Rat Model Of Necrotizing Enterocolitis Research Articles

Preclinical Neonatal Rat Studies of Heparin-Binding EGF-Like Growth Factor in Protection of the Intestines From Necrotizing Enterocolitis

We have previously demonstrated that enterally administered heparin-binding EGF-like growth factor (HB-EGF) produced in Escherichia coli decreases the incidence and severity of intestinal injury in a neonatal rat model of necrotizing enterocolitis (NEC). In preparation for upcoming human clinical trials, large-scale production of HB-EGF according to Good Manufacturing Practice (GMP) has been successfully accomplished using a Pichia pastoris yeast system. The current studies used a neonatal rat model of NEC to elucidate several important preclinical characteristics of HB-EGF therapy. We found that enteral administration of HB-EGF (800 microg/kg/dose) four times a day effectively reduced the incidence and severity of NEC, that Pichia-derived HB-EGF was not significantly different from E. coli-derived HB-EGF in preventing NEC, that EGF was not superior to HB-EGF in preventing NEC, and that prophylactic administration of HB-EGF added to formula starting with the first feed or 12 h later significantly reduced the incidence of NEC, with no change in the incidence of NEC noted if HB-EGF was added to the formula starting 24, 48, or 72 h after birth. Thus, large-scale production of GMP-grade HB-EGF in Pichia pastoris yeast produces a biologically active molecule suitable for human clinical trials.

Prenatal Lipopolysaccharide Increases Postnatal Intestinal Injury in a Rat Model of Necrotizing Enterocolitis

An increased incidence of necrotizing enterocolitis (NEC) has been noted in infants who are born to mothers with chorioamnionitis. Our objective was to test the hypothesis that newborn rat pups born to mothers exposed to prenatal lipopolysaccharide during pregnancy would be more susceptible to intestinal injury in a rat model of NEC and that the increased intestinal injury is mediated by dysregulation of inducible nitric oxide synthase. Time-dated pregnant Sprague-Dawley dams were given an intraperitoneal injection of either 2 mg/kg of lipopolysaccharide or vehicle. Rat pups from each group of dams were delivered at term and placed in a rat NEC model. A subset of pups was given either vehicle or aminoguanidine. Intestines were harvested and graded for degree of intestinal injury. Maternal prenatal lipopolysaccharide exposure increased the frequency and severity of intestinal injury in the neonatal rat NEC model. Treatment with aminoguanidine significantly decreased plasma nitric oxide levels. Additionally, aminoguanidine significantly decreased intestinal injury. Intestinal injury observed may be mediated via nitric oxide synthase dysregulation.

Role of LPS/CD14/TLR4-mediated inflammation in necrotizing enterocolitis: Pathogenesis and therapeutic implications

To establish the roles of lipopolysaccharide (LPS)/CD14/toll-like receptor 4 (TLR4)-mediated inflammation in a rat model of human necrotizing enterocolitis (NEC). Six pairs of intestinal samples from human NEC were collected before and after recovery for histological and molecular analysis of inflammatory cytokines and signaling components. In the rat NEC model, we isolated 10-cm jejunum segments and divided them into six groups (n = 6) for sham operation, treatment with LPS, bowel distension, combined bowel distension and LPS stimulation, and two therapeutic groups. The potential efficacy of a recombinant CD18 peptide and a monoclonal CD14 antibody was evaluated in the latter two groups. The serum and tissue levels of several inflammatory mediators were quantified by real-time polymerase chain reaction, ELISA and immunoblotting. Human acute phase NEC tissues displayed significant increases (P < 0.05) in levels of TLR4, CD14, myeloid differentiation protein (MD)-2, tumor necrosis factor (TNF)-alpha and nuclear factor-kappaB when compared to those after recovery. The histological and inflammatory picture of human NEC was reproduced in rats that were treated with combined bowel distension and LPS, but not in the sham-operated and other control rats. Serum levels of interleukin-6 and TNF-alpha were also elevated. The NEC pathology was attenuated by treating the NEC rats with a monoclonal CD14 antibody or an LPS-neutralizing peptide. LPS and distension are required to produce the histological and inflammatory features of NEC. A potential treatment option is blocking LPS activation and leukocyte infiltration.

Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii -Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis

Enterobacter sakazakii is an emerging pathogen that has been associated with outbreaks of necrotizing enterocolitis (NEC) as well as infant sepsis and meningitis. Our previous studies demonstrated that E. sakazakii induces NEC in a newborn rat model by inducing enterocyte apoptosis. However, the mechanisms responsible for enterocyte apoptosis are not known. Here we demonstrate that E. sakazakii induces significant production of nitric oxide (NO) in rat intestinal epithelial cells (IEC-6) upon infection. The elevated production of NO, which is due to increased expression of inducible NO synthase, is responsible for apoptosis of IEC-6 cells. Notably, pretreatment of IEC-6 cells with Lactobacillus bulgaricus (ATCC 12278) attenuated the upregulation of NO production and thereby protected the cells from E. sakazakii-induced apoptosis. Furthermore, pretreatment with L. bulgaricus promoted the integrity of enterocytes both in vitro and in the infant rat model of NEC, even after challenge with E. sakazakii. Infection of IEC-6 cells with E. sakazakii upregulated several genes related to apoptosis, cytokine production, and various signaling pathways, as demonstrated by rat gene array analysis, and this upregulation was subdued by pretreatment with L. bulgaricus. In agreement with these data, L. bulgaricus pretreatment protected newborn rats infected with E. sakazakii from developing NEC, resulting in improved survival.

Assessment of a Neonatal Rat Model of Necrotizing Enterocolitis

A neonatal rat model of necrotizing enterocolitis (NEC) is useful to investigate this devastating and obscure disease. The aim of this study was to assess a neonatal rat model of NEC to evaluate whether the histological appearance of the damaged intestine could be predicted by the clinical behaviour of the animals and the macroscopic appearance of the gut. Neonatal rats were delivered at term and assigned either to a control group consisting of breastfeeding and no stress factors, or to a NEC group in which NEC was induced by gavage feeding + hypoxia + oral lipopolysaccharide (4 mg/kg/day once daily for the first 2 days of life). Clinical status was assessed on day 4 using a clinical sickness score (general appearance, response to touch, natural activity, body colour; 0 - 3 for each variable). Neonatal rats were sacrificed at 4 different time points: day 1, day 2, day 3, and day 4. At sacrifice, a macroscopic assessment of the gut was performed using a new scoring system based on: colour (0 - 2), consistency (0 - 2) and degree of dilatation (0 - 2). The resected gut was stained with haematoxylin/eosin, and evaluated microscopically by 2 independent blinded scorers, including a consultant histopathologist. The histology results were used to validate the macroscopic gut assessment. Results were compared by ANOVA and linear regression analysis. Ethics Committee and Home Office approvals were obtained. In the control group NEC was not present either macroscopically or histologically. The clinical sickness score was higher in the NEC group (median = 4.5; range = 2 - 6) compared to controls (median = 0; range = 0 - 1; p < 0.0001). In the NEC group the macroscopic appearance (from day 2) and histological score (from day 1) increased significantly (p < 0.0001) and were strongly correlated (r (2) = 0.74, p < 0.0001). The clinical behaviour and macroscopic appearance of the intestine are valid tools to assess gut damage in our neonatal rat model of NEC. This allows future studies that are not exclusively based on histology.

HB-EGF knockout mice have increased susceptibility to necrotizing enterocolitis

NTRODUCTION: Necrotizing enterocolitis (NEC) is currently the eading surgical cause of death in premature infants. We have previusly used a neonatal rat model of NEC to show that administration f heparin-binding EGF-like growth factor (HB-EGF) decreases inestinal injury and mortality. Here we utilize HB-EGF knockout KO) mice to examine the effects of HB-EGF loss-of-function on usceptibility to NEC.

Change of PAF and TNF-α in neonatal rat model of necrotizing enterocolitis

Objective To study the change of platelet-activating factor(PAF)and tumor necrosis fac-tor-α(TNF-α)density in neonatal rat model of necrotizing enterocolitis,and analyze the correlation between the PAF、TNF-α levd and the scoring of intestinal injury.The aim is to explore the function of PAF and TNF-α in NEC.Methods According to 2 x 2 factor analysis,32 neonate Sprague-Dewley rats(48 hours olds)were divided into 4 groups(A,B,C and D,8 rats each group).Group A was made into NEC models as follows:separate from mother rats and feeding with rat milk substitute,hypoxia(100%N2)for 90 second and 4℃cold exposure for 10 minutes,twice a day during 3 consecutive days;Group B was given formula feeding alone,not hypoxia and cold exposure.Group C was given rat milk feeding,the same hypoxia and cold exposure as group A,Group D was control group,given rat milk feeding,not hypoxia and cold exposure.On the 4th day all the subjects were sacrificed and the intestine around ileocecal junction were obtained to evaluated by stained with H&E for histological analysis and score.The mean scores more than 2 were considered NEC.The contents of PAF,TNF-α in homogenate of intestinal tissue were measured by ELISA(Kits bought from Shanghai Langka Company).Kruskal-Wallis H test,Spearman correlation analysis were used to analyze difference among various groups.α=0.05 was considered significant.Results The score of histopatholigical and the contents of PAF,TNF-α had significant difference among groups(P＜0.01).By spearman correlation analysis,the relations of the degree of intestinal injury and the contents of PAF,TNF-α in intestinal tissue were positive(rpAF=0.71,rTNF-α=0.81,P＜0.05).Conclusion Endogenous PAF and TNF-α in intestine tissue are probably the critical factors for the development of NEC.As risk factors,they are involed in the development of NEC.It indicated that the concentrations of PAF and TNF-α in intestine tissue had a positive correlation with the severity of NEC and can inflect the degree of intestine mucosa lesions. Key words: Necrotizing enterocolitis; Neonatal rat; Animal model; Platelet-activating factor; Tumor necrosis factor-α

Expression of TLR-2, TLR-4, NOD2 and pNF-κB in a Neonatal Rat Model of Necrotizing Enterocolitis

BackgroundThe etiology of necrotizing enterocolitis (NEC) results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC.Methodology/Principal FindingsWe specifically evaluated intestinal epithelial cell (IEC) expression of Toll-like receptor 2 (TLR-2), TLR-4, NOD2 and phosphorylated NF-κB (pNF-κB) after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1), neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2) or hand-gavaged with a protein rich formula after a hypoxia–reoxygenation procedure (group 3). Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-κB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia–reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-κB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged.Conclusions/SignificanceThese results show that this rat model of NEC induced mucosal injury, leading to a highly responsive IEC phenotype and suggesting that alterations in the innate immune system participates in the pathogenesis of NEC and are enhanced by prematurity.

Decreased development of necrotizing enterocolitis in IL-18-deficient mice

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-Il18(tm1Aki)/J (NEC IL-18(-/-)) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-alpha-, IL-12-, and IL-1beta-positive cells and macrophages were determined in both ileum and liver via immunohistology. IkappaB-alpha and IkappaB-beta were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18(-/-) mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1beta were significantly reduced in IL-18(-/-) mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-alpha, ileal IL-1beta, or IL-12. IkappaB-alpha and IkappaB-beta were significantly increased in NEC IL-18(-/-) mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.

Inhibition of Nuclear Factor-κB Ameliorates Bowel Injury and Prolongs Survival in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). It remains unknown, however, whether NF-kappaB mediates injury in neonatal NEC. We therefore examined the activation status of NF-kappaB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-kappaB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-kappaB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-kappaB inhibitor protein IkappaBalpha and IkappaBbeta at d 2. To determine the importance of elevated NF-kappaB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IkappaB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-kappaB activity. Our findings therefore lead us to conclude that selective NF-kappaB inhibition represents a promising therapeutic strategy for NEC.

Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.

Reduction of experimental necrotizing enterocolitis with anti-TNF-α

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-alpha in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-alpha; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-alpha antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-alpha groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-alpha. Hepatic TNF-alpha and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-alpha compared with those sham injected. In addition, ileal luminal levels of both TNF-alpha and IL-18 were significantly decreased in the anti-TNF-alpha-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-alpha group. These data show that hepatic TNF-alpha is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-alpha could be used as a potential therapy for human NEC.

101 Effect of EGF on the Intestinal Tight Junction Barrier in Experimental Necrotizing Enterocolitis

Background: Necrotizing enterocolitis (NEC) is the most common intestinal disease predominately of formula fed, premature babies. Epidermal growth factor (EGF) reduces the incidence of disease in a neonatal rat model of NEC. It has been suggested that increased mucosal permeability may play an important role in the pathogenesis of NEC. Claudin and occludin are the major structural and functional components of the tight junction barrier. These proteins may be altered in disease states. However, the role of tight junctions in NEC pathogenesis and EGF treatment is currently unknown.Objective: The aim of this study was to determine if EGF affects expression of tight junction genes and proteins in the ileum during the development of NEC.Methods: Neonatal rats, either dam fed (DF), milk formula fed (NEC), or fed with formula plus 500 ng/ml EGF (NEC+EGF) were exposed to asphyxia and cold stress to develop NEC. After 96 hours, ileal expression of claudin-3 and occludin were evaluated using Realtime-PCR, Western blot and immunohistochemistry.Results: Claudin-3 and occludin mRNA levels in the ileum were significantly increased 2–4 fold in NEC animals compared to DF animals (p<0.0001). Supplementation with EGF normalized mRNA expression to DF levels. Phosphorylated occludin was significantly decreased in NEC animals compared to DF animals (p<0.01) and EGF treatment increased expression to DF levels. Non-phosphorylated occludin was only present in NEC and NEC+EGF animals. Histological localization of claudin-3 and occludin proteins was disturbed in animals with NEC compared to DF animals. NEC animals had upregulated expression and disorganization of the tight junction proteins. EGF treatment resulted in localization of the proteins at the tight junction complex.Conclusion: The ability of EGF to normalize expression and localization of tight junction proteins may be one mechanism by which integrity of the mucosal barrier is maintained, thereby protecting the intestinal mucosa against NEC. The increased expression of mRNA and disorganization of protein in animals with NEC may represent a compensation for the inability to form functional tight junctions at the mucosal barrier.Supported by the APS Porter Physiology Fellowship (to J.C.) and NIH Grants HD-39657, HD-47237 (to B.D.)

Effect of intestinal trefoil factor on expression of iNOS, TNF-α and NO in neonatal rat model of necrotizing enterocolitis

目的 探讨肠三叶因子(intestinal trefoil factor,ITF)对坏死性小肠结肠炎(necrotizingenterocolitis,NEC)新生大鼠的肠黏膜组织中iNOS、TNF-α、NO的含量的影响,及ITF对NEC是否具有保护作用.方法建立NEC模型,对新生1日龄Wistar大鼠予100%二氧化碳,5 min后再予以100%氧气,5 min后放回母鼠身边喂养,第4天处死,取肠组织待检.新生鼠40只随机分为五组,每组8只,A组为NEC模型后予以腹腔注射ITF 0.5 mg;B组为NEC模型后予以皮下注射ITF 0.2mg C、D组为NEC模型后予以腹腔注射生理盐水分别为0.5 ml和0.2 ml;E组为正常对照组.取近回盲部1～2cm肠道组织固定包埋、切片、HE染色做病理学检查及免疫组化观察iNOS的表达,其他肠道组织制备组织匀浆取上清液检测TNF-α、NO的含量.结果A、B及E组iNOS呈弱阳性表达,C、D组iNOS呈强阳性表达;A、B组组织匀浆中TNF-α的含量各为[(30.515±2.731)和(32.229±4.978)pg/mg·pro]均较C、D组[(39.957±8.283)、(39.960±8.374)pg/mg·pro]明显下降(P＜0.01),与E组[(33.523±6.752)pg/mg·pro]差异无统计学意义(P＞0.05);A、B组组织匀浆中NO的含量各为[(0.37±0.07)和(0.54±0.08)μmol/mg tissue]较C、D组[(0.76±0.01)和(0.82±0.04)μmol/mg tissue]明显下降(P＜0.01),与E组[(0.41±0.02)μmol/mg tissue]差异无统计学意义(P＞0.05);A、B组间及C、D组间TNF-α、NO含量差异无统计学意义(P＞0.05);病理切片显示C、D组HE染色切片见肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理评分的中位积分为3分;A、B组肠上皮细胞少量脱落,顶端绒毛坏死,病理评分的中位积分为1分.结论通过腹腔和皮下注射ITF可以减轻NEC后的肠道炎症反应,ITF有可能为治疗NEC提供新的方法。

TIGHT JUNCTIONS FORMATION IN EXPERIMENTAL MODEL OF NECROTIZING ENTEROCOLITIS - EFFECT OF EGF TREATMENT

Background: Necrotizing enterocolitis (NEC) is the most common intestinal disease predominately of formula fed, premature babies. Epidermal growth factor (EGF) reduces the incidence of disease in a neonatal rat model of NEC. Claudin and occludin are the major structural and functional components of the tight junction barrier. However, the role of tight junctions in NEC pathogenesis and EGF treatment is currently unknown. Aim: The aim of this study was to determine if EGF affects expression of tight junction genes and proteins in the ileum during the development of NEC. Methods: Neonatal rats, either dam fed (DF), milk formula fed (NEC), or fed with formula plus 500 ng/ml EGF (NEC + EGF) were exposed to asphyxia and cold stress to develop NEC. After 96 hours, ileal expression of claudin-3 and occludin were evaluated using Realtime-PCR, Western blot and immunohistochemistry. Results: Claudin-3 and occludin mRNA levels in the ileum were significantly increased 2-4 fold in NEC animals compared to DF animals (p < 0.0001). Supplementation with EGF normalized mRNA expression to DF levels. Histological localization of claudin-3 and occludin proteins was disturbed in animals with NEC compared to DF animals. NEC animals had upregulated expression and disorganization of the tight junction proteins. EGF treatment resulted in localization of the proteins at the tight junction complex. Conclusion: The ability of EGF to normalize expression and localization of tight junction proteins may be one mechanism by which integrity of the mucosal barrier is maintained, thereby protecting the intestinal mucosa against NEC. The increased expression of mRNA and disorganization of protein in animals with NEC may represent a compensation for the inability to form functional tight junctions at the mucosal barrier. Supported by the APS Porter Physiology Fellowship (to J.C.) and NIH Grant HD-39657 (to B.D.)

Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.

O0029 ENTERAL ADMINISTRATION OF HB-EGF REDUCES THE INCIDENCE OF NECROTIZING ENTEROCOLITIS IN A RAT MODEL

Introduction: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. The protective role of maternal milk against NEC has been reported, however the components of milk responsible for this protection have not been identified. Previously, we have shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model (Dvorak et al., AJP 282:G156, 2002). Heparin-binding EGF-like growth factor (HB-EGF), another member of the EGF family, has been shown to be cytoprotective for intestinal epithelial cells. Recently, the presence of HB-EGF has been detected in human breast milk. The aim of this study was to examine the effect of enteral administration of HB-EGF on survival and the incidence and severity of NEC in a neonatal rat model. Methods: Neonatal rats delivered via abdominal incision on day 22 of gestation were artificially fed with rat milk substitute for 4 days. Four diets were used in this study: growth factor-free rat milk substitute (RMS) and RMS supplemented with 5, 50, and 500 ng/ml of HB-EGF (HB-5, HB-50, and HB-500, respectively). Experimental NEC was induced by daily exposure to asphyxia and cold stress. In addition, dam fed littermates were used as a control (DF). The development and severity of NEC was evaluated in the distal ileum using our histopathological scoring system. Results: In the NEC group, 91% of animals developed characteristic pathophysiological signs of NEC in the distal ileum. Supplementation of HB-EGF into milk formula significantly reduced the incidence of NEC and improved survival of the pups in a dose dependent manner.Table 1Conclusion: Milk-borne HB-EGF provides a protective effect on the development of neonatal NEC. The mechanism by which HB-EGF prevents NEC must be further investigated on the molecular level. However, supplementation of an optimized growth factor “cocktail” into infant formula for prematurely born babies might be a feasible strategy for the prevention of neonatal NEC. Supported by the NIH Grant HD-39657 (to B.D.).

Serum cytosolic beta-glucosidase activity in a rat model of necrotizing enterocolitis.

The diagnosis of necrotizing enterocolitis (NEC) is made from a combination of clinical and radiographic findings. There are no useful screening biochemical markers of intestinal injury. The serum concentration of cytosolic beta-glucosidase (CBG), an enzyme found primarily in enterocytes, is markedly elevated in animal models of ischemia and bowel obstruction. We hypothesized that in a rat model of NEC, serum CBG activity would significantly increase before microscopic evidence of severe intestinal injury. Cohorts of 2-wk-old Sprague-Dawley rats (n = 10/cohort) were anesthetized and underwent laparotomy with occlusion of the superior mesenteric artery (SMA). Platelet-activating factor (200 microg/animal) was injected in the proximal duodenum. Serum and intestinal samples were obtained at time 0 (control) and 30, 60, and 90 min of ischemia (I) and after 90 min of I followed by 60 min of reperfusion (I/R). Histopathologic injury was categorized as either no or minimal injury or mural necrosis by two masked investigators and CBG activity was measured by ELISA. Data were analyzed with Fisher's exact test and ANOVA. Only the I/R group had significantly greater mural necrosis compared with the control group (90% versus 0%, respectively, p < 0.001). In contrast, CBG activity was significantly elevated after only 90 min of I and after I/R (15.1 +/- 5.6 and 16.4 +/- 4.3 units/mL, respectively, p < 0.05). We conclude that serum CBG is elevated before transmural intestinal injury in this model and may have utility as an early marker of ischemia in patients at risk for NEC.

Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.

The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.

Dietary polyunsaturated fatty acid (PUFA) supplementation has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in a recent randomized, controlled trial. These compounds are known to modulate the inflammatory cascade and to influence intestinal health in a variety of ways. Although the pathophysiology of NEC is not well understood, recent evidence suggests that platelet-activating factor (PAF) is a key endogenous mediator of intestinal necrosis in animals. Using a neonatal rat model of NEC that includes the key risk factors of asphyxia and formula feeding, we investigated the role of dietary PUFA supplementation on the incidence and pathophysiology of NEC. Our findings suggest that PUFA reduce the incidence of NEC by modulating PAF metabolism and endotoxin translocation.