129 Quantitative analysis of intestinal perfusion with ICG and MB DISCUSSION In this preclinical animal study, we have successfully performed a quantitative analysis of NIRF imaging for bowel perfusion using MB with a commercially available fluorescence imaging system. This analysis showed a significant negative correlation between local lactate levels (as a marker for ischemia) and MB ingress values. This correlation was stronger than the correlation for ICG quantification values, although the absolute ingress values of ICG were higher compared to MB. This camera system solved two significant drawbacks of MB as discussed in the previous literature 14: (1) its absorption and emission in the vicinity of 700 nm, which is susceptible to increased background auto-fluorescence, and (2) the need for distinct equipment settings. To imitate the clinical scenario involving bowel ischemia and/or inadequate anastomotic perfusion, we generated ischemic bowel loops in our experiment. As our goal was to validate fluorescence signals, we used lactate levels as an indicator of the perfusion state of the different ROIs 11, 13, 15. It should be noted that some lactate levels decreased after one hour. A potential explanation could be linked to the existence of small overlapping vessels on the serosa, emanating from a neighboring intestinal segment with better perfusion. This setup might contribute to a slight reperfusion effect, a phenomenon recognized previously by Diana et al. 13. For both MB and ICG imaging results, we found a significant negative Spearman’s correlation for the local lactate and corresponding ingress values in the same ROI. The results indicate that both dyes are suitable to assess bowel perfusion. Interestingly, the correlation of MB was stronger compared to the one of ICG (Spearman’s rho of -7709 and -0.5367, respectively). The latter may suggest that the use of MB for bowel perfusion assessment with this camera system may be more accurate. In contrast, we observed higher absolute ingress values for ICG imaging compared to MB. The finding that MB has a stronger correlation as opposed to ICG which shows higher absolute values may be attributed to the fact that ingress values were calculated at different time points (T = 10min for MB and T = 60min for ICG). To compare absolute ingress values, we performed NIRF imaging in one animal immediately after injecting MB and ICG into a non-ischemic bowel loop. The ingress values were 7.02 i/s for MB and 11.89 i/s for ICG, still indicating a slight difference. However, our research team considers the differences in the absolute values and correlation negligible as the real-time images obtained during surgery were very clear and informative for both dyes. We therefore do not state that one dye is better than the other, but we can conclude that MB may be as good as ICG for bowel perfusion assessment based on our quantitative analysis. Adverse effects are important to consider when performing NIRF imaging with an optical dye. MB is a safe drug at a therapeutic dose below 2 mg/kg 9, 16, which is eight to four times higher than used in the current study (0.25 mg/kg and 0.5mg/kg). There are some known adverse effects when administering doses above 2 mg/kg, such as hypertension, dyspnea, hemolysis, methemoglobinemia, nausea and vomiting, and pain in the chest 17, and it may 6
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