Rats were killed 2, 4, and 8 days following surgery and bead injection. CN injury only (positive control, n = 10) rats served as a positive control for apoptosis following CN injury. Additional controls were performed as follows: heat-inactivated 1X (n — 3) and 2X (n — 3) SHH protein killed at 4 and 8 days post-CN. All penes were harvested and fixed in 4% paraformaldehyde for sectioning. TUNEL staining for apoptosis was performed as described above. Quantification of apoptosis was performed by counting the number of apoptotic cells in photographs of 3-5 fields per rat.
All cells in a given field were counted in a similar manner based on propidium iodide staining. Changes in the number of apoptotic cells were reported as the ratio of the average number of apoptotic cells to the average number of all cells. Significance was determined using a t-test. The ratio of apoptotic cells to all cells was difficult to quantify reliably, since it was dependent on the distance from Affi-Gel beads, the number of beads in a given area, and the amount of SHH protein delivered to a particular area of the penis. An attempt was made to alleviate these concerns as much as possible by quantifying apoptosis within a given distance relative to the Affi-Gel beads (330 im).