Intranasal administration
Atosiban is a competitive OT receptor antagonist24. Both OT and atosiban have been shown to be bioavailable when administered intranasally25,26. The intranasal dose of normal saline was 20μl/day. The intranasal dose of OT was 1mg/kg/day, diluted in 20μl saline. The intranasal dose of atosiban was 10mg/kg/day, diluted in 20μl saline. Mice were handled daily one week before the start of intranasal treatments to reduce the stress associated with the procedure. Two days before DSS-induced colitis, mice had been received an intranasal saline, OT or OT with atosiban. Mice were picked up by the scruff of the neck and exposed in a supine position to immobilize them for the intranasal administration. This position of head was maintained throughout procedure preventing drainage of drug solution to trachea and esophagus. The total volume of 20μl solutions was administered by pipette in 5μl drops in alternating naris every 30 seconds. The drop was placed at the naris opening while occluding the opposite naris allowing the animal to snort the drop into the nasal cavity.
Study design
Behavior and biochemical test comprised four groups: NC, DSS, DSS+IN OT, DSS+IN OT+IN atosiban. NC group was treated with plain drinking water for seven days; untreated DSS group was treated with 2% DSS powder in plain drinking water for 7days; DSS+IN OT group was treated with 2% DSS powder in plain drinking water and intranasal administration of OT for seven days. DSS+IN OT+IN atosiban group was treated with 2% DSS powder, intranasal administration of OT and atosiban forseven days. At seventh day, half of each group were submitted to a water avoidance stress test for 10 minutes. The other half were sacrificed to perform a biochemical test.
In the second series of experiments, chemical colitis was induced in mice 15 days after splenectomy. Splenectomy mice were assigned to the following experimental groups: splenectomy, DSS +splenectomy and DSS+IN OT +splenectomy. With NC, DSS and DSS+IN OT groups together, these three groups were performed the same experiments as the first series. The design can be seen in Supplementary figure.