Page 34 - Effects of radiotherapy and hyperbaric oxygen therapy on oral microcirculation Renee Helmers

Chapter 2
Microcirculatory analyses on vessels with diameters (Ø) <25 μm of total vessel density (TVD; mm vessel/mm2) and perfused vessel density (PVD; mm perfused vessel/mm2), proportion of perfused vessels (PPV; %),9 and microvascular flow index (MFI; score based on determination of the predominant flow type in four quadrants defined as either being absent (0), intermittent (1), sluggish (2), or normal (3))6,43 were performed on a computer with a 19-inch Samsung SyncMaster 932mv LCD monitor with a 1440×900 screen resolution using the Automated Vascular Analysis software package (AVA v3.02, MicroScan Video Microscope System, MicroVision Medical, Amsterdam, The Netherlands). Blood vessel diameter (Øbv) analysis was performed according to a method described elsewhere.49 In brief, blood vessel diameter data was obtained from the skeletonization performed on the blood vessel density procedures in AVA for the TVD data. Five blood vessels (Ø<25 μm; mix of arterioles, capillaries, and venules) were selected at random from each of the four quadrants (i.e. 20 vessels total/video clip). The same 20 blood vessels and their diameters were analyzed at specific locations that were selected on each vessel segment as a landmark (e.g. bifurcation or vascular crossing) to ensure reproducibility across all measurement time points.
Statistical analysis
Based on studies correlating systemic hemodynamic variables with lingual mucosal blood flow using LDF in rabbits23,31 in combination with our experience using SDFI in these animal models,18,29 a sample size of eight was derived. Gaussian distribution of all datasets was assessed with the Shapiro-Wilk Normality Test. Each dataset was separately and accordingly analyzed using either repeated measures analysis of variance or Friedman test to detect overall time effects. Assessment of significant main effects were subsequently identified between each time point using a Fisher’s Least Significant Difference (LSD) test or a Wilcoxon signed-rank test (nonparametric datasets). To adjust for inherent biological variations in each subject’s microcirculation at the sublingual ROI, TVD, PVD, and Øbv parameters were normalized with respect to baseline and converted into percentages for ease in data presentation and statistical examination. MFI was evaluated using the Friedman test with a Dunn’s multiple comparisons test for paired nonparametric repeated measurements. Differences detected with a p-value <0.05 were considered statistically significant. All data analysis was performed using IBM SPSS statistics software package (IBM® SPSS® Statistics version 23, IBM Corp. Armonk, NY, USA) and is presented accordingly as either mean±standard deviation (SD) or median (25th- 75th percentiles).
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