Intratumoral microvessel density (MVD) was recorded by counting CD34-positive vessels in the most vascularized area in four × 200 fields [ 10].
In cases where the most vascularized area was not obvious, two or more optical fields were photographed and the field with the highest MVD was finally chosen for further analysis.
Confocal image stacks with 1-µm spacing were collected from the most vascularized area in the middle of the tumors using a 20x lens with a Solamere micro-lensed spinning disk confocal microscope (Solamere Technologies, Salt Lake City UT) equipped with an intensified charge-coupled device (ICCD) camera (XRMega-10EX S-30, Stanford Photonics, Palo Alto, CA).
Blood vessels were stained with antibody to von Willebrand Factor and MVD was quantitated using two methods: average density throughout the section (a-MVD) and density in the most vascularized area or 'hot spot' (h-MVD).
The area of a tumor to be imaged was determined according to 3 conditions as reported previously [ 27, 29]: (a) the whole tumor, if possible, should be seen on the image; (b) the sectional plane should contain the solid part (wall, septa, and papillae) of the tumor; and (c) the most vascularized area was selected.
Microvessels in the five most vascularized areas in a 200× magnification field (0.74 mm2) were counted simultaneously by two observers, and the average value of the five fields was calculated.
The brain is one of the most vascularized organs and pericytes have a higher density in the brain, and the brain has a lower endothelial/pericyte ratio compared to other organs[38].
These observations corroborated the results that the PEG coverage leads to an improved systemic distribution and to the lighting of the most vascularized areas such as the lower limb and liver, which permanently shelters about 10%% of the total blood [29, 30].
Five of the most vascularized areas within a tumor ("hot spots") identified based on CD31 positivity were chosen at low magnification and vessels were counted in a representative high-power (x400) field in each of these areas.
Gliomas are among the most vascularized tumors.
