The effect of sildenafil on electrostimulation-induced erection in the rat model

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1 (2002) 14, ß 2002 Nature Publishing Group All rights reserved /02 $ The effect of sildenafil on electrostimulation-induced erection in the rat model N Ueno 1, Y Iwamoto 1 *, N Segawa 1, M Kinoshita 1, H Ueda 1 and Y Katsuoka 1 1 Department of Urology, Osaka Medical College, Takatsuki, Osaka, Japan This study was conducted to show the effect of sildenafil on electrostimulation-induced erection in the rat model. Fifteen 12-week-old male Wistar Kyoto rats were used. The intracavernous pressure and arterial blood pressure were simultaneously monitored through electric cavernous nerve stimulation before and after the administration of sildenafil (2 mg=kg). Statistical analysis was performed on maximal intracavernous pressure (MIP), mean arterial blood pressure (MAP), the MIP=MAP and detumescence time. MAP decreased significantly by about 20 mmhg after sildenafil administration. The MIP=MAP increased significantly after sildenafil administration. The effect of sildenafil on the MIP=MAP was marked especially at lower (2 3 Hz) frequencies. The detumescence time significantly increased after sildenafil administration. We have shown that sildenafil is effective for enhancing erection at lower frequencies and prolonging penile erection in rats. After the administration of sildenafil, penile erection would be induced by weak stimuli that will not cause penile erection under normal conditions. (2002) 14, doi: =sj.ijir Keywords: sildenafil; cavernous nerve stimulation; rat; Introduction Penile erection is evoked by sexual stimulation that causes relaxation of corpus cavernosum and vascular smooth muscles in the penis. Nitric oxide (NO) released from non-adrenergic non-cholinergic nerves, and cyclic guanosine monophosphate (cgmp), a second messenger of NO, play an important role in the relaxation of corporal smooth muscles. Sildenafil citrate, a novel drug for erectile dysfunction, enhances penile responses to sexual stimulation via selective inhibition of phosphodiesterase type V that accelerates the degradation of cgmp. 1 The effectiveness of sildenafil has been demonstrated in clinical studies in patients with erectile dysfunction of organic, psychogenic, or mixed causes. 2 However, the effectiveness of the drug was assessed by subjective evaluation using a questionnaire to patients. Objective evaluation of the effectiveness is still difficult. In this study, therefore, we demonstrated the effect of sildenafil *Correspondence: Y Iwamoto, Department of Urology, Osaka Medical College, 2-7, Daigakumachi, Takatsuki, Osaka , Japan. Received 16 October 2001; revised 4 January 2002; accepted 28 January 2002 on electrostimulation-induced erection in the rat model. The intracavernous pressure (ICP) and arterial blood pressure (BP) were simultaneously monitored through electric cavernous nerve stimulation before and after the administration of sildenafil. Materials and Methods Experimental animals The experimental animals consisted of 15, 12-weekold male Wistar Kyoto rats bred under a constant temperature (23 1 C) and constant humidity (55 5%). Surgery Under ether anesthesia, incisions were made in the perineum and neck. A 23-gauge needle connected with a silicon tube ( N, Kaneka Medics Co., Japan) was placed into the corpus cavernosum via a perineal incision. The same silicon tube was inserted into the external cervical vein and common carotid artery via the cervical incision. The lumen of each tube was filled with heparinized saline (250 U=ml) before use. After exposing the bladder

2 252 and dissecting the lateral surface of the prostate via an incision in the inferior abdomen, a bipolar platinum microelectrode (MS-001, Unique Medical Co., Japan) with an inter-polar distance of 1 mm was placed on the cavernous nerve 2 mm peripheral to the pelvic ganglion. The electrode was placed on both sides to stimulate the cavernous nerve bilaterally. Electric stimulation Electric stimulation consisting of rectangular pulse trains of a constant amplitude (suprathreshold voltage of 3 V, pulse width of 5 ms, stimulation frequency of 1 32 Hz, stimulation duration of 1 min) was given using an electric stimulator (DPS- 1100D, Isolator DPS-105 D, Daiya Medical System Co., Japan). A rat model of erection induced by electric stimulation of the cavernous nerve was first developed by Quinlan et al, in Although the rat model of erection has been utilized at various institutions, 4 7 the method of electric stimulation varies study by study. To establish a reproducible method of electric stimulation, we performed a preliminary experiment of bilateral electric stimulation of the cavernous nerve. After multiple trials, it was found that when the pulse width was fixed to 5 ms, the maximal erectile activity was induced by electric stimulation at a frequency of 12 Hz, and the minimal suprathreshold voltage was 3 V. When electric stimulation was performed with a suprathreshold voltage of 3 V and stimulation duration of 1 min, reproducible responses were obtained with the stimulation interval of 1 min. In the present study, therefore, electric stimulation was performed with a suprathreshold voltage of 3 V, pulse width of 5 ms, stimulation duration of 1 min and the stimulation interval of 1 min. Pressure measurement ICP and BP were measured using a polygraph (Type 365, NEC San-ei Instruments, Ltd.) connected with a pressure transducer (BIOKIT, NEC San-ei Instruments, Ltd.) and recorded on a pen oscillograph (8K21-1-L, NEC San-ei Instruments, Ltd., Japan). Drug administration Sildenafil (2mg=kg) was suspended in distilled water (0.5 ml) and injected into the stomach using a syringe. With respect to the drug administration, we used 2 mg=kg of sildenafil because the maximum therapeutic dose of sildenafil for man is approximately this dose. 8 Protocol of experiment Rats were anesthetized with continuous intramuscular injection (50 mg=kg per h) of ketamine hydrochloride accompanied by intravenous drip infusion (0.7 ml=h) of physiological saline. ICP and BP were measured during electric stimulation of rectangular pulse trains with a suprathreshold voltage of 3 V, pulse width of 5 ms, stimulation duration of 1 min and stimulation frequencies at 1 Hz, 2 Hz, 3 Hz, 4 Hz, 6 Hz, 8 Hz, 12 Hz, 16 Hz and 32 Hz. Electric trains were given with 1-min intervals. The measurement was repeated 10 min after the administration of sildenafil (2mg=kg). Since it was reported that the half-lives of sildenafil after p.o. administration in rat was 0.4 h, 9 we determined that 10 min after sildenafil administration was the optimal time for evaluation. Statistical analysis Statistical analysis was performed on: (1) maximal intracavernous pressure (MIP): the maximal value of ICP during electric stimulation; (2) mean arterial blood pressure (MAP): the mean value of the systolic blood pressure and diastolic blood pressure at the time of MIP; (3) the MIP=MAP (%) and (4) detumescence time: the period from the termination of electric stimulation at 12 Hz to the time exhibiting ICP of 20 mmhg. Data of the above parameters were compared between before and after sildenafil administration. Repeated-measures analysis of variance was used to evaluate the significance of differences in MIP, MAP and the MIP=MAP. Wilcoxon s signed rank test was used to evaluate the significance of differences in the detumescence time. This study was performed following the guidelines for animal experiments in Osaka Medical College. Results MAP exhibited an almost uniform value irrespective of stimulation frequencies before and after sildenafil administration. No marked differences in MAP were noted between stimulation frequencies. MAP was mmhg before sildenafil administration and mmhg after sildenafil administration. MAP decreased by about 20 mmhg after sildenafil administration with significant differences (P < 0.01).

3 The MIP=MAP increased to a maximum value with the initial increase in stimulation frequency from 1 Hz to 12 Hz and then decreased with the following increase in stimulation frequency from 12 Hz to 32 Hz before sildenafil administration. The MIP=MAP increased significantly after sildenafil administration (Figure 1, P < 0.01). The effect of sildenafil on the MIP=MAP was marked at lower (2 3 Hz) frequencies. MIP increased to a maximum value with the initial increase in stimulation frequency from 1 Hz to 12 Hz and then decreased with the following increase in stimulation frequency from 12 Hz to 32 Hz before sildenafil administration. After sildenafil administration, MIP increased at lower (2 3 Hz) frequencies and slightly decreased at higher (6 16 Hz) frequencies (Figure 2). There were no statistical differences between before and after sildenafil administration for MIP. The detumescence time was s before sildenafil administration and s after sildenafil administration. The detumescence time significantly increased after sildenafil administration (Figure 3, P < 0.01). Original tracings from a typical experiment are shown in Figure 4. Only distilled water administration into the stomach did not affect MIP, MAP, the MIP=MAP and the detumescence time (data not shown). 253 Discussion Figure 1 Effect of sildenafil on the MIP=MAP. Each point is mean s.e. (n ¼ 15). *P < 0.01 vs absence of sildenafil. The effect of sildenafil on the intracavernous pressure during electric stimulation was marked especially at lower frequencies. The analytical results of the detumescence time revealed that sildenafil prolonged penile erection. The increasing effect of sildenafil on the intracavernous pressure was not evident at higher frequencies probably because cavernous smooth muscles and penile deep arteries were already in the maximally relaxed state at higher frequencies and could not respond further Figure 2 (n ¼ 15). Effect of sildenafil on MIP. Each point is mean s.e. Figure 3 Duration of detumescence time in absence or presence of sildenafil. Data are expressed as mean s.e. (n ¼ 15). *P < 0.01 vs absence of sildenafil.

4 254 Figure 4 Original tracing stimulated at frequency of 2 Hz before sildenafil (A), 2 Hz after sildenafil (B), 12 Hz before sildenafil (C) and 12 Hz after sildenafil (D). to sildenafil. The reason why the detumescence time was prolonged by sildenafil is because the drug inhibited the degradation of cgmp that caused the relaxation of corporal smooth muscles. The MIP=MAP indicates the extent of relaxation of corpus cavernosum and deep arteries in the penis, while MAP represents the capacity of pumping blood into the penis. Generally, drugs with an effect of increasing MAP will decrease the MIP=MAP because of associated contraction of corpus cavernosum and deep arteries in the penis. On the contrary, drugs with an effect of decreasing MAP will increase the MIP=MAP because of associated relaxation of corpus cavernosum and deep arteries in the penis. MIP should be determined by a balanced condition of changes in the MIP=MAP and MAP. The present study in rats showed that MIP decreased slightly at higher frequencies after sildenafil administration probably because the decrease in the blood pressure by about 20 mmhg offset the effect of sildenafil on increasing the MIP=MAP. Since sildenafil has modest effects on blood pressure in normal subjects, producing an average decrease of approximately 10 mmhg after a single oral dose of 100 mg, 10 an excessive decrease in the blood pressure may attribute to the ineffectiveness in patients who are refractory to the drug. We have shown that sildenafil is effective for achieving a more pronounced change in MIP=MAP in the rat model for particularly weak stimuli and that the detumescence time is prolonged. After the administration of sildenafil, penile erection would be induced by weak stimuli that will not cause penile erection under normal conditions and penile rigidity would be maintained even if sexual stimulation is stopped. References 1 Boolell M et al. Sildenafil: an orally active type 5 cyclic GMPspecific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 1996; 8: Goldstein I et al. Oral sildenafil in the treatment of erectile dysfunction. New Engl J Med 1998; 338: Quinlan DM et al. The rat as a model for the study of penile erection. J Urol 1989; 141: Mills TM, Wiedmeier VT, Stopper VS. Androgen maintenance of erectile function in the rat penis. Biol Reprod 1992; 46:

5 5 Martinez-Pineiro L et al. Rat model for the study of penile erection: pharmacologic and electrical-stimulation parameters. Eur Urol 1994; 25: Zvara P et al: Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. Int J Impot Res 1995; 7: Penson DF et al. Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat. Biol Reprod 1996; 55: Zusman RM et al. Overall cardiovascular profile of sildenafil citrate. Am J Cardiol 1999; 83: 35C 44C. 9 Walker DK et al. Pharmacokinetics and metabolism of sildenafil in mouse, rat, rabbit, dog and man. Xenobiotica 1999; 29: Jackson G et al. Effects of sildenafil citrate on human hemodynamics. Am J Cardiol 1999; 83: 13C 20C. / 255