In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring coloni c crypt function in vivo in mice. Na+ concentration in the extracellular pe ricryptal space of descending colon was measured using a low-affinity Na+-s ensitive fluorescent indicator consisting of an Na+-sensitive chromophore ( sodium red) and an Na+-insensitive chromophore (Bodipy-fl) immobilized on 2 00-nm-diameter polystyrene beads. The Na+ indicator beads accumulated in th e pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na+ concentration ([Na +]) in the pericryptal space was 491 +/- 62 mM (n=4). After a 70-min exposu re to amiloride (0.25 mM), pericryptal [Na+] was reduced to 152 +/- 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [ Na+]to 204 +/- 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 k Da) led to rapid accumulation of the dye into the crypt lumen with a half t ime of 19.8 +/-1.0 s, which was increased to 77.9 +/-6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measur e colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The peri cryptal space represents the first example of a hypertonic extracellular co mpartment in mammals that is not created by a countercurrent amplification mechanism.