ORIGINAL RESEARCH—BASIC SCIENCE
ORIGINAL RESEARCH—BASIC SCIENCE: Effect of Chronic Tadalafil Administration on Penile Hypoxia Induced by Cavernous Neurotomy in the Rat

https://doi.org/10.1111/j.1743-6109.2006.00208.xGet rights and content

ABSTRACT

Background

Numerous men develop postprostatectomy erectile dysfunction (PPED), due to surgery‐related nervous damage. PPED is often refractory to phosphodiesterase type 5 (PDE5) inhibitors therapy.

Aim

To verify whether chronic tadalafil (CT) preserves bilateral cavernous neurotomy (BCN)‐induced penile damage and hypo‐oxygenation.

Methods

In a rat model of BCN we evaluated in vitro and ex vivo effect of CT treatment (2 mg/kg, daily for 3 months).

Results

Bilateral cavernous neurotomy induced massive hypoxia and decreased muscle/fiber ratio, completely restored by CT. Hypersensitivity of hypoxic tissues to the relaxant effect of the endothelin type B receptor (ETB) agonist IRL‐1620 was observed, along with ETB mRNA and protein overexpression. CT restored sensitivity to IRL‐1620, and normalized ETB expression. Hypoxic penis showed increased sensitivity to the relaxant effect of the nitric oxide donor sodium nitroprusside (SNP), while acute tadalafil (100 nM) did not amplify the SNP effect. Accordingly, PDE5 mRNA and protein were reduced in BCN penile tissues. By restoring PDE5, CT decreased SNP‐induced relaxation and rescued sensitivity to acute tadalafil. However, in hypoxic penis, CT normalizes neither acetylcholine hyporesponsiveness nor neuronal nitric oxide synthase‐endothelial nitric oxide synthase expression.

Conclusion

Chronic tadalafil restores some of the investigated BCN‐induced alterations, including PDE5 and tadalafil efficacy. Vignozzi L, Filippi S, Morelli A, Ambrosini S, Luconi M, Vannelli GB, Donati S, Crescioli C, Zhang X‐H, Mirone V, Forti G, and Maggi M. Effect of chronic tadalafil administration on penile hypoxia induced by cavernous neurotomy in the rat. J Sex Med 2006;3:419–431.

Introduction

Prostate cancer is one of the most common male malignancies, which, in its localized form, is usually treated by radical retropubic prostatectomy (RRP). After RRP, even following the introduction of nerve‐sparing techniques [1], postprostatectomy erectile dysfunction (PPED) remains a persistent, severe problem for most patients [2, 3, 4, 5, 6], often crucial to men's self‐esteem and quality of life. In addition, in PPED, responsiveness to oral phosphodiesterase type 5 (PDE5) inhibitors (PDE5i) was variable, but overall lower than in patients with other forms of erectile dysfunction [7, 8, 9, 10]. Nonetheless, after the introduction of Walsh's technique of nerve preservation, the rate of postprostatectomy sexual recovery greatly improved. This strongly indicates that surgery‐related nerve injury, with the consequent degeneration of cavernous nerves, plays a major role in PPED. In fact, cavernous nerves supply the important autonomic signals needed for erection induction. Bilateral cavernous neurotomy (BCN) in the rat is a well‐established model to study effects of PPED on penile tissue [11, 12]. In this model it has been demonstrated that cavernous nerve injury is coupled to precocious apoptosis of penile smooth muscle cells and persistent penile atrophy [13, 14]. Accordingly, even after long‐term (12–15 weeks) cavernous nerve injury, an increase in collagen/smooth muscle ratio and transforming growth factor (TGF)‐β1 expression was recorded, along with an increase in hypoxia inducible factor (HIF)‐1α, a transcriptional factor strictly related to hypoxia [13, 15, 16]. Hence, it is generally assumed, but never directly demonstrated, that cavernous neurotomy‐induced penile injury is coupled to prolonged hypoxia [17].

Hypoxia is a rather physiological event in the penis, which, in the flaccid state, resides at a very low oxygen tension (PO2 = 25–40 mm Hg) [18]. However, in normal men, penile hypoxia is daily interrupted by discrete (total 1.5–3 hours), sleep‐related, erectile episodes [19], which substantially reoxygenate the penis (PO2 = 90–100 mm Hg). The lack of these daily erectile episodes, because of cavernous nerve injury, might underlie the neurotomy‐induced penile atrophy and fibrosis [17]. Accordingly, we reported that in human penile smooth muscle cell (hPSMC) culture prolonged (more than 24 hours) hypoxia resulted in an increased endothelin‐1 (ET‐1) production and activity [20]. ET‐1 is a profibrotic peptide [21], whose synthesis in the penis is TGF‐β1‐dependent [20]. In addition, as originally demonstrated by Saenz de Tejada et al. [22] and confirmed in subsequent studies [20, 23, 24, 25], ET‐1 is the most potent stimulator of penile smooth muscle cell contractility through the endothelin type A (ETA). Interestingly, we previously found by in vitro [20] and ex vivo [23] experiments that penile prolonged (more than 24 hours) hypoxia was associated with the upregulation of the vasorelaxant ETB receptor subtype, probably in order to counteract the contractile ET‐1 effects.

In the past few years, there has been a growing interest in penile rehabilitation after RRP to prevent—or decrease—the incidence of PPED and to increase the low responsiveness rate to PDE5i [5]. Montorsi and colleagues first employed successfully intracorporeal injections of alprostadil two to three times per week with the aim to stimulate penile tumescence and oxygenation [26]. Later on, early, daily based, administration of PDE5i was suggested, with the purpose to increase nitric oxide (NO) signaling and therefore vasodilatation and oxygenation. Preliminary results with 100 mg sildenafil, a PDE5i, were somehow encouraging, being statistically significantly better than those obtained with placebo [27]. However, the overall rate of return to normal erectile function in that study was only 27%[27]. Another recent report indicates that early use of 100 mg sildenafil every other day was able to preserve the loss of penile smooth muscle content [28], a critical step in the pathogenesis of permanent erectile dysfunction [29].

The aim of the present study was to evaluate morphological and biochemical alterations in rat penile tissue after long‐term (3 months) BCN, a lag time compatible with the spontaneous sexual recovery described in humans after RRP (12–24 months). The effect of a chronic treatment with oral tadalafil (2 mg/kg in drinking water) was also studied. We previously demonstrated in the rat the lack of tachyphylaxis and the effectiveness of chronic tadalafil (CT) dosing, when compared with an acute tadalafil administration (6 mg/kg), in terms of penile erection induced by electrostimulation of the cavernous nerve [30].

Results from this study demonstrate that CT administration allows tissue oxygenation and restores several, but not all, of the investigated neurotomy‐induced penile damages.

Section snippets

Chemicals

Phenylephrine HCl (Phe), acetylcholine (Ach), sodium nitroprusside (SNP), N wnitro‐l‐arginine methyl ester hydrochloride (L‐NAME), reagents for immunocytochemistry and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), phosphate‐buffered saline (PBS), bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). ET‐1 and the ETB selective agonist Suc‐(Glu [9], Ala [11, 15])‐endothelin‐1 (IRL‐1620) were purchased from Novabiochem (Switzerland). Hypoxyprobe™‐1 Kit

Results

Figure 1 shows the morphological effect of long‐term neurotomy on penile oxygenation. In sham‐operated rats only scanty cells reacted with the hypoxyprobe pimonidazole (Figure 1a). Conversely, positive hypoxyprobe staining dramatically increased in penile section of BCN rats (Figure 1b). Hypoxia was detected in the endothelial and muscular compartments of cavernous spaces. CT dosing almost completely restored penile oxygenation, as determined by the virtual absence of hypoxyprobe labeling in

Discussion

This study demonstrates, for the first time, that, in an experimental model of RRP‐induced erectile dysfunction (BCN), CT treatment restores several of the investigated neurotomy‐induced penile alterations, including PDE5 expression and in vitro responsiveness to tadalafil. This is probably due to an increased cavernous oxygenation, which preserves penile smooth muscle content and function.

In this study we demonstrate that BCN induces a severe penile hypoxia. In fact, hypoxyprobe labeling, a

Conflict of Interest

None declared.

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