An additional band at 38 kDa was observed that was blocked using the antigenic peptide for SHH. From the size of the product it is hypothesized that this band represents a multimer of the active SHH protein. SHH multimerization has been reported in the literature. The 38-kDa product was unchanged with CN injury (control — 1.070 6 0.04, CN-injured — 1.070 6 0.03, P-value — 0.5, Fig. 5). The localization of SHH and PTCH1 remain unchanged after CN injury by IHC analysis.
Localization of Shh and Ptch1 RNA by In Situ Hybridization Remained Unchanged Following CN Injury
The RNA localization of Shh and Ptchl was examined by in situ hybridization in control and CN-injured penes 21 days after CN injury. Shh and Ptchl RNA expression was abundant in the smooth muscle of the corpora cavernosal sinusoidal tissue of both control and CN-injured penes (Fig. 4). A change in RNA localization for Shh and Ptchl was not observed following CN injury.
The localization of the C-terminal SHH protein (precursor form only, Fig. 1) was similar to N-terminal SHH, with two notable differences. Precursor SHH protein was abundant in the perineurium of the nerves, but was not present in the Schwann cells or the veins, as was the active form of SHH protein. SHH/PECAM1 and PTCH1/PECAM1 dual staining shows that SHH and PTCH1 do not colocalize with PECAM1, indicating that SHH and PTCH1 are not present in endothelial cells.
Localization of SHH and PTCH1 in the Adult Penis by IHC
Antibodies that recognize the active and precursor forms of SHH protein and PTCH1 were used to determine their localization in adult Sprague Dawley penis tissue. N-terminal SHH protein (active and precursor forms) was localized within the corpora cavernosal sinusoidal tissue, a layer adjacent to the tunica, Schwann cells of the nerves, veins (Fig. 1), and the urethra. Colocalization of SHH and ACTA1 by dual IHC identifies SHH in smooth muscle cells of the corpora cavernosal sinusoidal tissue.
Quantitative RT-PCR was performed as described previously using noncompetitive methodology and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an endogenous internal standard. All measurements were made in the linear range for Shh and Gapdh (n = 9 control and 9 CN-injured penes) and Ptchl and Gapdh (n = 4 control and n = 4 CN-injured penes). Bmp4 expression was also measured (n = 7 control and n = 8 CN-injured penes) in the linear range for Bmp4 and ribosomal protection subunit 32 (Rpl32), which was used as an endogenous internal standard.
Quantification of apoptosis was performed by counting the number of apoptotic cells in photographs of 3-5 fields per rat and was reported as the average number of apoptotic cells. Significance was determined using a t-test. The number of apoptotic 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 or SHH inhibitor 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 (130 im).
Effect of SHH Inhibition and Supplementation on VEGFA Signaling in Intact Rats
Affi-Gel beads (100-200 mesh, Bio-Rad Laboratories, Hercules, CA) were equilibrated with either 5E1 SHH inhibitor (n = 5, 1-3 ig/ml, Jessel, Hybridoma bank at the University of Iowa), recombinant mouse SHH peptide (n = 5, 7.5 ig per animal, R & D Systems), or mouse IgG (control, n = 5, 3 ig/ml) overnight at 4°C. Approximately 30^0 beads were injected directly into the corpora cavernosa of P120 rat penes. Sprague Dawley rats used for these experiments were normal rats that had not undergone CN injury.
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.
Thin sections were cut and stained with 2% uranyl acetate and 3% lead citrate. Electron microscopy was performed using a JEOL 100CX Transmission Electron Microscope to identify in which cell type apoptosis was taking place. Apoptosis was identified by the presence of condensed chromatin, nuclear fragmentation, and cytoplasmic blebbing, which are common in cells undergoing apoptosis.
Secondary antibodies used were Alexa Fluor 488 rabbit anti-goat (1/300, Molecular Probes) and Alexa Fluor 594 chicken antimouse (1/300, Molecular Probes). Negative controls were performed with secondary only (without primary) to test for nonspecific staining and autofluorescence. Sections were mounted using Pro-Tex Mounting Medium (Baxter Diagnostics, Inc., Pittsburgh, PA). Microscopy was performed using a dual light and fluorescent microscope (Leitz) and photographed using a Nikon digital camera.