115 Simultaneous fluorescence imaging of bowel perfusion and ureter delineation using methylene blue One of the main limitations of this animal study is the small sample size. However, for the feasibility of our hypothesis and to respect the 3R principle (replace, reduce, refine) in animal research as described by Russell and Burch 28, the current number of animals was deemed sufficient. The current results must be interpreted with caution and human studies are necessary to evaluate the reproducibility of our finding in a clinical setting. In addition, while most of the elective abdominal clinical procedures are performed laparoscopically, in this study due to logistical reasons the camera system used was an open camera system (for use after laparotomy). Fortunately, a laparoscopic variant of the camera used in this study is commercially available. The duration of the study per animal was not exactly the same. The study setup was to have a follow-up time of 120 minutes (most related to a clinical setting) of observation in all animals. This requirement was met in all animals. As an interesting additional finding, we were also interested in the fluorescence signal of the ureter over time. The difference in the timings in the various animals after 120 minutes can be simply explained due to logistical reasons. As we performed more than one operation per day, the timing of the start of the procedure was the most important factor in the maximum time of follow-up after dye injection. This feasibility animal study has provided the basis for further, larger human studies evaluating dual-imaging camera systems, using only one single dye (MB) for ureteral imaging and bowel perfusion assessment. In line with ongoing animal intestinal perfusion quantification research by our team, we believe a next step should be to further investigate the use of MB in assessing bowel perfusion, as this is not well described 19 and take this research to the human setting in colectomy procedures. CONCLUSION Our study shows the feasibility of simultaneous fluorescence angiography with ICG and ureteral imaging with MB in the same surgical procedure, using the same commercially available NIRF imaging equipment. As both dyes can be used in humans, we believe that there is high potential for clinical translational. Additionally, this dual camera system allows for the simultaneous assessment of bowel perfusion and ureteral visualization, using a single dye (MB). Further human studies are necessary to translate our findings to clinical application. 5
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