Page 80 - Effects of radiotherapy and hyperbaric oxygen therapy on oral microcirculation Renee Helmers

Chapter 4
(MFI). MFI was determined by describing the type of flow in each quadrant of the clip using the following scoring system: absent (0), intermittent (1), sluggish (2) or normal (3).5,32
Mandibular histopathology
Following sacrifice, the mandible of each subject was carefully dissected and fixed using 4% buffered formaldehyde. Mandibular bone cross- sections were prepared as 4 mm thick slices, which were demineralized in ethylenediaminetetraacetic acid (EDTA) and processed into standard, paraffin embedded, 8 μm thick histological slides. All samples were (immuno-) histochemically stained with hematoxylin and eosin (H&E), Elastica van Gieson (EVG) and analyzed under a light microscope (BX50, Olympus, Tokyo, Japan) at x100 magnification. The samples were analyzed for general histological parameters for tissue integrity and reactive changes such as hemorrhage, acute and chronic inflammation, osteoblast and osteoclast activity, loss of vascular structures, fibrosis and necrosis. Current study samples were compared to the histology of 2 healthy age-matched rabbit mandibular material obtained from a tissue bank of a former study (Ref. No. DFL101932). All histological analyses were performed by the same investigator (HHdB).
Statistical analysis
As no standardized experimental IR rabbit model was available in literature, this pilot study aimed to determine which dose could induce measurable late IR damage in tissue. Since research data on the effects of late IR effects on in vivo microcirculation was not available, this study served as a basis for orientation and design. We aimed to detect an estimated difference between baseline and post- IR in TVD of approximately 8-10% in the proposed study groups. A minimum of 2 animals per group were used in order to achieve procedural endorsement. As all study groups consist of just 2 rabbits the Friedmann test, a non-parametric test with no correction for multiple comparisons, was applied to all groups to detect a statistical difference (p-value <0.05) over time compared to baseline. The data of all parameters were separately analyzed (TVD, PVD, PPV, MFI, body weight, whole blood counts) and presented in mean±SD. Mean TVD and PVD measurements were converted into percentages and to correct for innate biological variations datasets were normalized with respect to baseline. Data analysis was performed with Prism 8.3.1 for macOS (GraphPad Software, LLC, San Diego, California, USA).
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