We conducted a pilot experimental study to assess the top drug prediction in TNBS mice, amrinone. The study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University. We purchased twenty 6-week-old C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME). This species was chosen to match the species used from the source data used for drug prediction from Dohi et al [17], though this species is known to be more resistant to the development of TNBS colitis as compared to others [25,26]. The mice were divided evenly into drug treatment and control groups. TNBS 5% w/v in methanol was purchased from Fisher Scientific Company, LLC (Hampton, NH). TNBS at a dosage of 100mg/kg in 50% ethanol in a volume of 150μL was administered intrarectally to each mouse on day 0. For control mice, 150μL of 50% ethanol solution was used. Amrinone was purchased from Sigma-Aldrich, Inc (St. Louis, MO) and was administered at 10mg/kg in 1% v/v DMSO with saline at a dilution of 1mg/mL via intraperitoneal injection. Control mice received a 10mg/kg injection of saline. The treatment injections were administered daily from day 1 to day 4.

Mice were sacrificed on day 4, which was chosen to further match the experiments from the source data in Dohi et al [17], where mice were sacrificed on day 4. The colon was then dissected and harvested from each mouse. The tissue was fixed in formalin and paraffin-embedded. A longitudinal cross section slice of the colon was mounted on a slide and stained with hematoxylin and eosin.

Each slide was assessed in a blinded and randomized fashion by an independent veterinary pathologist. Each slide was graded based on ulceration, inflammation, edema, and fibroplasia. Ulceration, edema, and fibroplasia were considered as present or absent. Inflammation was graded as follows: 0 = no inflammation; 1 = small, focal areas limited to the lamina propria; 2 = multifocal or coalescing areas extending into the submucosa; 3 = transmural.

Given the heterogeneous and patchy nature of TNBS colitis, we assessed for the presence of fibroplasia, or wound healing, while taking into account the degree of induced colitis. Using R 3.6.1, we constructed a logistic regression model to assess the presence of fibroplasia. We used the degree of inflammation, the presence of ulceration, the presence of edema, and the treatment group as the covariates in our model.

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