Animal models of gastrointestinal and liver diseases. Animal models of necrotizing enterocolitis: pathophysiology, translational relevance, and challenges

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<ul><li><p>Animal models of gastrointestinal and liver diseases. Animal models ofnecrotizing enterocolitis: pathophysiology, translational relevance,and challenges</p><p>Peng Lu,1 Chhinder P. Sodhi,1 Hongpeng Jia,1 Shahab Shaffiey,1 Misty Good,3 Maria F. Branca,1</p><p>and David J. Hackam1,21Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 2Division of PediatricSurgery, Childrens Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania; 3Division of Newborn Medicine, Departmentof Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania</p><p>Submitted 9 December 2013; accepted in final form 18 April 2014</p><p>Lu P, Sodhi CP, Jia H, Shaffiey S, Good M, Branca MF,Hackam DJ. Animal models of gastrointestinal and liver diseases.Animal models of necrotizing enterocolitis: pathophysiology, transla-tional relevance, and challenges. Am J Physiol Gastrointest LiverPhysiol 306: G917G928, 2014. First published April 24, 2014;doi:10.1152/ajpgi.00422.2013.Necrotizing enterocolitis is the lead-ing cause of morbidity and mortality from gastrointestinal disease inpremature infants and is characterized by initial feeding intoleranceand abdominal distention followed by the rapid progression to coag-ulation necrosis of the intestine and death in many cases. Although therisk factors for NEC development remain well accepted, namelypremature birth and formula feeding, the underlying mechanismsremain incompletely understood. Current thinking indicates that NECdevelops in response to an abnormal interaction between the mucosalimmune system of the premature host and an abnormal indigenousmicroflora, leading to an exaggerated mucosal inflammatory responseand impaired mesenteric perfusion. In seeking to understand themolecular and cellular events leading to NEC, various animal modelshave been developed. However, the large number and variabilitybetween the available animal models and the unique characteristics ofeach has raised important questions regarding the validity of particularmodels for NEC research. In an attempt to provide some guidance tothe growing community of NEC researchers, we now seek to reviewthe key features of the major NEC models that have been developedin mammalian and nonmammalian species and to assess the advan-tages, disadvantage, challenges and major scientific discoveriesyielded by each. A strategy for model validation is proposed, theprincipal models are compared, and future directions and challengeswithin the field of NEC research are explored.</p><p>necrotizing enterocolitis; animal model; TLR4; innate immunity;microflora</p><p>NECROTIZING ENTEROCOLITIS (NEC) is the leading cause of deathfrom gastrointestinal disease in premature infants and is nowrecognized as a major cause of morbidity in patients who arefortunate to survive the initial disease. NEC develops in 1 in1,000 live births (51), and since there were 3.95 million livebirths in 2012 according to the Centers for Disease Control andPrevention, 4,000 infants were expected to develop thisdevastating disease in the past year alone. The typical infantwith NEC is a premature child born at around 30 wk gestation,who was offered infant formula for nutrition, and who at 23</p><p>wk of age develops feeding intolerance, abdominal distention,and bloody stools. In approximately half of cases, the childbecomes progressively ill, with systemic sepsis and cardiovas-cular collapse requiring aggressive resuscitation. Clinical de-terioration leads to abdominal exploration in nearly 30% ofcases, where the hallmarks of NEC, i.e., death of parts or all ofthe small and/or large intestine, are encountered. Risk factorsfor NEC development include prematurity and the administra-tion of formula feeds, and treatment is relatively nonspecific,which includes a combination of broad-spectrum antibioticsand removal of the diseased intestine (78). Although a fulldescription of the clinical course and management of NEC arebeyond the scope of the present review, the current consensusindicates that the incidence of NEC is either stable or increas-ing in frequency, that the overall survival for patients with thisdisease has not changed since the initial description 30 yearsago (12, 72), and that the societal costs for patients whodevelop the disease, especially due to the long-term neurode-velopmental and intestinal effects, are enormous (33).</p><p>Insights into the pathophysiology of NEC have been gainedthrough the study of clinical specimens and the careful selec-tion and usage of animal models. Although the pathophysiol-ogy of NEC is generally agreed to be multifactorial (40, 78),our understanding of the cellular and physiological mecha-nisms that lead to NEC has changed significantly in the pastdecade or so. Initially, NEC was thought to develop as a resultof an ischemic event leading to a disruption of an immatureintestinal epithelium and mucosal barrier, which leads to bac-terial translocation and the development of systemic sepsis(77). Although this explanation could account for the presenceof intestinal epithelial disruption that is characteristic of NEC,it could not account for the presence of intestinal necrosis thatis seen in patients with this disease, nor could this explanationreadily reconcile the observation that most patients who de-velop NEC do not actually have an antecedent ischemic event(104). Previous authors have reviewed the major mechanisticfactors thought to lead to the development of NEC (46, 78),and although a thorough evaluation is beyond the scope of thepresent review, current thinking suggests that formula feedingof the premature host that is colonized with a select microbialflora leads to an enhanced cellular and innate immune responsein the context of host genetic factors and an impaired intestinalmicrovascular network, resulting in aggressive mucosal in-flammation and reduced mesenteric perfusion. The ensuingtissue death leads to a cascade of bacterial translocation,systemic sepsis, and multisystem organ failure. There are many</p><p>Address for reprint requests and other correspondence: D. J. Hackam,Division of Pediatric Surgery, Childrens Hospital of Pittsburgh of UPMC,One Childrens Hospital Dr., 4401 Penn Ave., Pittsburgh, PA 15224 (</p><p>Am J Physiol Gastrointest Liver Physiol 306: G917G928, 2014.First published April 24, 2014; doi:10.1152/ajpgi.00422.2013. Themes</p><p>0193-1857/14 Copyright 2014 the American Physiological Society G917</p><p></p></li><li><p>important pathogenetic questions that remain to be answeredincluding how the process leading to mucosal inflammation isinitiated, whether the abnormal bacterial colonization that isobserved in the premature infant with NEC is a cause or aconsequence of the disease, how the inflammatory responsewithin the intestinal epithelium is coordinated, and the deter-minants of when a tipping point is reached, beyond whichreversibility and restoration of the intestinal mucosal barrierare unachievable. To address these (among others) importantgaps in our knowledge, careful study of clinical specimensfrom infants with and without NEC alongside carefully vali-dated animal models of this disease is required. This reviewwill now summarize the considerations in the selection ofanimal models for NEC research to address these importantquestions, will assess the key challenges associated with ani-mal models in this field, and will then discuss the key animalmodels available to researchers with an assessment of the keycontributions already gained from each, their strengths andtheir weaknesses. A summary of the major results from animalmodels that have provided a unifying approach to our under-standing of the causes of NEC and the strategies that preventthis disease is shown in Fig. 1.</p><p>General Considerations and Challenges in the Selection ofAnimal Models for NEC Research</p><p>There are some unique challenges associated with the selec-tion of appropriate animal models for use in NEC research.Many of these challenges pertain to the unique features of NECthat distinguish this disease from other inflammatory or septicprocesses, and which together reduce the number of reliable</p><p>options that are available for animal-based NEC research. Forinstance, NEC is known to primarily affect premature infantswho receive formula as opposed to breast milk. Thus, if onewishes to reliably reproduce the clinical disease in animals, amodel in an animal at an age that shows features of immaturityneeds to be selected. Furthermore, although models in smallanimals such as mice and rats offer the advantages of being ofrelatively low cost and readily available in large numbers, theylack the clinical robustness that is seen in larger animal modelssuch as piglets, in which the comparison to a premature humanis more readily obvious (a premature human infant that devel-ops NEC can be of a similar size and weight as a prematurepiglet). More important considerations for the selection of aparticular animal model relate to the specific question that isbeing asked, and a subsequent determination of how reduc-tionist the particular animal model needs to be. For instance,if the intent is to test a particular molecular pathway that ishypothesized to play a role in the pathogenesis of NEC, orperhaps a new therapeutic agent for the prevention or treatmentof NEC, then an animal model should be selected in which thispathway is affected under conditions that mimic the keyfeatures of NEC as seen in humans. By contrast, if the inves-tigator wishes to test a reagent that may be capable of inhib-iting or enhancing the signaling through a specific pathway thatis already known to be important in NEC pathogenesis, then areductionist approach can be selected in which this particularpathway is manipulated and subsequent effects on remotetissues including the gut can be assessed. Furthermore, asknowledge of the underpinnings of NEC have evolved, theanimal models that researchers employ to study the diseasemust necessarily evolve. For instance, with a greater under-standing that an abnormal community of enteric bacteria playsan important role in the pathogenesis of NEC in humans,comes the need to actively incorporate enteric bacteria associ-ated with NEC development in animal models of this disease.Additional considerations for the selection of a particularmodel include the cost, ease of implementation of the strategyfor induction of NEC, need for gene deletion, and availabilityof the required reagents. However, the overriding determina-tion of a particular model should be its relevance to the diseasein question, and for this reason it is important to understandhow a particular model of NEC should be validated, as will bediscussed below. An overview of each of the commonly usedanimal models used in NEC research is provided in Tables 13,and a comparative analysis is summarized in Table 4.</p><p>How Should Animal Models of NEC Be Validated?</p><p>The relatively large number of animal models that investi-gators have used to study NEC suggests that an approach isrequired to clearly define what constitutes a model of NEC inthe first place and to then apply a validation scheme so that theresults of studies can be compared within an appropriatecontext. At a minimum, animal models should replicate mostof the cardinal histopathological features that are seen inhuman NEC, which include the presence of mucosal edema,pneumatosis intestinalis (i.e., the presence of gas within thewall of the intestine due to microbial activity), epithelialsloughing/villous atrophy, evidence of enterocyte apoptosis,vascular thrombosis, and discontinuous necrotic segments ofintestine (so called skip lesions; Ref. 78). In addition, as a</p><p>Prematurity </p><p>Increased TLR4 </p><p>Stressed microbiome </p><p>Breast milk Amniotic fluid TLR4 blockade </p><p> Increased mucosal injury </p><p> Reduced intestinal perfusion </p><p> Reduced mucosal repair </p><p> Intestinal stem cell loss </p><p> Increased mucosal injury </p><p> Reduced intestinal perfusion </p><p> Reduced mucosal repair </p><p> Intestinal stem cell loss </p><p>Premature infant with NEC </p><p>TLR9 agonists Cytosolic Hsp70 </p><p>Immature immunity </p><p>Probiotics </p><p>Clinical Features of NEC: abdominal distention ventilatory support ill appearing feeding intolerance </p><p>Fig. 1. Pathogenesis of necrotizing enterocolitis (NEC). Graphical representa-tion of the mechanisms that lead to the development of NEC, as well asprotection from NEC, based on findings from experiments performed in animalmodels as discussed in this review.</p><p>Themes</p><p>G918 ANIMAL MODELS OF NECROTIZING ENTEROCOLITIS</p><p>AJP-Gastrointest Liver Physiol doi:10.1152/ajpgi.00422.2013</p></li><li><p>validation strategy, it should be possible to attenuate theseverity of NEC in a particular animal model through the useof experimental strategies that have been shown to attenuateNEC in humans, such as the administration of broad-spectrumantibiotics (57) or the substitution of formula with breast milk(44). Furthermore, the mechanisms by which the models areinduced should parallel the clinical condition in human NEC tothe extent that is possible. In this regard, models that includethe simple occlusion of the blood supply to the bowel (90) ormechanical obstruction of the distal bowel (27) are likely tohave a reduced overlap with models that meet all of the abovecriteria. By contrast, models that include factors associatedwith NEC development, including prematurity, formula ga-vage, manipulation of the microbiome, and external stressorssuch as hypothermia or hypoxia, are more likely to result in aphenotype that resembles the clinical condition.</p><p>Experimental Necrotizing Enterocolitis in Rats</p><p>The earliest rat NEC model, which was developed by for-mula feeding and hypoxia in newborn rats, was described byBarlow and colleagues in 1974 (7), the results of which led tothe conclusion that the gut flora and absence of breast milkwere suggested to play important roles in NEC pathogenesis.One year later, this model was modified to include the combi-nation of formula feeding, hypoxia, and hypothermia (6) toincrease the severity of the histological changes that were seen.In this model, formula feeding and hypoxia-hypothermia(100% nitrogen for 50 s followed by exposure to 4C for 10min) are the two critical instigating factors, since all maternallyfed animals or animals without exposure to hypoxia-hypother-mia showed normal intestinal histology. To prevent any ma-ternal milk exposure, researchers have studied premature neo-natal rats delivered by the cesarean section 1 day before thescheduled birth (14) or rat pups collected immediately afterbirth (29). This rat NEC model includes the widely acceptedmajor risk factors of NEC, i.e., intestinal immaturity, enteralfeeding, and inappropriate bacterial colonization (78), and istherefore widely considered to be very applicable for the studyof pathogenetic mechanisms associated with this disease.</p><p>Many laboratories have used this model to study variousaspects of the pathogenesis of NEC. These include the expres-sion of the cytokines...</p></li></ul>