Volume 133, Issue 6, Pages 1769-1778 (December 2007) Identification of Epithelial Gaps in Human Small and Large Intestine by Confocal Endomicroscopy  Ralf Kiesslich, Martin Goetz, Elizabeth M. Angus, Qiuping Hu, Yanfang Guan, Chris Potten, Terry Allen, Markus F. Neurath, Noah F. Shroyer, Marshall H. Montrose, Alastair J.M. Watson  Gastroenterology  Volume 133, Issue 6, Pages 1769-1778 (December 2007) DOI: 10.1053/j.gastro.2007.09.011 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Colocalization of acriflavine with nuclear stain. Living mouse small intestine was stained with dyes and visualized by 2-photon microscopy as described in Patients and Methods. All images were collected simultaneously in response to 810-nm excitation. Images of (A and D) Hoechst 33258 (435–485 nm), (B and E) acriflavine (500–530 nm), and (C) confocal reflectance are overlayed in panel F. Arrows indicate epithelial gaps lacking nuclei, and the arrowhead indicates the intervillous (extracellular) space containing 0.5 mmol/L acriflavine. Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Gaps in human terminal ileum. Images were obtained by confocal endoscopy as described in Patients and Methods. A–C and D–F are series from 2 z-stacks of terminal ileal epithelium. The boxed area in A is shown at higher magnification in B and C. B is a superficial plane, while C is from a deeper plane of focus. D–F are similarly associated. The white arrows indicate epithelial gaps. Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Gaps in human rectum. Images were obtained by confocal endoscopy as described in Patients and Methods. A–C and D–F are series from 2 z-stacks of rectal epithelium. The boxed area in A is shown at higher magnification in B and C. B is a superficial plane, while C is from a deeper plane of focus. D–F are similarly associated. The white arrows indicate epithelial gaps. Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Epithelial gaps are distinct from goblet cells. Images from confocal endoscopy of the terminal ileum (A) group of goblet cells; (B) goblet with nucleus (black arrow) and a nearby epithelial gap distinguished by the absence of a nucleus at the basolateral pole (white arrow). (C) Goblet cells shown in standard H&E staining with nuclei indicated. (D and E) Epithelial gaps shown in side view. Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Goblet cells have a distinct appearance. (A) Diagram illustrating the appearance of a goblet cell in the en face view. (B) Goblet cells in the en face view displaying their characteristic “target appearance.” (C) En face view of epithelial gaps. (D) Low and (E) high magnification of epithelial gap (white arrow) with nearby goblet cells (black arrow). Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Epithelial gaps in the ileum of wild-type and Math1ΔIntestine mice. (A) Epithelial gaps in wild-type mouse ileum are indicated (white arrows). The boxed area in A is shown magnified in B. A goblet cell exhibiting the “target” appearance in mice is also shown (black arrow). (C) Ileum from Math1 mutant intestine with a number of epithelial gaps indicated (white arrows). Note the absence of goblet cells. The area in the solid square is shown magnified in D and E. D is a superficial plane of focus, and E is a deep plane of focus. (F) An epithelial gap underneath a shedding cell with condensed nuclear chromatin is shown, which is a magnification of the dashed white square. Horizontal white arrows indicate epithelial gaps. Vertical black arrows indicate goblet cells. Bar = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Scanning electron microscopy of epithelial gaps in wild-type and Math1ΔIntestine mice. Epithelial gaps are marked by white arrows, and goblet cells are marked by black arrows. (A) An epithelial gap and goblet cells in a wild-type (WT) mouse. Boxed area is magnified in B. (C and E) Epithelial gaps in Math1ΔIntestine mouse. Boxes in C and E are magnified in D and F, respectively. Scale bars: C = 200 μm, D = 20 μm, E = 80 μm, F = 20 μm, A = 30 μm, B = 5 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Effect of TNF on cell shedding. (A) TNF-α or saline control (n = 8) was given by intraperitoneal injection (0.16 or 0.33 μg/L g mouse wt; n = 4 for both groups). The anesthetized mouse was then imaged on a confocal microscope as described before. Images were collected at 5-minute intervals at 4 planes 0, 10, 20, and 30 μm from the villus tip. The number of cell shedding events occurring up to 150 minutes after TNF injection was counted. Data are given as the mean ± SEM. (*P < .04, **P < .007 vs control, t test). (B) A single cell (arrow) being shed 17 minutes after 0.16 μg/g TNF-α. (C) This is followed 68 minutes later by shedding of a large number of adjacent cells (arrows and red ellipse). (D) Image of epithelial gaps 19 minutes after 0.33 μg/g. Epithelial gaps maintain barrier function against luminal LY (arrows). In some cases, the integrity of epithelial gaps is lost (arrowheads). The area in the square is enlarged in E. Bars = 20 μm. Gastroenterology 2007 133, 1769-1778DOI: (10.1053/j.gastro.2007.09.011) Copyright © 2007 AGA Institute Terms and Conditions