Mouse Models of Intestinal Fibrosis

Abstract

Mouse models are essential for investigation of underlying disease mechanisms that drive intestinal fibrosis, as well as assessment of potential therapeutic approaches to either prevent or resolve fibrosis. Here we describe several common mouse models of intestinal inflammation and fibrosis, including chemically driven colitis models, a bacterially triggered colitis model, and spontaneous intestinal inflammation in genetically susceptible mouse strains. Detailed protocols are provided for dextran sodium sulfate (DSS) colitis, 2,4,6-trinitro-benzene sulfonic acid (TNBS) colitis, adherent-invasive Escherichia coli (AIEC)-triggered colitis, the interleukin-10 knockout (IL-10KO) mouse model of spontaneous colitis, and the SAMP/YitFc model of spontaneous ileocolitis.

1. Introduction

Over half of Crohn’s disease (CD) patients develop disease complications during their disease course that can include fibrosis-induced intestinal obstruction and result in debilitating symptoms [1] and ultimately require surgery [2]. Although less appreciated, intestinal fibrosis also occurs in ulcerative colitis (UC) patients [3]. Short gut syndrome, need for hospitalizations, as well as the tremendous financial burden of fibrosis-associated complications pose significant concerns to these patients and their healthcare providers. No specific anti-fibrotic therapies are currently available, which emphasizes the critical need to obtain a mechanistic understanding of the drivers of intestinal fibrosis. The goal remains to develop novel preventive and effective therapeutic approaches.

Human studies have been limited in this field due to lack of available tissue for in vitro investigations and clinical trials endpoints. Hence, well-characterized animal models of intestinal fibrosis are filling this void to better define and understand the pathophysiology of fibrosis. Animal models enable us to perform mechanistic studies, and facilitate the testing of novel, pathophysiology-based therapeutic approaches at the same time.

This chapter provides detailed protocols for several frequently used animal models of intestinal fibrosis. These models include chemically induced intestinal inflammation, such as dextran sodium sulfate (DSS) [4–6] and 2,4,6-trinitro-benzene sulfonic acid (TNBS) [7–9], that when applied repetitively, result in the development of intestinal fibrosis. Another model described is chronic inflammation and fibrosis induced in response to colonization with a specific adherent-invasive Escherichia coli (AIEC) strain [10, 11]. Finally, genetically driven spontaneous interleukin-10 (IL-10) knockout [12–14] colitis, as well as the SAMP1/YitFc [15] spontaneous ileocolitis model are discussed (Table 1).

Table 1.

Summary of mouse models

	Summary	Advantages	Disadvantages
DSS colitis model	Multiple cycles of DSS induces chronic colitis and fibrosis	Easy to perform, good reproducibility	Limited human IBD relevance; limited cell types involved
TNBS colitis model	Intrarectal application of hapten TNBS: results in chronic colitis leading to fibrosis	Widely used for studying intestinal fibrosis	Requires susceptible mice strain to induce colitis; great variability
AIEC colitis model	Oral gavaging of AIEC induces gut inflammation	Mimics the inflammation pattern of human CD; Great tool for microbiota research in IBD	Require ABSL2 facility
IL-10 knockout colitis model	Spontaneous chronic intestinal fibrosis model	Spontaneous colitis model, while chemicals and infectious components could be added to accelerate the onset of disease	Disease development is slow
SAMP1/YitFc ileocolitis model	Spontaneous colitis models resemble all stages of human CD	Strain closely mimics all stages of stricture formation in CD patients; symptoms start as early as 4 weeks	Low breeding rate; not commercialized currently

Unless otherwise noted, these models are performed with mice of the ages of 6–8 weeks and between 18 and 20 g of weight at the start of the experiments. For all these models, disease severity is strongly influenced by genetic background and animal housing facility. Therefore, if performing these models using mice with a mixed strain background, littermate controls are required for robust interpretation of the results. Additionally, each model will require optimization for each animal facility. Generally, a sample size of 10 mice per group repeated in two independent experiments provides robust statistical power analysis. However, it is recommended that a formal power analysis is performed using the degree of effect and experimental variation expected for the hypothesis being tested. All models require the review and approval of the institutional animal care and use committee (IACUC) and must comply with the local regulations of the animal care facility.

2. Materials

2.1. DSS Colitis Model

1. DSS (30,000–50,000 colitis grade) solution: Prepare 1–10% (w/v) DSS solutions in sterile deionized water in 1% steps and store at room temperature. Confirm that the DSS powder is completely dissolved in water; otherwise it may clog the water bottle sipper tube.

2. Animals: 6–8-week-old male mice of preferred strains. This model can be performed in both male and female mice; however, the estrus cycle influences disease activity in this model and increases the variability of response. DSS induces colitis in a broad range of standard inbred mouse strains (e.g., C57BL/6 [16], BALB/c [17], as well as CD1 mice [18]), but severity of colitis is influenced by genetic background.

3. Hemoccult Sensa Single Slides(Beckman Coulter, Indianapolis, IN, USA).

2.2. TNBS Colitis Model

1. Pre-sensitization solution: Prepare pre-sensitization solution freshly on the day of pre-sensitization. Mix acetone and olive oil in a 4:1 volume ratio by vortexing rigorously. Prepare 1% (w/v) TNBS solution: Mix 4 volumes of acetone/olive oil with 1 volume of 5% TNBS solution to obtain 1% (w/v) TNBS. Mix the solution by rigorous vortexing (see Note 1).

2. Control pre-sensitization solution: prepare acetone and olive oil in a 4:1 volume ratio.

3. TNBS solution for intrarectal administration: Combine ethanol, double-distilled water (ddH₂O), and TNBS to create 0, 0.5, 1, 2, 3, and 4% (w/v) TNBS in 70% ethanol. Ensure mixing through vigorous vortexing. Prepare enough solution to administer 100 μL/mouse with an additional 300 μL volume to account for loss during administration.

4. Vehicle solution for intrarectal administration: 70% ethanol diluted in ddH2O. Prepare enough solution to administer 100 μL/mouse with an additional 300 μL volume to account for loss during administration.

5. French (F) catheter at least 6 cm in length.

6. Lubricant to help inserting catheter.

7. Electric razor.

8. 6–8-week-old mice with TNBS-sensitive genetic background [19].

9. Isoflurane for anesthesia.

2.3. AIEC Colitis Model

1. Bacterial strain: AIEC strain NRG857c (O83:H1); isolated from an ileal tissue biopsy from a CD patient in the Charité Hospital (Berlin, Germany) [10] (see Note 2).

2. Bacterial culture reagents: NRG857c is cultured in LB broth and is resistant to chloramphenicol (34 μg/mL) and ampicillin (100 μg/mL). Selective LB agar plates should be prepared with one or both antibiotics for monitoring colonization of mice by fecal AIEC levels and determination of tissue colonization and dissemination.

3. Animals: C57BL/6, CD1, DBA/2, C3H, or 129e mice housed in an animal biosafety level 2 (ABSL2) facility. Due to variation in colonization levels, we recommend performing the experiment with n = 4–5/group, independently repeating the experiment 3–4 times.

4. Antibiotics: Streptomycin (100 mg/mL) or vancomycin (17.5 mg/mL) diluted in sterile water for gavage into mice to disrupt the intestinal microbiota and provide a niche for AIEC colonization [10].

5. Commercially available phosphate-buffered saline.

6. Enzyme-linked immunosorbent assay (ELISA) reader.

2.4. IL-10-Deficiency Colitis Model

1. Mice: The strain background and housing conditions of the IL-10KO mouse are major contributing factors for the severity of disease and the time to onset. Another factor to consider is the age of the mouse at the time of intervention or study. To examine factors that accelerate disease onset (e.g., diet), mice should be challenged between 3 and 5 weeks of age. For experiments involving chemical or infectious agents to result in earlier or more severe colitis, animals should be between 8 and 10 weeks of age at the initiation of the accelerant.

2. ELISA reader.

2.5. SAMP1/YitFc Ileocolitis Model

1. Mice: Specific pathogen-free (SPF) environment does not affect the time of onset of disease; however, the severity of disease could be less, characterized by less inflammation [20]. For prevention of fibrosis, therapy can be started at 6–8 weeks of age. This stage is characterized by intense immune response without severe lesions histologically [21]. The long duration of this model to 30 weeks of age, however, is making daily administration of drugs bothersome or in case of intraperitoneal (IP) injections may even lead to risk of peritonitis over time. Alternative approaches could be implantation of drug pumps [22]. For therapeutic approaches to already established fibrosis, mice should be treated starting week 30 onward for approximately 8–10 weeks [15, 21].

2. ELISA reader.

3. Methods

3.1. DSS Colitis Model

Mice drinking the sugar polymer DSS (36,000–50,000 molecular weight fragments) for several days develop a highly reproducible colitis with bloody diarrhea, ulcerations, and weight loss [23, 24]. DSS is believed to have a toxic effect on intestinal epithelial cells leading to disruption of the epithelial barrier. Subsequently, activation of intestinal macrophages might contribute to intestinal inflammation. DSS-induced colitis is characterized by weight loss, bloody diarrhea, ulcer formation, loss of epithelial cells, and infiltration of neutrophils, which resembles human UC [24]. In certain strains, chronic, intermittent administration of low concentrations DSS for several cycles results in chronic colitis with the development of a substantial amount of fibrosis [6]. From a technical perspective, this model is easy and yields reproducible results, explaining its popularity and widespread use [25]. The main disadvantage of this model is the questionable relevance to human IBD, as IBD is not caused by a chemical and more cell types than epithelial cells and macrophages are involved [26]. Mouse genetic background and microbiota [27] affects the degree of induced colitis as well, which makes it essential to perform a DSS dose titration in a specific animal facility.

3.1.1. DSS Titration (5–8 d)

DSS dose titration is recommended for each mouse strain and housing facility prior to deciding on the DSS dose for the chronic experiments. Disease severity in this model is strongly influenced by strain background and housing conditions (e.g., environmental microbes and/or intestinal microbiota).

1. On day 7, start training the mice to use water bottles as their only source of water. Acclimatization to the water bottles will reduce stress and prevent weight loss due to the change of water source.

2. On day 0, mark the mice for identification and determine their weight and baseline water volume (e.g., bottle weight). Collect stool to check occult blood and stool consistency.

3. Administer DSS solution (0% as control, then 1%, 2%, 3%, etc.) to the cage water. Estimated water consumption is ~5 mL of DSS solution per mouse per day. Control mice should receive sterile deionized water without DSS.

4. Determine the mouse weight, water consumed, assess stool consistency and stool occult blood every day when the mice are on DSS as well as control mice (see Note 3). The above indicators determine mouse clinical disease severity, which is also referred to as clinical score. Briefly, weight loss of 1–5%, 5–10%, 10–20%, and more than 20% can be scored as 1, 2, 3, and 4, respectively. Stool consistency is scored as 0 for well-formed pellets, 2 for pasty and semi-formed stools, which does not stick to the anus, 3 for pasty unformed stool, and 4 for liquid stools that remain adhesive to the anus. Bleeding is scored 0 for no blood in hemoccult, 2 for positive hemoccult, 3 for obvious blood stick to the surface of stool, and 4 for gross bleeding from the rectum. Occult blood is tested by Hemoccult Sensa Single Slides(Beckman Coulter, Indianapolis, IN, USA). Weight, stool consistency, and bleeding sub-scores will be added and divided by 3, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis).

5. At the time points of choice (generally on days 5–8), euthanize the mice and proceed to harvest mouse colons. We measure the colon length, and collect terminal ileum, proximal colon, transverse colon, and rectum segments. For each segment, we collect one piece immersed in HistoChoice (VWR, PA, USA) overnight for paraffin-embedded tissue sections, freeze one piece in OCT for frozen sections, one piece for mRNA extraction, and one piece for protein extraction.

6. After harvesting the tissue, use the paraffin-embedded tissue sections for hematoxylin and eosin (H&E) staining, as well as Masson Trichrome staining, to evaluate the level of inflammation and fibrosis. The degree of inflammation and fibrosis should be evaluated ideally by a trained histopathologist blinded to treatment conditions using a standardized scoring systems [28, 29]. The most preferable DSS concentration is determined by weight loss between 15% and 20% within 5–8 d of DSS administration with 100% viability of the animals. The preferable DSS dosage will be used in the chronic colitis model.

3.1.2. DSS Induction of Chronic DSS Colitis (30–42 d)

1. On day 7, start training the mice to use water bottles as their only source of water.

2. On day 0, mark the mice for identification and determine their weight and water bottle weight. Collect stool to check occult blood and stool consistency.

3. Administer the preferred DSS dose in cage water bottles. Estimate water consumption as ~5 mL of DSS solution per mouse per day. Control mice should receive sterile deionized water without DSS.

4. Determine the mice weight and water bottle weight and check the stool consistency and stool occult blood every day when the mice are on DSS as well as control mice (see Note 4). Mouse disease severity is also determined by the clinical score, combining the score of weight loss, stool consistency, and occult blood as above mentioned. Briefly, weight loss of 1–5%, 5–10%, 10–20%, and more than 20% can be scored as 1, 2, 3, and 4, respectively. Stool consistency is scored as 0 for well-formed pellets, 2 for pasty and semiformed stools, which does not stick to the anus, 3 for pasty unformed stool, and 4 for liquid stools that remain adhesive to the anus. Bleeding is scored 0 for no blood in hemoccult, 2 for positive hemoccult, 3 for obvious blood stick to the surface of stool, and 4 for gross bleeding from the rectum. Occult blood is tested by Hemoccult Sensa Single Slides (Beckman Coulter, Indianapolis, IN, USA). Weight, stool consistency, and bleeding sub-scores will be added and divided by 3, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis).

5. The preferred DSS dose is administered in the drinking water for a cycle of 5–7 d, followed by 10 d of recovery on water. As mentioned above there are differences in susceptibility of different strains to DSS (e.g., C57B6 background mice are more sensitive to DSS, while BALB/c are more resistant) and disease activity is also influenced by housing conditions. Change the water bottle to sterile deionized water in all experimental animals for recovery. Continue monitoring the mice for disease activity as described in step 4 throughout the experiment.

6. A second cycle of DSS is performed, administering the same concentration of DSS for the same length of time as cycle one followed by 10 d of recovery. Harvest according to Subheading 3.1.1, steps 5 and 6, after two cycles of DSS and two cycles of recovery.

3.2. TNBS Colitis Model

Colitis can be induced in susceptible murine strains by intrarectal instillation of the hapten TNBS diluted in ethanol [30]. It is critical to check in the literature the susceptibility of the mouse strain to TNBS [19]. For instance, BALB/c is considered to be susceptible to TNBS, while C57BL/6 is considered resistant [19]. Comparable to the DSS models, a dose titration is strongly recommended, as disease severity is influenced by genetic background and housing facility (e.g., environmental microbes and/or microbiota). Mechanistically, ethanol breaks the epithelial barrier and TNBS is believed to modify colonic proteins and activate a delayed-type hypersensitivity reaction. This depends on T cells and leads to transmural inflammation [9]. When given weekly for 6 weeks of escalating doses of TNBS (chronic model), chronic colitis leads to fibrosis. This model is one of the most widely used to study intestinal fibrosis in vivo. Its limitations are the high variability [31] and again the nature of a chemical inducing colitis.

3.2.1. TNBS Titration (5–8 d)

1. Weigh and mark the mice to allow identification.

2. Lightly anesthetize the mouse via a nose cone with 1.5% isoflurane-mixed gas while it is resting on an isothermal warming pad.

3. Pre-sensitization: On day 1, carefully shave a 1.5 × 1.5 cm field of the skin of the mouse using an electric razor. To avoid the mouse from grooming TNBS off the skin (may potentially induce oral tolerance), we preferentially select an area on the back between the shoulders. While holding the lightly anesthetized mouse with one hand, we apply 150 μL pre-sensitization solution using a 200 μL pipette to the shaved back skin. The solution is absorbed by the skin quickly. Control mice are treated with pre-sensitization solution without TNBS.

4. On day 8, weigh the mouse.

5. Anesthetize the mouse by using 1.5% isoflurane-mixed gas. Attach a 1-mL syringe to a 3.5 F catheter, and load it with 0, 0.5, 1, 2, 3, and 4% (w/v) TNBS solution in 70% EtOH. Lubricate the catheter tip and insert the catheter through the anus into the colon ~4 cm (see Note 5).

6. Slowly inject 100 μL of TNBS solution into the lumen of the colon, and remove the catheter from the colon, and position the mouse head down for 1 min (see Note 6).

7. Closely monitor the animals for signs of distress, perform daily measurement of weight, stool consistency, and blood in stool [32].

8. At the timepoint of choice, euthanize the animal and proceed to harvest mouse colons. We measure the colon length, collect transverse colon and rectum. For each segment, we collect one piece immersed in HistoChoice overnight for paraffin-embedded tissue sections, one piece for optimal cutting temperature (OCT) compound embedded frozen sectioning, one piece for mRNA extraction, and one piece for protein extraction.

9. After harvesting the tissue, use the paraffin-embedded tissue sections for H&E staining, as well as Masson Trichrome staining, to evaluate the level of inflammation and fibrosis. The degree of inflammation and fibrosis should be evaluated ideally by a trained histopathologist blinded to treatment conditions using a standardized scoring system [28, 29]. The most preferable TNBS concentration is determined by weight loss between 15% and 20% within 5–8 d of TNBS administration with 100% viability of the animals.

3.2.2. Chronic TNBS Colitis (56 d)

Mice tolerize to TNBS over the duration of the experiment. For the chronic model we hence use escalating doses of TNBS with starting with the optimal dose from the titration experiments and increase by 0.5 mg increments every 2 weeks for up to 6 weeks.

1. Weigh and mark the mice to allow identification.

2. Lightly anesthetize the mouse via a nose cone with 1.5% isoflurane-mixed gas while it is resting on an isothermal warming pad.

3. Pre-sensitization: On day 1, we carefully shave a 1.5 × 1.5 cm field of the skin of the mouse using an electric razor. To avoid the mouse from grooming TNBS off the skin (may potentially induce oral tolerance), we preferentially select an area on the back between the shoulders. While holding the lightly anesthetized mouse with one hand, we apply 150 μL pre-sensitization solution using a 200 μL pipette to the shaved back skin. The solution is absorbed by the skin quickly. Control mice are treated with pre-sensitization solution without TNBS.

4. On day 8, weigh the mouse.

5. Anesthetize the mouse by using 1.5% isoflurane-mixed gas. Attach a 1-mL syringe to a 3.5 F catheter and load it with either (1) the optimal TNBS dose, (2) vehicle control solution for administration rectally. Lubricate the catheter tip and insert the catheter through the anus into the colon ~4 cm (see Note 7).

6. Closely monitor the animals for signs of distress, perform daily measurement of weight, stool consistency, and blood in stool [32].

7. Repeat steps 5 and 6 weekly, escalating the TNBS dose by 0.5 mg every 2 weeks, up to 6 weeks. Harvest at 8 weeks according to Subheading 3.2.1, steps 8 and 9.

8. At week 8, euthanize the animal and proceed to harvest mouse colons. We measure the colon length, collect transverse colon and rectum. For each segment, we collect one piece immersed in HistoChoice overnight for paraffin-embedded tissue sections, one piece for OCT embedded frozen sectioning, one piece for mRNA extraction, and one piece for protein extraction.

9. After harvesting the tissue, use the paraffin-embedded tissue sections for H&E staining, as well as Masson Trichrome staining, to evaluate the level of inflammation and fibrosis. The degree of inflammation and fibrosis should be evaluated ideally by a trained histopathologist blinded to treatment conditions using a standardized scoring system [28, 29].

3.3. AIEC Colitis Model

The microbiota plays a key role in the initiation and perpetuation of chronic inflammation in IBD [33]. A singular pathogenic microbial species has not be identified; rather, it is thought that the combination of host genetic susceptibility and the opportunistic expansion of commensal microbes initiates and perpetuates IBD [33]. Escherichia coli, a Gram-negative commensal member of the Proteobacteria phyla, is a normal component of the intestinal microbiota and contributes to intestinal homeostasis [10]. However, CD patients have been found to harbor higher than normal levels of E. coli that have acquired virulence factors [34, 35]. AIEC is one such mucosa-associated bacterium often found in ileal biopsies of CD patients [34, 36]. Several strains of AIEC have been shown to exacerbate or even initiate experimental colitis in murine models [10, 37–41]. However, several of these models require specific genetically altered mice [38–41] and/or mono-colonization [40, 41] to induce chronic intestinal inflammation. The exception to this is a model that utilizes the AIEC strain NRG857c that can colonize a broad variety of standard inbred mouse strains after antibiotic treatment [10]. AIEC strain NRG857c induces a strong Th17 response in the mouse intestine, which drives chronic intestinal inflammation and transmural fibrosis, mainly in the cecum, with limited mortality [10]. Extracellular matrix deposition can be detected by Masson trichrome stain or picrosirius red stain as early as 7 d post-infection and persists up to 63 d post-infection. These features have been observed in multiple standard inbred mouse strains, such as CD1, DBA/2, C3H, 129e, and C57BL/6, adding to its utility as a model of intestinal fibrosis [10, 11].

3.3.1. Induction of AIEC Colitis (7–63 d)

1. Day-4: Weigh mice and identify individual mice by ear punch or ear tag. If not already housed in an ABSL2 facility, transfer mice to the ABSL2 facility.

2. Day-2: Prepare a fresh AIEC NRG857c stock plate on selective LB agar.

3. Day-1: Antibiotic pretreatment: Remove food and water from the cage 4 h prior to gavage and weigh the mice. Orally gavage mice with 20 mg streptomycin or 3.5 mg of vancomycin in 200 μL sterile water. Sedation or anesthesia is not recommended before performing oral gavage, as it may increase the risk of aspiration pneumonia. Return food and water to cage after gavage.

4. Day-1: Inoculate duplicate aliquots of 4 mL sterile LB containing chloramphenicol/ampicillin with a single colony of AIEC NRG857c from the stock plate. Incubate overnight at 37 °C while shaking at 220 rpm.

5. Day 0: Mouse preparation: Remove food and water from the cage 4 h prior to gavage and weigh the mice.

6. Day 0: AIEC inoculation preparation: Measure the density of the overnight AIEC bacterial culture by reading the absorbance at 600 nm in a spectrophotometer. Calculate the volume of culture required to deliver 2 × 10⁹ colony forming units (cfu) to twice the number of mice intended for colonization. Concentrate overnight culture by centrifuging for 10 min at 4000 × g. Aspirate supernatant and gently, but thoroughly, resuspend bacterial pellet in sterile PBS at a volume of 200 μL per mouse. Inoculum should look milky white, but easily pass through the gavage needle. Use a small aliquot of this final inoculum to dilute and plate on duplicate selective LB agar plates to confirm the actual concentration of the inoculum. Incubate plate overnight at 37 °C and count colonies the next morning to calculate the actual amount of AIEC delivered to each mouse.

7. Day 0: Bacterial inoculation: Orally gavage mice with 2 × 10⁹ cfu in 200 μL volume inside a biosafety cabinet. Return food and water after gavage. As AIEC NRG857c is a human pathobiont, mouse handling must take place as specified in an approved biosafety protocol (e.g., within a biosafety cabinet during inoculation, and all subsequent work with these mice must also take place in a biosafety cabinet using BSL2 precautions).

8. Monitoring: Mouse weight and activity is evaluated daily as a gross measure of disease activity. The productivity of AIEC colonization can be monitored by AIEC cfu in stool homogenates as early as day 1 post-inoculation and every 3–4 d after inoculation. Stool pellets are collected fresh from mice and placed in a pre-weighed screw-cap tube containing 500 μL of sterile PBS. Tubes are then weighed to determine the amount of stool/mL. Stool samples are then homogenized in a biosafety cabinet by vortexing vigorously. Stool homogenates are then serially diluted in a sterile 96-well plate containing sterile PBS and 5 μL of the dilutions spot plated on duplicate selective LB agar plates to quantify the number of AIEC cfu per weight of the stool sample. Approximately 10⁷–10⁹ AIEC cfu/g of stool can be expected on day 1 post-infection [11]. Fecal loads generally peak at day 3, then steadily decline over the next 1–3 weeks post-inoculation depending upon the mouse strain background [10].

9. Endpoint collections (Day 0, 7, 14, 21, 63 post-inoculation): After euthanasia, collect blood for the analysis of systemic inflammation markers by ELISA (e.g., serum amyloid A). Collect intestinal tissue, including jejunum, terminal ileum, cecum, proximal colon, transverse colon as well as distal colon segments for fixation and histologic analysis of tissue damage, fibrosis, and inflammation. Stool, and segments of the colon, cecum, small intestine, liver, spleen, and mesenteric lymph nodes can be collected into pre-weighed screw-cap tubes containing sterile PBS for analysis of AIEC tissue colonization and dissemination. These tubes are weighed before processing to determine tissue weight and then homogenized in a biosafety cabinet using a tissue grinder. The volume of PBS for homogenization ranges between 0.5 and 5 mL depending on the day of collection and tissue type (use a lower volume for non-intestinal tissues or later in the experimental time course). Serially dilute and plate supernatants in duplicate from tissue homogenates on selective LB agar plates as performed with stool samples. Final AIEC tissue or stool levels are reported as cfu/g.

10. Analysis of inflammation and fibrosis: Sections from fixed tissues embedded in paraffin blocks are stained with H&E for evaluation of tissue histology and inflammation or Masson Trichrome stain to evaluate the deposition of extracellular matrix/fibrosis. The degree of inflammation and fibrosis should be evaluated ideally by a trained histopathologist blinded to treatment conditions using a standardized scoring system [10, 42, 43].

3.4. IL-10-Deficiency Colitis Model

IL-10 is an important immunoregulatory cytokine with anti-inflammatory properties that are normally employed to ramp down inflammation caused by the host immune response to pathogens [44]. Therefore, IL-10 is a major regulator of mucosal homeostasis and IL-10-deficient mice (IL-10KO) have been shown to develop chronic enterocolitis in response to even commensal microbiota stimuli [44, 45]. The severity and scope of colitis developed by IL-10KO mice is dependent on the milieu of microbes they are exposed to; germ-free IL-10KO mice will not develop inflammation, SPF mice will develop mild colitis generally restricted to the large intestine, and conventional or experimentally infected mice will develop severe enterocolitis. The other major contributor to disease activity and time to disease onset in this model is genetic background. The strains most susceptible to disease development are C3H/HeJBir and 129/SvEv, while the C57BL/6 J and C57BL/10 strains are the most resistant [14, 25, 44]. Taken together, depending on the strain background and the housing conditions, development of robust colitis in the IL-10KO model can vary between 12 and 20 weeks of age and must be experimentally determined in each animal facility.

The gradual onset of disease in the IL-10KO model is ideal for testing the contribution of environmental factors (such as food products or lifestyle choices like smoking tobacco) or specific immune system components to the acceleration or amelioration of disease onset [45–47]. However, since the development of spontaneous colitis in IL-10KO mice is slow, alternate methods are being investigated to accelerate the onset of disease. One approach relies on the administration of an anti-mouse IL-10R neutralizing antibody, rather than genetic deletion, to initiate disease more effectively [48–50]. Other approaches include the addition of intestinal irritants (e.g., DSS [51] or piroxicam [13]) or infectious agents (e.g., Helicobacter spp. [48, 49, 52] or AIEC [41]) to accelerate disease.

3.4.1. Spontaneous Colitis in IL-10 Knockout Mice (~20 Weeks)

1. Mice between 3 and 5 weeks of age should be assigned to groups and identified (e.g., ear punch or ear tag) for monitoring.

2. Measure the weight of the mice weekly and monitor stool consistency, occult blood in stool, and any sign of stress of the mice. Biomarkers of inflammation, such as fecal lipocalin-2 (Lcn-2) or circulating serum amyloid A can monitored weekly by ELISA to assess the levels of intestinal and systemic inflammation, respectively [53].

3. At weeks 12, 16, and 20, euthanize animals, collect blood for the analysis of systemic inflammation markers by ELISA (e.g., serum amyloid A), and proceed to excise the intestine. Observe any gross pathology of the intestine (e.g., intestinal thickening, strictures, intestinal shortening, poorly formed stool/diarrhea) and measure the colon length. Collect sections from the terminal ileum, proximal colon, transverse colon, and rectum for fixation and paraffin-embedding for histologic analysis of tissue damage, fibrosis, and inflammation. A key component of the histologic analysis is to select tissue from the same position (e.g., x cm from the cecum) in every mouse and every experiment, due to the heterogeneity of disease severity along the length of the intestine [54]. Collect additional pieces of ileal and colon segments for RNA extraction and/or for protein extraction, if desired. Collect stool in pre-weighed screw-cap tubes containing 500 μL PBS for homogenization and measurement of fecal Lcn2 levels (pg/mg stool) by ELISA.

4. Analysis of inflammation and fibrosis: Sections from fixed tissues embedded in paraffin blocks are stained with either H&E for evaluation of tissue histology and inflammation or Massons’ Trichrome stain to evaluate the deposition of extracellular matrix/fibrosis. The degree of inflammation and fibrosis should be evaluated by a trained IBD histopathologist blinded to treatment conditions using a standardized scoring system [42, 43, 54, 55].

3.5. SAMP1/YitFc Ileocolitis Model

While the abovementioned models develop mainly colitis, the SAMP1/Yit mouse exhibits spontaneous inflammation at 10–20 week of age with almost 100% penetrance after 30 weeks [20]. The strain was created by breeding a senescence-accelerated mouse line with inflammation being most severe in the terminal ileum [56]. What makes this model attractive is the spontaneous development of ileocolitis, the location in the terminal ileum, the segmental and transmural nature of the inflammation, and granulomas on histopathology. Foremost from a fibrosis perspective, this model develops accumulation of extracellular matrix in the small and large bowel and a thickening of the muscularis mucosa, which resemble features of human CD-associated intestinal strictures [15, 56]. Drawbacks of the SAMP/Yit mouse are the poor breeding ability, which makes very large colonies necessary, as well as the inconsistent location and severity of inflammation in younger mice in this model.

3.5.1. Spontaneous Colitis in SAMP1/YitFc Mice (~40 Weeks)

1. Mice between 3 and 7 weeks of age should be assigned to groups and identified (e.g., ear punch or ear tag) for monitoring (see Note 8).

2. Measure the weight of the mice weekly and monitor stool consistency, occult blood in stool, and any sign of stress of the mice [32].

3. At the timepoints delineated above, euthanize animals, collect blood for the analysis of systemic inflammation markers by ELISA, and proceed to harvest the intestine. Observe any gross pathology of the intestine (e.g., intestinal thickening, intestinal shortening, poorly formed stool/diarrhea) and measure the colon length. Collect sections from the terminal ileum, cecum, proximal colon, transverse colon, and rectum [56] for fixation and paraffin-embedding for histologic analysis of tissue damage, fibrosis, and inflammation. A key component of the histologic analysis is to select tissue from the same position (e.g., x cm from the cecum) in every mouse and every experiment, due to the heterogeneity of disease severity along the length of the intestine [54]. Collect additional pieces of ileal and colon segments for RNA extraction and/or for protein extraction, if desired. Collect stool in pre-weighed screw-cap tubes for further experiments.

4. Analysis of inflammation and fibrosis: Sections from fixed tissues embedded in paraffin blocks are stained with either H&E for evaluation of tissue histology and inflammation or Masson Trichrome stain to evaluate the deposition of extracellular matrix/fibrosis. The degree of inflammation and fibrosis should be evaluated by a trained IBD histopathologist blinded to treatment conditions using a standardized scoring system [20].