Intestinal inflammation constitutes the fundamental characteristic of celiac disease and inflammatory bowel diseases (IBD), such as Crohn's disease or ulcerative colitis, which usually involve severe diarrhea, pain, fatigue, and weight loss. In that context, it is considered to be the consequence of chronic dysregulated immune response in the gastrointestinal tract, stemming from the host's genetic predisposition, as well as inciting environmental factors, including gut microbes and possibly dietary components. Several intestinal inflammation animal models are available, deepening our understanding of enteric pathogenesis and the ways to combat it. These can be categorized as spontaneous colitis, chemically inducible colitis (Table 2.4.1), genetically modified (Table 2.4.2), and adoptive transfer models^17,18. Chemically induced colitis models are widely exploited because they effectively resemble human intestinal pathologies morphologically, symptomatically, and histologically, therefore they will be briefly presented.

The most extensively utilized animal model is that of DSS colitis, which relies on epithelial damage induction by dextran sodium sulfate (DSS), a chemical colitogen with anticoagulant properties that is added to drinking water (table). The latter's concentration and frequency of administration can be adjusted in order to simulate acute or chronic intestinal inflammation, as well as relapsing versions of it. DSS proposedly behaves as a chemical toxin that disrupts the epithelial monolayer lining of the intestine, resulting in the ingress of proinflammatory luminal content, including microbes and their metabolites, in the underlying tissue. Symptoms include gross bleeding in the stool, diarrhea, and weight loss. Elevated TNF-α levels is the hallmark of DSS-induced colitis, accompanied by changes in Th1/Th2 cytokine profile. Generally, the particular model of intestinal inflammation exhibits plenty of benefits, as the followed protocols are fast, simple, and reproducible.

DSS can also be exploited to cause colitis-associated cancer (CAC). Colorectal cancer (CRC) is the third most common cancer in the world and it has been demonstrated that individuals with colitis are predisposed to colorectal tumor formation. Therefore, it is worth considering a very well established model of inflammation-induced intestinal carcinogenesis which combines DSS with azoxymethane (methyl-methylimino-oxidoazanium, AOM): the AOM/DSS model, whose core principle is to chemically induce DNA damage and repeated cycles of colitis. AOM is converted by cytochrome p450 into the carcinogenic form of methylazocymethanol (MAM), a highly reactive alkylating species, which is received by enteric epithelial cells when excreted into the bile, causing DNA mutations. The AOM/DSS model is reproducible, relatively inexpensive with a moderate timeline (~10 weeks), and simulates accurately CAC. The protocol is initiated with an intraperitoneal injection of mice with AOM and then follow cycles of DSS uptake though water intervened by recovery phases. A detailed approach is presented in "AOM/DSS Model of Colitis-Associated Cancer" by Parang et al. (2017).

Another vastly used model of both acute and chronic intestinal inflammation, especially Crohn's disease, constitutes TNBS colitis, which utilizes hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS), a typical skin contactant (Table 2.4.1). This chemical is mixed with ethanol, which disrupts the intestinal barrier, and administered intra-rectally. Thus, TNBS interacts with colon tissue proteins, resulting in increased leukocyte infiltration and excessive Th1 inflammation, which involves IL-12 and TNF-α as effector cytokines. Due to the hypersensitivity immune response, the above proteins are rendered immunogenic to the host immune system. After treatment, animals present various symptoms of acute colitis, such as edema, ulceration, inconsistent stool formation, and bloody diarrhea.

Due to the polygenic and heterogeneous nature of intestinal inflammation, numerous genetic models have been established (Table 2.4.2). One of the primarily focused areas of research is the epithelial barrier function and involve mice deficient in essential structural elements of the protective mucosal layer (e.g. Muc2 or O-glycans), in transporters and exchangers of intestinal epithelial cells (e.g. Octn2, a carnitine transporter), and in pattern recognition receptors (e.g. Nod2, TLR4). Immune regulation is also extensively examined, highly involving cytokine responses and their regulators. For example, there are widespread models of deficiencies in T cells (like TGF-β1 and its signal transducer Smad3, IL-2, and IL-10), as well as models with modifications in important signal-transduction factors, like STAT3, NF-κΒ, and SOCS1, which exhibit types of spontaneous enterocolitis.

Moreover, models of intestinal stress responses have also been developed, since the latter are fundamental for maintaining enteric homeostasis. A representative example would be the double Gpdx1-Gpx2 (antioxidant enzyme glutathione peroxidase) knockouts, which lead to ileocolitis and adenocarcinoma, or XBP1 transcription factor deficiency in gut epithelial cells, resulting in intestinal inflammation18. Regarding intestinal carcinogenesis, a widely used for over 25 years genetically engineered model is the ApcMin^Min/+ mouse, which carriers an autosomal dominant loss of function mutation in the Apc - a tumor suppressor gene and the most common driver mutation for colorectal carcinoma in humans.

A number of factors are examined while testing the effectiveness of nutritional supplements in the context of intestinal inflammatory diseases. These are briefly described below:

• Disease activity index (DAI) score: body weight and disease symptoms, such as diarrhea and hematochezia are tactically monitored.
• Colon length: right after animal sacrifice, the intestine is isolated and measured
• Inflammatory cytokines: suitable parts of stool-free colon (e.g. 10mg from the ascending colon and 10mg from the descending colon) are homogenized in appropriate ice-cold lysis buffer. Suspensions are centrifuged (e.g. 13,000 rpm, 15min) and the resulting supernatant is used for ELISA assay of the desired inflammatory cytokines. Alternatively, the expression of relative genes can be measured with real-time PCR.
• Histopathological analysis: proximal and distal colonic sections are fixed and embedded in paraffin. Haematoxylin and eosin are used for staining. Microscopic colonic epithelial damage is assessed, alongside with inflammatory cell infiltration. For a detailed approach, refer to "Intestinal Preparation Techniques for Histological Analysis in the Mouse" by Williams et al. (2016).
• Cell population analysis: cell populations expressing distinct levels of suitable markers are isolated by flow cytometry or fluorescence-activated cell sorting (FACS). These can then be used for gene expression profiling with real-time PCR.