Chronic partial bladder outlet obstruction does not impair erectile function in male rats

Abstract

OBJECTIVE

To evaluate, in a well-controlled study, the effect of surgically induced partial bladder outlet obstruction (PBOO) on male erectile function in a rat model.

MATERIALS AND METHODS

PBOO was created in 17 adult male Sprague-Dawley rats by partial ligation of the proximal urethra. Sham-operated and PBOO rats were evaluated for urodynamic and erectile function at 4–8 weeks after surgery. Erectile responses to electrical field stimulation (EFS) to the major pelvic ganglion, and to erectogenic agents (1,1-diethyl-2-hydroxy-2-nitroso-hydrazine, DEA-NO, and Y-27632) were evaluated and the area under the curve (AUC, a product of the intracavernous pressure and duration) was used to denote the erectile response.

RESULTS

Experimental PBOO in rats significantly increased the mean (sem) bladder weight, to 256 (25) mg in PBOO rats vs 123 (24) mg in sham controls, and the voiding frequency to 1.01 (0.1) voids/min vs 0.72 (0.14) voids/min in sham controls (P < 0.05). There was no significant difference between the erectile response to EFS, with a mean AUC in sham control rats at 1.5, 3.0 and 4.5 V of 2603 (372), 3200 (332) and 3357 (166), respectively, vs 2273 (183), 3794 (211) and 4177 (306) in PBOO rats (P > 0.05); or to the erectogenic agents, the AUC for DEA-NO being 9000 (975) in PBOO rats vs 13 201 (2756) in sham controls, and the AUC for Y-27 632 being 44 915 (2462) and 45 907 (7408), respectively (P > 0.05). There was greater immunoreactivity to RhoA in bladder and penile tissues of PBOO than control rats.

CONCLUSION

PBOO does not affect erectile function in rats. Additional mechanisms or pathways might be involved in lower urinary tract symptom-related erectile dysfunction in humans.

Abbreviations

PBOO

partial BOO

ED

erectile dysfunction

NO(S)

nitric oxide (synthase)

CC(SM)(P)

corpus cavernosum (smooth muscle) (pressure)

ICP

intracavernous pressure

MAP

mean arterial pressure

CMG

cystometrogram

EFS

electrical field stimulation

MPG

major pelvic ganglion

DEA-NO

1,1-diethyl-2-hydroxy-2-nitroso-hydrazine

AUC

area under the curve.

INTRODUCTION

The relationship between LUTS and sexual dysfunction has received increased attention recently. Erectile dysfunction (ED) and LUTS coexist in the ageing male population. With a prevalence of 18.4% in men in the USA aged ≥20 years, it is estimated that 16–20 million men in the USA are affected by ED [1]. Other comorbidities of ED include depression, diabetes, hypertension and heart disease [2]. ED and LUTS are reported to be highly prevalent, frequently associated in ageing men, and to contribute significantly to overall quality of life [3]. While several studies, both epidemiological and clinical, have attempted to explain the relationship between them, a strong causal association between LUTS and ED has yet to be shown convincingly. Epidemiological studies suggest a strong association between LUTS and sexual dysfunction, independent of age and comorbidities. According to one such study, the prevalence of LUTS in men with ED men was 72.2%, vs 37.7% in men who did not have ED. When multivariate analysis was used, LUTS was shown to be an age-independent risk factor for developing ED [4]. The Multinational Survey of the Aging Male concluded that BPH/LUTS is an independent risk factor for ED and other types of sexual dysfunction [2,5].

Several theories have been postulated to explain the biological linkage between ED and LUTS [3,6–8]; these theories can be grouped into four major categories: increased Rho-kinase activation; prostate and penile ischaemia; decreased or altered levels of nitric oxide (NO) synthase (NOS)/NO in penile and prostate smooth muscle; and autonomic hyperactivity effecting ED, prostate growth, and LUTS [3]. However, these theories need validation in a well-established animal model.

Our laboratory has successfully used the rat as an animal model for urodynamic and erectile function studies [9,10]. The rat is a well-established and useful animal model for the study of male erectile function, physiologically and for pathophysiological evaluation [10–13]. Surgically induced partial BOO (PBOO) in the rat model is another technique that has been commonly used by researchers in our laboratory [14]. PBOO is a useful means of simulating LUTS in animals. In humans, BPH is the primary cause of LUTS in men aged >50 years and has repeatedly been reported as an independent risk factor for ejaculatory dysfunction and ED [5]. Specific molecular and functional changes in corpus cavernosum smooth muscle (CCSM) in rabbits with PBOO have also been documented in attempts to link PBOO with ED in previous ex vivo studies [15]. In the present well-controlled study, we aimed to evaluate the causal relationship between experimental PBOO and erectile function in a rat model.

MATERIALS AND METHODS

We used two groups of adult male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA; 325–350 g); the study was approved by our Institutional Animal Care and Use Committee and all the experimental procedures were in accordance with the Guide and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

PBOO in rats is a well established technique used to simulate functional changes in the bladder [14]. After a few days of acclimation, two groups of rats (17 experimental and six sham-operated) were anaesthetized with xylazine (5 mg/kg, i.m.) and ketamine (13 mg/kg, i.e.) and either the bladder outlet obstructed (PBOO group) or sham surgery (control). The bladder and urethra were exposed by a suprapubic midline incision and the obstruction created by placing a 3/0 silk ligature around the catheterized urethra. The sham group was subjected to the same operation but without tying the thread and thus with no obstruction created. In both groups the catheter was then removed and the incision was closed [14].

The rats were allowed to recover from surgery and evaluated urodynamically at 4–8 weeks after surgery. For the urodynamic evaluation, each rat was anaesthetized with 1.2 g/kg urethane (s.c.). The bladder was then exposed by laparotomy and a 25 G needle was inserted through the bladder dome. This needle was connected in turn to a pressure transducer, by polyethylene tubing, and a syringe pump via a three-way stopcock, to record intravesical pressure and to take a cystometrogram (CMG). Through this system, warm saline could be infused into the bladder. The bladder was returned to the abdomen with the line escaping through the incision. The muscle wall was sutured together using 4–0 tapered polypropylene, and the skin was sutured using 4–0 Nylon. The catheter was connected to a pressure transducer (UFI, Morro Bay, CA, USA) and in turn connected to an infusion pump (Harvard Apparatus, Holliston, MA, USA). During continuous filling bladder cystometry, the pressure was recorded with the transducer using LabView (National Instruments, Austin, TX, USA).

Baseline continuous-fill CMGs were taken by infusing warm saline (37 °C) into the rat bladder at 0.11 mL/min for at least 1 h, and a minimum of five micturition cycles were recorded visually and by the pressure curve. Both systemic and bladder pressure data were recorded [9].

Rats were assessed for erectile function after completing the urodynamic studies, using well-established techniques previously reported from our laboratory [10]. In brief, erectile responses to two methods of stimulation were used: electrical field stimulation (EFS) to the major pelvic ganglion (MPG), and pharmacological stimulation to two vasoactive agents, 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA-NO) and the Rho-A kinase inhibitor, Y-27632.

The intracavernous pressure (ICP) was recorded by cannulating either of the corporal bodies 3–5 mm above the base of the penis, using a 25 G i.v. butterfly needle filled with 100 U/mL heparinized saline, and connected to a pressure transducer. The MPG was identified by dissecting the fibrous capsule posterior to the intersection of the lateral lobes of the prostate and vas deferens. The MPG was subjected to EFS via two platinum electrodes delivered from a model S48 stimulator (Grass-Teledactor, West Warwick, RI, USA). Stimulus levels from 1.5 to 4.5 V were used for a duration of 1 min each with a 5-min rest period between stimulations to monitor the erectile response. Pressure (mean arterial pressure, MAP, and ICP) was measured using two pressure transducers (UFI) placed in series via a calibrated amplifier connected to a personal computer. The data were acquired and saved by data acquisition software (Labview, National Instruments Corporation, Austin TX, USA).

Each rat, after evaluation by EFS, was given DEA-NO (10 µg in 5 µL of saline) intracavernosally, followed by Y-27632 (50 nmol in 5 µL of saline). The erectile response was recorded based on previously described methods.

All drug solutions were stored in a freezer in amber bottles; working solutions were prepared frequently and kept cold until injected. In both sham and PBOO rats the drugs were administered intracavernosally in small volumes (5 µL) when the CC pressure (CCP) was at a baseline value. The effect of a single injection agent on CCP was measured until the CCP returned to pre-injection levels. The next injection, when applicable, was made at least 10–15 min after the CCP had returned to a stable baseline. An injection of 5 µL of saline vehicle had no significant sustained effect on CCP. The area under the curve (AUC) was used to compare the erectile responses [16]. All the rats were evaluated both urodynamically and for erectile function, with the urodynamic evaluation first. In the same way, rats were exposed to EFS before any pharmacological studies using vasoactive agents.

The protein expression of RhoA in PBOO and control rats was evaluated to assess the involvement of RhoA signalling pathways in bladder and penile tissues of PBOO rats, using immunohistochemical methods with a commercially available monocolonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), which has been validated as reacting with rat tissues [9,12]. For these evaluations, 5-µm paraffin tissue sections of bladder/penile tissue were applied to charged slides, deparaffinized, and hydrated with PBS containing 0.3% Triton X-100 for 10 min. After incubating for 30 min with normal horse serum to block nonspecific binding sites, the slides were incubated overnight with specific antibody (RhoA or nitrotyrosine; 1:200 dilution) at 4 °C. A conventional avidin-biotin complex kit (Vector Laboratories, Burlingame, CA, USA) was used to immunostain the sections and reaction products were visualized under a light microscope [12]. The slides were examined by a microscopist unaware of sample origin, and digital images were quantified using computer software (Image J, National Institutes of Health) [12].

Quantitative data are expressed as the mean (sem), with statistical analysis by anova followed by the Newman-Keuls multiple comparison test where relevant, and by paired or unpaired t-test; in all tests P < 0.05 was considered to indicate statistical significance.

RESULTS

PBOO rats had several hallmarks of BOO; there was a significant increase in bladder weight and an increase in frequency of micturition compared with the control group, indicating that PBOO was successfully created (Figs 1,2). Bladder weight increased from 115.40 (8.6) mg in the sham controls to 256.6 (28.6) mg in PBOO rats (P < 0.05). The voiding frequency also increased from 0.56 (0.09) voids/min in sham rats to 1.01 (0.1) voids/min in PBOO rats (P < 0.05).

Figure 1. Representative tracings of spontaneous bladder contractions in (A) control and (B) PBOO rats (five to seven in each group).

Figure 2. Comparison of (A) bladder weight (B) frequency of bladder contractions in sham and PBOO rats (five to seven in each group). An asterisk indicates a significant difference (P < 0.05) from sham rats.

The erectile response was quantified by using the AUC; at 1.5, 3 and 4.5 V EFS, the AUCs were 2603 (372) and 2273 (183), 3200  (332) and 3794 (211) and 3357 (166) and 4177 (306), in control and PBOO rats respectively. There were no significant differences in erectile response to EFS between the control and PBOO rats (Fig. 3).

Figure 3. Comparison of EFS-induced erectile response (the AUC) in sham and PBOO rats (five to seven in each group).

Intracavernous injections with both erectogenic agents (DEA-NO and Y-27632) induced an erectile response in both sham and PBOO rats, but there were no significant differences in the AUCs between the groups. DEA-NO injection produced an AUC of 9000 (975) and 13 201 (2756) in sham and PBOO rats, respectively (P > 0.05). After Y-27632 injection, sham rats had an AUC of 44 915 (2462) and PBOO rats an AUC of 45 907 (7408) (P > 0.05; Fig. 4). In addition, the correlation between the erectile response (AUC) and duration after obstruction and bladder weight was analysed (Figs 5,6); there was no significant correlation between these variables. Changes in RhoA protein expression were present in both bladder and penile tissue after PBOO, as shown by increased immunoreactivity to Rho-A (Fig. 7).

Figure 4. Comparison of (A) DEA-NO- and (B) Y-27632-induced erectile response (as the AUC) in sham and PBOO rats (five to seven in each group).

Figure 5. A scatterplot showing the correlation of erectile response (AUC) with the duration (days) after obstruction in PBOO rats.

Figure 6. A scatterplot showing the correlation of erectile response (AUC) with the bladder weight in PBOO rats.

Figure 7. Representative photomicrographs showing immunolocalization of RhoA proteins in (A) bladder tissues from sham control rats; (B) bladder tissues from PBOO; (C) penile tissues from sham rats and (D) penile tissues from PBOO rats (three in each group). ×20. Arrows indicate densely immunostained areas.

DISCUSSION

Ageing men commonly have urogenital disorders such as LUTS and ED. Many epidemiological studies have shown a strong correlation between them, but as of yet no temporal causality has been confirmed. In the present well-controlled study we sought to find causality between them by surgically creating PBOO in an in vivo rat model and then evaluating the erectile response in the same rat. Our results showed the successful creation of PBOO in the rat model, as evidenced by urodynamic and bladder weight changes. However, erectile function studies do not support a causal relationship between PBOO and erectile response. Rat models have been used by several investigators to evaluate obstruction-related pathophysiology and novel pharmacological therapies [14,17]. PBOO restricts the flow of urine from the bladder and simulates the LUTS associated with BPH in men. Both involve similar consequences on the LUT, such as instability of bladder contractions, incomplete emptying, and frequency. The rat model has also been recognized as one of the ideal animal models for evaluating sexual function in humans [11]. Rats have also been used as chronic models in previous BOO studies aiming to simulate bladder function and effects of alterations to the bladder and urethra in humans [18].

Some major effects of PBOO are well known and were present in this study. A major hallmark of PBOO is an increase in bladder weight. As more force is required to expel the urine past the obstruction, there is hyperplasia and/or hypertrophy of the detrusor muscle [19]. This PBOO- induced hyperplasia leads to an increase in bladder weight and explains the significant differences in bladder weight between the PBOO and sham groups. Immunohistochemical detection of RhoA in bladder and penile smooth muscle supports one of the existing theories [8].

EFS is a commonly used technique in both in vivo and ex vitro muscle contraction studies [11]. Pharmacological stimulation by erectogenic agents is another well-established method of inducing erectile response [10,11]. We used an NO donor (DEA-NO) as well as a Rho-kinase inhibitor (Y-27632), as these pathways are implicated in LUTS-related ED [8]. DEA-NO is a NO donor commonly used to induce erections in in vivo studies [20]. DEA-NO, as well as other NO derivatives, has been confirmed in previous studies to increase pressure in the CCSM in various animal models [20–22]. Y-27632 is a well-established inhibitor of the Rho-A-Rho kinase pathway, the overactivity of which is implicated in ED. Recent studies have shown that Y-27632, as an inhibitor of this pathway, can be very successful in attenuating ED in rat models [23]. The AUC is a useful variable or index for quantifying the sexual response and has been well documented as such [16]. This is considered a more objective means of evaluating erectile response than taking the ratio of ICP/systemic MAP, although both are commonly used [11]. Experimental data for erectile function studies showed no significant differences in the AUC between control and the PBOO rats. As erectile function was not significantly altered, PBOO as such could not be established as a causal factor in these rats.

On the contrary, a few recently published studies have reported varying levels of correlation of LUTS and ED in rat models. For example, Rahman et al.[24] described a rat model showing a correlation of ED and LUTS in rats fed with a high-fat diet. After 6 months of a high-fat diet containing 2% cholesterol and 10% lard, hyperlipidaemic rats developed LUTS, such as bladder overactivity and increased frequency of bladder contractions, accompanied by a decreased erectile response. Christ et al.[25], in a diabetic rat model, showed bladder dysfunction and ED in the same rat, but the correlation between these variables was not consistent. These observations suggest that it is possible to find ED and LUTS in the same animal, but the correlation was not always consistent. The methods in the present study are fundamentally different from those of the previous studies [24,25], in that LUTS was induced by PBOO.

There are major reasons that could explain the disparity between the results of the present study and the strong correlations between LUTS and ED reported in many human epidemiological studies. The first is in relation to the rat model; although the rat is a good model for erectile function studies, the present results cannot be totally extrapolated to humans. The protocol required the use of anaesthesia, and the effect of anaesthesia on the erectile response cannot be ignored. Another important caveat to the present findings is the period of assessment after surgery; the maximum interval that a rat was allowed before evaluating the erectile response was 8 weeks. The hallmarks of successful PBOO, e.g. increased bladder weight, increased voiding frequency, and increased tissue expression of RhoA, were certainly present, but it is possible that a longer duration of obstruction might be required to produce ED in rats. We propose to address this in our future research. It might also be possible that the mechanisms that lead to PBOO-related ED are different in humans than in rats. McVary et al.[3,6–8] postulated several possible physiological mechanisms that link LUTS and ED in humans. These mechanisms include: (i) decreased production of NO/NOS, a vasodilator, in the pelvic region; (ii) LUTS associated with a metabolic syndrome and related more to autonomic hyperactivity; (iii) increased Rho-kinase activity and subsequent Ca²⁺ sensitization; and/or (iv) chronic penile ischaemia associated with pelvic atherosclerosis [3,8]. While it is likely that these mechanisms are important in linking LUTS and ED in humans, it appears that these hypotheses might not be applicable to the rat model.

CONFLICT OF INTEREST

None declared.