Necrotizing Enterocolitis Model Research Articles

Intestinal Alkaline Phosphatase Prevents the Systemic Inflammatory Response Associated With Necrotizing Enterocolitis

Introduction: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates with an incidence of 0.5-2.4 cases per 1000 live births. the mortality rate is between 10-50% and has not improved significantly in the past two decades. Neonates affected by NEC develop a septic injury that is associated with an increased risk of chronic lung disease and neurological impairment due to intraventricular bleeding. Intestinal alkaline phosphatase (IAP) is an endogenous protein that has been shown to inactivate the endotoxin lipopolysaccharide (LPS), and has recently been used successfully as an adjunct to treat sepsis in adult patients. Our hypothesis is that systemic, exogenous IAP will mitigate the inflammatory response as measured by serum levels of pro-inflammatory cytokines in a rat model of NEC. Methods: Newborn Sprague Dawley rats were divided into groups. Control pups were breast-fed. in the remaining pups, NEC was induced by feeding formula containing LPS and exposure to intermittent hypoxia. NEC pups were also given intra-peritoneal injections of 4 or 40 glyceine units (U) of IAP or placebo twice daily. Rats were sacrificed on days 1-3. Intestine was harvested to grade intestinal injury and serum was collected for cytokine analysis using a bioplex assay as well as measurement of alkaline phosphatase (AP) activity. Results: Systemic IAP administration had no effect on increasing intestinal AP activity or preventing NEC as measured by histology. However, systemic IAP administration significantly increased serum AP activity in a dose and time dependent fashion. the serum AP activity for the 40 U IAP treated groups on days of life 1-3 were 0.285, 0.344, and 0.496 U/mL respectively, while the controls and NEC groups had serum activities ranging from 0.007-0.0489 U/mL on days 1-3 (p<0.05 for all). We also found that the pro-inflammatory cytokines (measured in pg/mL) TNF-a, IL-6, and IL-1b were significantly increased in NEC rats as compared to controls on days 2 and 3 (TNF-a: 1390 vs. 25 on DOL 2 and 2896 vs. 1332 on DOL 3; IL-1b: 3295 vs. 66 DOL 2 and 7010 vs. 2394 on DOL3; IL-6: 1718 vs. 21 on DOL2; (p<0.05 for all). Importantly, we found that treatment with 40 U of systemic IAP not only increased serum AP activity, but also decreased these pro-inflammatory cytokines back to near-control levels. (TNF-a: 26 on DOL 2 and 1018 on DOL3; IL-1b 183 on DOL 2 and 1339 on DOL 3; IL-6: 381 on DOL 3; (p<0.05 for all). Figure 1 shows serum TNF-a levels in control, NEC, and 40 U IAP treated pups. Conclusions: Systemic IAP administration appears effective in preventing the systemic inflammatory response associated with NEC, and may prove to be a valuable adjunctive treatment for NEC.

Bacterial translocation and intestinal injury in experimental necrotizing enterocolitis model

To study the occurrence of bacterial translocation and to assess the impact of breastfeeding on bacterial translocation in the animal model of necrotizing enterocolitis. A total of 20 neonate Sprague-Dawley rats were enrolled in the study. Rats were randomly allocated into either control or study group just after birth. Ten newborn rats in the control group were left with their mother to be breast-fed. In contrary, necrotizing enterocolitis group consisted of neonates that were separated from their mothers, housed in an incubator and were gavaged with a special rodent formula three times daily. Survival rates, weight changes, and morphologic scoring obtained after microscopic evaluation were determined as microbiologic evaluation criteria. All the rats in the control group survived, while 1 (10 %) rat died in the necrotizing enterocolitis group. Mortality rates of the two groups were similar. All the formula-fed animals in the necrotizing enterocolitis group had significant weight loss compared to the breast milk-fed rats in the control group (p<0.05). A total of 7 (70 %) and 2 (20 %) E. coli growths were identified in the bowel lumen, liver, and spleen of necrotizing enterocolitis and control groups, respectively. This difference was statistically significant. In peritoneal smear cultures, a total of 3 (30 %) growths were detected in the necrotizing enterocolitis group and 1 (10 %) growth in the control group. As the result of a disturbance in the intestinal flora and impairment of the intestinal barrier in necrotizing enterocolitis, microrganisms in the bowel pass through the intestinal barrier and reach the liver and the spleen via the hematogenous route. This condition is closely related to the impairment of physiological and functional features of the intestinal barrier and is independent from the degree of intestinal injury. Bacterial translocation should be remembered in cases suspected of necrotizing enterocolitis, and a rapid and effective treatment algorithm should be applied in such circumstances (Tab. 3, Fig. 3, Ref. 21). Full Text in PDF www.elis.sk.

Lactobacillus reuteristrains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of mortality and morbidity in the premature infant. Premature infants have a delay in intestinal colonization by commensal bacteria and colonization with potentially pathogenic organisms. Lactobacillus reuteri is a probiotic that inhibits enteric infections, modulates the immune system, and may be beneficial to prevent NEC. In previous studies, L. reuteri strains DSM 17938 and ATCC PTA 4659 differentially modulated inflammation in vitro; however, the strains had equivalent anti-inflammatory responses in LPS feeding-induced ileitis in neonatal rats in vivo. The impact of these two strains in the prevention of NEC has not been previously investigated. NEC was induced in newborn rats by orogastric formula feeding and exposure to hypoxia. L. reuteri was added to the formula to prevent NEC. NEC score, Toll-like receptor (TLR)-signaling genes, phospho-IκB activity, and cytokine levels in the intestine were examined. Both strains significantly increased survival rate and decreased the incidence and severity of NEC, with optimal effects from DSM 17938. In response to probiotic, mRNA expression of IL-6, TNF-α, TLR4, and NF-κB was significantly downregulated, while mRNA levels of anti-inflammatory cytokine IL-10 were significantly upregulated. In parallel, L. reuteri treatment led to decrease intestinal protein levels of TLR4 and cytokine levels of TNF-α and IL-1β in newborn rats with NEC. Both strains significantly inhibited not only intestinal LPS-induced phospho-IκB activity in an ex vivo study but also decreased the levels of phospho-IκB in the intestines of NEC rat model. Cow milk formula feeding produced a similar but milder proinflammatory profile in the intestine that was also ameliorated by 17938. Our studies demonstrate that each of the two L. reuteri strains has potential therapeutic value in our NEC model and in enteritis associated with cow milk feeding. These results support the concept that L. reuteri may represent a valuable treatment to prevent NEC.

Probiotic Bacteria Induce Maturation of Intestinal Claudin 3 Expression and Barrier Function

An immature intestinal epithelial barrier may predispose infants and children to many intestinal inflammatory diseases, such as infectious enteritis, inflammatory bowel disease, and necrotizing enterocolitis. Understanding the factors that regulate gut barrier maturation may yield insight into strategies to prevent these intestinal diseases. The claudin family of tight junction proteins plays an important role in regulating epithelial paracellular permeability. Previous reports demonstrate that rodent intestinal barrier function matures during the first 3 weeks of life. We show that murine paracellular permeability markedly decreases during postnatal maturation, with the most significant change occurring between 2 and 3 weeks. Here we report for the first time that commensal bacterial colonization induces intestinal barrier function maturation by promoting claudin 3 expression. Neonatal mice raised on antibiotics or lacking the toll-like receptor adaptor protein MyD88 exhibit impaired barrier function and decreased claudin 3 expression. Furthermore, enteral administration of either live or heat-killed preparations of the probiotic Lactobacillus rhamnosus GG accelerates intestinal barrier maturation and induces claudin 3 expression. However, live Lactobacillus rhamnosus GG increases mortality. Taken together, these results support a vital role for intestinal flora in the maturation of intestinal barrier function. Probiotics may prevent intestinal inflammatory diseases by regulating intestinal tight junction protein expression and barrier function. The use of heat-killed probiotics may provide therapeutic benefit while minimizing adverse effects.

Mesenchymal Stem Cell Therapy in Necrotizing Enterocolitis: A Rat Study

We evaluated the potential therapeutic use of exogenous human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in an experimental rat model of necrotizing enterocolitis (NEC). Thirty-six newborn Sprague-Dawley rats were randomly divided into three groups: NEC, NEC + hBM-MSC, and a control (control and control + hBM-MSC). NEC was induced by enteral formula feeding, exposure to hypoxia-hyperoxia, and cold stress. After NEC was induced, iron-labeled hBM-MSCs were administered by intraperitoneal injection. All pups were killed on the fourth day following injection, and the terminal ileum was excised for a histopathological and immunohistochemical evaluation. The pups in the NEC + hBM-MSC group showed significant weight gains and improvements in their clinical sickness scores (p < 0.01). Bowel damage severity observed in the histopathological evaluation was significantly lower in the NEC + hBM-MSC group than that in the NEC group (p = 0.012). The number of MSCs homing to the bowel was significantly higher in the NEC + hBM-MSC group than that in the control + hBM-MSC group. In conclusion, this is the first study that has evaluated the effectiveness of hBM-MSCs in a neonatal rat NEC model. MSCs reduced histopathological damage significantly.

The Effects of Insulin Like Growth Factor-1 and Erithropoetin on Apoptosis and Telomerase Activity in an Experimental Necrotizing Enterocolitis Model

Background and Aims: Apoptosis, that occurs after hypoxia/reoxygenation (H/R), has an important role in the pathogenesis of necrotizing enterocolitis (NEC) which is a major cause of morbidity and mortality in premature infants. Telomerase activity, showing the regenaration capacity, may also be important in the recovery process. We aimed to see the effects of insulin-like growth factor-1 (IGF) and erithropoetin (EPO) on apoptosis and telomerase activity in a H/R model.

Establishment and evaluation of three necrotizing enterocolitis models in premature rats

Necrotizing enterocolitis (NEC) is a gastrointestinal disease that usually affects premature infants and has high morbidity and mortality rates. Reliable animal models aid further study of the etiological factors, pathogenesis, prevention and treatment of NEC. The present study aimed to establish NEC models in premature rats using three common methods, and to determine the optimal model establishment method. The study consisted of six groups; in group A, rats were raised with simulated milk and subjected to inhalation of 100% nitrogen gas (hypoxia) for 90 sec, followed by exposure to cold ambient conditions (4˚C) for 10 min twice daily for 3 days. In group B, rats were exposed to 100% nitrogen gas for 5 min and 100% oxygen for 5 min twice daily for 3 days. Group C rats were intraperitoneally administered 5 mg/kg lipopolysaccharide. Group D and E rats did not receive any intervention. Group F rats were intraperitoneally administered 1 ml/kg physiological saline. Groups D-F served as the control groups corresponding to groups A-C, respectively. Following hematoxylin and eosin staining, intestinal tract, liver, lung and kidney tissues were observed under optical microscopy and were scored. Successful NEC induction was measured by a score of ≥2. Rats from groups A-C exhibited reduced movement, abdominal distention, diarrhea, intestinal tract expansion, and congestion to varying degrees. The pathological scores of intestinal injury in groups A-F were 3.13±0.64, 1.40±0.52, 2.00±0.42, 0.30±0.48, 0.30±0.48, and 0.40±0.52 points, respectively. Significant differences were found between the model groups and their corresponding control groups (p<0.01). Among the model groups, the histological score of group A was higher than that of groups B (p<0.01) and C (p<0.05). The morbidity rate of NEC in groups A-C was 75, 20 and 50%, respectively. There was no morbidity in groups D-F. Compared with groups A and B, injury to the liver, kidney and lung was more severe in group C. Similar symptoms were not observed in groups D-F. Compared with methods of simple hypoxia-reoxygenation or intraperitoneal administration of lipopolysaccharide, the combination of artificial feeding and hypoxia plus cold stimulation most resembles the pathological causes of neonatal NEC. This method resulted in high morbidity, reproducibility and specificity, and was therefore considered an ideal model for establishing NEC.

Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22-28 wk GA. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation, and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 overactivation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here, we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage) versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3-4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 overactivation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC.

Protective effects of caffeic acid phenethyl ester (CAPE) on intestinal damage in necrotizing enterocolitis

To determine the preventative effect of caffeic acid phenethyl ester (CAPE) in necrotizing enterocolitis (NEC) in an experimental rat model of NEC. Thirty newborn Sprague-Dawley rats were randomly divided into three groups; as NEC, NEC + CAPE and control. NEC was induced by enteral formula feeding, subjected to hypoxia-hyperoxia and cold stress. Pups in the NEC + CAPE group were treated with CAPE at a dose of 30 mg/kg daily by intraperitoneal route from the first day to the end of the study. All pups were executed on the fourth day. Proximal colon and ileum were allocated for histopathologic and biochemical evaluation, including xanthine oxidase (XO), total antioxidant status (TAS), total oxidant status (TOS), malonaldehyde (MDA) and myeloperoxidase (MPO) activities. The pups in the NEC + CAPE group had better histopathologic and apoptosis evaluations (TUNEL and caspase-9) and the severity of bowel damage was significantly lower in the NEC + CAPE group compared to the NEC group (P < 0.01). The clinical sickness scores and body weight in the NEC + CAPE group was significantly better compared to the NEC group (P < 0.05). Tissue MDA, MPO, XO levels and TOS were remarkably reduced in the NEC + CAPE group, however, TAS was significantly increased in the NEC + CAPE group (P < 0.05). Treatment with CAPE reduces the intestinal damage in NEC.

Recruitment of Dendritic Cells Is Responsible for Intestinal Epithelial Damage in the Pathogenesis of Necrotizing Enterocolitis by Cronobacter sakazakii

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA(+) C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA(-) C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA(+) C. sakazakii/DC coculture supernatant revealed significantly greater TGF-β production compared with the levels produced by OmpA(-) C. sakazakii infection. TGF-β levels were elevated in the intestinal tissue of mice infected with OmpA(+) C. sakazakii. Cocultures of CaCo-2 cells and DCs in a "double-layer" model followed by infection with OmpA(+) C. sakazakii significantly enhanced monolayer leakage by increasing TGF-β production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA(+) C. sakazakii/DC supernatant. Blocking TGF-β activity using a neutralizing Ab suppressed iNOS production and prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-β production.

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.

Gut Environmental Factors May Shape the Persistence of Faecalibacterium Prausnitzii in the Healthy and Diseased Large Intestine

Background: Exposure to Helicobacter hepaticus, common enterohepatic bacteria, increases the incidence of experimental colitis and promotes colonic tumorigenesis. However, the role of H. hepaticus in pathogenesis of necrotizing enterocolitis (NEC) is not known. Neonatal NEC is a devastating disease of prematurely born babies. The cause of NEC is not clear, but inappropriate bacterial colonization plays an important role in NEC pathogenesis. Autophagy protects organisms against diverse pathologies, such as bacterial infections. However, uncontrolled activation of autophagy may lead to cellular injury. Aim: To test the effect of H. hepaticus on development of intestinal injury and expression of inflammatory and autophagic regulators in the rat model of NEC. Methods: Pregnant rats and newborn pups were kept either in H. hepaticus-free or in the H. hepaticus-contaminated environment. Prematurely born rat pups were hand-fed with formula and exposed to asphyxia/cold stress to develop NEC. After 96 hours, all animals were sacrificed and ileal tissue was collected. Incidence of NEC injury, expression of inflammatory cytokines (IL-6, IL-8, IL-18 & TNF-α) and protein levels of autophagy regulators (Beclin 1, LC3II & p62) were evaluated in the ileum. Results: Results: The incidence of NEC was significantly increased to 71% (25/35) in rats exposed to H. hepaticus compared with the H. hepaticus-free group with NEC incidence of 33% (13/39; p < 0.001). The survival rates were not affected by H. hepaticus. The presence of H. hepaticus was detected in the GI tract of the H. hepaticus group. IL-8 and IL-18 gene expression was significantly altered in the H. hepaticus group, whereas IL-6 and TNF-α levels were not changed. Protein levels of Beclin 1 and LC3II were markedly increased, whereas p62 levels were significantly decreased in the ileum from the H. hepaticus group. Conclusions: Helicobacter hepaticus exacerbates intestinal injury and induces intestinal inflammation in the rat NEC model. Exposure to H. hepaticus is also associated with a strong induction of the autophagy pathway in the site of injury the terminal ileum. Inappropriate activation of intestinal autophagy by bacterial insult may facilitate massive cell death, leading to severe mucosal injury. Supported by the NIH Grant HD-39657 (to B.D.)

Gram Negative Bacteria Are Associated with the Early Stages of Necrotizing Enterocolitis

IntroductionNecrotizing enterocolitis (NEC) affects 5–10% of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and elevated inflammatory mediators. Evaluation of NEC at earlier, less lethal stages of disease will allow investigation of initial disease triggers and may advance our understanding of temporal relationships between factors implicated in NEC pathogenesis. In this manuscript, we describe our investigation of early NEC and test the hypothesis that bacteria and inflammatory mediators differ between animals with early NEC and disease free animals.MethodsOn DOL7 C3HeB/FeJ pups were fed liquid formula with 1×104 Streptococcus thoraltensis, Serratia marcescens, and Pseudomonas aeruginosa every 3 h. To initiate NEC, pups underwent asphyxia (100% N2 for 90 s) and hypothermia (4°C for 10 min) after feeding. Pups were euthanized at 72 h. Intestines were collected for histologic NEC scoring and DNA/RNA extraction. Bacterial populations were identified by 16S rRNA pyrosequencing and principal component analysis (PCA). RNA isolates underwent QRT-PCR for Toll-like Receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS).ResultsDespite histologic, intestinal damage in mice with NEC, no gross necrosis was observed suggesting early disease. QRT-PCR yielded no difference between groups in TLR4 or iNOS mRNA levels. PCA demonstrated relative clustering of microbial communities based on presence or absence of NEC. 16S pyrosequencing demonstrated similar phyla between groups (Firmicutes and Proteobacteria predominated in all animals). However, the colonic microbiota of animals with NEC had more Citrobacter (p<0.01), Klebsiella (p<0.05), and Tatumella (p<0.05), while that of animals without NEC had more Streptococcus (p<0.01) and Enterococcus (p<0.01).Conclusion Citrobacter, Klebsiella, and Tatumella are associated with NEC. Differential colonic bacteria were identified despite the lack of inflammatory mediator elevation traditionally associated with NEC. This suggests a temporal relationship between bacteria and inflammatory mediators such that alterations in gut microbiota are associated with early NEC, while inflammatory mediator elevation is associated with advanced NEC.

Ileal Immune Dysregulation in Necrotizing Enterocolitis: Role of CD40/CD40L in the Pathogenesis of Disease

CD40, a co-stimulatory molecule, plays a critical role in coordinating enteric inflammatory immune responses. In necrotizing enterocolitis (NEC), upregulation of IL-10, a CD40-modulated cytokine, has been described, but the role of the IL-10 receptor (IL-10Rβ), CD40, and its ligand CD40L in disease pathogenesis is unknown. The study herein investigates ileal expression of CD40, CD40L, and IL-10R in a rat model of NEC. NEC was induced in newborn rats using established methods of formula feeding, asphyxia, and cold stress. Expression of CD40, CD40L, IL-10Rβ, and other proinflammatory molecules, including Toll-like receptor-4 (TLR-4) and IL-18, was assessed by immunoblotting. Tissue infiltration by macrophages, monocytes, and T cells was examined by confocal immunohistochemistry. Ileum from rat pups with NEC showed increased expression of TLR-4, IL-18, and IL-10Rβ. Sections from both NEC and control pups demonstrated preservation of ileal cells expressing CD40/CD40L. The distal ileum of controls expressed both CD40 and CD40L; conversely, neither molecule was detected in ileal tissue from NEC pups. Additional studies showed that treatment with epidermal growth factor (EGF), previously shown to ameliorate the severity of NEC in an animal model, did not restore CD40 expression. Ileal cytokine dysregulation, manifested by decreased CD40/CD40L and increased IL-10Rβ expression, may be involved in the pathogenesis of NEC. Dampened CD40 signaling may be related to enhanced IL-10 expression and a suppressed T-cell response to injury. We speculate that augmenting CD40-CD40L interactions may achieve a protective effect in this NEC model.

The Incidence of Necrotizing Enterocolitis Is Increased Following Probiotic Administration to Preterm Pigs 3

AbstractPreterm birth and necrotizing enterocolitis (NEC) is associated with inappropriate gut colonization and immunity, which may be improved by probiotic bacteria. Using a preterm pig model of NEC, we investigated the effects of probiotics on intestinal structure, function, microbiology, and immunology in the immediate postnatal period. Just after birth, caesarean-delivered preterm pigs were inoculated with Lactobacillus paracasei, Bifidobacteria animalis, and Streptococcus thermophilus (total 2.4 1010/d) either as live (ProLive, n = 14) or gamma-irradiated dead bacteria (ProDead, n = 12) and compared with controls (n = 14). All pigs received parenteral nutrition for 2 d followed by enteral formula feeding until tissue collection on d 5. Compared with control pigs, intestinal weight was lower and NEC incidence was higher in both groups given probiotics (64–67 vs. 14%; P<0.01). Hexose absorption, brush border enzyme activities, and gut barrier function were lower in the ProDead group compared with the other groups (P < 0.05), whereas live probiotics induced higher expression of the proinflammatory cytokines IL-1a and IL-6 (P < 0.05). Probiotics minimally affected gut colonization, except that live probiotics induced a higher density of B. animalis and lower bacterial diversity in the distal intestinal mucosa and lower SCFA concentrations in the colon (P < 0.05). The detrimental effects of probiotic bacteria in this study may relate to the specific strain and dose combination and may have involved the very immature gut immune system and low NEC incidence in the control group. It remains to be determined whether similar adverse responses to probiotics occur in preterm infants.

Bacterial Outer Membrane Protein A Prevents the Maturation of intestinal Dendritic Cells in the Pathogenesis of Enterobacter Sakazakii Induced NEC

Introduction: Enterobacter Sakazakii (ES) is a highly virulent gram-negative opportunistic pathogen implicated in clinical outbreaks of necrotizing enterocolitis (NEC). Our previous studies have demonstrated that feeding ES-contaminated formula to neonatal rats exacerbates intestinal inflammation in an experimental model of NEC. We also observed that the OmpA, ubiquitous to gram-negative bacteria, is an important virulence factor allowing ES to survive inside dendritic cells (DCs) and incite an inflammatory response.

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.

Comparison of different methods in the establishment of necrotizing enterocolitis models in premature rats

Objective To establish and evaluate three different necrotizing enterocolitis models,established by combination of formula feeding, hypoxia and cold exposure, hypoxia-reoxygenation, and intraperitoneal injection of lipopolysaccharides (LPS) in premature rats. Methods Group A was given formula feeding, hypoxia by exposing to 100% N2 for 90 s and 4 ℃ cold stress for 10 minutes, the hypoxia and cold stress were given twice a day for 2 d. Group B was put into 100% N2 for 5 min and then 100% O2for 5 min, twice a day for 3 d. Group C was injected intraperitoneally 5 mg/kg LPS. Group D, E and F were served as the corresponding controls for group A, B and C. Ileocecal junction, liver, kidney and lung tissues were harvested and evaluated by HE staining for histological analysis, histological changes of ileal tissues were scored, and rats with score higher than two were diagnosed with NEC. Results Premature rats in group A, B and C showed various degrees of decreasing activity, abdominal distention, diarrhea,intestinal dilatation and congestion. Histological score in group A to F were 3. 13 ± 0. 64, 1.40 ±0. 52,2. 00±0. 42,0. 30±0. 48, 0. 30±0. 48 and 0. 40±0. 52, respectively. There were significant differences between model groups and their corresponding control groups (P＜0. 01 ). Among the model groups, the histological score of group A was higher than group B (P＜0.01) and group C (P＜0.05). The incidences of NEC in group A, B and C were 6/8, 20% (5/10) and 4/8, respectively, while of zero in all control groups. Liver, kidney and lung injures were more serious in group C compared with the other groups.Conclusions Compared with the single-factor modeling approaches of intraperitoneal injection of LPS and hypoxiareoxygenation, the NEC animal model in preterm rats established by formula feeding, repeated hypoxia and cold exposure, is more similar to the etiological factors of neonatal NEC in human, with higher incidence, better reproducibility and specificity. Key words: Infant,premature; Enterocolitis,necrotizing; Disease models,animal; Evaluation studies

Dual Roles of Endogenous Platelet-Activating Factor Acetylhydrolase in a Murine Model of Necrotizing Enterocolitis

Human preterm infants with necrotizing enterocolitis (NEC) have increased circulating and luminal levels of platelet-activating factor (PAF) and decreased serum PAF-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF. Formula supplemented with recombinant PAF-AH decreases NEC in a neonatal rat model. We hypothesized that endogenous PAF-AH contributes to neonatal intestinal homeostasis and therefore developed PAF-AH mice using standard approaches to study the role of this enzyme in the neonatal NEC model. After exposure to a well-established NEC model, intestinal tissues were evaluated for histology, proinflammatory cytokine mRNA synthesis, and death using standard techniques. We found that mortality rates were significantly lower in PAF-AH pups compared with wild-type controls before 24 h of life but surviving PAF-AH animals were more susceptible to NEC development compared with wild-type controls. Increased NEC incidence was associated with prominent inflammation characterized by elevated intestinal mRNA expression of sPLA2, inducible NOS, and CXCL1. In conclusion, the data support a protective role for endogenous PAF-AH in the development of NEC, and because preterm neonates have endogenous PAF-AH deficiency, this may place them at increased risk for disease.

Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E(2) (PGE(2)). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE(2), and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE(2) and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum-treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum. Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE(2) in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.