Pathogenesis of Necrotizing Enterocolitis
2014; Elsevier BV; Volume: 185; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2014.08.028
ISSN1525-2191
AutoresScott Tanner, Taylor F. Berryhill, James Ellenburg, Tamás Jilling, D Cleveland, Robin G. Lorenz, Colin Martin,
Tópico(s)Clinical Nutrition and Gastroenterology
ResumoNecrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant's intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice. Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant's intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice. Necrotizing enterocolitis (NEC) is a disorder characterized by intestinal necrosis in premature infants that results in significant morbidity and mortality.1Neu J. Walker W.A. Necrotizing enterocolitis.N Engl J Med. 2011; 364: 255-264Crossref PubMed Scopus (1450) Google Scholar Approximately 7% of infants with a birth weight between 500 and 1500 g develop NEC.1Neu J. Walker W.A. Necrotizing enterocolitis.N Engl J Med. 2011; 364: 255-264Crossref PubMed Scopus (1450) Google Scholar The pathogenesis is characterized by intestinal inflammation that can progress to systemic infection/inflammation, multiorgan failure, and death. The bowel is distended and hemorrhagic on gross inspection. On microscopic examination, signs of inflammation, mucosal edema, epithelial regeneration, bacterial overgrowth, submucosal gas bubbles, and ischemic transmural necrosis are seen (Figure 1, A–E ).2Ballance W.A. Dahms B.B. Shenker N. Kliegman R.M. Pathology of neonatal necrotizing enterocolitis: a ten-year experience.J Pediatr. 1990; 117: S6-S13Abstract Full Text PDF PubMed Scopus (223) Google Scholar Currently the pathogenesis of NEC is believed to have multifactorial causes, including intestinal immaturity and microbial dysbiosis. Intestinal immaturity leads to a compromised intestinal epithelial barrier, an underdeveloped immune defense, and altered vascular development and tone. The compromised epithelial barrier and underdeveloped immune system, when exposed to luminal microbiota that have been shaped by formula feedings, antibiotic exposure, and Cesarean delivery, can lead to intestinal inflammation and sepsis. Despite therapeutic success in animal model systems, there are relatively few therapeutic strategies that have allowed for significantly improved outcomes in infants with NEC. Two hurdles that persist are our incomplete understanding of the developing immune system in premature infants and our inability to adequately replicate these complex factors in animal models.3Lu P. Sodhi C.P. Jia H. Shaffiey S. Good M. Branca M.F. Hackam D.J. Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: pathophysiology, translational relevance, and challenges.Am J Physiol Gastrointest Liver Physiol. 2014; 306: G917-G928Crossref PubMed Scopus (65) Google Scholar, 4Dowling D.J. Levy O. Ontogeny of early life immunity.Trends Immunol. 2014; 35: 299-310Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar This review summarizes the complex intestinal immune system in premature infants and details what is known about the involvement of innate immune factors in NEC, both in animal models and in human disease. The neonatal intestinal ecosystem is extremely fragile. At birth, the newborn is exposed to the external environment for the first time, and the immune response must begin to distinguish between self and nonself. In particular, the recognition of food antigens and commensal microbiota must be distinguished from that of potential pathogens. This process is even more challenging in premature infants, as they are typically placed on broad-spectrum antibiotics that disrupt both the timing and the diversity of the initial bacterial colonization of the intestinal tract.1Neu J. Walker W.A. Necrotizing enterocolitis.N Engl J Med. 2011; 364: 255-264Crossref PubMed Scopus (1450) Google Scholar In addition, the immature epithelial barrier appears to be more sensitive to the detection of bacteria5Jilling T. Simon D. Lu J. Meng F.J. Li D. Schy R. Thomson R.B. Soliman A. Arditi M. Caplan M.S. The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis.J Immunol. 2006; 177: 3273-3282Crossref PubMed Scopus (318) Google Scholar, 6Leaphart C.L. Cavallo J. Gribar S.C. Cetin S. Li J. Branca M.F. Dubowski T.D. Sodhi C.P. Hackam D.J. A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair.J Immunol. 2007; 179: 4808-4820Crossref PubMed Scopus (353) Google Scholar and more susceptible to bacterial translocation,7Udall J.N. Pang K. Fritze L. Kleinman R. Walker W.A. Development of gastrointestinal mucosal barrier. I. The effect of age on intestinal permeability to macromolecules.Pediatr Res. 1981; 15: 241-244Crossref PubMed Scopus (177) Google Scholar allowing for both an unwarranted inflammatory response to commensals and the translocation of pathogens, both of which may contribute to intestinal damage. The innate immune system is the first line of defense against infections. Innate immune cells respond in a nonspecific manner and do not confer long-lasting immunity to the host.8Kayama H. Nishimura J. Takeda K. Regulation of intestinal homeostasis by innate immune cells.Immune Netw. 2013; 13: 227-234Crossref PubMed Google Scholar The major components are cells (including macrophages, neutrophils, dendritic cells (DCs), natural killer cells, B1 B cells, innate lymphoid cells, and γδ T cells) and anatomical barriers (such as the intestinal epithelium and the gastrointestinal mucus layer). In addition, the presence of commensal microbiota can serve as a part of the innate immune system by preventing the colonization by pathogenic bacteria (colonization resistance). Several major components of the human mucosal immune system are in place before birth. These components include IgM+ B cells; γδ T cells found in the intraepithelial lymphocyte compartment, which are seen as early as 12 to 15 weeks of embryonic age; and Peyer's patches, which can be seen by about 30 weeks of gestation.9Strunk T. Currie A. Richmond P. Simmer K. Burgner D. Innate immunity in human newborn infants: prematurity means more than immaturity.J Matern Fetal Neonatal Med. 2011; 24: 25-31Crossref PubMed Scopus (156) Google Scholar This development contrasts with that of the murine mucosal system, in which the same B cells and intraepithelial lymphocytes are seen only about 1 day before birth and macroscopic Peyer's patches are not seen until at least 1 week after birth.10McCracken V.J. Lorenz R.G. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota.Cell Microbiol. 2001; 3: 1-11Crossref PubMed Scopus (253) Google Scholar These differences in intestinal immune system development imply that a newborn mouse may have intestinal immaturity very similar to that of a premature infant—one reason that murine models are widely used to study the immune mechanisms that predispose to NEC development. Separation of the intestinal lumen from the rest of the organism is accomplished through a physical barrier established by the intestinal epithelial cells. All intestinal epithelial cells, including enterocytes, Paneth cells, and goblet cells, play an important role in maintaining barrier integrity. The barrier is maintained by the presence of tight junctions between the epithelial cells. In humans, tight junctions are formed by 10 weeks of gestation and are composed of claudins, junctional adhesion molecules, and zonula occludins.11Utech M. Brüwer M. Nusrat A. Tight junctions and cell-cell interactions.Methods Mol Biol. 2006; 341: 185-195PubMed Google Scholar Goblet cells, positioned throughout the intestine, are involved in the secretion of mucin glycoproteins, which generate the mucus layer of the intestine.12Kim Y.S. Ho S.B. Intestinal goblet cells and mucins in health and disease: recent insights and progress.Curr Gastroenterol Rep. 2010; 12: 319-330Crossref PubMed Scopus (915) Google Scholar Goblet cells can be found as early as 9 to 10 weeks of gestation,12Kim Y.S. Ho S.B. Intestinal goblet cells and mucins in health and disease: recent insights and progress.Curr Gastroenterol Rep. 2010; 12: 319-330Crossref PubMed Scopus (915) Google Scholar and mucins reach adult levels by 27 weeks of gestation.13Buisine M.P. Devisme L. Savidge T.C. Gespach C. Gosselin B. Porchet N. Aubert J.P. Mucin gene expression in human embryonic and fetal intestine.Gut. 1998; 43: 519-524Crossref PubMed Scopus (141) Google Scholar Animal model studies suggest that mucus gene expression is influenced by bacterial colonization, that mucus protein glycosylation patterns are developmentally regulated, and that glycosylation alters interactions with bacterial pathogens; however, equivalent studies in humans have not been reported.14Chu S.H. Walker W.A. Developmental changes in the activities of sialyl- and fucosyltransferases in rat small intestine.Biochim Biophys Acta. 1986; 883: 496-500Crossref PubMed Scopus (67) Google Scholar, 15Dai D. Nanthkumar N.N. Newburg D.S. Walker W.A. Role of oligosaccharides and glycoconjugates in intestinal host defense.J Pediatr Gastroenterol Nutr. 2000; 30: S23-S33Crossref PubMed Google Scholar, 16Bergström A. Kristensen M.B. Bahl M.I. Metzdorff S.B. Fink L.N. Frøkiaer H. Licht T.R. Nature of bacterial colonization influences transcription of mucin genes in mice during the first week of life.BMC Res Notes. 2012; 5: 402Crossref PubMed Scopus (42) Google Scholar Mucus forms in two distinct layers; the outer, thicker layer prevents intestinal bacteria from reaching the epithelial layer, whereas the inner layer is vital for cell signaling if a disruption occurs.12Kim Y.S. Ho S.B. Intestinal goblet cells and mucins in health and disease: recent insights and progress.Curr Gastroenterol Rep. 2010; 12: 319-330Crossref PubMed Scopus (915) Google Scholar, 17McElroy S.J. Weitkamp J.H. Innate Immunity in the Small Intestine of the Preterm Infant.Neoreviews. 2011; 12: e517-e526Crossref PubMed Scopus (57) Google Scholar In addition to preventing bacterial interaction with the epithelium, the mucus layer also provides scaffolding for antimicrobial peptides (AMPs) and secretory IgA (SIgA), which are discussed in the following section. Should pathogenic or commensal bacteria penetrate the mucus layer and reach the epithelial cells, pattern-recognition receptors (PRRs) are able to sense the presence of the bacteria and initiate an appropriate response. The most common PRRs are Toll-like receptors (TLRs) and nucleotide-binding oligomerization domains (NODs), which are expressed by both epithelial cells and immune cells, particularly macrophages and DCs, throughout the intestinal compartment. The PRRs detect common bacterial structures, such as lipopolysaccharide (LPS), flagella, and CpG-rich DNA. Current dogma indicates that abnormal TLR4 signaling in the premature intestinal epithelium is associated with NEC, perhaps by increasing platelet-activating factor (PAF) levels. However, on detection of microbial components, protective PRRs can initiate signaling pathways leading to the production of AMPs, IgA, and epithelial healing factors. Paneth cells—found at the base of the intestinal crypts—are yet another secretory cell type that provides protection to the host. Paneth cells are present at around 13 weeks of gestation and produce AMPs, which function to disable or kill microorganisms that have entered the digestive tract. Enterocytes, macrophages, and human neutrophils can also produce AMPs, including α-defensins, β-defensins, lysozyme, and type IIA secretory phospholipase A2, with α-defensins and lysozyme being the most common in the neonatal intestine.17McElroy S.J. Weitkamp J.H. Innate Immunity in the Small Intestine of the Preterm Infant.Neoreviews. 2011; 12: e517-e526Crossref PubMed Scopus (57) Google Scholar Type IIA secretory phospholipase A2 hydrolyzes the phospholipids in bacterial cell walls, which results in the production of arachidonic acid and lyso-PAF.18Gallo R.L. Hooper L.V. Epithelial antimicrobial defence of the skin and intestine.Nat Rev Immunol. 2012; 12: 503-516Crossref PubMed Scopus (620) Google Scholar As lyso-PAF is converted to the highly active inflammatory mediator PAF, type IIA secretory phospholipase A2 provides a mechanism that is at the intersection of bactericidal and inflammatory host defenses. As the newborn immune system begins its development, SIgA is transferred via maternal breast milk. Additionally, growth factors, cytokines, and other immune modulators are components of human milk, including IL-6, tumor necrosis factor α, IL-10, transforming growth factor (TGF)-β, and lysozyme.19Goldman A.S. Modulation of the gastrointestinal tract of infants by human milk. Interfaces and interactions. An evolutionary perspective.J Nutr. 2000; 130: 426S-431SPubMed Google Scholar Newborns acquire IgA through passive transfer via the ingestion of human milk after birth. The levels of IgA in human milk remain high throughout the breastfeeding period and help to provide protection and aid in the colonization of commensal bacteria.20Peitersen B. Bohn L. Andersen H. Quantitative determination of immunoglobulins, lysozyme, and certain electrolytes in breast milk during the entire period of lactation, during a 24-hour period, and in milk from the individual mammary gland.Acta Paediatr Scand. 1975; 64: 709-717Crossref PubMed Scopus (48) Google Scholar Although neonatal IgA production begins at approximately 2 weeks after birth, it does not reach adult values until 2 to 6 years of age.21Burgio G.R. Lanzavecchia A. Plebani A. Jayakar S. Ugazio A.G. Ontogeny of secretory immunity: levels of secretory IgA and natural antibodies in saliva.Pediatr Res. 1980; 14: 1111-1114Crossref PubMed Scopus (101) Google Scholar Maternal IgA in milk is thought to react with the microbiota present in the maternal gastrointestinal tract, and the passive transfer of these antibodies not only is thought to prevent the adherence of bacteria to mucosal surfaces and bacterial penetration into the mucosa but also is vital for the formation and composition of the newborn's intestinal microbiota.19Goldman A.S. Modulation of the gastrointestinal tract of infants by human milk. Interfaces and interactions. An evolutionary perspective.J Nutr. 2000; 130: 426S-431SPubMed Google Scholar Maternal milk has been shown to promote colonization with bificobacteria.22Sharma R. Tepas 3rd, J.J. Microecology, intestinal epithelial barrier and necrotizing enterocolitis.Pediatr Surg Int. 2010; 26: 11-21Crossref PubMed Scopus (38) Google Scholar As NEC cases are more highly associated with formula feeding, lower numbers of bifidobacteria and bacteroidetes, and higher proportions of proteobacteria and actinobacteria, it would appear that the shaping of the intestinal microbiota by the maternal IgA is one of the crucial factors in preventing NEC.23Torrazza R.M. Ukhanova M. Wang X. Sharma R. Hudak M.L. Neu J. Mai V. Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis.PLoS One. 2013; 8: e83304Crossref PubMed Scopus (150) Google Scholar Discussion of the multiple studies that have investigated the dysbiosis seen in NEC are outside of the scope of this review, but those studies were recently reviewed by Torrazza and Neu.24Torrazza R.M. Neu J. The altered gut microbiome and necrotizing enterocolitis.Clin Perinatol. 2013; 40: 93-108Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar IL-10 and TGF-β are anti-inflammatory cytokines involved in regulating T-cell responses and in down-regulating proinflammatory secretions from macrophages and neutrophils. Neonatal monocyte and T cell–derived IL-10 levels are significantly lower than are adult levels; however, IL-10 has been detected in human milk, suggesting an important role for milk-derived IL-10 in neonatal immune development.25Fituch C.C. Palkowetz K.H. Goldman A.S. Schanler R.J. Concentrations of IL-10 in preterm human milk and in milk from mothers of infants with necrotizing enterocolitis.Acta Paediatr. 2004; 93: 1496-1500Crossref PubMed Google Scholar A recent study has indicated that a low blood TGF-β level is associated with a higher risk for NEC.26Maheshwari A. Schelonka R.L. Dimmitt R.A. Carlo W.A. Munoz-Hernandez B. Das A. McDonald S.A. Thorsen P. Skogstrand K. Hougaard D.M. Higgins R.D. Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research NetworkCytokines associated with necrotizing enterocolitis in extremely-low-birth-weight infants.Pediatr Res. 2014; 76: 100-108Crossref PubMed Scopus (96) Google Scholar Numerous animal models are currently used for studying NEC disease pathogenesis. Although each model has specific advantages in mimicking certain aspects of the human disease, it has limitations,3Lu P. Sodhi C.P. Jia H. Shaffiey S. Good M. Branca M.F. Hackam D.J. Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: pathophysiology, translational relevance, and challenges.Am J Physiol Gastrointest Liver Physiol. 2014; 306: G917-G928Crossref PubMed Scopus (65) Google Scholar including the fact that laboratory animal models lack genetic diversity, are subject to static environmental conditions, and do not have innate immune and intestinal development identical to that in humans.27van der Worp H.B. Howells D.W. Sena E.S. Porritt M.J. Rewell S. O'Collins V. Macleod M.R. Can animal models of disease reliably inform human studies?.PLoS Med. 2010; 7: e1000245Crossref PubMed Scopus (928) Google Scholar However, human tissue is also not optimal, as surgical specimens are often necrotic and may not reflect the mechanisms that lead to the development of NEC. An ideal model of NEC would: i) be based on the mediators thought to contribute to and induce human disease, ii) take into account the developmental differences specific to the neonatal period, iii) have impaired intestinal restitution and development of necrosis, iv) include factors shown to play a role in the human disease (prematurity, hypoxic stress, formula feeding, dysbiosis), and v) have increased severity in the ileum. As this review will demonstrate, there is no model that perfectly replicates NEC; however, each of the following models is a useful tool for evaluating the specific mechanisms of the disease (Table 1): gavage/hypoxia (G/H) model, ischemia/reperfusion (I/R) model, PAF administration, Paneth cell depletion, xenograph model, infection model, and chemical injury models.Table 1Accuracy of Current Animal Models in Incorporating the Components of Human NECComponents of an optimal animal modelNecrotizing enterocolitis modelsG/HI/RPAF administrationPaneth cell depletionXenograftInfection and chemical injuryBased on predicted mediators of NEC pathogenesisYesYesYesYesYesReflects developmental differences specific to the neonatal period/prematurityYesImpaired intestinal restitutionYesYesYesDevelopment of necrosisYesYesYesYesYesHypoxic stressYesYesTemporal relationship to feeding/formula feedingYesDysbiosisYesYesYesYesMore severe disease in the ileumYesYesThe Institutional Care and Use Committee and the institutional review board of the University of Alabama at Birmingham approved all experiments.G/H, gavage/hypoxia; I/R, ischemia/reperfusion; NEC, necrotizing enterocolitis; PAF, platelet-activating factor. Open table in a new tab The Institutional Care and Use Committee and the institutional review board of the University of Alabama at Birmingham approved all experiments. G/H, gavage/hypoxia; I/R, ischemia/reperfusion; NEC, necrotizing enterocolitis; PAF, platelet-activating factor. Perhaps the most commonly cited animal model of NEC is the G/H model, which has been described in mice, rats, and piglets.28Sodhi C. Richardson W. Gribar S. Hackam D.J. The development of animal models for the study of necrotizing enterocolitis.Dis Model Mech. 2008; 1: 94-98Crossref PubMed Scopus (85) Google Scholar, 29Tian R. Liu S.X. Williams C. Soltau T.D. Dimmitt R. Zheng X. De Plaen I.G. Characterization of a necrotizing enterocolitis model in newborn mice.Int J Clin Exp Med. 2010; 3: 293-302PubMed Google Scholar This model links the altered vascular development and tone that are thought to be components of intestinal immaturity with early exposure to cold stress and a nonmilk diet. Rats are popular for use in these studies because they are easier to gavage than are mouse pups and are easier and less expensive to maintain than are piglets. The G/H models are characterized by sloughing epithelial cells, severe edema in the submucus and muscle layers, villous damage and destruction, separation of the mucosa and lamina propria, and, ultimately, complete destruction and necrosis of all epithelial structures, representing a presentation similar to that of human NEC (Figure 1, F–J).30Jilling T. Lu J. Jackson M. Caplan M.S. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis.Pediatr Res. 2004; 55: 622-629Crossref PubMed Scopus (163) Google Scholar This model has been adapted and more fully understood over the years, and it is now known that hypoxia, formula gavage, and intestinal bacteria are required for the development of NEC-like pathophysiology and histological factors.31Caplan M.S. Hedlund E. Adler L. Hsueh W. Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis.Pediatr Pathol. 1994; 14: 1017-1028Crossref PubMed Scopus (187) Google Scholar The mouse G/H model was originally described with pups delivered by Cesarean section (E20-21) but has recently been simplified to use newborn pups gavaged with adult commensal bacteria.28Sodhi C. Richardson W. Gribar S. Hackam D.J. The development of animal models for the study of necrotizing enterocolitis.Dis Model Mech. 2008; 1: 94-98Crossref PubMed Scopus (85) Google Scholar, 29Tian R. Liu S.X. Williams C. Soltau T.D. Dimmitt R. Zheng X. De Plaen I.G. Characterization of a necrotizing enterocolitis model in newborn mice.Int J Clin Exp Med. 2010; 3: 293-302PubMed Google Scholar This simplified model will encourage the use of the plethora of available knockout and transgenic mice to further our understanding of NEC. In addition, the G/H model has also has been used for linking several components of the innate immune system to the pathogenesis of NEC. Mucin production, SIgA, TLR4 signaling, AMP expression, and epithelial junctional proteins have all been shown to impact the degree of intestinal injury in animals that are subjected to the G/H model (details in Proposed Innate Immune Mechanisms Leading to NEC); however, the role of innate immune cells in the pathogenesis of NEC has not been investigated to date. Challenges associated with the G/H model include significant variability across research groups regarding the specific age at which the protocol is started, the type of formula administered, whether the formula is supplemented with bacteria and/or LPS, and the modality of hypoxia treatment. These challenges make it difficult to reproduce the model between laboratories and to unify the knowledge gained in the multitude of studies. Although the piglet model is not widely used because of costs and a lack of reagents, the piglet gastrointestinal system offers the resemblance perhaps closest to that in humans, particularly in the neonatal and newborn stages.32Sangild P.T. Thymann T. Schmidt M. Stoll B. Burrin D.G. Buddington R.K. Invited Review: the preterm pig as a model in pediatric gastroenterology.J Anim Sci. 2013; 91: 4713-4729Crossref PubMed Scopus (176) Google Scholar In pigs, the hypoxia and gavage are not always paired because one insult (either hypoxia/hypothermia or formula feedings) can result in NEC-like illness.33Sangild P.T. Siggers R.H. Schmidt M. Elnif J. Bjornvad C.R. Thymann T. Grondahl M.L. Hansen A.K. Jensen S.K. Boye M. Moelbak L. Buddington R.K. Weström B.R. Holst J.J. Burrin D.G. Diet- and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs.Gastroenterology. 2006; 130: 1776-1792Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar The I/R model of intestinal injury is caused by occluding the superior mesenteric artery, thus obstructing blood flow to the small bowel for a short time, followed by a period of reperfusion.34Musemeche C.A. Baker J.L. Feddersen R.M. A model of intestinal ischemia in the neonatal rat utilizing superior mesenteric artery occlusion and intraluminal platelet-activating factor.J Surg Res. 1995; 58: 724-727Abstract Full Text PDF PubMed Scopus (28) Google Scholar In the intestine, the initial ischemia results in damage to the epithelial cells, whereas the subsequent reperfusion injury causes significant damage to the rest of the mucus layer.35Mallick I.H. Yang W. Winslet M.C. Seifalian A.M. Ischemia-reperfusion injury of the intestine and protective strategies against injury.Dig Dis Sci. 2004; 49: 1359-1377Crossref PubMed Scopus (547) Google Scholar Histologically, this model is typically evaluated based on the extent of necrosis in the intestine, ranging from necrosis of the villus tip to transmural necrosis penetrating the muscle layer (Figure 1, K–O).36Clark D.A. Fornabaio D.M. McNeill H. Mullane K.M. Caravella S.J. Miller M.J. Contribution of oxygen-derived free radicals to experimental necrotizing enterocolitis.Am J Pathol. 1988; 130: 537-542PubMed Google Scholar This model attempts to replicate the ischemia that is thought to occur before NEC; however, it is more accurately classified as a model of intestinal injury rather than as a true model of NEC. The I/R model is particularly useful for investigating the role of free-radical damage in intestinal injury, as high levels of oxidative damage are induced after reperfusion (mimicking the potential reperfusion after an ischemic event in newborn humans). The primary strength of the I/R model is that it leads to defined and reproducible mucosal injury and barrier dysfunction. Both TLR4 and junctional protein expression have been shown to impact intestinal injury in the I/R model.37Dimmitt R.A. Staley E.M. Chuang G. Tanner S.M. Soltau T.D. Lorenz R.G. Role of postnatal acquisition of the intestinal microbiome in the early development of immune function.J Pediatr Gastroenterol Nutr. 2010; 51: 262-273PubMed Google Scholar The administration of PAF, combined with bacterial LPS, has been reported to initiate NEC-like pathologies in both mice and rats.38Sun X. Caplan M.S. Liu Y. Hsueh W. Endotoxin-resistant mice are protected from PAF-induced bowel injury and death. Role of TNF, complement activation, and endogenous PAF production.Dig Dis Sci. 1995; 40: 495-502Crossref PubMed Scopus (21) Google Scholar, 39Gonzalez-Crussi F. Hsueh W. Experimental model of ischemic bowel necrosis. The role of platelet-activating factor and endotoxin.Am J Pathol. 1983; 112: 127-135PubMed Google Scholar PAF has been identified as a proinflammatory phospholipid, causing vascular permeability, enhanced leukocyte degranulation, adhesion, and chemotaxis, as well as the production of tumor necrosis factor α, IL-6, and IL-8. PAF is primarily produced by innate immune cells, such as neutrophils, basophils, and monocytes, as well as endothelial cells and platelets. To induce NEC-like necrosis, PAF and LPS are injected directly into the mesenteric vascular circulation. In rats, the hemorrhagic lesions of necrosis observed in the jejunum and ileum are similar to those observed in human NEC. On microscopic examination, severe cases demonstrate transmural necrosis, with loss of mucosal architecture and sloughing of epithelial cells.40Maheshwari A. Kelly D.R. Nicola T. Ambalavanan N. Jain S.K. Murphy-Ullrich J. Athar M. Shimamura M. Bhandari V. Aprahamian C. Dimmitt R.A. Serra R. Ohls R.K. TGF-β2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine.Gastroenterology. 2011; 140: 242-253Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Dithizone, when administered i.v. to rats, leads to cell death in 25% to 30% of Paneth cells.41Sawada M. Takahashi K. Sawada S. Midorikawa O. Selective killing of Paneth cells by intravenous administration of dithizone in rats.Int J Exp Pathol. 1991; 72: 407-421PubMed Google Scholar This technique was used in conjunction with the administration of enteric pathogens (Escherichia coli, Klebsiella pneumonia) to induce NEC-like pathology in one study.42Sherman M.P. Bennett S.H. Hwang F.F. Sherman J. Bevins C.L. Paneth cells and antibacterial host defense in neonatal small intestine.Infect Immun. 2005; 73: 6143-6146Crossref PubMed Scopus (52) Google Scholar That study revealed gross necrotic lesions as well as damage to the intestinal tissue, including mucosal edema, loss of villous integrity, areas of transmural nec
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