ORIGINAL ARTICLE
Changes in erectile organ structure and function in a rat model of chronic prostatitis/chronic pelvic pain syndrome X.-J. Wang1, L.-L. Xia1, T.-Y. Xu1, X.-H. Zhang1, Z.-W. Zhu1, M.-G. Zhang1, Y. Liu2, C. Xu2, S. Zhong1 & Z.-J. Shen1 1 Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 Department of Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China
Keywords Animal model—chronic prostatitis/chronic pelvic pain syndrome—erectile dysfunction— erectile function—penis structure Correspondence Prof. Zhou-Jun Shen, No. 197 Ruijin Er Road, District of Huangpu, Shanghai 200025, China. Tel.: +86 21 64315370, or +86 13 761059626; Fax: +86 21 64315370; E-mail: [email protected] Xian-Jin Wang and Lei-Lei Xia contributed equally to this work. Accepted: March 29, 2015 doi: 10.1111/and.12437
Summary There is a growing recognition of the association between chronic prostatitis/ chronic pelvic pain syndrome (CP/CPPS) and erectile dysfunction (ED); however, most of the reports are based on questionnaires which cannot distinguish between organic and functional ED. The purpose of this study was to determine the exact relationship between CP/CPPS and ED, and to investigate the changes in erectile organ structure and function in a rat model of CP/CPPS. We established a rat model of experimental autoimmune prostatitis (EAP), which is a valid model for CP/CPPS. Erectile function in EAP and normal rats was comparable after cavernous nerve electrostimulation. The serum testosterone and oestradiol levels, ultrastructure of the corpus cavernosum and expression of endothelial nitric oxide synthase and neuronal nitric oxide synthase in the two groups were similar; however, there was a decrease in smooth muscleto-collagen ratio and alpha-smooth muscle actin expression and an increase in transforming growth factor-beta 1 expression was observed in EAP rats. Thus, organic ED may not exist in EAP rats. We speculate that ED complained by patients with CP/CPPS may be psychological, which could be caused by impairment in the quality of life; however, further studies are needed to fully understand the potential mechanisms underlying the penile fibrosis in EAP rats.
Introduction Chronic prostatitis/chronic pelvic pain syndrome (CP/ CPPS) is one of the most widely urological illnesses in men (Krieger et al., 1999; Habermacher et al., 2006; Schaeffer, 2006; La Vignera, 2012). CP/CPPS can cause substantial morbidity through the associated constellation of pelvic pain, urinary symptoms and sexual dysfunction. Erectile dysfunction (ED), defined as the consistent inability to obtain and/or maintain a penile erection sufficient for adequate sexual intercourse, is also a common problem in men (McVary, 2007; Shamloul & Ghanem, 2013; Muneer et al., 2014). Recently, there has been growing apprehension of an association between CP/CPPS and ED. Studies have shown that the prevalence of ED ranges from 13.8% to 48.3% in patients with CP/CPPS (Liang et al., 2004; Lee et al., 2008; Schultheiss, 2008; Hao et al., 2011; Chung et al., 2012). Most of these studies were based on questionnaires
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using assessment scales, such as the National Institutes of Health Chronic Prostatitis Symptom Index score, International Index of Erectile Function-5 (IIEF-5), Erection Hardness Grading Scale scores and Brief Sexual Functioning Questionnaire; however, organic and functional ED cannot be distinguished based on questionnaires. In fact, patients with CP/CPPS are frequently burdened with psychological disturbances due to pain and voiding dysfunction, which tend to reduce the frequency of sexual intercourse (Mehik et al., 2001; Aubin et al., 2008; Anderson et al., 2009; Riegel et al., 2014). Thus, erectile function in patients with CP/CPPS has not been fully described. In addition, no studies have investigated the penile structure of CP/CPPS in humans or animals. Therefore, we cannot confirm whether or not organic changes exist in the penises of CP/CPPS human or animal models. The purpose of our study was to determine the exact relationship between CP⁄CPPS and ED, and to investigate
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the changes in erectile organ structure and function in a rat model of CP⁄CPPS. We first established a rat model of experimental autoimmune prostatitis (EAP), which is considered to be a valid model for human CP⁄CPPS, and then compared the erectile function of EAP rats and control rats by measuring the intracavernous pressure (ICP) divided by the mean arterial pressure (MAP) during cavernous nerve electrostimulation. In addition, the structural changes of the penises in EAP rats were determined using Masson’s trichrome staining, immunohistochemistry, Western blotting and transmission electron microscopy (TEM). Indeed, our study will be conducive to distinguishing between organic and functional ED, and perfecting the therapeutic strategies for CP⁄CPPS.
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control group was injected with an equivalent volume of PBS. Three immunisations were administered on days 0, 15 and 30. Measurement of erectile responses in vivo On the 45th day after the first immunisation, the erectile function of the experimental rats was evaluated, as previously reported (Zhang et al., 2011, 2012, 2013). In brief, the erectile response was elicited by cavernous nerve electrostimulation and quantified by calculating the maximal ICP/MAP ratio. The electrical stimulation was repeated in triplicate after 10-min intervals, and the mean maximal ICP/MAP values were considered to represent the erectile function of rats.
Material and methods Serum testosterone and oestradiol assays Rats and materials Fifty male Sprague-Dawley rats (2 months old and weighing 180–210 g) and 10 male Sprague-Dawley rats (4 months old and weighing 400–440 g) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animals were bred and maintained in specific pathogen-free conditions in the Laboratory Animal Centre of Ruijin Hospital, which is affiliated with Shanghai Jiao Tong University School of Medicine. The Animal Experiment Committee of Ruijin Hospital approved this study, which was conducted in accordance with the ethical standards of the responsible Institutional Committee on Animal Experimentation. Establishment of a rat model of experimental autoimmune prostatitis (1) Preparation of an autologous prostate tissue homogenate supernatant (PTHS) from rats: Ten 4-month-old rats were sacrificed. Prostate tissues were collected under aseptic conditions, homogenised in 0.5% Triton X-100 saline and maintained in an ice water bath. The homogenate was centrifuged at 12 000 g for 30 min at 4 °C, and the supernatant was collected. The protein concentration in the PTHS was measured using a microplate reader and diluted into concentrations of 20 mg ml 1 with phosphate buffer solution (PBS; 0.1 mol l 1 [pH 7.4]). (2) Rats grouping and immunisation: fifty 2-month-old rats were randomly divided into EAP and control groups (25 rats each). For immunisation, each rat in the EAP group was administered 1.0 ml of PTHS emulsified with complete Freund’s adjuvant by subcutaneous injection into the abdominal flank and simultaneously immunised with 0.5 ml of a pertussis–diphtheria–tetanus vaccine by intraperitoneal injection. Each rat in the 244
The circulating levels of testosterone and oestradiol were determined using an enzyme-linked immunosorbent assay kit (Westang Bio-tech Co., Ltd., Shanghai, China) and an ELISA plate reader (Multiskan Ex Primary LIA V. 2.3; Thermo, Vantaa, Finland). The operation was conducted according to the ELISA protocol. Tissue weights of prostates and penises The ventral and dorsolateral lobes of the prostate glands were removed. The corpus cavernosum was removed from the glans penis to the crus penis. All tissues were weighed immediately after removal. The ratios of prostate wet weight to body weight and corpus cavernosum weight to body weight were calculated. Histopathologic examination After quantifying the erection function, prostate and corpus cavernosum were quickly removed and placed in Bouin’s solution for at least 24 h, dehydrated in alcohol, cleared in xylol and then embedded in paraffin. Paraffinembedded specimens were sliced into 5-mm sections and mounted on glass slides, followed by deparaffinisation and rehydration. Ten slides of different sections were evaluated per rat. The sectioned prostate tissues were stained with haematoxylin–eosin (H&E). To evaluate the ratio of smooth muscle to collagen (SM/C) of penises, the specimens were stained for Masson’s trichrome (MT) with a MT staining kit (Sigma-Aldrich, St. Louis, MO, USA), as previously described (Zhang et al., 2012, 2013). The areas of smooth muscle (red stain) and collagen (blue stain) were analysed in 2009 magnification images of the penis, using Olympus Biological Microscope (BX53; Olympus, Tokyo, © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
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Japan) and IMAGE PRO PLUS 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
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An unpaired Student’s t-test was used to analyse the differences between the EAP and control groups. A twotailed P < 0.05 was considered statistically significant.
Transmission electron microscopy Specimens of the ventral lobe of the prostate and corpus cavernosum were quickly removed and fixed with ice-cold glutaraldehyde (2% in 0.1 mol 1 1 sodium cacodylate buffer [pH 7.4]) overnight. The specimens were postfixed in osmium tetroxide and embedded in Epon 618 (TAAB Laboratories Equipment, Berks, UK). The specimens were sectioned into 0.1-mm sections, stained with uranyl acetate/lead citrate (Sigma-Aldrich) and viewed using Philips CM-120 transmission electron microscopy (Philips, Eindhoven, the Netherlands). Tissues obtained from three EAP and three control rats were examined according to the morphologic results, and at least 10 visual fields were observed for each sample. Immunohistochemistry analysis Paraffin-embedded specimens were used for immunohistochemical staining. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are erectile function indicators, alpha-smooth muscle actin (a-SMA) is used to assess the smooth muscle content, and transforming growth factor-beta 1 (TGF-b1) is used to assess fibrosis of the corpus cavernosum. Primary antibodies of anti-eNOS (1 : 200; Abcam, Cambridge, MA, USA), anti-nNOS (1 : 50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-a-SMA (1 : 200; Abcam) and anti-TGF-b1 (1 : 50; Santa Cruz Biotechnology, Inc.) were used for immunohistochemical staining. Quantitative image analyses were performed on each slide at 2009 magnification using the IMAGE PRO PLUS 6.0 software. Western blot analysis Frozen corpus cavernosum samples were lysed, and Western blot analysis was performed. Primary antibodies included anti-eNOS (1 : 500; Abcam), anti-nNOS (1 : 200; Santa Cruz Biotechnology, Inc.), anti-a-SMA (1 : 500; Abcam) and anti-TGF-b1 (1 : 200; Santa Cruz Biotechnology, Inc.). Anti-GAPDH (1 : 2000; SigmaAldrich Inc.) was used to assess protein loading equivalence. Densitometry of the gels was performed by GEL-PRO ANALYZER 4.0 software (Media Cybernetics).
Results Body weights, and tissue weights of prostates and penises Compared to control rats, the body weights of EAP rats were comparable (P = 0.094), but the prostate weights were heavier (P = 0.034), and the ratio of the prostate weight to body weight was increased (P = 0.007). No difference was observed between the two groups with respect to the penis weights (P = 0.818) or the ratio of the penis weight to the body weight (P = 0.417; Table 1). Serum concentration of testosterone and oestradiol There were no differences in serum testosterone and oestradiol concentrations between the control and EAP groups (P = 0.204, P = 0.381; Table 1). Morphologic features of prostate tissues in EAP rats Grossly, the prostate tissues of EAP rats on the 45th day were characterised by severe congestion and oedema, adhesions to the surrounding tissues and destruction of the prostate capsule. H&E staining revealed that the glandular epithelium structure of prostate glands in control rats was integrated and clear; no inflammatory cell infiltration or oedema was observed (Fig. 1a,b). In the EAP group, diffusely inhomogeneous tissue hyperplasia and ductal dilation or damage were observed, and a portion of the basal lamina was seriously destroyed by considerable chronic inflammatory cell infiltration (Fig. 1d,e). TEM revealed that the rough endoplasmic reticulum was decreased and the morphology of mitochondria was regular in the prostate epithelium cells of control rats (Fig. 1c). In the EAP group, the prostate epithelium cells showed high-response performance. Abundant rough endoplasmic reticulum in the cytoplasm was expanded into the pool. Numerous mitochondria exhibited moderate-to-severe swelling, the inner and outer membranes were disrupted, and the intracristal spaces were dilated (Fig. 1f). These morphologic features of prostate tissues suggested successful establishment of chronic prostatitis.
Statistical analysis Data were analysed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA), and continuous variables are expressed as the mean standard error of mean (SEM). © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
Erectile function of EAP rats Erectile responses to cavernous nerve electrostimulation revealed that the ICP, corresponding MAP and the ICP/ 245
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Variable
Control
EAP
P value
Body weight (g) Prostate weight (mg) Prostate/body weight (%) Penis weight (mg) Penis/body weight (%) Serum testosterone (ng ml 1) Serum estradiol (pg ml 1) Erectile function in vivo ICP (mmHg) MAP (mmHg) ICP/MAP (%) SM/C (Masson’s staining) Immunohistochemical staining eNOS/GAPDH nNOS/GAPDH a-SMA/GAPDH TGF-b/GAPDH Densitometry of Western blot eNOS/GAPDH nNOS/GAPDH a-SMA/GAPDH TGF-b/GAPDH
425.45 491.73 0.116 372.45 0.088 4.75 18.82
7.54 11.39 0.002 15.82 0.004 0.53 1.96
408.60 532.70 0.131 377.40 0.093 3.84 20.69
5.62 14.11 0.005 13.76 0.004 0.45 1.10
0.094 0.034* 0.007** 0.818 0.417 0.204 0.381
91.18 129.12 70.44 0.26
3.48 2.61 2.04 0.02
85.45 125.13 68.07 0.16
3.08 2.41 1.84 0.02
0.221 0.265 0.388 0.003**
13.92 17.48 18.54 7.49
1.44 1.99 2.75 1.44
13.32 19.05 9.58 13.07
1.88 1.47 1.41 2.00
0.804 0.534 0.009** 0.036*
0.21 0.83 0.49 0.21
0.04 0.06 0.09 0.02
0.20 0.82 0.12 0.46
0.05 0.11 0.02 0.07
0.838 0.975 0.015* 0.022*
Table 1 Comparison in serum sex hormone, erectile function, histologic staining and immunoblot data among the two groups (mean SEM)
EAP, experimental autoimmune prostatitis; ICP, intracavernous pressure; MAP, mean arterial pressure; SM/C, the ratio of smooth muscle to collagen; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; a-SMA, alpha-smooth muscle actin; TGF-b1, transforming growth factor-b1; *P < 0.05, **P < 0.01 vs. control group using the unpaired t-test.
MAP ratio in the EAP rats were lower after cavernous nerve electrostimulation compared to control rats, but there were no statistical differences (P = 0.221, P = 0.265, P = 0.388; Table 1 and Fig. 2a). Immunohistochemistry and Western blot analysis revealed that protein expressions of eNOS and nNOS in the two groups were similar (Table 1; Fig. 2c,d,g). Histopathologic features of the corpus cavernosum in EAP rats According to the Masson’s trichrome staining, there was a decrease in the area of smooth muscle (red stain) and an increase in the area of collagen (blue stain) in the corpus cavernosum of EAP rats compared to control rats. The EAP group had a decreased SM/collagen ratio vs. the control group (P = 0.003; Table 1 and Fig. 2b). Immunohistochemistry and Western blot analysis also revealed the EAP group had lower a-SMA expression and higher TGF-b1 expression compared with normal rats (Table 1; Fig. 2e,f,g). Ultrastructural features of the corpus cavernosum in EAP rats TEM revealed that the ultrastructure of the corpus cavernosum was similar between the two groups; no obvious 246
abnormalities were noted. Lower magnification TEM images showed that interstitial cells (ICs) had a narrow perinuclear cytoplasm and an oval nucleus and were often situated near smooth muscle cells (SMCs; Fig. 3a,d). Higher magnification micrographs suggested that the ultrastructure of ICs is characterised by abundant caveolae along the cell membranes, numerous mitochondria and dense bodies, and a small amount of rough endoplasmic reticulum in the cytoplasm (Fig. 3b,e). In the SMCs, the myofilaments were arranged orderly, the cell membranes were integrated, and the internal organelles were abundant (Fig. 3c,f). Discussion In the current study, we showed that the erectile responses to the cavernous nerve electrostimulation in EAP rats were not significantly decreased compared with control rats. The expressions of eNOS and nNOS, which are erectile-related indicators, in the corpus cavernosum were similar between the two groups. Our results are in contrast to a number of clinical studies reporting a high prevalence of ED among patients with CP/CPPS (Liang et al., 2004; Marszalek et al., 2007; Lee et al., 2008; Hao et al., 2011; Chung et al., 2012). The underlying reasons for this discrepancy may be as follows. First, the methods of evaluating erectile function between humans and © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
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(a)
(b)
(c)
(d)
(e)
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Fig. 1 Histologic and ultrastructural features of prostate tissues. (a, b, d and e) Histologic features of prostate tissues in the two groups. The prostate tissues of EAP rats had diffusely inhomogeneous tissue hyperplasia and ductal dilation or damage. A portion of the basal lamina was seriously destroyed by chronic inflammatory cell infiltration. (c and f) Transmission electron micrographs of prostate tissues in the two groups. The prostate epithelium cells of EAP rats exhibited a high-response performance. Abundant rough endoplasmic reticulum (white arrows) in the cytoplasm was expanded into the pool. Numerous mitochondria (black arrows) had moderate-to-severe swelling, the inner and outer membranes were disrupted, and the intracristal spaces were dilated. N, nuclei. Scale bars = 200 lm (1009), scale bars = 50 lm (4009) and scale bars = 2 lm (13 5009).
animals differ significantly and may in turn lead to different results. Questionnaires are the main tools to evaluate erectile function in humans. Among these questionnaires, the IIEF is the most widely used multidimensional selfreport instrument (Rosen et al., 2002; Bayraktar & Atun, 2013). Despite strong psychometric properties and wide adoption, the IIEF has limitations or weaknesses in specific areas. Indeed, the IIEF focuses only on current sexual functioning and provides superficial assessment of domains of sexual functioning other than erection. The IIEF lacks information about the aetiology of sexual disorders (Rosen et al., 1997), and the IIEF cannot differentiate between organic and functional ED. Besides, in the evaluation of human erectile function, nocturnal penile tumescence (NPT) monitoring is one of the most reliable methods to diagnose ED and to differentiate psychogenic from organic ED (Elhanbly & Elkholy, 2012). However, there have been no relevant studies that assessing erectile function in patients with CP/CPPS using NPT monitoring. In animals, including rats, mice and rabbits, the most common and accurate method with which to evaluate erectile function is to induce erectile responses through cavernous nerve electrostimulation. The ratio of the maximal ICP to the mean MAP represents the erectile © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
function of animals. The ICP monitoring in animals is parallel to NPT monitoring in human and can accurately determine whether or not organic ED exists. Thus, it is generally recognised that decrease in ICP/MAP ratio demonstrates the presence of ED. However, this method can accurately determine whether or not organic ED exists, but hardly evaluate the psychological factors. Second, many reports suggest that CP/CPPS impairs the overall quality of life (QoL), and it is the QoL that contributes to or causes ED (Muller & Mulhall, 2006; Rosen et al., 2009). Prostatitis-like symptoms, such as pelvic discomfort or pain and irritative or obstructive voiding symptoms, may affect the global emotional well-being of males. Patients with CP/CPPS tend to have lower mental health scores compared to the general population and also tend to present with psychological adaptation problems related to stress, depression, anxiety, hysteria, hypochondriasis and somatisation disorders (Mehik et al., 2001). The psychological factors have a negative impact on health-related QoL and ultimately influence the sexual lives of men with CP/CPPS (Aubin et al., 2008; Tran & Shoskes, 2013; Tripp et al., 2013; Wagenlehner et al., 2013). Third, some media, medical workers or research studies may exaggerate the severity of CP/CPPS and 247
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Fig. 2 Erectile function and histopathologic features of the corpus cavernosum. (a) Erectile responses to cavernous nerve electrostimulation. (b) Masson’s trichrome staining of corpus cavernosum. (c–f) Immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), alpha-smooth muscle actin (a-SMA) and transforming growth factor-beta 1 (TGF-b1) respectively. Dark brown colour represents positive staining. (g) Western blot analysis of eNOS, nNOS, a-SMA and TGF-b1 with GAPDH as the loading control. Values are presented as the ratio standard deviation. *P < 0.05, **P < 0.01 vs. control group using the unpaired t-test. Scale bars = 100 lm (2009).
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(a)
Fig. 3 Ultrastructural features of the corpus cavernosum. Transmission electron microscopy revealed similar ultrastructure of the corpus cavernosum in the two groups. (a–c) Control rats; (d–f) EAP rats. Lower magnification TEM (74009) images showed that interstitial cells (ICs) had a narrow perinuclear cytoplasm and an oval nucleus (N) and were often situated near smooth muscle cells (SMCs) (a and d). Higher magnification micrographs (24 5009) suggested that the ultrastructure of ICs is characterised by abundant caveolae (short white arrows) along the cell membranes, numerous mitochondria (long white arrows) and a small number of rough endoplasmic reticulum (long black arrows) in the cytoplasm (b and e). In the SMCs, the myofilaments were arranged orderly, the cell membrane was integrated, and its internal organelles were abundant (c and f). Scale bars = 2 lm (74009), scale bars = 1 lm (24 5009).
(c)
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effects on erectile function. All of these factors will increase sexual anxiety, decrease sexual activity and ultimately result in sexual disturbances or ED. Therefore, our results suggest that organic ED may not exist in patients with CP/CPPS. Although our results, which were based on a rat model, cannot be completely applied to humans, we believe that this study is still meaningful. In clinical practice, clinicians should not overuse pharmacotherapy, such as a-blockers, antibiotics and anti-inflammatories (Magri et al., 2011). Instead, the strong relationship between psychological factors and sexual dysfunction in men with CP/CPPS reinforces the need for a comprehensive approach to managing CP/CPPS. In addition to proper medical therapies, the interventions addressing psychological factors should be integrated in clinical trials as one possible way to improve sexual function and satisfaction in patients with CP/CPPS. Besides, more high-quality research is needed to understand the interplay of psychosocial factors in CP/CPPS. In the current study, no differences in serum concentrations of testosterone and oestradiol were detected between the EAP and normal groups. The ultrastructure of the corpus cavernosum was similar between the two groups; however, compared to control rats, there was a decrease in smooth muscle content, an increase in collagen and a © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
(b)
(e)
(f)
decreased SM/collagen ratio of the corpus cavernosum in EAP rats. Decreased expression of a-SMA and increased expression of TGF-b1 also suggested fibrogenesis in the corpus cavernosum of EAP rats. In most cases, penile fibrosis will lead to ED (Castiglione et al., 2013; Hannan et al., 2014); however, in the current study, the EAP rats had a fibrotic corpus cavernosum, but did not have ED. The possible reasons for this finding are as follows: the degree of corporal fibrosis was slight and not sufficient to result in ED; corporal fibrosis is related to a reduction in the cavernous sinuses, which will increase the intracavernosal pressure and the ICP/ MAP ratio may not decrease; and erectile function is a complex process involving biological and psychological factors, and other confounding factors may counteract the effect of prostatitis on erectile function. Further studies are warranted to fully understand the potential mechanisms of penile corporal fibrosis in EAP rats. In conclusion, erectile responses to cavernous nerve electrostimulation in EAP rats were not significantly decreased compared to control rats. Our results suggest that organic ED may not exist in patients with CP/CPPS. We speculate that CP/CPPS impairs the overall QoL and that a low QoL contributes to or causes ED. In clinical practice, clinicians should not overuse pharmacotherapy. 249
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Instead, psychological interventions should be considered to improve sexual function and satisfaction in males with CP/CPPS. In addition, further studies are needed to fully understand the potential mechanisms underlying the penile corporal fibrosis in EAP rats. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81270695), and the Key Project of Science and Technology Commission of Shanghai (SHD09411950400). We thank Dr Yan-Qin Hu (Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China), Shanghai WEIAO BioTech Co., Ltd.; and Shanghai Showbio, Biotech Co., Ltd. for their expert technical assistance. We also thank International Science Editing, Ltd. for assisting in the preparation of our report. References Anderson RU, Orenberg EK, Morey A, Chavez N, Chan CA (2009) Stress induced hypothalamus-pituitary-adrenal axis responses and disturbances in psychological profiles in men with chronic prostatitis/chronic pelvic pain syndrome. J Urol 182:2319–2324. Aubin S, Berger RE, Heiman JR, Ciol MA (2008) The association between sexual function, pain, and psychological adaptation of men diagnosed with chronic pelvic pain syndrome type III. J Sex Med 5:657–667. Bayraktar Z, Atun I (2013) Impact of physician assistance on the reliability of the International Index of Erectile Function. Andrologia 45:73–77. Castiglione F, Hedlund P, Van der Aa F, Bivalacqua TJ, Rigatti P, Van Poppel H, Montorsi F, De Ridder D, Albersen M (2013) Intratunical injection of human adipose tissuederived stem cells prevents fibrosis and is associated with improved erectile function in a rat model of Peyronie’s disease. Eur Urol 63:551–560. Chung SD, Keller JJ, Lin HC (2012) A case-control study on the association between chronic prostatitis/chronic pelvic pain syndrome and erectile dysfunction. BJU Int 110:726– 730. Elhanbly S, Elkholy A (2012) Nocturnal penile erections: the role of RigiScan in the diagnosis of vascular erectile dysfunction. J Sex Med 9:3219–3226. Habermacher GM, Chason JT, Schaeffer AJ (2006) Prostatitis/ chronic pelvic pain syndrome. Annu Rev Med 57:195–206. Hannan JL, Kutlu O, Stopak BL, Liu X, Castiglione F, Hedlund P, Burnett AL, Bivalacqua TJ (2014) Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats. J Sex Med 11:1442–1451. Hao ZY, Li HJ, Wang ZP, Xing JP, Hu WL, Zhang TF, Zhang XS, Zhou J, Tai S, Liang CZ (2011) The prevalence of
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with chronic prostatitis/chronic pelvic pain syndrome. Eur Urol 63:953–959. Zhang MG, Shen ZJ, Zhang CM, Wu W, Gao PJ, Chen SW, Zhou WL (2011) Vasoactive intestinal polypeptide, an erectile neurotransmitter, improves erectile function more significantly in castrated rats than in normal rats. BJU Int 108:440–446. Zhang MG, Wu W, Zhang CM, Wang XJ, Gao PJ, Lu YL, Shen ZJ (2012) Effects of oral finasteride on erectile function in a rat model. J Sex Med 9:1328–1336. Zhang MG, Wang XJ, Shen ZJ, Gao PJ (2013) Long-term oral administration of 5alpha-reductase inhibitor attenuates erectile function by inhibiting autophagy and promoting apoptosis of smooth muscle cells in corpus cavernosum of aged rats. Urology 82:743.e9–743.e15.
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Changes in erectile organ structure and function in a rat model of chronic prostatitis/chronic pelvic pain syndrome X.-J. Wang1, L.-L. Xia1, T.-Y. Xu1, X.-H. Zhang1, Z.-W. Zhu1, M.-G. Zhang1, Y. Liu2, C. Xu2, S. Zhong1 & Z.-J. Shen1 1 Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2 Department of Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China
Keywords Animal model—chronic prostatitis/chronic pelvic pain syndrome—erectile dysfunction— erectile function—penis structure Correspondence Prof. Zhou-Jun Shen, No. 197 Ruijin Er Road, District of Huangpu, Shanghai 200025, China. Tel.: +86 21 64315370, or +86 13 761059626; Fax: +86 21 64315370; E-mail: [email protected] Xian-Jin Wang and Lei-Lei Xia contributed equally to this work. Accepted: March 29, 2015 doi: 10.1111/and.12437
Summary There is a growing recognition of the association between chronic prostatitis/ chronic pelvic pain syndrome (CP/CPPS) and erectile dysfunction (ED); however, most of the reports are based on questionnaires which cannot distinguish between organic and functional ED. The purpose of this study was to determine the exact relationship between CP/CPPS and ED, and to investigate the changes in erectile organ structure and function in a rat model of CP/CPPS. We established a rat model of experimental autoimmune prostatitis (EAP), which is a valid model for CP/CPPS. Erectile function in EAP and normal rats was comparable after cavernous nerve electrostimulation. The serum testosterone and oestradiol levels, ultrastructure of the corpus cavernosum and expression of endothelial nitric oxide synthase and neuronal nitric oxide synthase in the two groups were similar; however, there was a decrease in smooth muscleto-collagen ratio and alpha-smooth muscle actin expression and an increase in transforming growth factor-beta 1 expression was observed in EAP rats. Thus, organic ED may not exist in EAP rats. We speculate that ED complained by patients with CP/CPPS may be psychological, which could be caused by impairment in the quality of life; however, further studies are needed to fully understand the potential mechanisms underlying the penile fibrosis in EAP rats.
Introduction Chronic prostatitis/chronic pelvic pain syndrome (CP/ CPPS) is one of the most widely urological illnesses in men (Krieger et al., 1999; Habermacher et al., 2006; Schaeffer, 2006; La Vignera, 2012). CP/CPPS can cause substantial morbidity through the associated constellation of pelvic pain, urinary symptoms and sexual dysfunction. Erectile dysfunction (ED), defined as the consistent inability to obtain and/or maintain a penile erection sufficient for adequate sexual intercourse, is also a common problem in men (McVary, 2007; Shamloul & Ghanem, 2013; Muneer et al., 2014). Recently, there has been growing apprehension of an association between CP/CPPS and ED. Studies have shown that the prevalence of ED ranges from 13.8% to 48.3% in patients with CP/CPPS (Liang et al., 2004; Lee et al., 2008; Schultheiss, 2008; Hao et al., 2011; Chung et al., 2012). Most of these studies were based on questionnaires
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using assessment scales, such as the National Institutes of Health Chronic Prostatitis Symptom Index score, International Index of Erectile Function-5 (IIEF-5), Erection Hardness Grading Scale scores and Brief Sexual Functioning Questionnaire; however, organic and functional ED cannot be distinguished based on questionnaires. In fact, patients with CP/CPPS are frequently burdened with psychological disturbances due to pain and voiding dysfunction, which tend to reduce the frequency of sexual intercourse (Mehik et al., 2001; Aubin et al., 2008; Anderson et al., 2009; Riegel et al., 2014). Thus, erectile function in patients with CP/CPPS has not been fully described. In addition, no studies have investigated the penile structure of CP/CPPS in humans or animals. Therefore, we cannot confirm whether or not organic changes exist in the penises of CP/CPPS human or animal models. The purpose of our study was to determine the exact relationship between CP⁄CPPS and ED, and to investigate
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the changes in erectile organ structure and function in a rat model of CP⁄CPPS. We first established a rat model of experimental autoimmune prostatitis (EAP), which is considered to be a valid model for human CP⁄CPPS, and then compared the erectile function of EAP rats and control rats by measuring the intracavernous pressure (ICP) divided by the mean arterial pressure (MAP) during cavernous nerve electrostimulation. In addition, the structural changes of the penises in EAP rats were determined using Masson’s trichrome staining, immunohistochemistry, Western blotting and transmission electron microscopy (TEM). Indeed, our study will be conducive to distinguishing between organic and functional ED, and perfecting the therapeutic strategies for CP⁄CPPS.
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control group was injected with an equivalent volume of PBS. Three immunisations were administered on days 0, 15 and 30. Measurement of erectile responses in vivo On the 45th day after the first immunisation, the erectile function of the experimental rats was evaluated, as previously reported (Zhang et al., 2011, 2012, 2013). In brief, the erectile response was elicited by cavernous nerve electrostimulation and quantified by calculating the maximal ICP/MAP ratio. The electrical stimulation was repeated in triplicate after 10-min intervals, and the mean maximal ICP/MAP values were considered to represent the erectile function of rats.
Material and methods Serum testosterone and oestradiol assays Rats and materials Fifty male Sprague-Dawley rats (2 months old and weighing 180–210 g) and 10 male Sprague-Dawley rats (4 months old and weighing 400–440 g) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Animals were bred and maintained in specific pathogen-free conditions in the Laboratory Animal Centre of Ruijin Hospital, which is affiliated with Shanghai Jiao Tong University School of Medicine. The Animal Experiment Committee of Ruijin Hospital approved this study, which was conducted in accordance with the ethical standards of the responsible Institutional Committee on Animal Experimentation. Establishment of a rat model of experimental autoimmune prostatitis (1) Preparation of an autologous prostate tissue homogenate supernatant (PTHS) from rats: Ten 4-month-old rats were sacrificed. Prostate tissues were collected under aseptic conditions, homogenised in 0.5% Triton X-100 saline and maintained in an ice water bath. The homogenate was centrifuged at 12 000 g for 30 min at 4 °C, and the supernatant was collected. The protein concentration in the PTHS was measured using a microplate reader and diluted into concentrations of 20 mg ml 1 with phosphate buffer solution (PBS; 0.1 mol l 1 [pH 7.4]). (2) Rats grouping and immunisation: fifty 2-month-old rats were randomly divided into EAP and control groups (25 rats each). For immunisation, each rat in the EAP group was administered 1.0 ml of PTHS emulsified with complete Freund’s adjuvant by subcutaneous injection into the abdominal flank and simultaneously immunised with 0.5 ml of a pertussis–diphtheria–tetanus vaccine by intraperitoneal injection. Each rat in the 244
The circulating levels of testosterone and oestradiol were determined using an enzyme-linked immunosorbent assay kit (Westang Bio-tech Co., Ltd., Shanghai, China) and an ELISA plate reader (Multiskan Ex Primary LIA V. 2.3; Thermo, Vantaa, Finland). The operation was conducted according to the ELISA protocol. Tissue weights of prostates and penises The ventral and dorsolateral lobes of the prostate glands were removed. The corpus cavernosum was removed from the glans penis to the crus penis. All tissues were weighed immediately after removal. The ratios of prostate wet weight to body weight and corpus cavernosum weight to body weight were calculated. Histopathologic examination After quantifying the erection function, prostate and corpus cavernosum were quickly removed and placed in Bouin’s solution for at least 24 h, dehydrated in alcohol, cleared in xylol and then embedded in paraffin. Paraffinembedded specimens were sliced into 5-mm sections and mounted on glass slides, followed by deparaffinisation and rehydration. Ten slides of different sections were evaluated per rat. The sectioned prostate tissues were stained with haematoxylin–eosin (H&E). To evaluate the ratio of smooth muscle to collagen (SM/C) of penises, the specimens were stained for Masson’s trichrome (MT) with a MT staining kit (Sigma-Aldrich, St. Louis, MO, USA), as previously described (Zhang et al., 2012, 2013). The areas of smooth muscle (red stain) and collagen (blue stain) were analysed in 2009 magnification images of the penis, using Olympus Biological Microscope (BX53; Olympus, Tokyo, © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
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Japan) and IMAGE PRO PLUS 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
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An unpaired Student’s t-test was used to analyse the differences between the EAP and control groups. A twotailed P < 0.05 was considered statistically significant.
Transmission electron microscopy Specimens of the ventral lobe of the prostate and corpus cavernosum were quickly removed and fixed with ice-cold glutaraldehyde (2% in 0.1 mol 1 1 sodium cacodylate buffer [pH 7.4]) overnight. The specimens were postfixed in osmium tetroxide and embedded in Epon 618 (TAAB Laboratories Equipment, Berks, UK). The specimens were sectioned into 0.1-mm sections, stained with uranyl acetate/lead citrate (Sigma-Aldrich) and viewed using Philips CM-120 transmission electron microscopy (Philips, Eindhoven, the Netherlands). Tissues obtained from three EAP and three control rats were examined according to the morphologic results, and at least 10 visual fields were observed for each sample. Immunohistochemistry analysis Paraffin-embedded specimens were used for immunohistochemical staining. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are erectile function indicators, alpha-smooth muscle actin (a-SMA) is used to assess the smooth muscle content, and transforming growth factor-beta 1 (TGF-b1) is used to assess fibrosis of the corpus cavernosum. Primary antibodies of anti-eNOS (1 : 200; Abcam, Cambridge, MA, USA), anti-nNOS (1 : 50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-a-SMA (1 : 200; Abcam) and anti-TGF-b1 (1 : 50; Santa Cruz Biotechnology, Inc.) were used for immunohistochemical staining. Quantitative image analyses were performed on each slide at 2009 magnification using the IMAGE PRO PLUS 6.0 software. Western blot analysis Frozen corpus cavernosum samples were lysed, and Western blot analysis was performed. Primary antibodies included anti-eNOS (1 : 500; Abcam), anti-nNOS (1 : 200; Santa Cruz Biotechnology, Inc.), anti-a-SMA (1 : 500; Abcam) and anti-TGF-b1 (1 : 200; Santa Cruz Biotechnology, Inc.). Anti-GAPDH (1 : 2000; SigmaAldrich Inc.) was used to assess protein loading equivalence. Densitometry of the gels was performed by GEL-PRO ANALYZER 4.0 software (Media Cybernetics).
Results Body weights, and tissue weights of prostates and penises Compared to control rats, the body weights of EAP rats were comparable (P = 0.094), but the prostate weights were heavier (P = 0.034), and the ratio of the prostate weight to body weight was increased (P = 0.007). No difference was observed between the two groups with respect to the penis weights (P = 0.818) or the ratio of the penis weight to the body weight (P = 0.417; Table 1). Serum concentration of testosterone and oestradiol There were no differences in serum testosterone and oestradiol concentrations between the control and EAP groups (P = 0.204, P = 0.381; Table 1). Morphologic features of prostate tissues in EAP rats Grossly, the prostate tissues of EAP rats on the 45th day were characterised by severe congestion and oedema, adhesions to the surrounding tissues and destruction of the prostate capsule. H&E staining revealed that the glandular epithelium structure of prostate glands in control rats was integrated and clear; no inflammatory cell infiltration or oedema was observed (Fig. 1a,b). In the EAP group, diffusely inhomogeneous tissue hyperplasia and ductal dilation or damage were observed, and a portion of the basal lamina was seriously destroyed by considerable chronic inflammatory cell infiltration (Fig. 1d,e). TEM revealed that the rough endoplasmic reticulum was decreased and the morphology of mitochondria was regular in the prostate epithelium cells of control rats (Fig. 1c). In the EAP group, the prostate epithelium cells showed high-response performance. Abundant rough endoplasmic reticulum in the cytoplasm was expanded into the pool. Numerous mitochondria exhibited moderate-to-severe swelling, the inner and outer membranes were disrupted, and the intracristal spaces were dilated (Fig. 1f). These morphologic features of prostate tissues suggested successful establishment of chronic prostatitis.
Statistical analysis Data were analysed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA), and continuous variables are expressed as the mean standard error of mean (SEM). © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
Erectile function of EAP rats Erectile responses to cavernous nerve electrostimulation revealed that the ICP, corresponding MAP and the ICP/ 245
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Variable
Control
EAP
P value
Body weight (g) Prostate weight (mg) Prostate/body weight (%) Penis weight (mg) Penis/body weight (%) Serum testosterone (ng ml 1) Serum estradiol (pg ml 1) Erectile function in vivo ICP (mmHg) MAP (mmHg) ICP/MAP (%) SM/C (Masson’s staining) Immunohistochemical staining eNOS/GAPDH nNOS/GAPDH a-SMA/GAPDH TGF-b/GAPDH Densitometry of Western blot eNOS/GAPDH nNOS/GAPDH a-SMA/GAPDH TGF-b/GAPDH
425.45 491.73 0.116 372.45 0.088 4.75 18.82
7.54 11.39 0.002 15.82 0.004 0.53 1.96
408.60 532.70 0.131 377.40 0.093 3.84 20.69
5.62 14.11 0.005 13.76 0.004 0.45 1.10
0.094 0.034* 0.007** 0.818 0.417 0.204 0.381
91.18 129.12 70.44 0.26
3.48 2.61 2.04 0.02
85.45 125.13 68.07 0.16
3.08 2.41 1.84 0.02
0.221 0.265 0.388 0.003**
13.92 17.48 18.54 7.49
1.44 1.99 2.75 1.44
13.32 19.05 9.58 13.07
1.88 1.47 1.41 2.00
0.804 0.534 0.009** 0.036*
0.21 0.83 0.49 0.21
0.04 0.06 0.09 0.02
0.20 0.82 0.12 0.46
0.05 0.11 0.02 0.07
0.838 0.975 0.015* 0.022*
Table 1 Comparison in serum sex hormone, erectile function, histologic staining and immunoblot data among the two groups (mean SEM)
EAP, experimental autoimmune prostatitis; ICP, intracavernous pressure; MAP, mean arterial pressure; SM/C, the ratio of smooth muscle to collagen; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; a-SMA, alpha-smooth muscle actin; TGF-b1, transforming growth factor-b1; *P < 0.05, **P < 0.01 vs. control group using the unpaired t-test.
MAP ratio in the EAP rats were lower after cavernous nerve electrostimulation compared to control rats, but there were no statistical differences (P = 0.221, P = 0.265, P = 0.388; Table 1 and Fig. 2a). Immunohistochemistry and Western blot analysis revealed that protein expressions of eNOS and nNOS in the two groups were similar (Table 1; Fig. 2c,d,g). Histopathologic features of the corpus cavernosum in EAP rats According to the Masson’s trichrome staining, there was a decrease in the area of smooth muscle (red stain) and an increase in the area of collagen (blue stain) in the corpus cavernosum of EAP rats compared to control rats. The EAP group had a decreased SM/collagen ratio vs. the control group (P = 0.003; Table 1 and Fig. 2b). Immunohistochemistry and Western blot analysis also revealed the EAP group had lower a-SMA expression and higher TGF-b1 expression compared with normal rats (Table 1; Fig. 2e,f,g). Ultrastructural features of the corpus cavernosum in EAP rats TEM revealed that the ultrastructure of the corpus cavernosum was similar between the two groups; no obvious 246
abnormalities were noted. Lower magnification TEM images showed that interstitial cells (ICs) had a narrow perinuclear cytoplasm and an oval nucleus and were often situated near smooth muscle cells (SMCs; Fig. 3a,d). Higher magnification micrographs suggested that the ultrastructure of ICs is characterised by abundant caveolae along the cell membranes, numerous mitochondria and dense bodies, and a small amount of rough endoplasmic reticulum in the cytoplasm (Fig. 3b,e). In the SMCs, the myofilaments were arranged orderly, the cell membranes were integrated, and the internal organelles were abundant (Fig. 3c,f). Discussion In the current study, we showed that the erectile responses to the cavernous nerve electrostimulation in EAP rats were not significantly decreased compared with control rats. The expressions of eNOS and nNOS, which are erectile-related indicators, in the corpus cavernosum were similar between the two groups. Our results are in contrast to a number of clinical studies reporting a high prevalence of ED among patients with CP/CPPS (Liang et al., 2004; Marszalek et al., 2007; Lee et al., 2008; Hao et al., 2011; Chung et al., 2012). The underlying reasons for this discrepancy may be as follows. First, the methods of evaluating erectile function between humans and © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1 Histologic and ultrastructural features of prostate tissues. (a, b, d and e) Histologic features of prostate tissues in the two groups. The prostate tissues of EAP rats had diffusely inhomogeneous tissue hyperplasia and ductal dilation or damage. A portion of the basal lamina was seriously destroyed by chronic inflammatory cell infiltration. (c and f) Transmission electron micrographs of prostate tissues in the two groups. The prostate epithelium cells of EAP rats exhibited a high-response performance. Abundant rough endoplasmic reticulum (white arrows) in the cytoplasm was expanded into the pool. Numerous mitochondria (black arrows) had moderate-to-severe swelling, the inner and outer membranes were disrupted, and the intracristal spaces were dilated. N, nuclei. Scale bars = 200 lm (1009), scale bars = 50 lm (4009) and scale bars = 2 lm (13 5009).
animals differ significantly and may in turn lead to different results. Questionnaires are the main tools to evaluate erectile function in humans. Among these questionnaires, the IIEF is the most widely used multidimensional selfreport instrument (Rosen et al., 2002; Bayraktar & Atun, 2013). Despite strong psychometric properties and wide adoption, the IIEF has limitations or weaknesses in specific areas. Indeed, the IIEF focuses only on current sexual functioning and provides superficial assessment of domains of sexual functioning other than erection. The IIEF lacks information about the aetiology of sexual disorders (Rosen et al., 1997), and the IIEF cannot differentiate between organic and functional ED. Besides, in the evaluation of human erectile function, nocturnal penile tumescence (NPT) monitoring is one of the most reliable methods to diagnose ED and to differentiate psychogenic from organic ED (Elhanbly & Elkholy, 2012). However, there have been no relevant studies that assessing erectile function in patients with CP/CPPS using NPT monitoring. In animals, including rats, mice and rabbits, the most common and accurate method with which to evaluate erectile function is to induce erectile responses through cavernous nerve electrostimulation. The ratio of the maximal ICP to the mean MAP represents the erectile © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
function of animals. The ICP monitoring in animals is parallel to NPT monitoring in human and can accurately determine whether or not organic ED exists. Thus, it is generally recognised that decrease in ICP/MAP ratio demonstrates the presence of ED. However, this method can accurately determine whether or not organic ED exists, but hardly evaluate the psychological factors. Second, many reports suggest that CP/CPPS impairs the overall quality of life (QoL), and it is the QoL that contributes to or causes ED (Muller & Mulhall, 2006; Rosen et al., 2009). Prostatitis-like symptoms, such as pelvic discomfort or pain and irritative or obstructive voiding symptoms, may affect the global emotional well-being of males. Patients with CP/CPPS tend to have lower mental health scores compared to the general population and also tend to present with psychological adaptation problems related to stress, depression, anxiety, hysteria, hypochondriasis and somatisation disorders (Mehik et al., 2001). The psychological factors have a negative impact on health-related QoL and ultimately influence the sexual lives of men with CP/CPPS (Aubin et al., 2008; Tran & Shoskes, 2013; Tripp et al., 2013; Wagenlehner et al., 2013). Third, some media, medical workers or research studies may exaggerate the severity of CP/CPPS and 247
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Fig. 2 Erectile function and histopathologic features of the corpus cavernosum. (a) Erectile responses to cavernous nerve electrostimulation. (b) Masson’s trichrome staining of corpus cavernosum. (c–f) Immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), alpha-smooth muscle actin (a-SMA) and transforming growth factor-beta 1 (TGF-b1) respectively. Dark brown colour represents positive staining. (g) Western blot analysis of eNOS, nNOS, a-SMA and TGF-b1 with GAPDH as the loading control. Values are presented as the ratio standard deviation. *P < 0.05, **P < 0.01 vs. control group using the unpaired t-test. Scale bars = 100 lm (2009).
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(a)
Fig. 3 Ultrastructural features of the corpus cavernosum. Transmission electron microscopy revealed similar ultrastructure of the corpus cavernosum in the two groups. (a–c) Control rats; (d–f) EAP rats. Lower magnification TEM (74009) images showed that interstitial cells (ICs) had a narrow perinuclear cytoplasm and an oval nucleus (N) and were often situated near smooth muscle cells (SMCs) (a and d). Higher magnification micrographs (24 5009) suggested that the ultrastructure of ICs is characterised by abundant caveolae (short white arrows) along the cell membranes, numerous mitochondria (long white arrows) and a small number of rough endoplasmic reticulum (long black arrows) in the cytoplasm (b and e). In the SMCs, the myofilaments were arranged orderly, the cell membrane was integrated, and its internal organelles were abundant (c and f). Scale bars = 2 lm (74009), scale bars = 1 lm (24 5009).
(c)
(d)
effects on erectile function. All of these factors will increase sexual anxiety, decrease sexual activity and ultimately result in sexual disturbances or ED. Therefore, our results suggest that organic ED may not exist in patients with CP/CPPS. Although our results, which were based on a rat model, cannot be completely applied to humans, we believe that this study is still meaningful. In clinical practice, clinicians should not overuse pharmacotherapy, such as a-blockers, antibiotics and anti-inflammatories (Magri et al., 2011). Instead, the strong relationship between psychological factors and sexual dysfunction in men with CP/CPPS reinforces the need for a comprehensive approach to managing CP/CPPS. In addition to proper medical therapies, the interventions addressing psychological factors should be integrated in clinical trials as one possible way to improve sexual function and satisfaction in patients with CP/CPPS. Besides, more high-quality research is needed to understand the interplay of psychosocial factors in CP/CPPS. In the current study, no differences in serum concentrations of testosterone and oestradiol were detected between the EAP and normal groups. The ultrastructure of the corpus cavernosum was similar between the two groups; however, compared to control rats, there was a decrease in smooth muscle content, an increase in collagen and a © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 243–251
(b)
(e)
(f)
decreased SM/collagen ratio of the corpus cavernosum in EAP rats. Decreased expression of a-SMA and increased expression of TGF-b1 also suggested fibrogenesis in the corpus cavernosum of EAP rats. In most cases, penile fibrosis will lead to ED (Castiglione et al., 2013; Hannan et al., 2014); however, in the current study, the EAP rats had a fibrotic corpus cavernosum, but did not have ED. The possible reasons for this finding are as follows: the degree of corporal fibrosis was slight and not sufficient to result in ED; corporal fibrosis is related to a reduction in the cavernous sinuses, which will increase the intracavernosal pressure and the ICP/ MAP ratio may not decrease; and erectile function is a complex process involving biological and psychological factors, and other confounding factors may counteract the effect of prostatitis on erectile function. Further studies are warranted to fully understand the potential mechanisms of penile corporal fibrosis in EAP rats. In conclusion, erectile responses to cavernous nerve electrostimulation in EAP rats were not significantly decreased compared to control rats. Our results suggest that organic ED may not exist in patients with CP/CPPS. We speculate that CP/CPPS impairs the overall QoL and that a low QoL contributes to or causes ED. In clinical practice, clinicians should not overuse pharmacotherapy. 249
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Instead, psychological interventions should be considered to improve sexual function and satisfaction in males with CP/CPPS. In addition, further studies are needed to fully understand the potential mechanisms underlying the penile corporal fibrosis in EAP rats. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81270695), and the Key Project of Science and Technology Commission of Shanghai (SHD09411950400). We thank Dr Yan-Qin Hu (Shanghai Key Laboratory of Reproductive Medicine, Shanghai, China), Shanghai WEIAO BioTech Co., Ltd.; and Shanghai Showbio, Biotech Co., Ltd. for their expert technical assistance. We also thank International Science Editing, Ltd. for assisting in the preparation of our report. References Anderson RU, Orenberg EK, Morey A, Chavez N, Chan CA (2009) Stress induced hypothalamus-pituitary-adrenal axis responses and disturbances in psychological profiles in men with chronic prostatitis/chronic pelvic pain syndrome. J Urol 182:2319–2324. Aubin S, Berger RE, Heiman JR, Ciol MA (2008) The association between sexual function, pain, and psychological adaptation of men diagnosed with chronic pelvic pain syndrome type III. J Sex Med 5:657–667. Bayraktar Z, Atun I (2013) Impact of physician assistance on the reliability of the International Index of Erectile Function. Andrologia 45:73–77. Castiglione F, Hedlund P, Van der Aa F, Bivalacqua TJ, Rigatti P, Van Poppel H, Montorsi F, De Ridder D, Albersen M (2013) Intratunical injection of human adipose tissuederived stem cells prevents fibrosis and is associated with improved erectile function in a rat model of Peyronie’s disease. Eur Urol 63:551–560. Chung SD, Keller JJ, Lin HC (2012) A case-control study on the association between chronic prostatitis/chronic pelvic pain syndrome and erectile dysfunction. BJU Int 110:726– 730. Elhanbly S, Elkholy A (2012) Nocturnal penile erections: the role of RigiScan in the diagnosis of vascular erectile dysfunction. J Sex Med 9:3219–3226. Habermacher GM, Chason JT, Schaeffer AJ (2006) Prostatitis/ chronic pelvic pain syndrome. Annu Rev Med 57:195–206. Hannan JL, Kutlu O, Stopak BL, Liu X, Castiglione F, Hedlund P, Burnett AL, Bivalacqua TJ (2014) Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rats. J Sex Med 11:1442–1451. Hao ZY, Li HJ, Wang ZP, Xing JP, Hu WL, Zhang TF, Zhang XS, Zhou J, Tai S, Liang CZ (2011) The prevalence of
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