And light circumstances and have been permitted absolutely free access to food and water, as described previously (Chen et al. 2012). The mice have been age and sex matched and used in complete adulthood, in between 10 and 16 weeks of age. All experiments involving animals had been authorized by the Hannover Healthcare School Committee on investigations involving animals and an independent committee assembled by the regional authorities.Measurement of in vivo fluid absorption and bicarbonate secretionSurgical procedures. The procedure for duodenal cannulation has been described in detail (Singh et al. 2012). The mice were anaesthetized by a spontaneous inhalation of isoflurane (Forene; Abbott Germany, Wiesbaden, Germany). The inhalation gas contained a mixture of 105 oxygen, 850 air, and 2.0 0.two isoflurane using the use of an isoflurane pump (Univentor 1250 Anaesthesia Unit; AgnTho, Liding, o Sweden) and, just after tracheal intubation, they had been mechanically ventilated (MiniVent Variety 845; Hugo Sachs Electronik, March-Hugstetten, Germany) and also a catheter was placed within the left carotid artery for continuous infusion of a solution in the following composition (mM) to right the systemic acid ase balance: 200 Na+ , one hundred HCO3 – , 0.005 K+ and 0.005 Cl- , at a price of 0.three ml h-1 . Animals have been maintained at 37 C working with a heating pad. Immediately after making a mid-line incision within the abdomen, the middle (beginning 2.five cm away from caecocolonic junction) and distal colon (last 1.five cm) with intact blood provide were selected for the experiments. The middle colon was opened about 2 cm distal for the caecocolonic junction. A smaller polyethylene tube (PE-100; inner diameter 1 mm) having a distal flange was inserted and secured by a ligature, then the middle and distal colon was perfused gently to remove the contained faeces. The outlet tube (PE-200; inner diameter 2 mm) was place through the rectum and fixed in the anal canal. The middle and distal colon was perfused (Gilson minipuls evolution, Villiers, France) at a price of 30 ml h-1 with typical saline using a single pass. The abdomen was closed right after the surgery, and then the mice were left to stabilize for 20 min for fluid equilibration after the surgical trauma. Measurement of colonic fluid absorption and HCO3 – secretion. Measurement of fluid absorption began afterduring the perfusion period.Inavolisib Following each and every experiment, the blank fluid loss as a result of water evaporation, adhesion around the tube tip and also other non-intestinal absorptive motives was measured inside the exact same perfusion situations (37 C water bath, 30 ml h-1 perfusion speed, 20 min period) but without intestinal perfusion.Hyaluronic acid The difference amongst total fluid loss and blank fluid loss was taken because the absorptive fluid loss.PMID:24275718 Just after the experiment, the mice had been killed by cervical dislocation along with the colonic segment was measured. The fluid absorption prices (in microlitres per centimetre per hour) have been calculated in the absorptive fluid loss (assuming a fluid density of 1 ml mg-1 ), colonic length and perfusion time. The measurement of in vivo colonic HCO3 – secretion was very equivalent to that for duodenal HCO3 – secretion (Xiao et al. 2012a).Two-photon in vivo measurementsMeasurement of pHi within the duodenocytes. The duodenum in the anaesthetized mouse was exteriorized with an intact blood provide, opened near the mesenteric axis and mounted on a custom-made perfusion chamber as previously described (Atuma et al. 2001; Sjblom o et al. 2009). Immediately after surgery, the exposed duodenal segment was incubated with 1 ml of 6 mM acet.
