In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy - PubMed (original) (raw)

In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy

C Becker et al. Gut. 2005 Jul.

Abstract

Background: Mouse models of colitis and cancer are indispensable for our understanding of the pathogenesis of these diseases. In the past, mice had to be sacrificed in order to analyse colitis activity and tumour development. We have developed a safe method for high resolution endoscopic monitoring of living mice.

Methods: Mice developing colitis or colonic tumours were anaesthetised using avertine and repeatedly examined by endoscopy. A novel miniendoscope (1.9 mm outer diameter), denoted Coloview, was introduced via the anus and the colon was carefully insufflated with an air pump before analysis of the colonic mucosa. An extra working channel allowed the introduction of biopsy forceps or injection needles as well as surface staining with methylene blue in order to visualise the surface of the crypts and the pit pattern architecture.

Results: Endoscopic pictures obtained were of high quality and allowed monitoring and grading of disease. Scoring of colitis activity as well as tumour size and growth was possible. In addition, pit pattern analysis using chromoendoscopy permitted discrimination between inflammatory and neoplastic changes. Biopsies yielded enough tissue for molecular and histopathological analyses.

Conclusions: In summary, chromoendoscopy in mice allows monitoring of the development of colitis and colon cancer with high resolution. Manipulations such as local injection of reagents or taking biopsies can be performed easily.

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Figures

Figure 1

Figure 1

Experimental setup of the Coloview miniendoscopic system. (A) Schematic diagram of the endoscopic setup. Endoscopic tools used for mouse examinations: straightforward telescope, examination sheath, manipulation sheath, biopsy forceps, and injection tube. (B) Experimental procedure used to induce colon carcinogenesis in FVB mice. Mice were injected intraperitoneally with a single dose (7.4 mg/kg) of the mutagenic agent azoxymethane (AOM) followed by three cycles of dextran sodium sulphate (DSS) in drinking water for one week and normal drinking water for two weeks. E = endoscopic examination.

Figure 2

Figure 2

In vivo high resolution endoscopy and chromoendoscopy of mice treated with azoxymethane and dextran sodium sulphate. Mice were anaesthetised by intraperitoneal injection of avertine. The colon mucosa was stained with methylene blue to visualise the crypt pattern. (A) Representative endoscopic pictures showing the colon of a healthy mouse. Note the smooth and transparent mucosa, normal vascular pattern, and regular crypt pattern in the methylene blue stained colon. (B) Representative endoscopic pictures showing signs of severe inflammation. Note the close up pictures of bleeding mucosa, altered vascular pattern, transparent mucosa, and fibrin. (C) Endoscopic colitis score based on the observed signs of inflammation. The modified murine endoscopic index of colitis severity (MEICS) consisted of five parameters, as indicated. (D) Methylene blue staining of the colonic mucosa during endoscopy at day 20. Shown are representative pictures of chromoendoscopic signs of inflammation and early neoplasias (aberrant crypt foci (ACF)).

Figure 3

Figure 3

Endoscopic scoring of tumour development in mice. (A) Representative endoscopic pictures showing the development of colon tumours during the course of the experiment. The grading of tumour size was performed as indicated in materials and methods. (B) Number of lesions in each mouse was counted and compared during the course of the experiment (left panel). Alternatively, the size of each tumour was graded at the indicated time points and summed up for each mouse (right panel).

Figure 4

Figure 4

Molecular investigation and manipulation of tumour development in mice. (A) Biopsy sampling (upper left panel) of a tumour during routine endoscopic monitoring of azoxymethane/dextran sodium sulphate treated animals. Tumour biopsies were snap frozen in liquid nitrogen and cut for staining. Subsequent haematoxylin-eosin staining (upper middle panel) demonstrated high grade intraepithelial neoplasias. Immunohistochemistry for tumour necrosis factor α (TNF-α) (upper right panel) of cross sections was performed using the TSA Cy3 system. Cryosections were fixed in acetone. Slides were then incubated with primary antibodies specific for TNF-α. Before examination, nuclei were counterstained with Hoechst 3342. Lower left panel: gel electrophoretic picture showing the high quality of RNA extracted from five tumour biopsies. Tumours typically yielded 1–2 µg RNA per biopsy. Lower right panel: reverse transcription-polymerase chain reaction analysis for TNF-α expressed in tumour biopsies. β-Actin served as a control. (B) Injection of fluorescein isothiocyanate (FITC) into a tumour during endoscopy. A needle mounted onto a thin tube was introduced through the instrument channel of the endoscope and FITC was slowly injected. Tumour samples were then analysed by immunofluorescence. One representative picture of a tumour injected with 20 µl of an FITC solution and a control tumour not injected is shown.

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