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ORIGINAL RESEARCH New Methodology for Investigating Ejaculation Dysfunction: Measuring Intraluminal Seminal Vesicle Pressure in Rats with a Telemetric Device Hyun Hwan Sung, MD, PhD,* Jung Jun Kim, MD,* Deok Hyun Han, MD, PhD,* Su Jeong Kang, BS,* Mee Ree Chae, MS,* Chul Young Kim, PhD,† Jong Kwan Park, MD, PhD,‡§ and Sung Won Lee, MD, PhD* *The Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; †College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Korea; ‡Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, Jeonju, Korea; §Research Institute and Clinical Trial Center of Medical Device, Chonbuk National University Hospital, Jeonju, Korea DOI: 10.1111/jsm.13025
ABSTRACT
Introduction. Ejaculation dysfunction is one of the most common male sexual disorders. Despite its prevalence and adverse impact on patients, little attention has been given to investigating ejaculation dysfunction. Aim. We introduce a new method for evaluating ejaculation dysfunction in rats with a telemetric device. Methods. A pressure transducer was surgically implanted in the seminal vesicles of 7-week-old male Sprague– Dawley rats. One week later, the rats were subcutaneously administered tamsulosin 3 μg/kg, and intra-seminal vesicle pressure (ISVP) was recorded in freely moving rats after an injection of apomorphine (80 μg/kg). Same rats repeated experiment with tamsulosin 10 μg/kg, silodosin 1 mg/kg, and normal saline with 3-day intervals. Main Outcome Measure. Sexual events were visually identified and recorded. Ejaculation was confirmed by visualization of a copulatory plug in the tip of the penis. We compared the maximal ISVP and area under the curve (AUC) of the ISVP. Results. Adequate ISVP data were easily recorded and available in 66.6% rats (10/15) over a 6-week telemetric recording period (12 recordings). The mean number of ejaculations during an inspection time of 30 minutes was 1.5 ± 0.1. The maximal ISVP values in rats receiving 3 μg/kg (30.0 ± 5.2 mm Hg) and 10 μg/kg tamsulosin (15.1 ± 1.6 mm Hg) and 1 mg/kg silodosin (12.9 ± 2.2 mm Hg) were significantly lower than that in control rats (61.4 ± 13.4 mm Hg, P < 0.05). The AUC values in rats receiving 3 μg/kg (72.7 ± 18.9 mm Hg × s) and 10 μg/kg tamsulosin (23.5 ± 6.1 mm Hg) and 1 mg/kg silodosin (23.9 ± 8.0 mm Hg) were also lower than that of control rats (162.6 ± 34.3 mm Hg, P < 0.05). Conclusions. Telemetric ISVP assessment is reliable and feasible for investigating apomorphine-induced ejaculation in rats. Tamsulosin (3 μg/kg and 10 μg/kg) and silodosin 1 mg/kg decreased the ISVP during ejaculation. Sung HH, Kim JJ, Han DH, Kang SJ, Chae MR, Kim CY, Park JK, and Lee SW. New methodology for investigating ejaculation dysfunction: measuring intraluminal seminal vesicle pressure in rats with a telemetric device. J Sex Med 2015;12:2134–2140. Key Words. Ejaculation Dysfunction; Telemetry; Telemetry Monitoring; Apomorphine
J Sex Med 2015;12:2134–2140
© 2015 International Society for Sexual Medicine
Intra-Seminal Vesicle Pressure Measuring with Telemetry Introduction
E
jaculation dysfunction is one of the most common male sexual disorders [1,2]. This disorder encompasses various types of ejaculatory problem such as premature ejaculation (PE), delayed ejaculation, and inability to ejaculate. Among these, PE is the most prevalent, accounting for approximately 20–30% of cases, depending on the definition for this condition [1–3]. PE has been clearly shown to exert a significant negative impact on the psychological statuses of patients with PE and their partners [4]. Medical conditions, including depression, anxiety, psychological distress, relationship problems, and general confidence, may be affected by PE [5–7]. In addition, many patients with PE suffer from concomitant sexual disorders. PE correlates with erectile dysfunction (ED) and coexists in approximately onethird of patients complaining of ED [8]. However, investigations of male sexual dysfunction have predominantly focused on ED, rather than ejaculation dysfunction. Accordingly, comprehensive research is essential to a better understanding of ejaculatory dysfunction. In vivo experimental studies of ejaculation physiology and PE have introduced procedures in which the intra-seminal vesicle pressure (ISVP) of an animal is measured [9–12]. Specifically, in rats, the intraluminal pressure of the seminal vesicle in response to electrical nerve stimulation was recorded to investigate the effects of particular drugs. This technique is usually performed under anesthesia in rats fixed on a surgical plate. However, this research has been limited by the unnatural characteristics of sexual behaviors in response to anesthesia and electrical stimulation [13,14]. Copulatory behavior, matched ejaculation, and confirmation of increased ISVP cannot be simultaneously obtained. Additionally, the animals must be sacrificed after the experiments, thus restricting repeated trials with different drugs. In a previous study, a telemetric device was introduced to measure the intra-cavernosal pressure (ICP), thus overcome the limitations associated with in vivo studies of ED [15]. In our previous studies, we successfully determined that ICP could be feasibly and reproducibly measured in rats using a telemetric device under minimally affected or unaffected conditions [16,17]. However, to our knowledge, no studies concerning ejaculation dysfunction have utilized a telemetric device. Therefore, the current study aimed to introduce new
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methodology with which evaluate the ISVP in rats with a telemetric device. Materials and Methods
Animals Seven-week-old male Sprague–Dawley (SD) rats (Orient Bio, Seoul, Korea) were housed 1 per cage at 22.8°C under 12-h light/12-h dark photoperiodic conditions (lights-off at 8:00 pm). Rats were fed a standard, fat-free pellet diet and water ad libitum throughout the experimental period. The rats were divided into four groups: tamsulosin (3 μg/kg or 10 μg/kg; Sigma-Aldrich Chemical Co, St. Louis, MO, USA), silodosin (1 mg/kg; JW Pharmaceutical, Seoul, Korea), and normal saline (control). A total of 15 rats were used. All animal experiments were conducted under the approval of the Institute for Animal Care and Use Committee of Samsung Medical Center. Telemetric Device Implantation Surgical implantation of the telemetric device was similar to the previously described technique used for telemetric ICP measurement [15,16]. The rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg; Yuhan Co, Seoul, Korea). A sterile telemetric sensor device (PA-C40; Data Sciences International, St Paul, MN, USA) was implanted as follows. A mid-abdominal incision was made to expose the seminal vesicle. The tip of a recording catheter was carefully inserted into the body of the right seminal vesicle. This tube was tightly secured to prevent seminal vesicle pressure leakage. A pressure transducer was placed subcutaneously at the lateral aspect of the abdominal wall (Figure 1). All telemetric devices were calibrated and zeroed before and after implantation. After surgery, rats were housed in cages in the animal facility for 7 days before the ISVP recordings were performed. No obvious immediate adverse events were observed in any rats. All rats exhibited spontaneous movement and eating behaviors immediately after implantation. After completing the experiments, the rats were killed with an intracardiac injection of air. The insertion site was then evaluated for displacement of the catheter tip and any damage to the catheter or obstructions. ISVP Measurements in Apomorphine-Induced Rats One week after surgery, experiments were performed between 1:00 and 6:00 pm. Each rat was J Sex Med 2015;12:2134–2140
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Figure 1 Surgical technique for implanting the telemetric device into the seminal vesicle of a Sprague–Dawley rat. A. Rat under anesthesia on a surgical plate (A); two small midline incisions following a surgical drape with povidone (B); pressure transducer implantation (C); exposure of the Rt seminal vesicle (D); catheter insertion into the seminal vesicle (E); fixation of the catheter to the seminal vesicle (F); incision closure (G and H). B. Magnified images of catheter insertion and fixation in the seminal vesicle
placed alone in the telemetric observation chamber for a 5- to 20-minute adaptation period until a stabilized ISVP appeared. The rats were then subcutaneously administered tamsulosin at 3 μg/kg. After a half-hour, a single injection of apomorphine (80 μg/kg, subcutaneously; SigmaAldrich Chemical Co) was administered. Continuous recordings for changes in ISVP and coordinated-erection behaviors were subsequently collected for 30 min. Penile erections were visually identified from recorded video collected by a camera installed in the telemetric observation chamber and connected to a computer, and ejaculation was confirmed by visualization of a copulatory plug in the tip of the penis. Erection behaviors and ejaculation were identified by a trained observer according to previously described behavioral criteria [18]. An erection behavior was identified in male rats as standing on the hind limbs, bending the head toward the genital region, and displaying hip movements. ISVP increases that coordinated with erection behaviors were analyzed. Pressure signals were amplified and telemetrically transmitted to an external receiver (model RPC-1; Data Sciences International) placed under the observation chamber (sampling at 500 Hz). After a 3-day washout period, tamsulosin (10 μg/kg), silodosin (1 mg/kg), and normal saline (sham group) were injected into the same rats in the stated order. Telemetric experiments were subsequently repeated as described earlier. Each group underwent three telemetric J Sex Med 2015;12:2134–2140
recordings. All rats completed 12 telemetric recordings after implantation over a total period of 6 weeks.
Data Analysis The feasibility and applicability of ISVP measurement with the telemetric device was evaluated in the SD rats. In the ISVP graph, the beginning of an ejaculation event was defined as the moment when the ISVP first exceeded the baseline ISVP ± 2 standard deviations, as described in a previous report of telemetry-based ICP measurement [17,19]. The end of an ejaculation event was defined as the moment when the pressure fell below the level of the first pressure rise in ISVP. The number of ejaculations during the recordings, maximal ISVP, and area under the curve (AUC, area under time × ISVP curves) were assessed to compare the effects of α-blockers on ejaculation among the four groups. Statistical Analysis All parameters were presented as means ± standard errors. One-way analysis of variance was performed to compare parameters among the four groups. Bonferroni’s correction was applied for multiple comparisons. Statistical analyses were conducted using SPSS software (version 22.0; Statistical Product and Services Solutions, Chicago, IL, USA). Differences were considered significant at a P value
ORIGINAL RESEARCH New Methodology for Investigating Ejaculation Dysfunction: Measuring Intraluminal Seminal Vesicle Pressure in Rats with a Telemetric Device Hyun Hwan Sung, MD, PhD,* Jung Jun Kim, MD,* Deok Hyun Han, MD, PhD,* Su Jeong Kang, BS,* Mee Ree Chae, MS,* Chul Young Kim, PhD,† Jong Kwan Park, MD, PhD,‡§ and Sung Won Lee, MD, PhD* *The Department of Urology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; †College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Korea; ‡Department of Urology, Medical School, and Institute for Medical Sciences, Chonbuk National University, Jeonju, Korea; §Research Institute and Clinical Trial Center of Medical Device, Chonbuk National University Hospital, Jeonju, Korea DOI: 10.1111/jsm.13025
ABSTRACT
Introduction. Ejaculation dysfunction is one of the most common male sexual disorders. Despite its prevalence and adverse impact on patients, little attention has been given to investigating ejaculation dysfunction. Aim. We introduce a new method for evaluating ejaculation dysfunction in rats with a telemetric device. Methods. A pressure transducer was surgically implanted in the seminal vesicles of 7-week-old male Sprague– Dawley rats. One week later, the rats were subcutaneously administered tamsulosin 3 μg/kg, and intra-seminal vesicle pressure (ISVP) was recorded in freely moving rats after an injection of apomorphine (80 μg/kg). Same rats repeated experiment with tamsulosin 10 μg/kg, silodosin 1 mg/kg, and normal saline with 3-day intervals. Main Outcome Measure. Sexual events were visually identified and recorded. Ejaculation was confirmed by visualization of a copulatory plug in the tip of the penis. We compared the maximal ISVP and area under the curve (AUC) of the ISVP. Results. Adequate ISVP data were easily recorded and available in 66.6% rats (10/15) over a 6-week telemetric recording period (12 recordings). The mean number of ejaculations during an inspection time of 30 minutes was 1.5 ± 0.1. The maximal ISVP values in rats receiving 3 μg/kg (30.0 ± 5.2 mm Hg) and 10 μg/kg tamsulosin (15.1 ± 1.6 mm Hg) and 1 mg/kg silodosin (12.9 ± 2.2 mm Hg) were significantly lower than that in control rats (61.4 ± 13.4 mm Hg, P < 0.05). The AUC values in rats receiving 3 μg/kg (72.7 ± 18.9 mm Hg × s) and 10 μg/kg tamsulosin (23.5 ± 6.1 mm Hg) and 1 mg/kg silodosin (23.9 ± 8.0 mm Hg) were also lower than that of control rats (162.6 ± 34.3 mm Hg, P < 0.05). Conclusions. Telemetric ISVP assessment is reliable and feasible for investigating apomorphine-induced ejaculation in rats. Tamsulosin (3 μg/kg and 10 μg/kg) and silodosin 1 mg/kg decreased the ISVP during ejaculation. Sung HH, Kim JJ, Han DH, Kang SJ, Chae MR, Kim CY, Park JK, and Lee SW. New methodology for investigating ejaculation dysfunction: measuring intraluminal seminal vesicle pressure in rats with a telemetric device. J Sex Med 2015;12:2134–2140. Key Words. Ejaculation Dysfunction; Telemetry; Telemetry Monitoring; Apomorphine
J Sex Med 2015;12:2134–2140
© 2015 International Society for Sexual Medicine
Intra-Seminal Vesicle Pressure Measuring with Telemetry Introduction
E
jaculation dysfunction is one of the most common male sexual disorders [1,2]. This disorder encompasses various types of ejaculatory problem such as premature ejaculation (PE), delayed ejaculation, and inability to ejaculate. Among these, PE is the most prevalent, accounting for approximately 20–30% of cases, depending on the definition for this condition [1–3]. PE has been clearly shown to exert a significant negative impact on the psychological statuses of patients with PE and their partners [4]. Medical conditions, including depression, anxiety, psychological distress, relationship problems, and general confidence, may be affected by PE [5–7]. In addition, many patients with PE suffer from concomitant sexual disorders. PE correlates with erectile dysfunction (ED) and coexists in approximately onethird of patients complaining of ED [8]. However, investigations of male sexual dysfunction have predominantly focused on ED, rather than ejaculation dysfunction. Accordingly, comprehensive research is essential to a better understanding of ejaculatory dysfunction. In vivo experimental studies of ejaculation physiology and PE have introduced procedures in which the intra-seminal vesicle pressure (ISVP) of an animal is measured [9–12]. Specifically, in rats, the intraluminal pressure of the seminal vesicle in response to electrical nerve stimulation was recorded to investigate the effects of particular drugs. This technique is usually performed under anesthesia in rats fixed on a surgical plate. However, this research has been limited by the unnatural characteristics of sexual behaviors in response to anesthesia and electrical stimulation [13,14]. Copulatory behavior, matched ejaculation, and confirmation of increased ISVP cannot be simultaneously obtained. Additionally, the animals must be sacrificed after the experiments, thus restricting repeated trials with different drugs. In a previous study, a telemetric device was introduced to measure the intra-cavernosal pressure (ICP), thus overcome the limitations associated with in vivo studies of ED [15]. In our previous studies, we successfully determined that ICP could be feasibly and reproducibly measured in rats using a telemetric device under minimally affected or unaffected conditions [16,17]. However, to our knowledge, no studies concerning ejaculation dysfunction have utilized a telemetric device. Therefore, the current study aimed to introduce new
2135
methodology with which evaluate the ISVP in rats with a telemetric device. Materials and Methods
Animals Seven-week-old male Sprague–Dawley (SD) rats (Orient Bio, Seoul, Korea) were housed 1 per cage at 22.8°C under 12-h light/12-h dark photoperiodic conditions (lights-off at 8:00 pm). Rats were fed a standard, fat-free pellet diet and water ad libitum throughout the experimental period. The rats were divided into four groups: tamsulosin (3 μg/kg or 10 μg/kg; Sigma-Aldrich Chemical Co, St. Louis, MO, USA), silodosin (1 mg/kg; JW Pharmaceutical, Seoul, Korea), and normal saline (control). A total of 15 rats were used. All animal experiments were conducted under the approval of the Institute for Animal Care and Use Committee of Samsung Medical Center. Telemetric Device Implantation Surgical implantation of the telemetric device was similar to the previously described technique used for telemetric ICP measurement [15,16]. The rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg; Yuhan Co, Seoul, Korea). A sterile telemetric sensor device (PA-C40; Data Sciences International, St Paul, MN, USA) was implanted as follows. A mid-abdominal incision was made to expose the seminal vesicle. The tip of a recording catheter was carefully inserted into the body of the right seminal vesicle. This tube was tightly secured to prevent seminal vesicle pressure leakage. A pressure transducer was placed subcutaneously at the lateral aspect of the abdominal wall (Figure 1). All telemetric devices were calibrated and zeroed before and after implantation. After surgery, rats were housed in cages in the animal facility for 7 days before the ISVP recordings were performed. No obvious immediate adverse events were observed in any rats. All rats exhibited spontaneous movement and eating behaviors immediately after implantation. After completing the experiments, the rats were killed with an intracardiac injection of air. The insertion site was then evaluated for displacement of the catheter tip and any damage to the catheter or obstructions. ISVP Measurements in Apomorphine-Induced Rats One week after surgery, experiments were performed between 1:00 and 6:00 pm. Each rat was J Sex Med 2015;12:2134–2140
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Sung et al.
Figure 1 Surgical technique for implanting the telemetric device into the seminal vesicle of a Sprague–Dawley rat. A. Rat under anesthesia on a surgical plate (A); two small midline incisions following a surgical drape with povidone (B); pressure transducer implantation (C); exposure of the Rt seminal vesicle (D); catheter insertion into the seminal vesicle (E); fixation of the catheter to the seminal vesicle (F); incision closure (G and H). B. Magnified images of catheter insertion and fixation in the seminal vesicle
placed alone in the telemetric observation chamber for a 5- to 20-minute adaptation period until a stabilized ISVP appeared. The rats were then subcutaneously administered tamsulosin at 3 μg/kg. After a half-hour, a single injection of apomorphine (80 μg/kg, subcutaneously; SigmaAldrich Chemical Co) was administered. Continuous recordings for changes in ISVP and coordinated-erection behaviors were subsequently collected for 30 min. Penile erections were visually identified from recorded video collected by a camera installed in the telemetric observation chamber and connected to a computer, and ejaculation was confirmed by visualization of a copulatory plug in the tip of the penis. Erection behaviors and ejaculation were identified by a trained observer according to previously described behavioral criteria [18]. An erection behavior was identified in male rats as standing on the hind limbs, bending the head toward the genital region, and displaying hip movements. ISVP increases that coordinated with erection behaviors were analyzed. Pressure signals were amplified and telemetrically transmitted to an external receiver (model RPC-1; Data Sciences International) placed under the observation chamber (sampling at 500 Hz). After a 3-day washout period, tamsulosin (10 μg/kg), silodosin (1 mg/kg), and normal saline (sham group) were injected into the same rats in the stated order. Telemetric experiments were subsequently repeated as described earlier. Each group underwent three telemetric J Sex Med 2015;12:2134–2140
recordings. All rats completed 12 telemetric recordings after implantation over a total period of 6 weeks.
Data Analysis The feasibility and applicability of ISVP measurement with the telemetric device was evaluated in the SD rats. In the ISVP graph, the beginning of an ejaculation event was defined as the moment when the ISVP first exceeded the baseline ISVP ± 2 standard deviations, as described in a previous report of telemetry-based ICP measurement [17,19]. The end of an ejaculation event was defined as the moment when the pressure fell below the level of the first pressure rise in ISVP. The number of ejaculations during the recordings, maximal ISVP, and area under the curve (AUC, area under time × ISVP curves) were assessed to compare the effects of α-blockers on ejaculation among the four groups. Statistical Analysis All parameters were presented as means ± standard errors. One-way analysis of variance was performed to compare parameters among the four groups. Bonferroni’s correction was applied for multiple comparisons. Statistical analyses were conducted using SPSS software (version 22.0; Statistical Product and Services Solutions, Chicago, IL, USA). Differences were considered significant at a P value
