Volume 56, Issue 4, Pages 716-726 (October 2009) Apoptosis and Effects of Intracavernous Bone Marrow Cell Injection in a Rat Model of Postprostatectomy Erectile Dysfunction  Papa Ahmed Fall, Mohamed Izikki, Li Tu, Salem Swieb, Francois Giuliano, Jacques Bernabe, Rachid Souktani, Claude Abbou, Serge Adnot, Saadia Eddahibi, René Yiou  European Urology  Volume 56, Issue 4, Pages 716-726 (October 2009) DOI: 10.1016/j.eururo.2008.09.059 Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 1 Electrical stimulation to induce penile erection after bilateral cavernous nerve ablation (BCNA) or sham operation. Examples of intracavernous pressure (ICP; ●) and mean arterial pressure (MAP; ○) values in response to nerve stimulation after (A) sham operation or (B) BCNA; the ratio of maximum ICP/MAP during electrical stimulation of the pelvic ganglion at (C) week 3 and (D) week 5; in sham-operated rats, ICP increased after electrical stimulation of the pelvic ganglion; in the rats with BCNA (n=20), the ICP response was completely abolished at (C) week 3 (n=10) and (D) week 5 (n=10) and was significantly decreased compared with the sham-operation group (n=20); no significant change in MAP was found in any of the groups, indicating that the changes in ICP were not related to a systemic effect. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 2 Expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), respectively, in cavernous tissue from sham rats (white bars, n=20) and rats subjected to bilateral cavernous nerve ablation (BCNA; black bars, n=20). Real-time quantitative polymerase chain reaction (RTQ-PCR) was used for relative quantification of eNOS and nNOS mRNA to r18S. Relative quantification of protein levels was achieved by Western blot analysis and normalised for β-actin. Results are expressed as mean±SEM. Significant decreases in mRNA and protein levels of eNOS and nNOS were observed at week 3 after BCNA (n=10) as compared with sham operation (n=10). At week 5, mRNA and protein levels of eNOS were normal, whereas nNOS levels were significantly increased after BCNA (n=10) compared with sham-operated rats (n=10), suggesting spontaneous nerve regeneration. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 3 Assessment of neuronal nitric oxide synthase (nNOS recovery and apoptosis after bilateral cavernous nerve ablation (BCNA); (A) immunostaining for nNOS (brown) 3 wk after BCNA shows a weak staining of nerves (black arrow) and arteries (red arrow) located around sinusoid spaces (asterisks) as compared with week 5 (see Fig. 1B) and normal controls (data not shown); (B) 5 wk after BCNA, dense immunostaining for nNOS is found in nerve (black arrow) of the corpus cavernosum; the walls of arteries (red arrow) also express nNOS, suggesting recovery of vascular autonomic innervation; (C) terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) 5 wk after BCNA, showing numerous apoptotic cells (brown nuclei); (D) higher power view of the same section, showing apoptotic cells lining the sinusoid spaces of the corpus cavernosum (asterisks show sinusoid spaces). Two adjacent cross-sections are immunostained with (E) antivimentin and (F) anti–α-actin to determine the nature of apoptotic cells; immunostaining for vimentin (brown) shows that most of the apoptotic cells in Fig. 3D are vimentin+ and were thus considered of mesenchymal origin (C); (F) immunostaining for α-actin (brown) of an adjacent cross-section to Fig. 3D; most of the apoptotic cells found in this area are not smooth muscle cells; (G) TUNEL 5 wk after BCNA showing apoptotic cells (brown) in the wall of an artery at the centre of the corpus cavernosum; both smooth muscle cells (red arrow) and endothelial cells (black arrow) are stained; (H) adjacent cross-section to Fig. 3G immunostained for α-actin showing that apoptotic cells are located in the smooth muscle layer of the artery. Magnification is ×100 for Fig. 3A–C and ×250 for Fig. 3D–H. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 4 Effect on intracavernous pressure of injecting bone marrow mononucleated cells (BMMNC) after bilateral cavernous nerve ablation (BCNA). Figures show the maximum intracavernosal pressure (ICP) and mean arterial pressure (MAP). BMMNC injections increased ICP compared with controls. The difference was significant at week 5 but not at week 3. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 5 Effect of injecting bone marrow mononucleated cells (BMMNC) after bilateral cavernous nerve ablation (BCNA) on expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). Both real-time quantitative polymerase chain reaction (RTQ-PCR) and Western blot showed significant increases in nNOS and eNOS at week 3 (n=10) and week 5 (n=10) after BMMNC injection (black bars) compared with controls (white bars) injected with saline (n=10 at each end point). At week 5, mRNA levels measured using RTQ-PCR were not significantly different between the control (n=10) and BMMNC groups (n=10), whereas Western blot showed significant increases in relative protein levels of nNOS and eNOS in the BMMNC group. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions

Fig. 6 Decreased apoptosis after bone marrow mononucleated cell (BMMNC) injection; (A) terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) performed 5 wk after bilateral cavernous nerve ablation (BCNA) and injection of BMMNCs stained with PKH-26 showed decreased numbers of apoptotic cells (compare with Fig. 3D; arrows point to apoptotic cells). Adjacent cross-section observed through (B) Nomarsky and (C) TRITC filters; PKH-26 dye (C, red) was detected in the connective tissue lining the sinusoid spaces (*) of the corpus cavernosum, indicating the persistence of injected BMMNC at week 5; the areas stained with PKH-26 were those containing decreased numbers of apoptotic cells, indicating that BMMNC protected against apoptosis; (D) shows superimposition of Fig. 6B and C. Magnification is ×250. European Urology 2009 56, 716-726DOI: (10.1016/j.eururo.2008.09.059) Copyright © 2008 European Association of Urology Terms and Conditions