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Vascular Remodeling and Angiogenesis: MATERIALS AND METHODS(7)

METHODS(7)

We have applied a relative scale in order to estimate the condition of the ovary on the basis of follicle growth and viability. The seven follicle types were scored as follows: the viable ones received a positive grade from 1 to 7; the smallest one received a score of 1 and the largest one a score of 7. The atretic follicles were negatively scored, the smallest one received a score of —7 and the largest one received a score of —1. The rationale for this scoring system is based on the probability that small follicles being atretic is normally low, while as the follicles grow, their probability of being atretic is increased. Therefore, we assume that excessive atresia of small follicles in the transplants is the result of ovarian stress due to insufficient blood supply. Thus, atretic primordial follicles received the most negative score (—7). Viable antral follicles received the highest positive score (+7). Each follicle type was graded as follows: follicle grade X (total number of follicles from the specified type/total area of the examined ovary [mm2]). The total score of an ovary was obtained by summing up the scores of each follicle type.

Histological Quantification of Vascular Permeability

The distribution of the biotinylated MR contrast agent in blood vessels and its extravasation in the ovarian graft and the adjacent muscle were measured by the fluorescence intensity of avidin-FITC. The samples were photographed at 100 X magnification at 1 sec of exposure and a gain of 10 dB. Pictures were converted to 256 gray scale by Adobe Photoshop 6.0 ME (San Jose, CA) and the intensity of the different regions was measured using NIH Image. Selected regions of interest of the graft and the muscle were of the same size, whereas the blood vessel was analyzed using a smaller region confined to a well-defined blood vessel. Intensity data were normalized to a scale of 0-1, where 1 represents intensity within blood vessels. The intensity of the contrast agent driven from the histology data is referred to as APShistology. generic paxil