Page 60 - Effects of radiotherapy and hyperbaric oxygen therapy on oral microcirculation Renee Helmers

Chapter 3
SDF measurements
All microcirculatory measurements were performed by the same investigator (RH) in the same room kept at a constant temperature of 22±1oC; data was obtained from all animals preoperatively (baseline), directly postoperatively and on days 2, 4, 7, 9, 11, 14, and 21. As the HBOT lasted until day 11, the last SDF measurement was carried out 10 days after the final tank session. After anesthetizing and withdrawing 1 mL of blood, the animals were placed on their back and a short (1 cm) cotton dental roll was wedged between the right first and second mandible and maxillary premolars to keep the jaws in an open configuration on an operating table. Using a small piece of sterile gauze, the tongue was rolled towards the back of the oral cavity to create free access to the palatine wound flap for positioning of the SDF imaging probe. The ROI was subsequently moistened with a drop of warm (37oC) sterile 0.9% NaCl (saline) solution to enable proper contact between the probe lens and the mucosal surface.
FCD measurements were performed by gently positioning the lens of the imaging probe, covered with a sterile disposable cap (MicroScan Lens, MicroVision Medical, Amsterdam, The Netherlands), perpendicularly over the palatine mucosal surface on the area between the 2 sutures. Without applying pressure and gently gliding the imaging probe at a 1 mm distance from the edge of the flap posterior border, a 2-min video recording of 5 adjacent sites in the length of the wound surface was obtained. A minimum of 10 seconds of stable image acquisition, with proper brightness and contrast adjustments, was recorded at each time point. To ensure no pressure artifacts were present during image acquisition, the SDF probe objective was briefly withdrawn and then advanced between image recordings on each of the 5 adjacent sites in the ROI.
Vessel enumeration
All microcirculatory data stored on DVI tapes were digitalized into AVI files for offline analysis with Adobe Premier Pro 1.5 (Adobe Systems Incorporated, San Jose, California, USA); image analysis was performed by isolating one image frame per adjacent site from each 2-min recording from all time points (45 in total). The selection criteria for isolation of individual image frames for analysis was based on image resolution, clarity, and elimination of pressure induced artifacts as described previously in a consensus meeting on evaluating microcirculation data for analysis.1 All microcirculation data were analyzed at random by the same investigator (RH).
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