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P009 Fluorescence mediated tomography detects and quantifies early intestinal neutrophil infiltration in experimental colitis

T. M. Nowacki*1,2, P. Lenz3, D. Bettenworth2, M. Brückner2, P. Tepasse2, A. Becker4, M. Wildgruber4, M. Eisenblätter4

1Josephs-Hospital Warendorf, Department of Medicine I, Warendorf, Germany, 2University Hospital Münster, Department of Medicine B, Gastroenterology and Hepatology, Münster, Germany, 3University Hospital Münster, Institute of Palliative Care, Münster, Germany, 4University Hospital Münster, Translational Research Imaging Center, Department of Clinical Radiology, Münster, Germany

Background

Recruitment, infiltration, and activation of inflammatory cells are crucial steps in the pathogenesis of IBD. The aim of this study was the visualisation of these processes in vivo and documentation of the kinetics of infiltration in experimental colitis.

Methods

Colitis was induced in C57BL/6 WT mice fed with 2.5% (w/v) dextran sodium sulphate (DSS) in their drinking water. Animals were monitored for weight loss and presence of blood in the stools by hemoccult testing. Intestinal neutrophil infiltration was measured by targeted fluorescence mediated tomography (FMT) after injection of a neutrophil-specific fluorescence labelled (Cyanine7, λexcitation: 750 nm, λemission: 776 nm) rat-anti-mouse Gr1 antibody or unspecific isotype control. FMT examinations and additional white light and fluorescence endoscopy were performed before (Day 0) and during (Day 5) colitis induction as well as at the end of the experiment (Day 10). Distribution of inflammatory cells in peripheral blood samples was determined by FACS staining for CD11b and Ly6C. Post mortem, intestinal neutrophil infiltration was quantified by immunohistochemistry for Gr1 and ELISA measurements of tissue myeloperoxidase (MPO) levels.

Results

Colitic animals showed decreasing body weight and faecal occult blood. FMT revealed a significantly increased level of fluorescence only 5 days after colitis induction when compared with pre-experiment healthy conditions (738.6 pmol tracer vs. 73.2 pmol tracer; p < 0.05) while neither clinical parameters nor endoscopy detected significant changes at this early time. Confirmatory, FACS analysis revealed a significant increase in inflammatory CD11bhighLy6Chigh monocytes (p < 0.05). At the end of the experiment, white light endoscopy showed significant colonic inflammation with confirmatory neutrophil infiltration in colon tissue indicated by significant tracer accumulation in FMT and fluorescence endoscopy (compared with pre experiment healthy conditions, p < 0.05) as well as increased numbers of Gr1 positive cells and elevated MPO levels in post mortem analysis of colonic tissue (compared with healthy control mice, p < 0.05).

Conclusion

Gr1-targeted FMT can detect early colonic infiltration of inflammatory neutrophils before clinical symptoms or endoscopic alterations occur. In vivo FMT and fluorescence endoscopy allow repetitive monitoring of inflammatory activity and kinetics of leucocyte emigration and can be employed in various models of inflammation providing a valuable non-invasive tool to visualise and quantify the accumulation of inflammatory cells or other desirable targets.