Sildenafil citrate improves erectile function after castration in a rat model

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1 Sildenafil citrate improves erectile function after castration in a rat model John P. Mulhall*, Nipun Verma, Serkan Deveci, Raanan Tal*, Keith Kobylarz and Alexander Müller* *Department of Urology, Memorial Sloan-Kettering Cancer Center, and Department of Urology, Weill Medical College of Cornell University, New York, USA Take Home Message The administration of phosphodiesterase 5 inhibitor commencing at the time of castration might preserve erectile function. Objective To determine if sildenafil citrate treatment could improve erectile function after castration. To determine if sildenafil citrate treatment reduces collagenisation and apoptosis in erectile tissue after castration. Materials and Methods In all, 60 Sprague-Dawley rats were studied; the rats were divided into the following groups: sham no orchidectomy (S), control orchidectomy only (O) and treatment orchidectomy plus sildenafil treatment (V), with 10 rats per group. Erectile haemodynamics assessment was done at 7 days (S7, O7, V7) and at 28 days (S28, O28, V28) yielding a total of six groupings. Functional assessment measured the mean maximum intracavernosal pressure mean arterial pressure (ICP/MAP) ratio. TUNEL assay was used to define apoptotic indices (AIs) and Masson s trichrome staining was used to evaluate smooth muscle collagen (SM C) ratios. Results The S28 group had the highest and the O7 group the lowest ICP/MAP ratio, at a mean (SD) of 70 (6)% and 36 (6)%, respectively. Both treatment groups, V7 [42 (12)%] and V28 [49 (13)%] showed statistically significant improvements over their corresponding control groups: O7 [36 (6)%] and O28 [37 (9)%] (P < 0.05). However, ICP/MAP values for V7 and V28 remained significantly below the S28 group (P < 0.001). There were no significant differences in ICP/MAP values between the 28-day and 7-day ICP/MAP ratios within each group (S, O, V). There were no significant differences in SM C ratio between the O and V groups (O7 vs V7, P = 0.45; O28 vs V28, P = 0.16). There were no significant differences in AIs between the O and V groups (O7 vs V7, P = 0.54; O28 vs V28, P = 0.8). Conclusions Daily treatment with sildenafil improved erectile function in rats after castration. ICP/MAP ratios increased significantly in the treatment groups compared with the control groups with the greatest erectile function occurring 28 days from administration. In this series of experiments the improved erectile function recovery with sildenafil after surgical castration cannot be explained by smooth muscle protection and decreased collagenisation. The improved erectile function with sildenafil after surgical castration cannot be explained by reduced apoptosis in erectile tissue. Keywords orchidectomy, rat, castration, phosphodiesterase type 5 inhibitor, sildenafil Introduction Androgens have a modulating effect on multiple pathways for erectile function, but their role in this area remains incompletely understood [1]. Castration either by surgical or chemical means results in negligible levels of testosterone in the body and is used as a standard therapy for metastatic prostate cancer [2]. The resulting testosterone depletion has been shown to have a deleterious effect on erectile function because of negative changes within the erectile tissue of the penis [3]. BJU Int 2014; 113: wileyonlinelibrary.com 2013 BJU International doi: /bju Published by John Wiley & Sons Ltd.

2 Sildenafil citrate and castration Castrated rabbits were shown to accumulate adipocytes in the sub-tunical region of the penis, suggesting that a lack of testosterone promotes the differentiation of progenitor stroma cells into fat cells [4]. Another hypothesised change in the penile structure is erectile tissue collagenisation [5]. The resulting imbalance between smooth muscle and collagen results in the loss of expandable trabecular smooth muscle in the corpora cavernosa, leading to the development of veno-occlusive dysfunction (venous leak). Experiments in castrated rats have shown decreased expression and activity of neuronal nitric oxide synthase (nnos) and endothelial NOS (enos) in the corpora cavernosa, which is associated with decreased response to cavernous nerve stimulation [6]. Castration also resulted in reduced expression and activity of phosphodiesterase type 5 (PDE5) [7]. The PDE5 inhibitor (PDE5i) sildenafil citrate (V), has been shown in several animal studies to minimise collagenisation of erectile tissue after cavernous nerve injury [8 10]. The aim of the present study was to define whether the use of sildenafil would result in preservation of erectile function and tissue structure in the rat penis after castration. Materials and Methods In all, 60 male Sprague-Dawley rats, initially weighing g were studied and randomly divided into six groups (10 rats in each group): (i) Sham rats (S) had no surgery but were exposed to an equivalent time of anaesthesia; control rats (O) underwent a bilateral orchidectomy (O); treatment groups (V) had a bilateral orchidectomy and were injected with sildenafil citrate (V) 20 mg/kg s.c. daily commencing on the day of castration. Within all three groups (S, O, V), erectile haemodynamics were measured at two time points, 7 and 28 days, resulting in six groups: S7, S28, O7, O28, V7, and V28. Our Institutional Animal Care Committee approved the study and the rats were cared for and housed under strict guidelines established by the Institutional Animal Care and Use Committee guidelines. Castration For the initial surgery, all rats were anaesthetised using 4% isoflurane and placed supine. The control and treated rats underwent surgical castration. A lower midline incision was made and the abdominal wall was retracted. Both testes were manipulated from the scrotum into the abdominal cavity. The vas deferens and associated vasculature was identified and separately ligated and removed. The rats in both treatment groups (V7, V28) received sildenafil citrate 20 mg/kg s.c. daily for either 7 or 28 days commencing immediately after castration. The last dose of sildenafil was given 24 h before the second surgery (erectile haemodynamics assessment). Haemodynamics Assessment Measurement of the maximum intracavernosal pressure mean arterial blood pressure (ICP/MAP) ratio is a standard measurement to assess erectile function [11]. At the second, non-survival surgery, at either 7 days (S7, O7, V7) or 28 days (S28, O28, V28), after anaesthesia induction, a lower midline incision exposed the bladder, prostate and cavernous nerves. The penis was denuded of skin. And the cavernous nerves were isolated for electrode placement. A stainless-steel bipolar electrode with parallel hooks (1-mm apart) was placed around cavernous nerve and positioned by a micromanipulator. A Grass S48 stimulator (Quincy, MA, USA) was used, with stimulation parameters of: 20 Hz, 5 ms duration, and 7.5 V. To measure ICP, a heparinized 24-G Angiocath (Insyte-N, Becton Dickinson Vascular Access, Sandy, UT, USA) attached to polyethylene-50 tubing was inserted into the rat right cavernous body and connected to a pressure transducer and an amplifier unit (Harvard Apparatus, Holliston, MA, USA). The amplifier was connected to a data acquisition module (DI-190, Dataq Instruments, Akron, OH, USA). The data was recorded on a computer with Windaq/Lite recording software (Dataq). Structural Analyses Immediately after nerve stimulation, penile tissue was harvested and prepared for structural analysis. Masson s trichrome (MT) staining was used to evaluate smooth muscle collagen (SM C) ratio on slides using a complete cross-section of the corpora cavernosa. Briefly, MT is a three-colour staining protocol staining keratin and muscle fibres red, collagen and cell nuclei blue. Montage on an Olympus Meta Morph Imaging System was used to capture the complete cross-section of the penile tissue of each rat. Terminal deoxynucleotidyl transferase-mediated biotin-dutp nick-end labelling (TUNEL) assay was used to calculate the proportion of apoptotic cells within the corpora cavernosa cross-section (apoptotic index, AI). Briefly, after preparing slides from 5 7 mm tissue sections, they were then deparaffinized with xylene, passed through graded ethanol solutions, and rehydrated with PBS. Nuclei were exposed by treatment with recombinant proteinase K, followed by citrate/triton X buffer on ice. TUNEL-positive cells were detected using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA). The ratio of cells stained with the fluorescent TUNEL method to the total number of cells stained with 4,6-diamidino-2-phenylindole (DAPI) was recorded. Two slides per rat were prepared counting five non-overlapping fields on each slide using a Zeiss axioplan 2 microscope system at ax20. Image Analysis Image analysis was done using ImageJ ver.1.33u (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, 2013 BJU International 657

3 Mulhall et al. Table 1 ICP/MAP ratios. Group 7 Days 28 Days P Fig. 1 SM C ratios. 45 S O V P 0.047* <0.001, <0.005* V, orchidectomy plus sildenafil treatment; S, sham no orchidectomy, O, orchidectomy only. *O vs V; SvsV. Maryland, USA, ). For IHC, the mean stained area percentage was calculated across 10 tissue sections (two rats per group, five sections per rat). For MT staining one slide per rat was used to calculate the ratio of the red staining smooth muscle to the blue staining collagen content in the cross-section of the corpora cavernosa. Smooth Muscle/Collagen ratio, % O7 V7 O28 V28 Statistical Analysis Mean values were calculated for each group and reported as themean(sd). Individual pair-wise comparison between groups was analysed with independent two-tailed Student s t-tests; a P < 0.05 was considered to indicate statistical significance. Results Table 1 gives an overview of the ICP/MAP ratios for all groups. As expected the sham group had the highest ICP/MAP ratios at 7 days, at 67 (9)%, and at 28 days, at 69 (6)%. The orchidectomy only control group had the lowest ICP/MAP ratios at 7 days, at 36 (6)%, and at 28 days, at 37 (9)%. Notably the ICP/MAP ratio of the sildenafil treatment group at 7 days, at 42 (12)%, was significantly greater than the ICP/MAP ratio of O7 (P = 0.047). And the ICP/MAP ratio of the sildenafil treatment group at 28 days, at 49 (13)%, was significantly greater than O28 (P < 0.001). Furthermore compared with the sham and orchidectomy groups the sildenafil treatment group had a greater improvement in ICP/MAP ratios between the 7 and 28 days measurements, although it was not a statistically significant change. However, the ICP/MAP ratios of the sildenafil treatment group remained lower than the sham group at both 7 (V7 vs S7, P < 0.001) and 28 days (V28 vs S28, P < 0.001). As in any experimental model, in particular animal models, inter-subject variability is not unexpected. Despite the significant standard deviations the data are statistically different and valid. Overall these results indicate that sildenafil treatment improved erectile function after castration, but was not sufficient to restore erectile function to pre-castration levels. The results for the SM C ratio are shown in Fig. 1. At 7 days the orchidectomy only and sildenafil treatment groups both had a SM C ratio of 7. At 28 days the SM C ratio for the Table 2 Ais. Group 7 Days 28 Days P S O V P 0.54* <0.005, 0.8* V, orchidectomy plus sildenafil treatment; S, sham no orchidectomy, O, orchidectomy only. *O vs V; SvsV. orchidectomy only group had increased to 39% and for the sildenafil treatment group had risen to 29%. The SM C ratio of V28 was significantly greater than the SM C ratio for the V7 (P = 0.04) and O7 groups (P = 0.046). However, the SM C ratio of V28 was not significantly different from O28 (P = 0.16). Table 2 shows the AI for the different groups. The AI for the S28 group was the lowest at 10 (8)%. The AI for O7 was 28 (11)% and for V7 was 22 (4)%, this difference was not statistically significant (P = 0.54). The AI for O28 was 28 (18)% and for V28 was 32 (20)%, and again this was not a statistically significant difference (P = 0.8). In summary, sildenafil treatment did not significantly decrease collagenisation or apoptosis after castration, thus other protective mechanisms must be involved in recovering erectile function after PDE5i administration. Discussion Castration either by surgical or chemical means is a recognised treatment for prostate cancer. Androgendeprivation therapy (ADT) is used as a first-line treatment for metastatic prostate cancer, and as an adjuvant therapy for recurrent disease, locally advanced disease or neoadjuvant treatment before radiotherapy. In a cohort study of 1485 men with newly diagnosed prostate cancer who had 12 months BJU International

4 Sildenafil citrate and castration follow-up evaluation, Meng et al. [12] found that 46% underwent ADT: 41% as primary therapy and 5% as secondary therapy or neoadjuvant therapy. The mechanism of the erectile response is dependent on arteriolar dilatation and increased blood flow to the corpora cavernosa, and veno-occlusion leading to a reduction in blood draining from the corpora cavernosa. This combination increases intracavernosal pressure and produces penile erection. In veno-occlusive erectile dysfunction (ED) due to trabecular smooth muscle dysfunction, a low outflow resistance results in a failure to increase intracavernosal pressure. Testosterone depletion has been shown to have a deleterious effect on erectile function accompanied by increased fibrosis [13] and apoptosis [14] within the erectile tissue of the penis, resulting in ED [15]. Erectile response declines 24 h after castration and continues to worsen 1 week after castration. The animal model to study veno-occlusive dysfunction (venous leak) is animal castration [16,17]. Previous studies have shown that castration produces a defect in veno-occlusion, which is improved with testosterone treatment. These studies show that testosterone regulates the rate of blood flow into the cavernous sinuses as well as blood outflow, possibly through the actions of nitric oxide (NO) [18 21]. ADT down-regulates the expression of PDE5. The mechanism of action of sildenafil is through PDE5 inhibition, resulting in the accumulation of intracellular cgmp. NO stimulates cgmp production, which is responsible for smooth muscle relaxation and subsequent erection. In addition to its inhibition of PDE5, sildenafil has significant effects on endothelial cell function and cellular apoptosis. Sildenafil has been shown to prolong erection in aged rats via AKT-dependent enos phosphorylation. Phosphorylation of enos is coincident with enzyme activation and increased eno release [22]. In rabbit models of cardiac ischaemia-reperfusion, sildenafil has been shown to reduce the area of ischaemia [23] and, in a followup study from the same institution, to reduce the amount of necrosis and apoptosis in mouse myocytes exposed to ischaemia [24]. In that study the authors showed that sildenafil increased inducible NOS (inos) and enos expression and increased the ratio of the anti-apoptotic protein Bcl-2 compared with the pro-apoptotic protein Bax. They postulated that sildenafil increases levels of NO and cgmp through its effects on inos and enos. The breakdown of cgmp is inhibited by sildenafil, which results in the opening of K ATP channels and subsequent cardio-protection [25]. Sildenafil has also been shown to ameliorate copulatory behaviour in castrated rats [26] and in a randomised clinical trial, sildenafil improved the return of spontaneous erections in men after radical prostatectomy [27]. Bilateral cavernous neurotomy (BCN) in the rat is an established model for post-prostatectomy ED. BCN induces penile hypoxia, down-regulation of PDE5, and increased expression and sensitivity of the pro-fibrotic endothelin-1 type B receptor (ETB). In this BCN model, acute sildenafil administration was shown to diminish hypoxia, ETB overexpression and ETB hypersensitivity [28] and chronic tadalafil restored PDE5 expression and increased responsiveness to acute tadalafil treatment [29]. In our own study we have shown that sildenafil improves preservation of erectile function in a cavernous nerve crush injury model, by reducing smooth muscle apoptosis and maintaining endothelial function [30]. In the present study, we wished to examine whether erectile response is improved in castrated rats treated with sildenafil compared with castrated rats that were not exposed to sildenafil. Daily sildenafil citrate treatment for 7 and 28 days improved erectile function in rats after castration. The ICP/MAP ratio increased significantly in both treatment groups (V7 and V28) compared with their control groups (O7 and O28). The greatest recovery in erectile function was seen after 28 days of sildenafil administration, but remained statistically lower than the sham rats (S28). The improved erectile function recovery for V28 after surgical castration compared with O7 and V7 appeared to be mediated partly by a higher SM C ratio. However, our structural analysis looking at apoptosis and SM C ratio does not fully explain our findings of erectile function improvement with daily sildenafil application after bilateral orchidectomy. Previous preclinical experiments were not able to show improved erectile function in androgen-deficient animals with the application of PDE5i. In the study by Traish et al. [15] it was reported that there was no restoration of erectile function after PDE5i administration in surgically or medically castrated rabbits. We think the reason for this finding was their use of only a single, low concentration dose of vardenafil. In contrast to our findings, Zang et al. [7] found that both acute and chronic tadalafil treatment was ineffective in ameliorating the electro-stimulated erectile response in castrated rats. They also showed that testosterone positively regulates PDE5 expression and in vivo responsiveness to tadalafil. The detrimental effects of androgen deficiency on erectile function are time dependent and appear to be cumulative over time. Although the beneficial effect of daily use of PDE5i on erectile function has been confirmed in previous studies, the first-line therapy in hypogonadal men remains unclear. Several studies have suggested that the first-line therapy in patients with hypogonadism should be androgen replacement. A meta-analysis showed that 64% of men with primary hypogonadism and 44% with secondary hypogonadism responded to testosterone treatment [31]. Buvat et al. [32] found that 42% of patients on testosterone monotherapy showed improvement in vascular parameters and 36% showed improved erectile function. Notably testosterone monotherapy for ED due to hypogonadism was far less effective in 2013 BJU International 659

5 Mulhall et al. middle-aged and elderly patients than in young men with profound organic hypogonadism. PDE5i may improve erectile function even in cases of severe hypogonadism. In eight patients with marked hypogonadism (total testosterone <2 ng/ml) and 16 patients with undetectable testosterone levels (total testosterone <0.5 ng/ml) Rochira et al. [33] showed that sildenafil normalised sleep-related erections with the same efficacy as testosterone therapy. It is thought that because NO and PDE5 are androgen dependent, restoring testosterone levels in patients with hypogonadism may increase the effectiveness of PDE5i treatment. In 44 patients with hypogonadism, Guay et al. [34] showed that 75% of hypogonadal men responded to sildenafil alone and 85% responded when androgen supplementation was combined with sildenafil treatment. In a placebo-controlled study of hypogonadal men with ED who did not respond to sildenafil alone, Shabsigh et al. [35] found that treatment with testosterone gel combined with sildenafil significantly improved erectile function, orgasmic function and overall satisfaction. Buvat et al. [36] investigated the efficacy of co-administration of testosterone with a PDE5i in men who were unresponsive to a PDE5i alone. In their multi-centre, placebo-controlled study they showed that the addition of testosterone improved erectile function in men with baseline testosterone levels of <3 ng/ml. Hwang et al. [37] showed that one-third of hypogonadal patients with ED who failed to respond to sildenafil, responded to testosterone alone, and another third responded to sildenafil after normalisation of testosterone. Thus, there is evidence that erectile function in some patients with hypogonadism can be improved with sildenafil treatment alone, while in others improvement requires testosterone replacement. The present study shows that the daily use of sildenafil improves erectile function even in rats with castrate levels of testosterone. A limitation of the present study was the lack of a group of castrated rats with androgen-replacement therapy to compare the differences in erectile function with the sildenafil-only treated group of rats. In addition, we lacked a control group in which castrated rats were given a placebo injection to ensure that the response we observed with sildenafil treatment was not due to handling and injection procedures. While these experiments need further elaboration, the significant improvement in the ICP/MAP ratio with sildenafil treatment provides evidence that early treatment with a PDE5i may be of benefit for erectile function preservation in men being treated with ADT. In conclusion, daily treatment with sildenafil may prevent or reduce the damage to penile tissue after castration. Androgen deprivation down-regulates the expression of PDE5, the molecular target of sildenafil [7]. Although the replacement of testosterone improves the effect of the PDE5i, sildenafil has an independent beneficial effect in restoring erectile function in the absence of androgens. Conflict of Interest None declared. References 1 Mazzola CR, Mulhall JP. Impact of androgen deprivation therapy on sexual function. J Androl 2012; 14: Smith AY, Gudziak MR. Hormonal therapy for stage D cancer of the prostate. WestJMed1994; 160: Nehra A, Goldstein I, Pabby A et al. Mechanisms of venous leakage: a prospective clinicopathological correlation of corporeal function and structure. J Urol 1996; 156: Traish AM, Toselli P, Jeong S, Kim NN. Adipocyte accumulation in penile corpus cavernosum of the orchiectomized rabbit: a potential mechanism for veno-occlusive dysfunction in androgen deficiency. J Androl 2005; 26: Sirad E, Hlaing S, Kovanecz I et al. 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