEC) were investigated to explore the protective mechanism of ITF against NEC. Experimental NEC was induced by exposure to hypoxia for 60 sec followed by cold stress at 4°C for 10 min. A total of 50 One-day-old Wistar rats were randomly divided into five groups : group A, NEC+NS; group B, NEC+ITF; group C,NEC+ wortmannin; group D ,NEC+ wortmannin + ITF; group E , Normal control. The animals were euthanized at development of NEC, and at 96 h the intestinal tissue was harvested and examined for histological changes of NEC, and then the PI3K content and Caspase-3/9 activity were detected using ELISA and spectrophotometry, respectively. The PI3K content (pg/ml) in group A was slightly higher than group E (P 0.05), but the PI3K content (pg/ml) in group B was significantly higher than the remaining groups (P<0.01), and the PI3K content (pg/ml) in group C was significantly lower compared with group E (P<0.01). Compared with group B and E, Caspase-3/9 activity was significantly higher in group A than in group B and E (P<0.01), but all lower than that in group C. So we concluded that ITF could activate the PI3K/Akt pathway to down-regulate Caspase-3/9 activity, and to protect against intestinal damage of NEC rats. Key words: Intestinal trefoil factor, necrotizing enterocolitis, PI3K/Akt signal transduction, Caspase-9, Caspase-3.

Platelet-activating factor induces TLR4 expression in intestinal epithelial cells: implication for the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in neonatal intensive care units, however its pathogenesis is not completely understood. We have previously shown that platelet activating factor (PAF), bacteria and TLR4 are all important factors in the development of NEC. Given that Toll-like receptors (TLRs) are expressed at low levels in enterocytes of the mature gastrointestinal tract, but were shown to be aberrantly over-expressed in enterocytes in experimental NEC, we examined the regulation of TLR4 expression and signaling by PAF in intestinal epithelial cells using human and mouse in vitro cell lines, and the ex vivo rat intestinal loop model. In intestinal epithelial cell (IEC) lines, PAF stimulation yielded upregulation of both TLR4 mRNA and protein expression and led to increased IL-8 secretion following stimulation with LPS (in an otherwise LPS minimally responsive cell line). PAF stimulation resulted in increased human TLR4 promoter activation in a dose dependent manner. Western blotting and immunohistochemical analysis showed PAF induced STAT3 phosphorylation and nuclear translocation in IEC, and PAF-induced TLR4 expression was inhibited by STAT3 and NFκB Inhibitors. Our findings provide evidence for a mechanism by which PAF augments inflammation in the intestinal epithelium through abnormal TLR4 upregulation, thereby contributing to the intestinal injury of NEC.

The Protective Role of Intestinal Alkaline Phosphatase in Necrotizing Enterocolitis

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