Animals and experimental protocol
All experiments were performed with wild type mice (Cftr+/+ ) and CFTR null mice (Cftr-/- ) (FVB/N-CFTRtm1CAM) (Ratcliff et al., 1993). BothCftr+/+ and Cftr-/-mice were bred and maintained at Hannover Medical School as previously described (Xiao et al., 2012), except for the use of an energy-rich diet for transgene animals (1414MOD141003, Altromin, Lage, Germany) instead the low-fiber diet (C1013, Altromin, Lage, Germany), after the mice had survived the first 2 months of age. Cftr -/-and their respective wild type littermates were co-housed (unless the males need to be separated due to fighting), and received identical diet (1414MOD141003, Altromin, Lage, Germany) and the osmotic laxative drinking solution (Oralav, 40 mmol l-1Na2SO4, 75 mmol l-1NaHCO3, 10 mmol l-1 NaCl, 10 KCl, 23 g l−1 PEG 4000) to prevent obstruction and enhance survival post weaning.
The experimental protocol was performed in age-matched mice (9-13 weeks) with similar percentage of males and females. Post withdrawal of Oralav,Cftr +/+ and Cftr-/-mice were intragastrically gavaged twice daily (with a gap of 10-12 hours) with either the vehicle (PBS, pH 6.0) or with Tenapanor (30mg kg-1) for 21 days. Mice were assigned into the vehicle or Tenapanor treated group randomly. The mice were monitored daily and sacrificed either at the end of the experiment or if obstruction was suspected. Mice were sacrificed when a suspicion of obstruction was present over two consecutive observation periods (1 observation period every 12 hours, lasting 12 hours). Suspicion of obstruction was present when the mice had a prominent abdomen, a hunched body composure, no passage of feces during one 12 hour observation period, or a score of 3 or higher in the mouse wellbeing score. The severity and scoring was assessed based on a published severity assessment system which is recommended by our institute for animal research (Bleich & Tolba, 2017). The abdomen was opened, the intestine was dissected, photographed and the site of obstruction was verified. Post sacrifice, the intestinal tissues were harvested for RT-PCR and histology.
The experimental protocol was approved by the Hannover Medical School Committee and an independent committee assembled by the local government. Animal studies are reported in accordance with the ARRIVE guidelines 2.0 and the guidelines from the British Journal of Pharmacology (Percie du Sert et al., 2020). The application and permission numbers are Az. 33.14-42502-04-14/1549 and Az. 33.12-42502-04-19/3197 for breeding “stressed strains” and Az. 33.9-42502-04-18/2829 for experimental procedures.