Charcoal Meal Test for Gastrointestinal Transit Analysis

Forty-eight hours before testing, mice were placed in cages with raised mesh wire to suspend them above their bedding and prevent ingestion of feces or bedding. The animals were habituated for 24 h in the presence of food and water and then fasted for 24 h with free access to water as previously reported.²⁴ This time frame was chosen to deplete the intestine and colon of any feces. To maintain caloric intake and to avoid hypoglycemia, mice had access to a sugar water solution consisting of a final concentration of 5% dextrose for the first 8 h of the fasting period. Mice were treated with either saline (10 μL/g s.c.) or morphine (10 mg/kg s.c.), and 20 min later they were given an oral gavage consisting of 5% aqueous suspension of charcoal in a 10% gum Arabic solution. At 30 min after the administration of the charcoal meal, the mice were euthanized by cervical dislocation, and the small intestine from the jejunum to the cecum was dissected and placed in cold saline to stop peristalsis. The distance traveled by the leading edge of the charcoal meal was measured relative to the total length of the small intestine, and the percentage of intestinal transit for each animal was calculated as percentage transit (charcoal distance)/(small intestinal length) × 100. This is referred to as intestinal transit in the text.