FIGURE 2.

Analysis of penile relaxation responses. (A) Time-lapse video observation criteria for dynamic relaxation. In order to analyze the relaxation process in vitro, time-lapse visualization analysis (the images were taken 10 s apart) was performed. For achieving an explant relaxation after 5 min of the pre-contraction, relaxation-enhancing compounds (RECs) were applied. After REC treatment, time-lapse pictures were taken for 10 min. RECs of the final concentration of each reagent were treated with 100 μM sodium nitroprusside, 1.0 μM acetylcholine, and 10 μM prostaglandin E₁. A time-lapse video analysis was performed by ImageJ software. (B) Graph was calculated from images of the movie and indicates the relative area of sinusoidal spaces by the prominent phase of the movie. The relaxed CC was significantly increased in its sinusoidal size compared with single SNP treatment. (C,C′) Representative 8-bit images of the PB dye-stained CC during relaxation responses. Many sinusoidal spaces in CC are indicated by yellow dotted lines. From sequential images (interval time was 10 s), the sinusoidal spaces were calculated before and after treatment by ImageJ software. Relaxation was represented as a ratio of the area of sinusoids treated by RECs and before treatment. The whole CC (indicated by the green dotted line and arrow) was extended, as well as the sinusoidal space. The red arrowheads show that the collagen area was stretched by the treatment of the RECs (see Supplementary Movie S2). Scale bar 100 μm.(D,D′) Image of Masson’s trichrome staining of the CC showing the collagen-rich regions. Sinusoidal spaces are separated by structures including collagen fibers (blue signal). Similar to the time-lapse images, the sinusoidal spaces were dilated after treatment with RECs, and the collagen area was extended toward the tunica albuginea. Scale bar 50 μm. (E) Schema shows the reproduction of the ‘erection state’ by the current system. The central collagen area near the deep arteries showed little movement while the collagen area extended outward around the adjacent collagen area. Therefore, the relaxation movement was observed to be region-dependent inside the CC. These results suggest that treatment with RECs reproduces the “erectile state” of the CC in vitro. All data shown are representative results of at least three independent experiments using adult specimens from different litters (n ≥ 3).