Volume 139, Issue 6, Pages 1837-1843.e1 (December 2010) Assessment of Tumor Development and Wound Healing Using Endoscopic Techniques in Mice  Markus F. Neurath, Nadine Wittkopf, Alexandra Wlodarski, Maximilian Waldner, Clemens Neufert, Stefan Wirtz, Claudia Günther, Christoph Becker  Gastroenterology  Volume 139, Issue 6, Pages 1837-1843.e1 (December 2010) DOI: 10.1053/j.gastro.2010.10.007 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 (A) Pictures of the endoscopic workstation (top) showing in detail the endoscopic instruments [(a) biopsy forceps, (b) endoscope sheath with working channel, (c) injection needle, (d) monitor, (e) camera module, (f) light source and air pump, (g) telescope with camera head] and the anesthetic setup [(h) isoflurane chamber, (i) gas flow tubes, (k) gas mixing chamber]. The endoscope has an outer diameter of 1.9 mm. The air pump provides a continuous air flow necessary for the inflation of the mouse colon. The digital camera is connected to a personal computer to allow recording of endoscopic videos. Instruments, including a biopsy forceps or injection needle, can be introduced through the working channel. (B) Development of tumors monitored by using the mouse endoscopic setup. Different tumor sizes and shapes can be distinguished. Colitis-associated colon cancer was induced by intraperitoneal injection with a single dose of the mutagenic agent AOM followed by 3 cycles of DSS in the drinking water for 1 week and normal drinking water for 2 weeks. For endoscopy mice were anesthetized by a mixture of air and isoflurane using the anesthetic setup and high-resolution colonoscopy was performed, as previously described.6 Black arrows indicate the tumor. (C) Comparison of normal colonoscopy of a healthy colon (left) and chromoendoscopy (right). Visualization of crypt pattern was achieved by flushing the mouse colon with 500 μL of a 1% solution of methylene blue. The arrow indicates an aberrant crypt focus. Gastroenterology 2010 139, 1837-1843.e1DOI: (10.1053/j.gastro.2010.10.007) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 (A) Stepwise procedure of taking a biopsy from a tumor in a living mouse using a biopsy forceps during mouse colonoscopy. (B) Longitudinal endoscopic and histologic analysis of a single tumor. Colonic tumor development was followed by colonoscopy and biopsies for histopathologic analysis were taken at the indicated time points. Black arrows indicate the tumor. The development of a single tumor over time is shown. (C) Measurement of IL-17 (left) and Cyclin D1 (right) expression in control colonic tissue. DSS-treated inflamed colonic tissue and colon tumors at different stages of tumor development were assessed using real-time polymerase chain reaction. Accordingly, RNA was isolated from tumor samples and real-time polymerase chain reaction was performed. *P < .05 compared with control; **P < .01 compared with control. Statistical analysis was performed using the 1-sided Student t-test. (D) Injection of a fluidic substance into a tumor during mouse colonoscopy using an injection needle coupled with a syringe. (E) Stepwise procedure of submucosal injection of reagents. Gastroenterology 2010 139, 1837-1843.e1DOI: (10.1053/j.gastro.2010.10.007) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 An endoscopy-guided, in vivo wound healing model. (A) Representative endoscopic pictures of a healthy mouse and from mice with colitis showing various signs of inflammation, including loose stool, an altered vascular pattern, a bleeding mucosa, and abundant fibrin. (B) Generation of a wound in the mucosal surface by using a biopsy forceps during mouse colonoscopy (left) and its appearance on days 0, 1, 3, and 6. The fresh wound bed usually had a diameter of approximately 800 μm. The black arrow indicates the wound bed. (C) The diagram shows an analysis of wound healing. The size of the wound bed was calculated relative to the diameter of the initial wound bed on day 0; histologic examination of the wound bed from a wild type mice on day 2 after the wounding by cryosections (stain: hematoxylin and eosin and myeloperoxidase antibodies; original magnification, left, 4×; right, 10×). Gastroenterology 2010 139, 1837-1843.e1DOI: (10.1053/j.gastro.2010.10.007) Copyright © 2010 AGA Institute Terms and Conditions