After 6 wk, smooth muscle lined sinusoidal tissue was abundant, the morphology of the corpora cavernosa was indistinguishable from PBS-treated controls (Fig. 6), and SHH protein was present in the sinusoidal tissue (data not shown).
CN Injury Induces Abundant Apoptosis
TUNEL staining was performed on control and CN-injured penes 2, 4, 7, and 21 days following CN injury in Sprague Dawley rat penes.
Apoptosis was abundant the first week following CN injury (Fig. 7) and remained elevated above basal levels at 21 days post-CN. Electron microscopy identified abundant apoptosis in smooth muscle cells and to a lesser extent in endothelial cells of the corpora cavernosa following CN injury (Fig. 7).
SHH Inhibition in the Corpora Cavernosa Causes Apoptosis
TUNEL staining for apoptosis was performed on penes from adult Sprague Dawley rats that had been treated with SHH inhibitor for 7 days via Affi-Gel beads or control rat penes treated with PBS. Apoptosis was abundant within the sinusoidal tissue and the tissue between the sinusoids of the corpora cavernosa following SHH inhibition (Fig. 7, number of apoptotic cells within 130 im of a bead in 5E1 SHH inhibitor = 44.3 6 5.2 and in PBS control = 3.8 6 0.8 treated penes, P-value = 0.0001).
FIG. 7. TUNEL staining of CN-injured, SHH inhibited, and SHH protein-treated/CN-injured rats. a) TUNEL staining of corpus cavernosal sinusoidal tissue from CN-injured penes 4 days post-CN injury (left). Apoptosis is abundant in and around the sinusoidal tissue. White arrows indicate apoptotic cells. Original magnification X400. Electron microscopy was used to examine apoptosis in CN-injured Sprague Dawley rat penes 21 days after CN injury (middle and right). Apoptosis was identifiable in endothelial cells (middle, original magnification X4400) and abundant in smooth muscle cells (right, original magnification X4400) of the corpora cavernosa. Apoptotic cells were identified by the appearance of condensed chromatin, membrane blebbing, and nuclear degradation. sm, Smooth muscle;e, endothelium;s, sinusoidal space. Arrows indicate apoptotic cells. b) TUNEL staining of SHH-inhibited (left) and PBS-treated control (right) corpora cavernosa from adult Sprague Dawley rat penes. Apoptosis was abundant following SHH inhibition in the corpora cavernosa, and the localization of apoptosis was very similar to that observed following CN injury. Apoptosis was not evident in PBS-treated control penes, indicating that the presence of the Affi-Gel bead vehicles used to deliver SHH inhibitor or PBS to the corpora cavernosa did not induce apoptosis. Original magnification X400. White arrow indicates apoptotic cells, and yellow arrows indicate red blood cells, which autofluoresce. c) TUNEL staining of Sprague Dawley corpora cavernosa that were treated with PBS (control, left) or SHH protein (right) via Affi-Gel bead vehicle at the time of bilateral CN injury. Four days following CN injury, apoptosis was abundant in the corpora cavernosa, as observed in the PBS-treated control penes (left). This shows that the presence of the bead vehicle itself did not decrease the amount of apoptosis induced by CN injury. In the SHH protein-treated corpora cavernosa, the presence of apoptotic cells was significantly decreased following CN injury (right). White arrows indicate apoptotic cells, and yellow arrows indicate red blood cells, which autofluoresce. b, Affi-Gel bead. Original magnification X100;bar — 10 im.