Ko Kobayashi M.D., Department of Urology, Sapporo Medical University School of Medicine, S1W16, Chuo-ku, Sapporo 060-8543, Japan. Email: firstname.lastname@example.org
Objectives: Lower urinary tract symptoms (LUTS) are frequently associated with erectile dysfunction (ED) in aged men, and both significantly influence quality of life. However, the mechanism linking LUTS to ED has not been clarified completely. The purpose of the present study was to establish an animal model of ED following LUTS/bladder outlet obstruction (BOO) and investigate the expression of molecules related to the cause of this type of ED.
Methods: Male Sprague-Dawley rats with partial BOO were used as an experimental model of LUTS. Sham-operated animals served as controls. Voiding and erectile function were evaluated 4, 8, and 16 weeks after obstruction or sham operation. The mRNA expression of penile tissue genes related to penile corporal smooth muscle relaxation was examined by quantitative real-time reverse transcription–polymerase chain reaction.
Results: Voiding and bladder function of BOO rats were significantly worse than in sham-operated rats 4, 8, and 16 weeks after obstruction. The erectile function of BOO rats was significantly decreased compared with that of the sham-operated controls (P < 0.01) 16 weeks after obstruction, although it was similar to that of sham-operated animals at 4 and 8 weeks after obstruction. The expression of endothelial nitric oxide synthase (eNOS) mRNA was significantly decreased 16 weeks after obstruction compared with that in sham-operated rats (P < 0.01).
Conclusion: An animal model for investigations into the association between LUTS and ED is described herein. Endothelial dysfunction induced by impaired eNOS function is likely to be involved in ED following LUTS/BOO.
Recent clinical studies have suggested an association between lower urinary tract symptoms (LUTS) and erectile dysfunction (ED).1–3 A large body of epidemiologic data supports this relationship.4–9 Both conditions are highly prevalent, frequently associated in the same aging group, and significantly influence quality of life. In previous studies, multiple regression analyses clearly showed that the severity of LUTS was the second most important risk factor for sexual dysfunction after age.10,11
Various hypothesis have been considered2,3 regarding the mechanism of ED induced by LUTS, including: (i) decreased or altered nitric oxide synthase (NOS)/nitric oxide (NO) levels in the prostate and penile smooth muscle; (ii) effects of autonomic hyperactivity on LUTS, prostate growth, and ED; (iii) increased Rho-kinase activation/endothelin activity; and (iv) prostate and penile ischemia. However, the causal relationship between LUTS and ED is not completely understood.3 One of the problems hindering complete understanding of this relationship was the lack of an appropriate experimental animal model of ED induced by LUTS/bladder outlet obstruction (BOO). Some reports demonstrated a causal relationship between LUTS and ED using animals in vitro,12,13 but there were few reports using an in vivo animal model to investigate this.14,15
The purpose of the present study was to establish an appropriate animal model of ED induced by LUTS and to assess the pathophysiological changes in this model to explain the relationship between LUTS and ED. In particular, we investigated the mRNA expression of molecules related to penile corporal smooth muscle relaxation, which may be involved in the causal relationship between LUTS and ED, as hypothesized previously.2,3
Sixty male Sprague-Dawley rats (8 weeks old) with BOO were used as an experimental model of LUTS/BOO. Rats were maintained on a 12 h light–dark cycle, with food and water freely available. A PE200 polyethylene catheter with an inner diameter of 1.40 mm (Clay Adams, Parsippany, NJ, USA) was used to create partial BOO as a model of LUTS. Rats were anesthetized with pentobarbital (45 mg/kg, i.p.). Then, the side of a 2-mm piece of the PE200 catheter was cut open to form a circle and placed around the urethra.16 The PE200 catheter was placed at the urethrovesical junction level proximal to the urethra after two prostate lobes had been retracted gently to expose the bladder neck and urethra.17 Sham-operated animals, in which the prostate was retracted only, served as controls.18 The experiment was approved by the Animal Experiment Committee of Sapporo Medical University. Animal care, housing and surgery followed the 1988 Guidelines of the Animal Experiment Committee of the University.
Evaluation for voiding and erectile function
Voiding and erectile function were evaluated 4, 8 and 16 weeks after obstruction or sham operation. Rats were randomly divided into 10 animals per group.
Water filling cystometry and measurements of post-void residual volume and bladder weight were performed to evaluate voiding and bladder function, as described previously.16,19 Briefly, a PE50 polyethylene catheter (Clay Adams) with a cuff was inserted through a small incision in the bladder dome and secured with a ligature while rats were under general anesthesia with 1%–2% isoflurane. After surgery, rats were placed in restraining cages and allowed to recover from the anesthesia for 1 h before cystometry was started. The other end of the intravesical catheter was connected via a T tube to a pressure and syringe pump for the infusion of physiological saline. Voiding volume (VV) was determined by collecting voided saline. After the VV had been determined, saline infusion was stopped and the residual urine volume (RU) was measured by withdrawing intravesical fluid through the catheter. Voiding efficiency was calculated as [VV/(VV + RU)] × 100.20
Intracavernous pressure (ICP) and arterial pressure (AP) during electrical field stimulation (EFS) were measured to evaluate erectile function, as described previously.20–22 Briefly, artificial erection was evoked by EFS of the cavernous nerve. Rats were anesthetized with pentobarbital (45 mg/kg, i.p.) and the AP was monitored with a 20-gauge cannula placed in the left carotid artery. A 23-gauge needle was inserted in the penile crus for measurement of ICP. The AP and ICP lines were connected to a pressure transducer, which was connected via a transducer amplifier to a data-acquisition board. The cavernous nerve was stimulated three times per side with a delicate stainless steel bipolar hook electrode (1 mA, 20 Hz for 1 min). Rats were fully anesthetized during the procedure and no body movement or distress was observed during EFS. The best ICP value was divided by simultaneous mean AP (ICP/AP).19–21
Intravesical, arterial, and ICP changes were measured using data-acquisition software (ADInstruments, Castle Hill, NSW, Australia) with a PowerLab system (ADInstruments). To confirm the severity of urethral obstruction, the whole bladder was dissected out and weighed after evaluation of voiding and erectile function.
Evaluation of mRNA expression using real-time reverse transcription–polymerase chain reaction
Penile tissue samples were freshly harvested for molecular experiments after functional evaluation. Total RNA was prepared from penile tissue samples from each group using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The concentration of the RNA was quantified by determination of optical density at 260 nm. The RNA (2 µg) was reverse-transcribed into cDNA using Superscript III (Qiagen). Aliquots from the reverse transcription (RT) reaction were used for real-time polymerase chain reaction (PCR) amplification with primer pairs ubiquitously expressing glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. All reagents for TaqMan PCR, including the primers for endothelial nitric oxide synthase (eNOS), Rho-kinase 1 (ROCK1), Rho-kinase 2 (ROCK2), endothelin-1 (ET-1), endothelin ETA receptors, endothelin ETB receptors, and GAPDH, were purchased from Applied Biosystems (Foster City, CA, USA). The reactions were run in an Applied Biosystems PRISM 7300 sequence detection system using the 96-well plate format. The cycling conditions included an initial phase at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, and 55°C for 60 s. The quantitative real-time PCR results were analyzed using Applied Biosystems Sequence Detection System software to determine the expression levels of the genes of interest relative to that of GAPDH.
All values are expressed as the mean ± SD and P < 0.05 was considered significant. The significance of changes in the parameters was tested using the Wilcoxon signed-rank test. Statistical analyses were performed with StatView 5.0 for Windows (SAS Institute, Cary, NC, USA).
Bladder weight in BOO rats was significantly increased compared with that in sham rats 4, 8 and 16 weeks after obstruction (Fig. 1a). Post-void residual volume in BOO rats was also significantly increased compared with that in sham-operated rats (Fig. 1b). Voiding efficiency was slightly, but significantly, decreased in BOO rats compared with sham-operated rats (Fig. 1c).
In BOO rats, ICP/AP was significantly decreased 16 weeks after obstruction compared with that in sham-operated rats (0.44 ± 0.12 vs 0.71 ± 0.17, respectively; P < 0.01), although it was similar to that of sham-operated rats 4 and 8 weeks after obstruction (Fig. 2). Figure 3 shows representative responses to EFS in rats that had undergone either sham operation or BOO. There was a decrease in ICP/AP along with absolute ICP in BOO rats 16 weeks after obstruction.
Results of quantitative real-time RT-PCR
Expression of eNOS mRNA was significantly decreased 16 weeks after obstruction in BOO rats compared with that in sham-operated rats (P < 0.01), although there were no significant differences in expression at 4 and 8 weeks (Fig. 4a). The mRNA expression of ROCK1, ROCK2, ET-1, and ETA and ETB receptors in each group did not change significantly during the experimental period (Fig. 4b–f).
Although LUTS does not necessarily imply BOO in the clinical setting, the BOO model can be used as an experiment model of LUTS/BOO. In the present study, the voiding function of BOO rats was impaired 4, 8, and 16 weeks after obstruction. The erectile function of BOO rats was also impaired 16 weeks after obstruction. These results indicate that this model can be used as an animal model of LUTS/BOO that results in impaired erectile function.
Several reports have hypothesized a link between LUTS and ED in animal experiments.1–3 Only one study clearly demonstrated ED induced by LUTS using animals in vivo;14 that study evaluated bladder weight to confirming BOO. In the present study, similar results were obtained for bladder weight and we further evaluated voiding function impaired by BOO. Although the methods used to create BOO differed between the two studies, it is of interest that ED induced by LUTS/BOO was demonstrated in two different animal experiments. In contrast, Aldredge et al. failed to produce ED following experimentally induced BOO.15 We propose to evaluate histological changes in the corporal smooth muscle in our next study to support the results of the present study.
Erectile dysfunction became apparent, along with decreased eNOS mRNA expression in cavernous smooth muscle. This coincides with one of the hypotheses that was reported in previous studies.1–3 It is widely accepted that NO is important in the relaxation of cavernous smooth muscle and eNOS is considered the essential factor responsible for maintaining the relaxation of cavernous smooth muscle.3 Conditions associated with reduced endothelial function in penile tissue result in ED. Therefore, decreased or altered NOS/NO levels in penile smooth muscle are strongly suggested to be one of the mechanisms linking LUTS with ED.
Another hypothesis for the mechanism linking LUTS with ED includes “increased Rho-kinase activation/endothelin activity”.1–3 In the present study, we investigated the mRNA expression of molecules related to penile corporal smooth muscle relaxation reported previously. However, in the present study the mRNA levels of Rho-kinase and endothelin did not changed. There may be some experimental differences between the studies that account for these apparent discrepancies; for example, differences in the animals used or in the way BOO was induced. Moreover, previous studies did not demonstrate the presence of ED.12,13 These differences in experimental conditions may also explain the different results. We plan to confirm Rho-kinase activation/endothelin activity, including protein levels, under various experimental conditions with this established LUTS/ED animal model in future studies. We also need to confirm the mRNA expression of these factors in other parts of the body (e.g. the bladder) to help to elucidate the systemic changes during LUTS/BOO-induced ED.
The degree of voiding dysfunction in the present animal model was speculated to be severe because of bad voiding efficiency, large residual urine volume, and heavy bladder weight. In a human epidemiologic study, actual voiding dysfunction in most aging men was not severe, although ED assessed by the International Index of Erectile Dysfunction score was strongly related to the severity of LUTS.9 Therefore, our animal model will not necessarily elucidate all the details of the relationship between LUTS and ED. However, our experimental model can be used in future studies to clarify the relationship between them. There are some limitations to the present study, one of which is that we investigated only the mRNA expression of molecules related to penile corporal smooth muscle relaxation. We are planning further investigations, including evaluation of the histological changes to the corporal smooth muscle and the major pelvic ganglion, related to erectile function using our animal model.
We have established an animal model for investigations into the association between LUTS and ED. This model can be used for future in vivo studies to elucidate the causal relationship between LUTS and ED.
The mRNA expression of eNOS is decreased in the corpus cavernosum with the corresponding onset of ED following LUTS. It appears that impaired eNOS function may induce endothelial dysfunction in ED after LUTS.
This study was supported, in part, by the Gohtaro Sugawara-Memorial Research Fund for Urological Disease and a Grant-in-Aid (20791116) from the Japan Society for the Promotion of Science.