326 Chapter 14 assessed with an intravenous dose (IV) of 0.2 mg/kg indocyanine green (ICG). Ureteral visualization was investigated in two pairs, each receiving an IV injection of methylene blue (MB) at doses of 0.75, 0.50, or 0.25 mg/kg. NIRF imaging was conducted using the Quest Spectrum Fluorescence Camera (Quest Medical Imaging, Middenmeer, The Netherlands). The NIRF imaging successfully visualized ureters and bowel perfusion in all animals. Ureters became visible within five to ten minutes and remained clear throughout each experiment (120 - 420 min). A mixed model analysis did not reveal significant differences between the three doses groups or over time. Notably, bowel perfusion could be visualized using MB as well, and no interference was observed between ICG and MB. Additionally, MB exhibited an earlier washout time, which may be clinically advantageous in situations where repeated dye injections are necessary during a surgical procedure. In chapter 6, a quantitative analysis of bowel perfusion assessment for both ICG and MB was performed in another animal model. Four mature female Landrace pigs underwent laparotomy under general anesthesia. An ischemic bowel loop with five regions of interest (ROIs) exhibiting varying perfusion levels was created in each animal. After 10 minutes, an intravenous injection of 0.25 mg/kg - 0.50 mg/kg MB was administered, followed by NIRF imaging in MB mode and measurement of local lactate levels in all corresponding ROIs. This procedure was repeated in ICG mode (IV dose of 0.2 mg/kg) after 60 minutes, utilizing the Quest Spectrum Fluorescence Camera (Quest Medical Imaging, Middenmeer, The Netherlands) for NIRF imaging of both MB and ICG. Intraoperative NIRF imaging of bowel perfusion assessment with MB and ICG proved successful in all studied animals. Ingress (i/s) levels were calculated and correlated with local lactate levels. Both MB and ICG ingress values exhibited a significant negative correlation (r = -0.7709; p < 0.001; r = -0.5367, p = 0.015, respectively) with local lactate levels. Notably, the correlation was stronger for MB compared to ICG, even though ICG analysis showed higher absolute ingress values. Therefore, this fluorescence quantification analysis validated the potential use of MB for bowel perfusion assessment alongside the well-established and widely used ICG. The imaging technology discussed in chapters 5 and 6 provides encouraging results with NIRF during colorectal surgery, notably not just with ICG but also with MB. This study revealed a new potential application of MB in assessing bowel perfusion, a use previously unexplored. A thorough review prior to our study outlined existing applications of MB, covering its use in visualizing ureters, identifying parathyroid glands, imaging pancreatic tumors, detecting margins of breast cancer tumors, and facilitating breast cancer sentinel node biopsies 24. A recent study also revealed its potential in identification of small intestinal neuroendocrine tumors and PM 25. Overall, MB finds application in numerous clinical procedures with a relatively low risk for patients 24, 25. Based on our analyses, it is evident that MB, when employed within a specialized imaging system, provides a variety of simultaneous and versatile functionalities including bowel perfusion assessment. However, the investigation of its fluorescent properties is still in its nascent stage, necessitating further pre-clinical
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