104 Chapter 5 MATERIALS AND METHODS This feasibility study was performed at the central animal facilities of Maastricht University (Maastricht, The Netherlands). Animals were used in compliance with Dutch regulations and legislation concerning animal research, and the study was performed according to a protocol approved by the Experimental Animal Committee of Maastricht University (DEC-UM) (approval number: 2017-021-001). Informed written consent was not applicable. Animals A total of six mature (35-45 kg) female Landrace pigs were used for this study. A pig model was chosen because of the anatomical similarities between humans and pigs, and previous successful application of NIRF imaging in pigs 13. Animals were used in compliance with the regulations of Dutch legislation concerning animal research, and the study was performed according to an approved protocol by the local animal ethics committee. Preparation of the dyes MB (Proveblue, Provepharm Life Solutions, Marseille, France) was diluted in a sterile phosphate-buffered saline (PBS) solution to a concentration of 1 mg/ml. In a previous review, MB doses ranging from 0.25 to 1 mg/kg were studied 14. A dose of 0.75 mg/kg resulted in the highest target-to-background ratio of the fluorescence image of the ureter 14. However, in another review by van Manen et al. 15, a dose of 0.25 mg/kg was recommended. In our own experience in a clinical pilot study using this dye, we found that NIRF imaging was strongly influenced by the dose/concentration of the dye 4. Consequently, in this study, we investigated three different MB doses: two pigs received a bolus IV injection of 0.75 mg/kg (group 1), two other pigs of 0.50 mg/kg (group 2), and the final two pigs 0.25 mg/kg (group 3). Additionally, ICG (Verdye, Diagnostic Green GmbH, Aschheim, Germany) was diluted in a sterile H2O solution to a concentration of 2.5 mg/ml. A dose of 0.2 mg/kg was given as a bolus IV injection. This dose is based on the current frequently clinically used dose range in patients as was previously found in our analysis of 1,240 patients registered in the EURO-FIGS registry on fluorescence angiography 16. Fluorescence imaging system The commercially available Quest Spectrum Fluorescence Camera (QUEST SPECTRUM®, Quest Medical Imaging, Middenmeer, The Netherlands) was used for NIRF imaging. To ensure standardized measurements and prevent potential movement of the camera, the camera was fixed with a custom-made mechanic, articulated arm, which was connected to the surgical table. The distance of the camera tip to the target organ was measured with a sterile paper ruler and was 15 cm in all procedures. During NIRF imaging, environmental lights were dimmed preventing ambient light interference. Because of this standardization, and prevention of motion of the camera and animal, we ensured to have high quality of images through the surgical procedure. The 800 nm channel was used to capture ICG fluorescence
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