AOM/DSS mouse model of colon cancer

Based upon a previously described technique alone (29), young (40–50 days old) female Swiss Webster mice were randomly assigned to treatment groups. Based on our previous experience, a minimum of five animals per group were needed to detect significant differences. They were administered a single i.p. injection of azoxymethane (AOM) at 7.5 mg/kg. One week after the AOM challenge, the mice were placed on drinking water containing dextran sulfate sodium (DSS) at 2%, 3%, or 4% for 7 days, and then provided normal diet and drinking water for the next 2 weeks. The DSS challenge was repeated two more times (in total 3-cycles of DSS) and the protocol took 10 weeks.

The test-drugs, aspirin and Aspirin-PC, at a daily aspirin dose (20 mg/kg) were administered intragastrically starting a week before the AOM challenge. The AOM control group was daily intragastrically administered the same volume of saline. The absolute control group did not receive AOM, DSS or test drugs. During the study, body weight was determined every week as a measure of general health and fecal samples were also collected weekly for hemoglobin analysis as a measure of gastrointestinal (GI) bleeding (potential side effect of aspirin). At the end of the 10-week study period, blood was collected, the mice were euthanized by overdose of isoflurane anesthesia followed by thoracotomy, and the colon measured in length and weighed and biopsies taken and snap frozen in liquid N₂ for biochemical analysis or fixed and stained for aberrant crypt foci (ACF) counts and immunohistochemical (IHC) staining; hematocrit was assessed to detect blood loss due to GI bleeding. Whole blood from the four mice groups were used to isolate PRP, count platelets and TXB₂ analysis. PRP was further challenged with 2.5 microgram/ml of collagen or left untreated (basal). The degree of platelet activation was assessed by flow cytometry using anti-P-selectin FITC and isotype control antibodies, as published (26).