Azoxymethane

All in vivo experiments were performed in male C57Bl/6 mice (20 to 25 g; Charles River Laboratories, Wilmington, MA, USA). Mice were given free access to water and rodent chow and were housed in constant temperature, humidity, and 12 h light-dark cycling. Acute liver failure was induced via a single intraperitoneal (ip) injection of 100 mg/kg of azoxymethane (AOM). In parallel, systemic inhibition of CCR2 and CCR4 activity was accomplished via pretreatment with INCB (1 mg/kg/day ip) or C021 (1 mg/kg/day ip) for 3 days prior to injection of AOM. Following injection, mice were placed on heating pads adjusted to 37°C and monitored frequently for signs of neurological decline. To reduce the impacts of hypoglycemia and dehydration, cage floors were supplied with hydrogel and rodent chow and after 12 h, and every subsequent 4 h, mice were injected subcutaneously with 5% dextrose in 250 μL of saline. If mice underwent a 20% or greater weight loss they were removed from the study. All animal experiments performed were approved by and complied with the Baylor Scott & White IACUC regulations on animal experiments (protocol #2011-052-R).
At 8 h following injection (and every 2 h after), body temperature, weight, and neurological assessments were measured. Neurological functioning was assessed by measuring the pinna reflex, corneal reflex, tail flexion reflex, escape response reflex, righting reflex, and ataxia, which were assessed and scored on a scale of 0 (no reflex) to 2 (intact reflex). The neurological score at each time point was defined as the summation of these reflex scores. In addition, time to coma (defined as a loss of all reflexes) was recorded. Tissue was flash frozen and collected at coma (loss of corneal and righting reflexes) for further analysis. Mice used for histochemical studies were transcardially perfused with PBS followed by 4% paraformaldehyde. Whole brains were removed and placed into paraformaldehyde for 24 h, after which they were moved to a 30% sucrose solution for cryoprotection. Brains were frozen and sectioned using a cryostat for immunofluorescence imaging.

Host mice received an intraperitoneal injection of azoxymethane (AOM; 10 mg/kg). 5 days later, mice received dextran sulfate sodium (DSS) at 2.5% ad libitum in drinking water for 7 days, followed by a second 5-day cycle at 2.5% and a third DSS cycle (5 days, 2.0%). DSS cycles were interspaced with a recovery cycle with sterile drinking water for 14 days. At the end of the third recovery cycle, mice were bled and received 25 μg of either isotype (IgG1, R&D Systems) or anti-GCSF (MAB414, R&D Systems) antibody intraperitoneally three times a week for 3 weeks. Colons were harvested, cleaned, washed and tumor load was assessed under a dissection microscope. Tissues were processed to a single-cell suspension as described previously (31 (link)). Each in vivo study contained at least four replicates and the study was repeated three times.

Azoxymethane (AOM, 10mg/kg, Sigma-Aldrich- A5486-25MG) in saline was injected intraperitoneally (IP) 2 days in advance of three cycles of 3.5% dextran sodium sulfate (DSS, MP Biomedicals- 160110, molecular weight: 36,000–50,000). Each cycle consisted of 5 days of 3.5% DSS in the drinking water, followed by 16 days of regular water. Mice were euthanized at various time points (“baseline”; at the end of each DSS treatment, designated as “colitis”; one week after each DSS cycle, designated as “recovery”, with the end of the 3^rd cycle being designated as “cancer”). This AOM-DSS colitis model leads to predictable and reproducible colitis and cancer [15 ]. Disease activity index (DAI) was recorded, a colonoscopy was performed 1 week after each DSS treatment, and mice were euthanized at various time points (colitis, recovery, cancer) with colon tissue either stored in liquid nitrogen or placed in formalin. Disease activity index was calculated based on weight loss (1 = 0–10%, 2 = 10–15%, 3 = 15–20%, 4 = >20%), stool consistency (1 = solid, 2 = loose, 3 = wet, 4 = diarrhea), and hematochezia (1 = no blood, 2 = slight blood, 3 = blood, 4 = significant hematochezia).

The male Sprague Dawley rats (30 animals) were grouped randomly into five groups (6 animals for each): Group 1 (Normal control), Group 2 (Cancer control), Group 3 (Reference control), Group 4 (200 mg/kg OME), and Group 5 (400 mg/kg OME). The rats were given anesthesia before the injection of Azoxymethane (AOM) as an inducer for aberrant crypt foci (ACF) into rats is a common laboratory method for colon cancer investigation. The rats (besides the normal rat group) received 15 mg/kg of colon carcinogen (AOM) once a week for two weeks by subcutaneous injection. After that, Group A (Normal control) received Normal saline 15 mg/kg and then treated with 10% Tween 20 (5 mL/kg). Group B (Cancer control) was subcutaneously injected with AOM and then orally administered with distilled water. Group C (Reference control) was injected with AOM and intravenously injected with 35 mg/kg of 5-FU (5-fluorouracil). Group D (Low dose) was injected with AOM and administered OME (200 mg/kg) orally. Group E (High dose) was injected with AOM and administered OME (400 mg/kg) orally.
All rats had free access to food and water and the body weight of the rats was measured throughout the procedure. After two months, the rats were sacrificed and colon tissues were obtained for the histopathological examination. The tissue samples from colons were prepared by freezing with liquid nitrogen and homogenization.