Quantitative Histology

To ensure data quality, we defined three standards as inclusion criteria for quantitative graphical analysis of the slices:

Intact lamination after the immunohistochemical processes,

Fascia providing a straight line of at least 4 mm length on the slide, and

Vivid coloring, homogenously spread.

We used a Leica microscope (Leica Microsystems Gmbh, Wetzlar, Germany) linked to the program CellP (Olympus Hamburg, Germany) with attached digital photograph camera to digitalize the slides. Pictures of each slide were taken under twofold magnification, and afterward joined together with the CellP merge function. Using Photoshop (ADOBE San Jose, Calif.), red-colored pixels were separated from the surrounding pixels. The selection was then inversed and cut out, resulting in a picture reduced to the red-colored endothelium in cross section on a white background. Tracing of the fascial rim was performed with the Pixelmator graphics software (ver. 2.0.2; Pixelmator Team Ltd., London, UK), and then combined with the picture of the vasculature. This procedure enabled the identification of the fascia on the sheer-vasculature-image and served as reference level for measuring distances.

Slammer (Ringce, Chromepet, Southern India) was used to subdivide the picture into rectangles of a predefined size with a grid. With ImageJ (http://rsbweb.nih.gov/ij/), the tresholding function was used to select all the pixels, except the pixels depicting the grid. A measuring field with the size of 0.2 mm height × 0.8 mm width was created according to the previously defined grid, and the percentage of pixels below the threshold was set. Thus, all red-colored pixels of each box were measured. Due to the undulating course of the fascia and the common distortion of the tissue layers caused by the processes of immunohistochemistry, we decided to measure a small range of 4 mm in width. In height, the slices were analyzed as far as the staining reached. Excel (Microsoft Office, Microsoft, Seattle, USA) was used for data collection and analysis.