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.