In vivo models of intestinal restitution typically involve the creation of ulcers over which the migration of surviving adjacent epithelium can be assessed. These ulcers can be produced in a variety of injury modalities, including mechanical wounding, chemical injury, genetic models, chemotherapy and radiation, and infection, that resemble human GI insults. Each injury model has its own nuances regarding the severity of injury and the types of epithelial responses that are triggered. Mild injury may lead to a transient loss of epithelial cells within individual crypts that are replenished with a proliferative burst of stem cells without necessitating the activation of cellular migration pathways. In some models, ulcerations are only seen with severe injury, but the tissue microenvironment associated with these injuries can vary regarding the general types of immune cells, hormones, microbial agents, and matrix factors involved in the response. When evaluating whether a specific agent or gene has effects on restitution, it is important to clarify what animal models were used. Moreover, when interpreting the animal’s overall outcome after exposure to these injury models, one must note that many other biological responses (e.g., epithelial survival and morphogenesis, immune cell signaling) are at play in addition to restitution, and without detailed examination of the specimen it can be virtually impossible to ascribe differential outcomes to restitution. Nonetheless, there are a few in vivo models where the epithelial restitutive response has an important role in dictating the overall outcome.

Mechanical wounding : Direct mechanical wounding of the rodent distal colon can be achieved by manual biopsy of inner colonic surface. The animal (usually a mouse) is sedated and a mini-colonoscope is passed into the rectum, and wounds are generated using biopsy forceps attached to the end of the colonoscope. Superficial wounds of approximately 1 mm 2 surface area each can be generated in the colon. Depending on the skill of the investigator, multiple wounds can be created in a single animal, allowing for observation of restitution at different sites along the proximal-distal axis. Wounds of this size in the mouse are typically healed within 7 days. Because the colonic ulcers in this model are all created at the same time, this model has been useful to identify the time-locked onset and duration of the restitutive response, the cellular markers defining the restitutive cellular front, and influence of immune and support cells on the wound healing response. However, this model requires specialized equipment and specially trained staff. A variation of the mechanical wounding model involves the insertion of a rotating brush into the rectum, obviating the need for an expensive miniature colonoscope, but this procedure poses a significant risk for colonic perforation. A sophisticated variation of the colonoscope method modifies protocols for confocal endomicroscopic imaging and utilizes a 2-photon laser to induce photodamage to the colonic epithelium; restitution and repair of the mucosal surface occurs within a week and can be monitored over time.

Chemical models : Chemical models of intestinal wounding are common because of their relative ease-of-use. The administration of ~ 40 kDa dextran sulfate sodium (DSS) in the drinking water of rodents over 3–7 days induces epithelial injury, ulceration, and inflammation of the distal colon. DSS-induced colitis is mediated by innate immune cells, as the lymphocyte lineage has been shown to be dispensable for injury. The animal suffers reversible weight loss, bloody stools, and diarrhea. Imaging studies have shown distinct phases of restitution and crypt regeneration in the DSS colitis model. While the exact pathogenic mechanisms underlying DSS-induced injury are not known, a primary cause may be increased epithelial apoptosis, stem cell ablation, and loss of epithelial integrity as early events, which pave the way for subsequent mucosal immune responses. DSS administration spares the small intestine and upper GI tract. Thus, the DSS model enables the observation of a strong epithelial regenerative response in the context of colonic inflammation in a relatively practical model. Other chemical colitis models useful for studying restitution in vivo include intrarectal administration of acetic acid, which induces mucosal changes resembling human ulcerative colitis, and of oxazolone and 2,4,6-trinitrobenzenesulfonic acid (TNBS), which are haptenating agents that induce an adaptive immune-mediated colitis similar to human inflammatory bowel disease.

Nonsteroidal antiinflammatory drugs : Administration of nonsteroidal antiinflammatory drugs (NSAIDs) to rodents is another method to induce intestinal injury. These models demonstrate the importance of prostaglandins for maintenance of epithelial integrity and mobilization of wound healing processes. Subcutaneous injections of indomethacin induce a Crohn’s-like disease in mice and rats specifically affecting the jejunum and ileum. Depending on the injection schedule, an acute or chronic disease can be modeled. Like DSS, the mechanism of action of indomethacin has not been elucidated, but it may involve synergistic effects with specific bile acids. Another NSAID, piroxicam, can be used to accelerate the onset of colitis in animals with susceptible genetic backgrounds. These models are useful for studying epithelial responses to NSAID-induced injury and may identify restitutive targets in iatrogenic disease throughout the GI tract.

Genetic models : There is a plethora of gene knockouts that induce spontaneous colitis in mice (e.g., Refs. ). These genes affect a variety of host processes, including immune tolerance of the intestinal microbiome, epithelial barrier function, T cell signaling, the unfolded protein response, and so forth. One commonly used genetic model of colitis is the knockout of the cytokine interleukin (IL)-10 (encoded by the Il10 gene). In humans, defects in the IL-10 pathway are associated with infant-onset IBD with extensive distal colonic and anal involvement. IL-10 is involved in the suppression of immune responses to the colonic microbiome; IL10 −/− mice raised in a germ-free environment do not develop colitis. In conventionally housed animals, the colitis usually begins after weaning and intensifies with age. However, there is considerable variability in colitis onset and severity between and within animal care facilities, likely reflecting the effects of differing microbial ecosystems. Coinhibition of signaling proteins, such as EGFR, involved in epithelial restitution along with IL-10 accelerates the onset and increases the intensity of disease. Thus, genetic colitis models combined with transgenic targeting to intestinal epithelium may identify pathways involved in restitution. One should note that the outcomes of colitis in these models may be influenced by factors other than restitution; direct examination of ulcer lesions combined with staining for specific markers of WAE or cytoskeletal proteins may be required to interpret results in relation to restitution.

Chemotherapy and radiotherapy : Mucositis in the GI tract is a major adverse event in patients receiving radio- and chemotherapy. Chemotherapeutic agents and radiation are also used to induce injury in the rodent intestine; restitution pathways can be studied in these models with the ultimate goal of reducing the burden of mucositis in cancer treatments. Administration of high-dose methotrexate, doxorubicin, or ionizing radiation induces crypt cell apoptosis, ulceration, and regenerative responses in the small intestine and colon. Radioablative models have been used to determine the identities of reserve stem cell populations in the mouse small intestine. Interestingly, differing results are obtained depending on the dose and schedule of radiation given. In addition, application of both chemotherapy and radiation will alter the number and function of immune cells, especially neutrophils; thus, these may not represent optimal models for understanding the crosstalk between epithelium and immune cells in restitution.

Infection models : Mouse infection models enable the study of intestinal epithelial restitution in a context mimicking human infectious colitis. In particular, Citrobacter rodentium infections in mice emulate enteropathogenic Escherichia coli and enterohemorrhagic E. coli infections in humans. Gavage of overnight cultures of C. rodentium induces acute colitis that can range from mild to severe depending on the inbred strain of mice. While mild disease induces epithelial hyperplasia, immune cell infiltration, and goblet cell depletion, severe disease causes crypt loss and marked erosion of the epithelial surface, thereby providing a context for restitution.