Regulated alkali secretion acts in tandem with unstirred layers to regulate mouse gastric surface pH  Heidi K. Baumgartner, Marshall H. Montrose  Gastroenterology  Volume 126, Issue 3, Pages 774-783 (March 2004) DOI: 10.1053/j.gastro.2003.11.059

Figure 1 Effect of superfusion rate on surface pH. The stomachs of anesthetized mice were exteriorized, and the gastric mucosa was exposed. The animal was placed on the confocal microscope stage (with the exposed mucosa facing the 10× objective lens) and superfused with a lightly buffered, Cl-NERF—containing saline solution of pH 3 at the indicated rates. Ratio images were taken 1 to 2 minutes after the superfusion rate was changed. (A ) Representative ratio image reporting the surface pH gradient at different superfusion rates. (B) Quantitative measurement of surface pH gradient thickness with an increased superfusion rate. In the same experiments, pH was measured at various distances tangential to the gastric surface. The thickness (d0.5) was defined as the distance from the gastric surface in which the pH was halfway between the pH directly at the tissue surface and pH 3.0. (C ) Quantitative measurement of surface pH with an increased superfusion rate. Results are compiled from the ratio images from the confocal microscope. The pH was measured directly at the gastric surface in response to increased rates of superfusion (mean ± SEM; n = 4–6 animals per superfusion rate). Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 2 Representative experiment showing the presence and absence of visible mucus. The stomach of an anesthetized mouse was exteriorized and the gastric mucosa exposed. Fluorescent beads (15 μm) were sprinkled on top of the gastric mucosa. The mucosa was then placed on a perfusion chamber on the stage of a confocal microscope and superfused with a lightly buffered saline solution of pH 3. The confocal microscope was used to visualize the tissue and the localization of the bead in the presence (A ) or absence (B) of visible mucus. The left panels are images of fluorescence, and the right panels are images of confocal reflectance images. Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 3 Representative washout experiment of Cl-NERF. The stomach of an anesthetized mouse was exteriorized, and the exposed gastric mucosa was placed on a perfusion chamber on the stage of confocal microscope containing a lightly buffered, Cl-NERF—containing solution of pH 3. The tissue was then imaged (every 2 seconds) in response to 458 nm of excitation while being superfused at 0.8 mL/min. At time 0, the tissue was exposed to a fresh saline solution of pH 3 that did not contain Cl-NERF. Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 4 Quantitative measurement of fluorescence intensity during washout of Cl-NERF (A ) or Cl-NERF/dextran (B). Results are compiled from images taken of fluorescence (Fl.) intensity while the indicator was washed out with fresh saline solution, as shown in Figure 3. Measurements of fluorescence intensity over time were taken from 5 to 10 μm adjacent to the gastric surface in the presence (○) and absence (▵) of visible mucus and at 90 μm away from the gastric tissue (•) (mean ± SEM; n = 5–10). Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 5 Representative images of the unstirred layer during Cl-NERF/dextran washout. Experiments were performed as in Figure 4B. Images presented (before the washout was started) show the confocal reflectance image of representative tissues with (A) or without (E) mucus and the corresponding overlay images with the (red) fluorescence of the Cl-NERF/dextran plus the reflectance image (B and F). The same tissues were imaged 8 seconds after washout was initiated with dye-free solution, to show the physical sites of residual fluorescence in the presence (C and D) or absence (G and H) of mucus. Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 6 Representative fluorescence profile of Cl-NERF/dextran washout. Confocal images of Cl-NERF/dextran washout were used to measure the fluorescence (Fl.) intensity of the dye at indicated distances tangential to the gastric surface. The fluorescence intensity profile is displayed over time for a representative experiment. The slow increase in intensity from 0 to 50 pixels (corresponding to 23 μm) is due to spatial averaging near a nonlinear surface, which smears the values at the interface. Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 7 Representative uncaging experiment. The stomach of an omeprazole-treated (60 mg/kg intraperitoneally) and anesthetized mouse was exteriorized, and the gastric mucosa was placed on the chamber on the confocal microscope stage. The nonperfused chamber contained a strongly buffered saline solution containing 200 μmol/L of CMNB-caged fluorescein (pH 5). A 2-photon microscope was used to uncage the fluorescein in a defined region near the tissue surface, and fluorescence intensity was imaged every 0.3 seconds thereafter. Images are fluorescence and reflectance images overlayed. The tissue is on the right side of the images (white), and the solution containing the caged fluorescein is on the left side (green). Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 8 Quantitative measurements of fluorescence intensity after uncaging fluorescein. Results are compiled from experiments such as those shown in Figure 7. The decay of fluorescence intensity within the uncaging region over time was measured in fresh solution in the absence of tissue (•) or near the gastric surface in the presence (○) and absence (▵) of mucus (mean ± SEM; n = 4–8). Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 9 Quantitative measurements of surface pH in response to a luminal pH of 2. Results are compiled from ratio images of extracellular pH during continuous superfusion (0.2 mL/min) of a pH 3 (•) or pH 2 (○) solution in the same tissues. pH was measured at various distances tangential to the gastric surface (mean ± SEM; n = 5; ∗P < 0.05). Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)

Figure 10 Effect of changing perfusate buffering capacity on the gastric surface pH. Results are compiled from ratio images from confocal measurements of extracellular pH during continuous superfusion (0.2 mL/min) of a pH 3 solution. (A ) pH was measured at various distances tangential to the mucosal surface during superfusion of NaCl 150 mmol/L and HOMOPIPES 4 mmol/L (•), 16 mmol/L (○), 50 mmol/L (□), or 100 mmol/L (▵) (mean ± SEM; n = 5–12; ∗P < 0.05). (B) Surface pH was measured over time from ratiometric images of Cl-NERF taken with the confocal microscope. The gastric surface was superfused with a solution containing NaCl 150 mmol/L and HOMOPIPES 4 mmol/L at 0.2 mL/min. The superfusate was then switched to NaCl 150 mmol/L, HOMOPIPES 4 mmol/L, and mannitol 110 mmol/L (4HP + MANNITOL) to increase osmolarity without changing the buffering capacity and was then switched to an NaCl 150 mmol/L and HOMOPIPES 100 mmol/L solution (100HP). (C ) Surface pH was measured over time during superfusion of NaCl 150 mmol/L and HOMOPIPES 4 mmol/L at 0.2 mL/min. The superfusate was then switched to NaCl 150 mmol/L and citrate 19 mmol/L. Gastroenterology 2004 126, 774-783DOI: (10.1053/j.gastro.2003.11.059)