Inflammopharmacol DOI 10.1007/s10787-014-0207-y

Inflammopharmacology

REVIEW

Animal models of inflammatory bowel disease: a review Nidhi Goyal • Ajay Rana • Abhilasha Ahlawat Krishna Reddy V. Bijjem • Puneet Kumar

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Received: 27 March 2014 / Accepted: 9 May 2014 Ó Springer Basel 2014

Abstract Inflammatory bowel disease (IBD) represents a group of idiopathic chronic inflammatory intestinal conditions associated with various areas of the GI tract, including two types of inflammatory conditions, i.e., ulcerative colitis (UC) and Crohn’s disease (CD). Both UC and CD are chronic inflammatory disorders of the intestine; in UC, inflammation starts in the rectum and generally extends proximally in a continuous manner through the entire colon. Bloody diarrhea, presence of blood and mucus mixed with stool, accompanied by lower abdominal cramping, are the characteristic symptoms of the disease. While in CD, inflammatory condition may affect any part of the GI tract from mouth to anus. It mainly causes abdominal pain, diarrhea, vomiting and weight loss. Although the basic etiology of IBD is unknown, there are several factors that may contribute to the pathogenesis of this disease, such as dysregulation of immune system or commensal bacteria, oxidative stress and inflammatory mediators. In order to understand these different etiological factors, a number of experimental models are available in the scientific research, including chemical-induced, spontaneous, genetically engineered and transgenic models. These models represent Collection of data For this review article, literature has been surveyed thoroughly, and the references that were relevant to the study have been selected without any kind of biasing. For the collection of data, we have searched on PubMed, Google Scholar, Science Direct, Plos One, etc., and approximately 2,997 articles were obtained for animal models of IBD, while approximately 900 articles were there each for UC and CD. But only the articles that were recent and relevant to our study have been selected for the compilation of data. N. Goyal  A. Rana  A. Ahlawat  K. R. V. Bijjem  P. Kumar (&) Department of Pharmacology, I.S.F College of Pharmacy, Moga 142001, Punjab, India e-mail: [email protected]

a major source of information about biological systems and are clinically relevant to the human IBD. Since there is less collective data available in one single article discussing about all these models, in this review an effort is made to study the outline of pathophysiology and various types of animal models used in the research study of IBD and other disease-related complications. Keywords Inflammatory bowel disease  Ulcerative colitis  Crohn’s disease  Pathogenesis  Inflammatory mediators

Introduction Inflammatory bowel disease (IBD) is an idiopathic, chronic and relapsing intestinal inflammatory disorder, characterized by abdominal pain and diarrhea. It may result in significant morbidity and mortality, with compromised quality of life and life expectancy (Yan 2012). The term IBD covers the two main diseases that are Crohn’s disease (CD) and ulcerative colitis (UC), and both have overlapping and distinct clinical and pathological features (Uhlig 2013). CD and UC are chronic remittent or progressive inflammatory conditions that may affect the entire GI tract and the colonic mucosa which may be associated with an increased risk for colon cancer (Arthur et al. 2010). Most of the clinical and pathological characteristics of CD and UC are same, but they also have some markedly different features with regard to the regions of the GI tract that may be affected as well as in the distribution and depth of inflammation (Hemstreet 2010). Crohn’s disease is an inflammatory condition that may affect any part of the GI tract from mouth to anus. It mainly causes abdominal pain, diarrhea, vomiting and weight loss

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(Baumgart and Sandborn 2012). On the other hand, UC is a condition in which the inflammatory response and morphological changes remain confined to the colon, invariably involving the rectum. It involves only the innermost lining or mucosa, manifesting as continuous areas of inflammation, ulceration, edema, and hemorrhage along the length of the colon (Orda´s et al. 2012). The most consistent feature of UC is the presence of blood and mucus mixed with stool, accompanied by lower abdominal cramping which is most intense during the passage of bowel movements (Orda´s et al. 2012). In Western Europe and USA, CD and UC have an incidence of approximately 6–8 cases per 100.000 populations and an estimated prevalence of approximately 70–150 per 100.000 populations. The peak occurrence is between ages 15 and 35. Both sexes are equally affected in case of these diseases (Burisch 2014). Since the basic etiology of IBD is not known, still there are several factors which may contribute to the pathophysiology of this disease, for example, dysregulation of innate and adaptive immune system, commensal bacteria, oxidative stress, inflammatory mediators and disruption of tight junctions. In order to study these different etiological factors, various experimental models are available in the scientific research, which includes chemical-induced, spontaneous, genetically engineered and transgenic models. These models represent a major source of information about biological systems and pathogenesis of the disease and are clinically relevant to both human UC as well as CD. Though there is less collective data available in any article discussing about all the models, in this review we have made an effort to study the outline of pathophysiology involved in the disease and various types of animal models that can be used in the research study of IBD and other disease-related complications.

Pathophysiology Although the exact cause of IBD remains undetermined, it appears to be related to both genetic and environmental factors. Among the pathological findings associated with IBD are dysregulated response of innate and adaptive immune system, loss of tolerance to commensal bacteria, disrupted mucosal barrier, an increase in inflammatory mediators and oxidative stress. Innate and adaptive immunity The innate and adaptive immune responses are the two types of effector classes of immune system. Innate immune response acts as a first line of defense and provides nonspecific protection, while adaptive immune response is highly specific in nature and gets activated by the innate immunity (Wallace et al. 2014).

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Innate immunity consists of epithelial barrier, neutrophils, macrophages, dendritic cells, natural killer cells (NK cells), etc. This form of immunity is activated by the microbial agents recognized by the pattern recognition receptors, such as toll-like receptors (TLRs) and NOD-like receptors (Pastorelli et al. 2013). Neutrophils along with other cells initiate inflammation by releasing pro-inflammatory cytokines, such as TNF-a, IL-a, IL-6 and IL-8, which further activate the adaptive immune response (Wallace et al. 2014). Adaptive immune system comprises of T lymphocytes and B-cells that on activation releases cytokines and antibodies. T cells are thought to be the main contributor of IBD due to the increased release of pro-inflammatory mediators. Th-1 cells produce a large amount of interferons (IFNs), induced by IL-12, and cause pathogenesis in CD patients. On the other hand, Th-2 cells are involved in the production of IL-4, IL-5, and IL-13 and cause inflammation in UC patients (Zhang and Li 2014). Recent reports have shown that a third type of T-helper cell is also involved in IBD pathogenesis, i.e., Th-17 cells (Siegmund and Zeitz 2011). These cells produce IL-17 and IL-22, both of which are pro-inflammatory cytokines capable of promoting local tissue destruction (Siegmund and Zeitz 2011). These cytokines further increase the gut permeability by disrupting epithelial barrier and results into uncontrolled inflammation. Commensal bacteria The commensal bacterial flora is the environmental factor most frequently implicated in the development of IBD because intestinal inflammation does not develop when animals are kept under sterile (germ-free) conditions. This led to the current theory of ‘‘no bacteria, no IBD’’ (Danese et al. 2004; Kamada et al. 2013). Although the large numbers of bacteria colonize the lumen of the intestine, several studies have revealed that under normal conditions, the intestinal mucosa is relatively free of adherent bacteria due to the presence of protective mucosal layer on epithelium (Zhang and Li 2014). However, in IBD patients, because of the alterations in the mucus-epithelial layer, a more intimate association of gut bacteria with the inner layer of mucosa is observed which acts as a pathogen and stimulates the inflammatory response by binding on antigen-presenting cells (APC) leading to mucosal injury (Wallace et al. 2014). Though the identities of the commensal bacteria that may be involved in the pathogenesis of IBD are unclear, some investigators have pointed to a role of anaerobic bacteria such as Bacteroides or Clostridium spp. and aerobic E. coli that are dramatically increased in IBD mucosa (Beisner et al. 2010).

Animal models of inflammatory bowel disease

Mucosal barrier The intestinal epithelium is a first line of defense against luminal bacteria, which consists of absorptive and secretory cells such as goblet cells and small intestinal Paneth cells (Beisner et al. 2010). The integrity of layer is maintained by several proteins which include tight junctions, adherens junctions and desmosomes. It does not only provide mechanical barrier but also release number of microbe-killing molecules (Beisner et al. 2010). Disruption of this barrier results in uncontrolled or dysregulated gut epithelial permeability, which can induce an overactive mucosal immune response and chronic intestinal inflammation (Pastorelli et al. 2013). The exact mechanism of the loss of barrier is not known, but several studies have reported that cytokines, TNF-a, interleukins, etc. involved in the pathogenesis of UC and CD may attribute to the increased epithelial permeability and alteration of tight junctions resulting into barrier disruption (McGuckin et al. 2009). Inflammatory mediators IBD is a condition in which several genetic, environmental and biological factors trigger inappropriate immune responses that involve the over-production of different proinflammatory mediators such as TNF-a, ILs, cytokines and chemokines (Pedersen et al. 2014). There are certain differences in the inflammatory mediator profile of CD and UC such as IL-2 and IFN-c are responsible for the inflammation in CD while IL-4, IL-5 and IL-10 are involved in UC pathogenesis (Monteleone et al. 2002). The concentration of these mediators is highly elevated in blood, stool and intestinal mucosa of IBD patients. The release of these inflammatory cells is regulated by different pathways involved in inflammation, i.e., NF-jB and MAPK pathway and JAK/STAT pathway (Pedersen et al. 2014). Nuclear factor-jB (NF-jB) and mitogen-activated protein kinases (MAPK) are activated by the toll-like receptors 4 (TLR-4) which further stimulate the release of TNF-a, IL-6 and IL-12 through macrophages, exacerbating the inflammatory response (Coskun et al. 2011). In addition to this, cytokines exert their signaling by activating JAK/ STAT pathway which is involved in the production of a number of interleukins. JAK when phosphorylate STAT proteins, they translocate from cytoplasm into nucleus to alter gene expression of target genes (Coskun et al. 2013). The pro-inflammatory mediators release through these pathways result in the progression of disease. Oxidative stress In IBD patients, an imbalance exists between oxidative species, such as superoxide ions, peroxynitrite ions and

hydrogen peroxide, and endogenous antioxidant levels, such as glutathione (GSH), catalase and superoxide dismutase (SOD), which results in the oxidative stress (Kruidenier and Verspaget 2002). These ROS are generated by the activated neutrophils and macrophages, and can be assessed by examining the amount of 8-hydroxy-20 deoxyguanosine (8-OHdG) in blood or malondialdehyde (MDA) in colonic biopsies, which are known biomarker for oxidative damage (Kim et al. 2012). These various factors are interlinked and functions in a viscous cycle which contributes to the progression of disease leading to uncontrolled inflammation. Various pathways involve in the pathogenesis of IBD as shown in Fig. 1.

Animal models of inflammatory bowel diseases Since the exact etiology of IBD is not known yet, a number of animal models have been developed over past decades in order to study the possible mechanism involved in pathogenesis of disease and new therapeutic targets. The different types of animal models are developed for the study of IBD, which are discussed here, emphasizing on their clinical and histological features showing relevance to human IBD (Tables 1, 2). Chemically induced model Dextran sulfate sodium (DSS) Dextran sulfate sodium is a synthetic sulfated polysaccharide composed of dextran and sulfated anhydro-glucose unit (Tran et al. 2012). Supplementing the drinking water of rodents with low molecular weight DSS (54,000 mol. wt.) results in symptomatic features resembling UC (Laroui et al. 2012). The effectiveness of DSS-induced colitis depends on several factors, including molecular weight (5 kDa for mild and 40 kDa for severe colitis), dosage (usually 1–5 %), duration (acute or chronic), strain of animals (C3H/HeJ and Balb/c mice strains are more susceptible), sex of animals (male mice are more susceptible) and microbial environment of animals (e.g., germ-free [GF] vs. specific pathogen-free [SPF]) (Low et al. 2013a). Procedure DSS is commonly administered in a dose range of 3–10 % for 7–10 days to induce an acute inflammation depending on the susceptibility of the species (such as Balb/c mice are more susceptible) or the molecular weight of DSS. By prolonging the DSS administration, acute colitis may be extrapolated to chronic colitis by administering in three to five cycles with a 1- to 2-week rest between cycles (Tran et al. 2012), while in some species like adult female mice of the outbred strain Him:

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N. Goyal et al. Fig. 1 Various pathways involve in the pathogenesis of IBD. APC antigen-presenting cell, TLR 4 toll-like receptor 4, Teff cells T-effector cells, Th1 T-helper 1, Th2 T-helper 2, NFjB nuclear factor-jB, MAPK mitogen-activated protein kinase, JAK/STAT pathway Janus kinase/signal transducers and activators of transcription pathway, TNF-a tissue necrosis factor-a, IL interleukin, ROS reactive oxygen species, IFN-c interferonX c

OF1, dose of less than 3 % can also be used to induce colitis (Mitrovic et al. 2010). Clinical features DSS colitis in acute phase shows weight loss, diarrhea and occult blood in stools, piloerection, anemia, and eventually death, whereas in chronic phase of colitis usually do not reflect severity of inflammation or histological features found in large bowel (Persˇe and Cerar 2012). Molecular changes DSS upregulates the different cytokines, chemokines, nitric oxide and inducible nitric oxide synthase (iNOS) levels which are implicated in the pathogenesis of IBD (Marshall and Swain 2011; Johnson et al. 2014). DSS causes progressive and severe colitis with increased activation of NF-jB which further worsens the colitis and also increases the expression of Gall-R expression that can be an important component of the excessive fluid secretion observed in IBD (Dou et al. 2013). There is also increased expression of inflammatory cytokines genes (IL-1, TNF-a, IL-6 and IL-8), and some adhesion molecules genes [endothelial leukocyte adhesion molecule-1 (ELAM- 1) and ICAM-1] in IBD and both of them are regulated by the transcription factor, nuclear factor jB (NF-jB) (Algieri et al. 2014). Histological features Histological changes in acute DSS-colitis include mucin depletion, epithelial degeneration and necrosis leading to disappearance of epithelial cells which further leads to the formation of cryptitis and crypt abscesses. These cryptitis and crypt abscesses formed due to the migration of neutrophils are the common histological feature of human IBD but rarely reported in DSS-

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induced colitis (Dou et al. 2013). Chronic phase consists of mononuclear leukocytes infiltration, crypt architectural disarray, increasing the distance (widening of the gap) between crypt bases and muscularis mucosa, deep mucosal lymphocytosis and transmural inflammation, and the changes appear after few weeks of DSS administration (Persˇe and Cerar 2012). There is increased apoptosis and decreased proliferation of epithelium that take place in the acute phase of DSS further causing relevant leaks in the epithelial barrier (Araki et al. 2010). 2,4,6-Trinitrobenzene sulfonic acid (TNBS) TNBS is a hapten, which when bound with a high molecular tissue protein, turns into an antigen. Previous studies have shown that it can elicit the number of immunologic responses (Zheng et al. 2000) and represents both form of IBD, predominantly CD (Motavallian-Naeini et al. 2012). The major inflammatory mediators involved in this model are arachidonate metabolites, such as leukotriene B4 (LTB4) and the monhydroxyl fatty acids 5-HETE, 12-HETE and 15-HETE (Hassan et al. 2010). TNBS colitis develops as a delayed-type hypersensitivity reaction to haptenized proteins, whereas DSS colitis is the result of a change in epithelial barrier function (Brenna et al. 2013). Procedure: TNBS can be administered through a trocar needle using a rubber catheter inserted via the anus. The recommended dose of TNBS is 0.5–4.0 mg in 45–50 % ethanol intra-rectally, which varies between different mouse strains, such as SJL and BALB/c mice are highly

Animal models of inflammatory bowel disease

susceptible, whereas C57Bl/6 and 10 mice are resistant (Kawada et al. 2007). Ethanol is required in a high concentration as a vehicle for the TNBS administration since it acts as a barrier breaker so that TNBS can enter the mucosa to induce colitis where it covalently binds to the E-amino group of lysine and modify cell surface proteins (Tran et al. 2012). Clinical features This model showed features such as progressive weight loss, bloody diarrhea, rectal prolaspe and large bowel wall thickening (Tran et al. 2012). Molecular changes TNBS increases the level of various inflammatory mediators such as prostaglandin E2, thromboxane B2, leukotriene B4, 6-keto-prostaglandin F1a, leukotriene C4 and interleukins which play an important role in the pathogenesis of IBD (Dothel et al. 2013). This increase is also correlated with the colonic myeloperoxidase activity (Barnett and Fraser 2011). The increased level of PAF can also be another potential mechanism of TNBSinduced colitis, which is not seen at 1–4 days of TNBS administration but can be seen after 1–3 weeks (Camuesco et al. 2012). Histological features In the case of microscopic evaluation, severe and intense transmural inflammation and/or diffuse necrosis, inflammatory granulomas and submucosal neutrophils infiltration were observed in mucosal and submucosal layers (Motavallian-Naeini et al. 2012). Moreover, signs of cryptitis and architectural distortion were seen in endoscopic biopsies (Brenna et al. 2013). Advantages and disadvantages This model has many advantages which include (1) a simple process, (2)

reproducible colonic damage, (3) short duration of the experiment and (4) long-lasting damage accompanied by inflammatory cell infiltration and ulcers (Zheng et al. 2000). Moreover, this model can mimic both acute and chronic phases of inflammation since it is characterized as both Th1- and Th2-type cytokine-mediated colitis. Apart from these advantages, this model also suffers from some disadvantages like the absence of spontaneous relapse which is the hallmark of human IBD (Motavallian-Naeini et al. 2012). Also, ethanol itself causes severe inflammation in the intestinal mucosa which makes it difficult to distinguish between the ethanol-induced inflammation and hapten-induced inflammation (Tran et al. 2012). Recurrent TNBS-induced colitis This model is also an animal model for human IBD which is induced by repeated intra-rectal administration of TNBS leading to the development of chronic intestinal inflammation (Wang et al. 2011). The inflamed colon shows increased weight and increased thickness, especially in the distal part. Histopathologically, it is associated with increased inflammatory cellular infiltration which consists of CD4? and CD8? T cells, macrophages, granulocytes and mast cells, irregular crypts and loss of Goblet cells (Kremer et al. 2012). Previous studies have also revealed that the serum levels of pro-inflammatory cytokines along with IL-17, IFN-c, IL-1b and MIP-1a, showing that both TH1 and TH17 cells play a role in the inflammatory response (Mariman et al. 2012).

Table 1 Classification of animal models of IBD Animal models Chemically induced models

Bacterial induced models

Spontaneous models

DSS

Salmonella- induced

C3H/HejBir mice model

Genetically engineered models

Transgenic Mouse models

Mutation knock- in models

Adoptive transfer models

IL-7 Tg mice

TNFDARE

CD45RB high-transfer model

Conventional IL-10 -/-

TNBS

TGF-b -/-

STAT 4

DNBS

IL-2 -/-

HLA-B27

Oxazolone Acetic acid Carrageenan

Adherent invasive E. coli

NOD2 -/SAMP1/4it mice model

A20 -/MDR1A -/-

Indomethacin

Gai2 -/-

Iodoacetamide

TCRa -/IL-23 -/-

Immunologic model

Conditional

DNCB

XBP1 -/-

DNN

NEMO -/-

Cadherin Tg mice

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N. Goyal et al. Table 2 Classification of IBD models according to specificity for UC or CD

Category

Specificity UCs

Chemically induced models

CD

DSS

TNBS/DNBS

TNBS/DNBS

Indomethacin

Oxazolone Acetic acid Carrageenan Iodoacetamide Immunologic model

2, 4-Dinitrochlorobenzene (DNCB)

Bacterial induced models

Salmonella-induced Adherent invasive E. coli

Spontaneous models Genetically engineered models

C3H/HejBir mice model TGF-b -/-

SAMP1/4it mice model IL-10 -/-

IL-2 -/-

NOD2 -/-

MDR1A -/-

A20 -/-

Gai2 -/-

IL-23 -/-

TCRa -/-

NEMO -/-

XBP1 -/NEMO -/Transgenic mouse models

IL-7 Tg mice

STAT 4

HLA-B27

HLA-B27 DNN Cadherin Tg mice TNFDARE

Mutation knock-in models Adoptive transfer models

Dinitrobenzene sulfonic acid (DNBS) DNBS is hapten, which is used to induce colonic inflammation, and the feature of colitis in this model is similar to that of the TNBS model with bloody diarrhea and significant loss of body weight evident but is comparatively less hazardous (Dothel et al. 2013). DNBS and TNBS both bind to proteins, but TNBS has an additional active nitro group and binds more readily at lower concentrations. However, DNBS is more selective and binds only to the e-amino group of lysine (Tran et al. 2012). Procedure Earlier, 2, 4-dinitrobenzenesulphonic acid (DNBS; 25 mg/rat), dissolved in 50 % ethanol (total volume, 0.8 ml), was administered as an enema to induce UC, but recently, method was modified where colitis was induced in lightly anesthetized mice by an intra-rectal injection of 3 mg of DNBS in 100 ll of 50 % ethanol, delivered 3 cm into the colon via a polyethylene catheter (Cuzzocrea et al. 2001; Tran et al. 2012). Clinical features Decreased food intake, body weight, increased colon weight and changed stool consistency are the common symptoms to be seen, while macroscopic inspection revealed the presence of mucosal congestion, erosion, hemorrhagic ulcerations and severe inflammation in the colon, cecum and rectum (Joshi et al. 2011).

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CD45RB high-transfer model

CD45RB high-transfer model

Molecular changes In the rat, DNBS causes an overproduction of nitric oxide (NO) due to induction of inducible nitric oxide synthase (iNOS), which contributes to the inflammatory process (Darji et al. 2013). As in the TNBS model, DNBS induces a strong inflammatory response and a significant increase in myeloperoxidase (MPO) activity compared to controls (Ko et al. 2005). Histological features The histopathological features in DNBS-induced colitis model includes a trans-mural necrosis along with extensive morphological disorientation, edema and a diffuse leukocyte cellular infiltrate as well as lymphocyte in the submucosa of colon section (Joshi et al. 2011). This model also showed elevated levels of NO, MDA and decreased levels of SOD and GSH as compared to normal control animals (Darji et al. 2013). Oxazolone The oxazolone is another hapten that when administered intra-rectally with ethanol produced Th2-mediated acute colitis. Unlike TNBS-induced colitis, this model resembles only UC and is limited to the distal part of the colon only (Kawada et al. 2007). Oxazolone-induced colitis has been suggested to be dependent on the presence of IL13 producing invariant NK-T cells (Wirtz and Neurath 2007).

Animal models of inflammatory bowel disease

Procedure Colitis can be induced by applying 100 lL of 1 % oxazolone dissolved in a mixture of four parts acetone to one part olive oil to the shaved abdominal skin of male C57BL/6CrSlc mice under pentobarbital anesthesia. Then, 1 week later, 100 lL of 50 % ethanol solution with 1 % oxazolone was instilled rectally under anesthesia with pentobarbital and atropine (Kaneko et al. 2013). Recently, oxazolone-induced colitis has also been established as a chronic model in Balb/c mice via repeated intrarectal administration of oxazolone in ethanol. This allows the model to be used to define specific features of the inflammatory milieu that favors tumor development (Schiechl et al. 2011). Clinical features The clinical manifestations of this model are weight loss, thin stool with pus and blood, bowel wall thickening, erosion, edema and small patches of ulcer on colon (Patel et al. 2012). Molecular changes Oxazolone-induced colitis is characterized by a marked increase in production of IL-4 and IL-5 accompanied by the clinical and histological features (Boirivant et al. 1998). Chronic oxazolone-induced colitis begins as severe inflammation with corresponding weight loss, which transforms into chronic inflammation and partial weight recovery (Schiechl et al. 2011). The inflammation is marked by the rapid increase in the production of IL13 in the lamina propria and the appearance of NK T cells, both of which are immunologic features of acute oxazolone-induced colitis (Schiechl et al. 2011). Histological features The microscopic studies in this model revealed neutrophil infiltration, fibrin deposition, submucosal neutrophil migration, submucosal edema, epithelial necrosis and epithelial ulceration with loss of epithelial villi in the colon section along with the increase in MPO activity and decrease in number of goblet cells (Patel et al. 2012). Acetic acid Acetic acid produces acute inflammation restricted to the colon and mimics characteristic features of UC. Ulceration and crypt abnormalities can be induced by luminal instillation of dilute acetic acid in a dose-responsive fashion (Low et al. 2013a). The injury in this model was related to the epithelial necrosis and edema that variably extended into the gastric mucosal layers, depending on the concentrations and length of exposure of acetic acid (Nakao et al. 2014). Transient local ischemia might contribute to the acute injury, but neutrophils were apparently not involved in very early phases. Mucosa and submucosal inflammation followed initial injury and was associated with activation of NF-jB and other inflammatory mediators (Niu et al. 2013). Procedure Colitis can be induced by using 0.5 ml of 10–50 % acetic acid diluted with water instilled into the

rectum of male Wistar rats. After 10 s of surface contact, the acidic solution was withdrawn, and the lumen was flushed three times with 0.5 ml saline (Low et al. 2013). Lately, the modifications were made in which 1.5 ml of 4 % acetic acid (pH 2.3) was slowly infused 8 cm into the colon through anus with a rubber cannula of a lightly anesthetized rat. After a 30-s exposure, excess fluid was withdrawn, and the colon was flushed with 1.5 ml PBS (Sotnikova et al. 2013). Many further modifications have been introduced throughout the year, and most subsequent studies have used 2 ml of 4 % acetic acid solution using a 2.7-mm soft pediatric catheter under light ether anesthesia, and animal was held horizontally for 2 min to avoid AA solution leakage because higher concentrations induced frequent perforations (Aleisa et al. 2014). Clinical features Weight loss, decreased mucous production and increased colonic weight are the common manifestations of this model (Al-Rejaie et al. 2013; Aleisa et al. 2014). Extensive hemorrhage, occasional ulceration and bowel wall thickening were also observed in some studies (Sotnikova et al. 2013). Molecular changes In rodents, the protonated form of the acid liberates protons within the intracellular space and causes massive intracellular acidification resulting in massive epithelial damage (Das et al. 2013). The administration of acetic acid results in colonic epithelial destruction without inflammation within 4 h, which is then followed by an influx of acute inflammatory cells, and reaches its maximum intensity at 12 h. The inflammatory response is caused by non-specific factors after disruption of the epithelial barrier. The chemical injury heals within days in mice or 2–3 weeks in rats (Kawada et al. 2007). Histological features The microscopic observation of histopathological slides revealed eroded mucosa with ulceration and necrosis, including edema, goblet cell depletion, lymphoid follicular hyperplasia and heavy infiltration of inflammatory cells (Darwish et al. 2012; AlRejaie et al. 2013). Advantages and disadvantages The advantages of acetic acid-induced colitis are its low cost and the ease of administration. Of note, the epithelial injury observed within the first 24 h of acetic acid induction is not immunologic in nature. Thus, designing drugs that target immune responses should be tested at a time point after 24 h post-induction (Low et al. 2013a). Carrageenan Carrageenan is a high molecular weight sulfated polygalactan, derived from several species of red seaweeds (Rhodophyceae), including Gigartina, Chondrus and Eucheuma (Borthakur et al. 2007). Carrageenan triggers innate immune pathways of inflammation that resembles

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UC in which TLR4 and BCL10 play critical role (Bhattacharyya et al. 2011). Procedure Delivery of 10 % carrageen (degraded carrageenan) for 10 days in the drinking water of CF1 mice can induce colitis exhibiting characteristic macro- and microscopic features (Tran et al. 2012). Clinical features Bloody diarrhea, colon length shortening and pericryptal inflammation, with marked dilatation of the cecum and ascending colon, are the clinical features observed in this model (Tran et al. 2012). Molecular changes NFjB is a key determinant of the intestinal epithelial inflammatory cascade and occupies a central role in the transcriptional activation of pro-inflammatory genes. Previous studies have suggested that activation of NFjB in the intestinal cells following carrageenan exposure is largely attributable to an increase in Bcl10 (Borthakur et al. 2012). Bcl10 (B cell leukemia/ lymphoma) and TLR4 (Toll-like receptor 4) that reside in the cytoplasm relay receptor-mediated signals to activate NFjB which further leads to increase in IL-8 production and other inflammatory mediators (Borthakur et al. 2012). Histological features These include the mucosal ulceration with distorted crypt architecture, inflammatory infiltration of the lamina propria and hyperplastic epithelium, conditions that were more pronounced in the proximal colon but were also present in the distal colon (Benard et al. 2010). Indomethacin-induced enterocolitis Indomethacin induces small intestinal and colonic ulceration in a dose-dependent fashion in rodents. Although small bowel ulceration and transmural inflammation have some similarity to CD, the chronic ulcerations are located in the mid-small intestine rather than the ileum. It involves small and large intestines and is associated with extraintestinal lesions (Stadnicki and Colman 2003). Procedure Fasted male Wistar rats were treated subcutaneously with indomethacin 7.5 mg/kg solubilized in 100 % alcohol and then diluted with 5 % w/v sterile sodium bicarbonate, causing an acute inflammatory response. This acute response reaches its maximum intensity at 24 h and is completely resolved within 7 days, whereas two daily subcutaneous injections of indomethacin produce a chronic inflammation that lasts at least 2 weeks (Pawar et al. 2011). Clinical features They include acute intestinal inflammation, characterized by a thickening of the bowel wall, mesenteric hemorrhage, mesentery adhesion and multiple mucosal ulcers of small intestine and colon (Pawar et al. 2011). Molecular changes Initially, the epithelial damage is mediated partly by synthesis inhibition of the protective

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prostaglandins PGE1, PGE2 and prostacyclin. Luminal bacteria and bacterial products clearly contribute to the exacerbation and perpetuation of the chronic phase of indomethacin-induced inflammation (Jurjus et al. 2004). Histological features In previous studies, histopathological analysis of the colon/ileum in indomethacin-induced enterocolitis clearly showed that there was a significant degree of lymphoid hyperplasia, neutrophillic infiltration, crypt damage and submucosal inflammation when compared with normal animals (Behera et al. 2012). Iodoacetamide Iodoacetamide is a blocker of SH compounds (Sulfhydryl) in the colon. An endogenous SH compounds, such as glutathione, play an important role in the protection of gastric mucosa which when blocked by the iodoacetamide is known to induce UC and cause injury to the mucosa by decreasing the amount of defensive SH compounds (Jurjus et al. 2004). Procedure A single dose of 6 % iodoacetamide induces well-reproducible colonic lesions in female Sprague– Dawley rats seen in 1–2 h, leading to erosions and ulcers at 6–12 h, and then followed by extensive acute and chronic inflammation on 7–14 days. In brief, 0.1 ml of 6 % iodoacetamide dissolved in 1 % methylcellulose administered once by enema (7 cm from anus) via a rubber catheter can be used for the induction of colitis (Paunovic et al. 2011). Clinical and Histopathological features The different clinical alterations in this model include diarrhea, dilatation, adhesion, mucosal damage, increased colon wet weight and inhibition of body weight gain (Paunovic et al. 2011), whereas histological examination of the intestines showed deep mucosal and submucosal ulcerations associated with an inflammatory infiltrate consisting mainly of lymphocytes and macrophages (Kenet et al. 2001). Immunologic model of colitis Dinitrochlorobenzene (DNCB) DNCB is a hapten which when bound to tissue proteins is capable of eliciting a cell-mediated (T-lymphocytedependent) immune response with significantly higher levels of CD4? CD29? cells. Animals can be sensitized to DNCB by placing it on their skin. After the initial sensitization, reapplication of DNCB will induce an immune response to DNCB at the site. However, the sites of DNCB sensitization and challenge do not have to be the same (Jiang and Cui 2000). But in order to continue the progression of disease to chronic conditions, the presence of antigen/DNCB is necessary. The clinical and histological features of DNCB include crypt abscesses, superficial

Animal models of inflammatory bowel disease

mucosal ulcerations and depletion of mucus in the cells lining the crypts, edema of both the mucosa and submucosa, and infiltration of the lamina propria with plasma cells, lymphocytes and polymorphonuclear leukocytes, including eosinophils. However, due to self-limited course of 2 weeks of DNCB, it is too short to be utilized as an ideal model for UC (Jiang and Cui 2000). In order to overcome these shortcomings, a new chronic UC model was established, i.e., by using DNCB and AA in combination because of the following advantages: (1) Like human UC, it manifests mucus in stools, bloody diarrhea and weight loss; (2) it reflects the pathologic characteristics of UC, such as continuous superficial colonic inflammation. Microscopically there exist mucosal edema and congestion, infiltration of lymphocytes, plasma cells and polymorphonuclear cells, crypt abscesses and ulceration; (3) also, it is an immune response model; immunology is well studied in UC (Jiang et al. 2000; Xia et al. 1998). Bacterial induction of colitis Salmonella-induced colitis The gram-negative Salmonella typhimurium and Salmonella dublin are food-borne enteric bacterial pathogens that can cause intestinal diseases by oral infection which results in systemic infection further causing intestinal inflammation. The inflammation has similar histopathological characteristics to human UC, including epithelial crypt loss, erosion and neutrophilic infiltration (Mizoguchi 2006; Papanikolaou et al. 2007). The colitis is induced usually after systemic infection within 5–7 days of infection in C57BL/6 mice. Therefore, it is perceived that S. typhimurium infection is a valuable model to study the acute phase, but not later stages, of colitis (Low et al. 2013a). Adherent–invasive E. coli The commensal Adherent– Invasive Escherichia Coli (AIEC) adheres to both small and large intestinal epithelial cells (IECs) with equal affinity (Jensen et al. 2011). Induction of colonic inflammation in animal models using AIEC infection requires mild epithelial damage, such as low-dose DSS treatment, during the entire course of the infection. The phenotype of the colonic inflammation mimics UC, including body weight loss, presence of blood in stool and colonic neutrophilic infiltrations (Mizoguchi 2006, Low et al. 2013b). Administration of chitin microparticles (1–10 lm in size) into C57Bl/6 WT mice ameliorates colonic intestinal inflammation, by blocking the interaction of bacterial-derived factors (such as AIEC chiA) with host CHI3L1 (Nagatani et al. 2012). Spontaneous model This category can be divided into two related subcategories: animals that spontaneously develop mucosal

inflammation, and models in which a gene defect or a transgene causes an inappropriate mucosal immune response (Michelle et al. 2004). Spontaneous models represent one of the most attractive model systems for studying intestinal inflammation because, similar to human disease, inflammation occurs without any apparent exogenous manipulations. For example, the C3H/HeJBir murine model of colitis is characterized by spontaneous and chronic focal inflammation localized to the right colon and cecal region (Pizarro et al. 2003). C3H/HejBir Mice model C3H/HejBir is a derivative of selective breeding of C3H/Hej mice with colitis known to develop occasionally perianal ulcers and colitis (Michelle et al. 2004). Colitis in this model is limited to ileocecal lesions and the right side of the colon and occurs spontaneously in the third to fourth week of life and disappears after 10–12 weeks. Ulcers, crypt abscesses and regeneration of epithelium are seen, but thickening of the intestinal wall and granulomas is not observed. Increased levels of IFN-c and IL-2 have been detected in the lamina propria lymphocyte, which shows that colitis in this model is a Th type-1 response, and thus represents CD. This model has also been used in combination with inducible colitis models and has proven to be valuable for studying and identifying genetic susceptibility factors (Cong et al. 1998; Jurjus et al. 2004). SAMP1/Yit mice model This model is an excellent experimental system to study disease mechanisms of CD, because the inflammation occurs spontaneously, and it is one of the few models with severe inflammation in the terminal ileum, the primary location of CD lesions (Matsumoto et al. 1998). Lesions in this model are characterized by transmural inflammation, granulomatas and alterations in epithelial morphology. Increased epithelial permeability precedes the onset of inflammation, and epithelial cell dysfunction is primarily responsible for the disease progression (Sugawara et al. 2005). Ileitis established as early as 10 weeks of age and the incidence of skin lesions inversely correlated with the occurrence of intestinal inflammation (Riviera-Nieves et al. 2003). So, mainly they developed colitis and ileitis, with the infiltration of eosinophils (Modi et al. 2012). Genetically engineered models of colitis Genetically engineered models are the knock-out models in which an existing gene is inactivated or ‘‘knocked-out’’ and replaced with an artificial piece of DNA, i.e., conventional gene knock-out models. The knockout strategy can also be achieved by the conditional knock-out method in which a specific gene is deleted from a single organ of the body rather than the whole body (Reilly et al. 2012).

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Conventional gene knock-out models IL-10-/- IL-10 is a pivotal cytokine that establishes the physiological inflammatory condition in the gut by maintaining a check on pro-inflammatory responses to normal antigens and beneficial bacteria (Unutmaz and Pulendran 2009). In IL-10-deficient mice spontaneously develop a transmural pancolitis and cecal inflammation by 2–4 months of age that appears to resemble CD in humans and the inflammation occurs in the whole intestine, mainly in the duodenum, proximal jejunum and ascending colon (Berg et al. 1996; Modi et al. 2012). TGF-b-/- TGF-b-/- is produced by number of cells like macrophages and develops multi-organ dysfunction due to macrophage hyper-activation and reduced regulatory T cell activity, including severe colitis, and these mice die within 4–5 weeks (Kulkarni et al. 1993; Shull et al. 1992). The exact mode of action of TGF-b in the intestine remains unclear, but it appears to involve increased production of IL-10 and down-regulation of IL-12 receptor expression (Boismenu and Chen 2000). IL-2-/- IL-2 is a four bundled a-helical regulatory cytokine produced mainly by activated T lymphocytes, and its synthesis is tightly regulated at the mRNA level by signals from the TCR and CD28. IL-2 binds to and signals through a receptor complex. The small intestine of IL2 -/- mice remains intact, whereas the colon (from rectum to cecum) was severely affected with ulcers and wall thickening (Modi et al. 2012). Mice deficient in the cytokine IL-2 develop a chronic inflammation of the colonic mucosa/submucosa at 8–9 weeks of age that resembles UC (Baumgart et al. 1998). NOD2-/- NOD2 (Nucleotide-binding Oligomerization Domain 2) that is also known as caspase recruitment domain-containing protein 15 (CARD15) has been shown to be expressed in myeloid cell line, including macrophages, monocytes, Paneth cells and dendritic cells and is induced by the pro-inflammatory cytokines such as TNF-a and IFN-c, exhibiting features of CD. Mutations of NOD2 result in an alteration in the ability of NOD2 to activate NF-kB (Ogura et al. 2001; Gutierrez et al. 2002; Rosenstiel et al. 2003). A20-/- A20 is an ubiquitin-modifying enzyme in mice induced by TNFR, IL-1R and even NOD2. A20 is an inducible and broadly expressed cytoplasmic protein that inhibits TNF-induced NFjB activity. A20-deficient mice develop spontaneous inflammation, cachexia and premature death due to failure of A20 deficient cells to terminate TNF-induced NFjB responses (Lee et al. 2000). So mice deficient in A20 develop spontaneous colitis indicating that NOD2 signaling alone is not sufficient to cause colitis (Hammer et al. 2011).

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MDR1A-/- MDR1A gene is an ATP-binding transporter limited to intestinal epithelial cells and mucosal lymphocytes. Deletion of this gene induces a severe spontaneous colitis by TH1 type inflammation (Panwala et al. 1998). Mdr1a KO mice are devoid of the proper ability to dispose of bacterial breakdown products in epithelial cells which increases abnormal antigen presentation to neighboring T cells, leading to a marked T cell activation that drives the colitis (Tanner et al. 2013). Histology includes pancolitis with enlarged crypts, immune cell infiltration, crypt abscesses and ulceration similar to the pathology of UC in humans (Wilk et al. 2005). Gai2-/- Gai2 protein is a member of signal transduction molecules expressed in many cell types, including intestinal epithelial cells, myofibroblasts and intestinal T and B lymphocytes (Dalwadi et al. 2003; Edwards and Smock 2006). Deficiency of Gai2 in mice results in a spontaneous severe colitis developing around 8–12 weeks of age. Gai2-deficient colitis resembles that of UC, with the most severe inflammation occurring in the distal colon. The clinical and histopathological features include colonic thickening, lymphocyte and neutrophilic infiltrations, crypt and goblet loss, and crypt abscesses (Rudolph et al. 1995). TCRa-/- T-cell receptor a chain (TCRa) KO mice spontaneously developed chronic colitis, mediated by Th2type immune response. It closely resembles the human UC with an inflammatory pattern restricted primarily to the colonic mucosa. TCRa-/- mice develop colitis only after 12–16 weeks of age (Mombaerts et al. 1993). It is characterized with soft stools, associated with loss of goblet cells, and a mixed cellular infiltration mainly consisting of lymphocytes and neutrophils (Nagatani et al. 2012). IL-23-/- IL-23 belongs to IL-12 family of heterodimeric cytokines and is specific to mucosal inflammation rather than systemic inflammation. IL-23 is an important cytokine required for the initiation, generation and development of TH17 cytokines such as IL-17 (Lees et al. 2011). Conditional knock-out models (cell type-specific gene alteration) XBP1-/- Xbp1 is a protein essential for the development of secretory cells such as goblet and Paneth cells which are involved in mucus secretion in the gut (Shaffer et al. 2004). Deletion of this protein from small intestinal epithelium results in total loss of Paneth cells and reduced size and number of goblet cells which make it susceptible to UC (Kaser et al. 2008). NEMO-/- NEMO (IKKc) is a regulatory unit of the IKK complex. NEMO-/- model exhibit T cell-mediated immune response. Targeted deletion of this gene leads to the intestinal epithelial integrity (Nenci et al. 2007). The characteristic histopathological features of this model are

Animal models of inflammatory bowel disease

severe transmural pancolitis with enlargement of crypts, goblet cell ablation and mononuclear cell infiltration into the mucosa, similar to that observed in IBD (Nenci et al. 2007). Transgenic mouse models of colitis Transgenic mouse models of colitis, unlike knock-out mice, express one or more copies of the gene of interest resulting in over expression of that particular gene. However, similar to genetically engineered mice, the transgenic mice can be either conventional or cell-specific targeted conditional transgenic animals. Conventional transgenic mouse models IL-7 Tg mice IL-7 is synthesized in intestinal epithelial cells that regulates proliferation and differentiation of lymphocytes within the gut mucosa and has been found to be up-regulated in case of patients with UC. The expression of IL-7 in the colonic mucosa of IL-7 transgenic mice is associated with chronic colitis caused by infiltrating CD4? T cells also with over-expression of IL-7 mRNA along with an infiltration of neutrophils and cd T cells in the intestine (Modi et al. 2012). An IL-7 Tg mouse model is useful to understand T cell-mediated pathogenesis of colitis for therapeutic interventions targeting mainly T cell functions characterized by a mixed cellular infiltration that includes neutrophils and lymphocytes. Therefore, this is a chronic colitis model that closely resembles human UC (Watanabe et al. 1998). STAT4 STAT4 is an important transcription factor involved in the development and regulation of T-helper 1 cells that regulates the IL-12 pathway which in turn is critical for the regulation of TH1 cytokines such as the pro-inflammatory interferon gamma (IFN-c). STAT4 transgenic mice develop weight loss, diarrhea and severe colitis resembling human CD (Simpson et al. 1998; Wirtz et al. 1999). HLA-B27 Human MHC class I allele HLA-B27 overexpression in both mice and rats results in intestinal inflammation. Rats transgenic for human HLA-B27 develops a spontaneous inflammatory bowel disease which affects the stomach, ileum and in particular the entire colon. Crypt hyperplasia and mucosal infiltration of mostly mononuclear inflammatory cells are characteristic features of the disease. This model has been used extensively to study the effect of resident intestinal bacteria for acute and chronic stages of gastrointestinal inflammation (Rath et al. 1999). Conditional (cell-specific) transgenic mouse models Transgenic (Tg) mouse models of intestinal significance with cell-specific deletions include T cell-specific SOCS1

Tg in which CTLA-4 mediated control of T cell proliferation resulted in spontaneous colitis (Inagaki-Ohara et al. 2006), T and B cell-specific CD40L (Kawamura et al. 2004) and inducible STAT4 Tg mice (Onizawa et al. 2009; Wirtz et al. 1999). DNN-cadherin transgenic mice/keratin 8-/- mice Keratin 8 is one of the major intermediate filament proteins present in intestinal enterocytes of patients with IBD (Owens et al. 2004). So, mice deficient for Keratin 8 develop colonic hyperplasia and colitis due to a primary epithelial rather than immune cell defect (Baribault et al. 1994).This model provides direct evidence for the importance of an intact epithelial barrier for mucosal homeostasis, expressing a dominant negative mutation of the cell adhesion molecule N-cadherin in intestinal epithelial cells along the crypt villus axis (Hermiston and Gordon 1995). Mutation knock-in mouse models of colitis TNFDARE In this model, there is targeted deletion of AUrich elements (ARE) of the TNF gene in the mice which further display overproduction of TNF-a resulting in colitis strikingly similar to CD. The pathology in these mice is mainly restricted to terminal ileum (Onizawa et al. 2009). Even due to overproduction of TNF, it leads to polyarthritis and chronic intestinal inflammation with infiltrating inflammatory cells and transmural inflammation which is mainly located in the terminal ileum and which is characterized by granulomata (Kontoyiannis et al. 2002). Adoptive transfer models of colitis Bowel inflammation is induced in adoptive transfer models by selective transfer of certain cell types to immunocompromised host animals. These models are versatile tools for unraveling many immunologic and genetic factors contributing to disease and have provided outstanding new insights into the predominant role of T cells for mucosal immune regulation (Wirtz et al. 2000). CD45RB High Transfer model Mucosal inflammation is induced in severe combined immunodeficient (SCID) mice by the adoptive transfer of naive (CD45RBhigh) T cells lacking regulatory cells (Michelle et al. 2004). Studies have supported that the bowel inflammation is caused by a proinflammatory IL-12 driven TH1 response of CD4? CD45RBHigh cells (Leach et al. 1996). CD4? CD45RBHigh T cells obtained from mice deficient for the signal transducer and activator of transcription (STAT) 4, a key component in IL-12 signal transduction, develop a less severe disease upon transfer than wild-type cells (Read et al. 2000). These mice develop severe small intestinal and pancolitic inflammation within 5–8 weeks of transfer. The

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severity of colitis resulting from the transfer may vary depending on the strain of both donor and recipient mice (Powrie 1995; Powrie et al. 1994).

Conclusion Animal models can be used to contribute to the understanding of the information about various mechanisms involved in IBD and for screening and developing new medications for their treatment that would be impossible in humans. So, the purpose of this review is to introduce the investigators to sources of information and to guiding principles regarding the choice of animal models for experimental studies on IBD. The evidence discussed in this review indicates that currently available animal models are relevant to human IBD if the models are chosen carefully (chronic, immune mediated). There are close similarities in pathological findings between experimental IBD and humans mechanisms of tissue injury, effector cell activity, T cell dependence, chronic drive by resistant luminal bacteria and response to pharmacological agents, and also, genetic heterogeneity and primary defects in mucosal barrier function are well-established in several experimental models of colitis. If animals are chosen appropriately, then the exact pathological mechanism can be explored, and it will also bring an ease to develop novel therapeutic agents and developing the hypothesis. So, the current review enlightens the various aspects of animal model of IBD, which may be used for the further research purpose and formulating safe and effective treatment of the disease.

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