- Email: [email protected]
Behavioral and Urological Evaluation of a Testicular Pain Model
Interstitial Cystitis, Chronic Pelvic Pain, and Infection Behavioral and Urological Evaluation of a Testicular Pain Model Katsuro Yoshioka, Masayuki Tanahashi, and Wataru Uchida OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To develop an animal model of testicular pain to examine the hypothesis that neural crosstalk between testicular nociceptors and bladder reflex pathways may underlie bladder overactivity. In chronic pelvic pain disorders, neural crosstalk is thought to underlie referred pain and functional interaction in pelvic organs, and patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) suffer from pain in multiple organs, including the testes and perineum, as well as increased urinary frequency. In male Wistar rats, acetic acid was injected into the testes, and behaviors and bladder functions with conscious cystometry were examined. The effects of indomethacin and capsaicin pretreatment on both behaviors and bladder functional changes induced by acetic acid injection were examined. The weight of the testes and bladder after the testicular injection were measured. Injection of acetic acid (1% and 3%) induced pain behaviors and bladder overactivity proportional to the concentration. Indomethacin reduced, and capsaicin pretreatment almost completely abolished, both pain behavior and bladder overactivity induced by acetic acid injection. Administration of acetic acid increased testis weight and blanched the tissue, but no apparent changes were observed in the bladder. Injection of dilute acetic acid into the testes produces a reproducible testicular pain model involving testicular inflammation and activation of primary afferent C fibers and suggests a neural pathway for interaction between testicular pain and bladder overactivity. This study may provide a simple method to evaluate testicular pain, related bladder overactivity, and insight into the pathophysiology of bladder overactivity in patients with CP/CPPS. UROLOGY 75: 943–948, 2010. © 2010 Elsevier Inc.
C
hronic urogenital pain is a severe problem in urology. Chronic pelvic pain (CPP) disorders are present in multiple pelvic organs and may represent viscerovisceral referred pain. Interstitial cystitis, one of the CPP disorders, is characterized by suprapubic pain and increased urinary frequency and urgency.1 Irritable bowel syndrome is also encompassed in CPP disorders, and the patients suffer from unexplained lower abdominal pain, discomfort, and bloating in association with altered patterns of defecation, which ranges from diarrhea to constipation.2 Many patients present with overlapping symptoms of both interstitial cystitis and irritable bowel syndrome.3 Recent experiments suggest that this comorbidity is due to cross-organ sensitization (ie, crosstalk) between bladder and colon primary afferent fibers.4,5 For example, Pezzone et al demonstrated in rats
Financial support for this study was provided by Astellas Pharma Inc. From the Pharmacology Research Labs, Astellas Pharma Inc, Ibaraki, Japan; and Clinical Pharmacology, Development, Astellas Pharma Inc, Tokyo, Japan Reprint requests: Katsuro Yoshioka, 21, Miyukigaoka, Tsukuba-shi, Ibaraki 3058585, Japan. E-mail: [email protected] Submitted: June 7, 2009, accepted (with revisions): August 14, 2009
© 2010 Elsevier Inc. All Rights Reserved
that abdominal wall electromyography to colorectal distention (a measure of colonic nociception) increased after acute bladder irritation, whereas in contrast, urinary frequency (a measure of bladder nociception) increased as a result of colonic irritation. This cross-organ sensitization can be mediated by the convergence of separate populations of afferent fibers to second-order neurons or dichotomizing afferent fibers that individually innervate both colon and bladder.4 In CPP disorders, neural crosstalk may underlie pain and functional changes in multiple pelvic organs. Chronic prostatitis/chronic pelvic pain syndrome (CP/ CPPS), also one of the CPP disorders, is characterized by pain in multiple organs such as perineum, penis, and lower abdomen with discomfort during voiding or ejaculation. In particular, 40%-50% of patients with CP/CPPS are reported to have pain or discomfort in the testes.6,7 In addition, CP/CPPS is often associated with increased urinary frequency.8 It has been speculated that the symptoms arise from abnormal activation of afferent nerves in the pelvis, neurogenic inflammation of pelvic nerves, pelvic floor spasticity, pelvic organ ischemia, and/or au0090-4295/10/$34.00 doi:10.1016/j.urology.2009.08.043
943
toimmune reactions. However, the etiology remains uncertain, and there is no effective treatment. The present study developed an animal model of testicular pain to examine the hypothesis that neural crosstalk between testicular nociceptors and bladder reflex pathways may underlie bladder overactivity. An earlier model of CP/CPPS was developed in mice by injection of rat prostate gland extract into the mouse prostate, which evoked prostatic inflammation and referred pain to the lower abdominal area.9 However, to our knowledge, there have been no reports regarding animal models of testicular pain and ones about the crosstalk between testis and bladder. The testes can be stimulated by external triggers without surgery or anesthesia, which allows behavioral analysis to evaluate noxious responses. First, the present study examined whether the injection of dilute acetic acid into the testes of rats was able to induce pain behaviors. Second, we examined whether the injection of acetic acid into the testes induced bladder overactivity, suggesting existence of crosstalk between the testes and the bladder as we hypothesized earlier. We also examined the effects of an analgesic-, indomethacin-, and capsaicininduced C-fiber desensitization on the behaviors and bladder functional changes induced by acetic acid injection. Finally, we also measured the weight of the testis and bladder after the injection of acetic acid to assess inflammation.
MATERIAL AND METHODS Animals Male Wistar rats (Charles River, Japan, Kanagawa, Japan) were used. For behavioral tests, rats weighing approximately 100 g were used, and for cystometry and tissue weight measurements, rats weighing 180-340 g were used, respectively. All rats were fasted the evening before experiments. All animal experimental procedures were approved by the Committee for Animal Experiments of Astellas Pharma, Inc., and followed the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.
Testis Injection A 27-gauge needle and 1-mL syringe was used to administer 0.3%, 1%, or 3% aqueous solution of acetic acid or distilled water (DW) into the center of both the left and right testes (1 mL/kg each) of the rats that were under conscious, but restrained, conditions. In preliminary studies, a dye (Evans blue, Wako Pure Chemical Industries, Osaka, Japan) was injected into the testis along with acetic acid or DW. Successful testicular injections (as opposed to extratesticular scrotal injection) produced a characteristic swelling of the testis observed by palpation. Thus, in subsequent procedures, injection into the testis was confirmed by palpating the swollen testis. If both testes were not swollen, the rat was excluded from this study.
diately after the testicular injection. The following behavior was scored as a pain response: contraction of oblique musculature with inward flexion of the hind limb, stretching of the body, and flattening of the lower abdomen against the floor. In the time-course experiment, the number of pain responses during the period of 5-20 minutes, 35-50 minutes, and 65-80 minutes, after the injection of acetic acid (0.3%, 1%, 3%) or DW were counted (n ⫽ 5 each). In the evaluation of indomethacin and capsaicin pretreatment, only the number of responses from 5-20 minutes after 1% acetic acid injection was counted (vehicle: n ⫽ 9; indomethacin: n ⫽ 8; vehicle pretreatment: n ⫽ 5; capsaicin pretreatment: n ⫽ 5). Indomethacin (5 mg/kg) or its vehicle (0.1% Na2CO3) was orally administered 60 minutes before the injection.
Cystometry Rats were anesthetized with diethyl ether. The bladder was exposed through an abdominal midline incision, and a PE-50 catheter (Becton Dickinson & Company, Tokyo, Japan) was implanted into the bladder through the bladder dome, which was permanently sutured with silk thread. The catheter was tunneled subcutaneously and externalized at the back of the neck. Water was removed overnight to minimize urine production influencing bladder capacity measurement, and the next day conscious cystometry was performed by placing individual rats in Bollman cages (KN-326, Natsume, Tokyo, Japan). The bladder catheter was connected to a three-way connector, which led to an infusion pump (TE-331, Terumo, Tokyo, Japan) and a pressure transducer (DX-100, Becton Dickinson, & Company, Tokyo, Japan). Physiological saline was continuously infused into the bladder at a rate of 4 mL/h at room temperature while the intravesical pressure was continuously measured. Before testicular injection, cystometry was performed for 2 to 3 hours and after the voiding pattern stabilized, control values were recorded for an additional 1-1.5 hours. Immediately after the last void during the control period, the rat was removed from the cage. Acetic acid or DW was then injected into the testes. Immediately after the injection, each rat was placed back in the Bollman cage and cystometry was repeated. Bladder capacity (average volume of infusion between each micturition) was recorded during each 30-minute period after injection, and expressed as a percentage of the control capacity. In the timecourse experiment, cystometry was recorded for 3 hours after the injection of acetic acid or DW (n ⫽ 6-8). In the evaluation of indomethacin and capsaicin-pretreatment, cystometry was recorded for 2 hours after the injection of 1% acetic acid (vehicle: n ⫽ 7; indomethacin: n ⫽ 8; vehicle-pretreatment: n ⫽ 5; capsaicin-pretreatment: n ⫽ 6). Indomethacin (5 mg/kg p.o.) or its vehicle was administered 15 minutes before the testicular injection.
Measurement of Tissue Weight For the measurement of testis weight, rats were killed by exsanguination 0.5, 1, or 4 hours after the testicular injection of 1% acetic acid or DW (n ⫽ 3-4). After laparotomy, testes were harvested, weighed, visually examined, and palpated. Bladders were also harvested, weighed, and visually inspected 0.5 and 1 hours after the testicular injection of 1% acetic acid or DW.
Behavioral Test
Capsaicin Pretreatment
Rats were placed individually in a clear, round, plastic container (diameter 24.5 cm, height 30 cm) for observation imme-
To eliminate primary afferent C-fiber function, rats were pretreated with capsaicin. Capsaicin (125 mg/kg) or its vehicle
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UROLOGY 75 (4), 2010
(10% ethanol, 10% Tween 80, and 80% saline) was subcutaneously administered over a 2-day period 4-7 days before the experiments. Rats received 25 mg/kg of capsaicin on the first day, and 50 mg/kg twice on the second day with an interval of 10 hours. Desensitization of C fibers was confirmed by an eye wipe test before the experiments.10 Briefly, a drop of 20 g/mL capsaicin solution was instilled into the eye and rats that showed defensive wiping movements were excluded.
Compounds Acetic acid was purchased from Kanto Chemical (Tokyo, Japan) and diluted with DW. Indomethacin was purchased from Sigma Aldrich, Japan (Tokyo, Japan) and dissolved in 0.1% Na2CO3 solution (5 mg/3 mL). Capsaicin was purchased from Sigma Aldrich, Japan (Tokyo, Japan) and dissolved in 10% ethanol, 10% Tween 80%, and 80% saline.
Data Analysis Results are presented as means ⫾ standard error of means. Data were statistically analyzed using an SAS software (version 8.2, SAS Institute, Tokyo, Japan). The differences between 2 groups were analyzed with Student t test except for the differences in bladder capacity in indomethacin- and capsaicin-treated groups from their vehicle groups, which were analyzed with two-way analysis of variance. The differences in bladder capacity in acetic acid (0.3%, 1%, 3%) groups from DW group for each time point were analyzed with one-way analysis of variance or Dunnett multiple comparison test. P ⱕ.05 was considered significant.
Figure 1. Number of pain behaviors (mean ⫾ standard error of mean, n ⫽ 5) at various time periods (15 minutes each) after injection of 1% or 3% acetic acid.
RESULTS Testicular Acetic Acid Injection-induced Pain Behaviors Rats injected with 1% or 3% acetic acid showed characteristic behaviors such as contraction of oblique musculature with inward flexion of the hind limbs, stretching of the body, and flattening of the lower abdomen against the floor. These pain behaviors appeared from 5 minutes and gradually diminished to near zero within 80 minutes, and were proportional to the concentration of acetic acid (Fig. 1). Injections of DW and 0.3% acetic acid produced no responses (data not shown). Testicular Acetic Acid Injection-induced Decreases in Bladder Capacity Rats injected with 1% or 3% acetic acid showed significant decreases in bladder capacity immediately after the injection and gradually recovered across the next 1.5 hour. The decrease in bladder capacity was proportional to the acetic acid concentration at 1% and 3%, but rats injected with DW or 0.3% acetic acid did not show significant changes in bladder capacity (Fig. 2). Control bladder capacities were not significantly different among the 4 groups (DW: 1.3 ⫾ 0.2, 0.3%: 1.5 ⫾ 0.2, 1%: 1.5 ⫾ 0.2, 3%: 1.5 ⫾ 0.1 mL, P ⫽ .77), and maximum micturition pressure did not change significantly after DW or acetic acid injection (data not shown). Figure 2B shows the typical charts of intravesical pressure before and after the injection of DW (top trace) or 1% acetic acid (bottom trace). UROLOGY 75 (4), 2010
Figure 2. Time course of the bladder capacity changes after injection of DW or acetic acid (0.3%, 1%, 3%) into the testes. (A) Changes in bladder capacity in animals injected with DW and 0.3%, 1%, and 3% acetic acid (n ⫽ 6-8). Vertical lines indicate the standard error of mean. * P ⬍.05, ** P ⬍.01 vs DW group at the same time point by Dunnett multiple comparison test. (B) Physiograph tracings of bladder pressure during continuous infusion cystometry showing an example of the increase in bladder contraction frequency (because of a decrease in the bladder capacity) resulting from injection (at asterisk) of DW (top trace) or 1% acetic acid (bottom trace).
Indomethacin and Capsaicin Pretreatment Reduced Effects of Acetic Acid Injection Into Testes Indomethacin (5 mg/kg p.o.) significantly decreased the pain behaviors induced by 1% acetic acid injection (Fig. 945
Figure 3. Data are expressed as the mean ⫾ standard error of mean. (A) Number of pain responses from 5 to 20 minutes after 1% acetic acid injection into the testes of the vehicle-treated (n ⫽ 9) or indomethacin-treated (n ⫽ 8) animals. (B) Bladder capacity after testicular 1% acetic acid injection in vehicle-treated (n ⫽ 7) or indomethacin-treated (n ⫽ 8) animals across time. (C) Number of pain responses in vehicle-pretreated (n ⫽ 5) and capsaicin-pretreated (n ⫽ 5) groups (Capsaicin-pretreated group had zero responses). (D) Bladder capacity of vehicle-pretreated (n ⫽ 5) and capsaicinpretreated (n ⫽ 6) animals across time. (A,C) * P ⬍.05, ** P ⬍.01 vs vehicle group by Student t test. (B,D) * P ⬍.05 vs vehicle group as assessed by two-way analysis of variance.
3A). Indomethacin also significantly reduced the decrease in bladder capacity induced by 1% acetic acid injection (Fig. 3B). There was no significant difference between control values of bladder capacity in either group (vehicle: 1.3 ⫾ 0.1, indomethacin: 1.4 ⫾ 0.1 mL, P ⫽ .37). Capsaicin pretreatment significantly and completely inhibited the pain behaviors induced by 1% acetic acid (Fig. 3C). Capsaicin pretreatment also significantly and almost completely inhibited the decrease in bladder capacity induced by 1% acetic acid (Fig. 3D). There was no significant difference between control values of bladder capacity in either group (vehicle: 1.4 ⫾ 0.3, capsaicin: 1.2 ⫾ 0.1 mL, P ⫽ .37).
Acetic Acid Injection Into Testes Increased Testis Weight At every time point (0.5, 1, 4 hours), testes injected with 1% acetic acid showed evidence of tissue damage that was apparent by whitening and hardening of the tissue compared with testes injected with DW. In addition, the weight of the testis were significantly increased in the acetic acid group at 1 and 4 hours (Fig. 4), whereas the body weights of each group were nearly the same (DW: 0.5 hour 196 ⫾ 6 g, 1 hour 191 ⫾ 3 g, 4 hours 195 ⫾ 3 g; 1% acetic acid: 0.5 hour 195 ⫾ 5 g, 1 hour 946
Figure 4. Mean testicular weight (⫾ standard error of mean) at various time points after injections of DW or 1% acetic acid into the testes (n ⫽ 3-4). * P ⬍.05 vs DW group at the same time point by Student t test.
190 ⫾ 4 g, 4 hours 190 ⫾ 4 g). In contrast to the testes, the visual appearance and the tissue weight of the bladder remained unchanged by acetic acid injection into the testes (DW: 0.5 hour 64.5 ⫾ 3.3 mg, 1 hour 73.0 ⫾ 6.6 mg; 1% acetic acid: 0.5 hour 68.0 ⫾ 4.7 mg, 1 hour 65.2 ⫾ 7.5 mg). UROLOGY 75 (4), 2010
COMMENT In the present study, injection of acetic acid (1% and 3%) into the testes of conscious rats produced characteristic pain behaviors such as contraction of oblique musculature with inward flexion of the hind limb, stretching of the body, and flattening of the lower abdomen against the floor. Similar behaviors have been reported in other pain models such as the artificial ureteral calculosis model, the visceral pain model induced by intracolonic administration of mustard oil or capsaicin, and writhing tests.11-13 The behaviors induced by 1% acetic acid were reduced significantly by the analgesic, indomethacin, which indicates that the behaviors may reflect inflammatory pain and prostaglandins may contribute to the behaviors. In addition, the behaviors were completely eliminated by capsaicin pretreatment, indicating that these behaviors were mediated by primary afferent C fibers, which have been previously described in the testes.14,15 The fact that the testes had obvious tissue damage and increased weight after 1% acetic acid injection suggests that inflammation occurred during the observation period. Taken together, it appears that acetic acid injection into testes produces inflammation with an increase in prostaglandin synthesis and activation of capsaicin-sensitive afferent C fibers, which leads to the pain behaviors. Writhing tests with intraperitoneal injection of acetic acid in rodents have been widely used to evaluate analgesics for visceral pain, and our model is similar to the writhing tests in terms of acetic acid stimulation and inducing similar spontaneous pain behaviors at some level. Indomethacin (5 mg/kg p.o.) and capsaicin pretreatment, which was effective in the present study, was also shown to inhibit writhing behaviors induced by intraperitoneal acetic acid.13,16 However, an important difference between these models is that intraperitoneal injection of acetic acid produces global visceral pain, whereas the intratesticular injection of acetic acid is localized to the testes, as evidenced by the injection of dye, and the fact that intraperitoneal injection of 1% acetic acid at the same volume as our model (total 2 mL/kg) did not evoke pain behaviors in a preliminary study (data not shown) also supports the pain behaviors in our model as those reflecting testicular pain. Injection of acetic acid (1% and 3%) into the testes decreased bladder capacity without direct stimulation of the bladder under conscious conditions. Measurement of bladder capacity is commonly used for measuring bladder sensitivity, and a reduction in bladder capacity is considered an indicator of the clinical condition of bladder overactivity. A reduction in bladder capacity without any effects on maximum micturition pressure suggests an effect on the afferent limb of the bladder reflex rather than the efferent limb. This suggestion is supported by the fact that capsaicin pretreatment nearly abolished the bladder overactivity and further suggests that testicular primary afferent C fibers mediate the bladder overactivity. The fact that neither bladder weight nor visual appearance of UROLOGY 75 (4), 2010
the bladder was changed also supports modification of the afferent or sensory limb of the bladder reflex resulting from testicular pain. The modification of bladder activity by testicular pain suggests that neural crosstalk may exist between the testes and the bladder. Qin and Foreman17 showed that about a third of L6-S2 spinal neurons of rats received convergent inputs of afferent nerves from both bladder and colon. Ustinova et al18 implicated afferent C fibers in mediating crosstalk between colon and bladder as capsaicin pretreatment abolished bladder overactivity after trinitrobenzene sulfonic acid-induced colitis in rats. Consequently, the crosstalk between colon and bladder can be explained at least partially by the convergence of C-fiber afferent nerves in the spinal cord. Contribution of afferent C-fibers to neural crosstalk is also supported by the report that colonic inflammation by dextran sulfate sodium enhanced the response of bladder sensory neurons to capsaicin by 60%.19 Neural crosstalk has also been reported between prostate and bladder in a study in which Vera and MeyerSiegler20 reported that intraprostatic formalin injection produced immediate bladder overactivity and increased inflammatory mediators in L6-S1 spinal cord and bladder. Prostatic and bladder crosstalk were also reported by Ishigooka et al21 who showed that injections of chemical irritants into the bladder or prostate produced convergent expression of c-Fos within second-order interneurons in the L6-S1 spinal cord. Scrotal afferent nerves of rats have been reported to enter the spinal cord through L5-S1 dorsal roots, and cutaneous plasma extravasation in the groin results from application of capsaicin to the L5-L6 intervertebral disk, indicating overlaps between bladder and groin dermatomes at the level of second-order neurons.22,23 The present study suggests that there is also convergence of testicular C fibers and bladder afferent fibers onto second-order interneurons in the lumbosacral spinal cord. This convergence is further supported by the parallel reduction in both pain behavior and bladder overactivity by indomethacin and capsaicin. However, there is a slight discrepancy between the duration of the pain behavior and bladder overactivity after testicular injection with the pain behavior disappearing at 1 hour, whereas bladder overactivity remained for at least 3 hours. This may be due to differences in endogenous control of pain perception vs visceral nociception or possibly secondary inflammatory changes in the bladder. Patients with CP/CPPS are reported to have higher sensitivity to capsaicin and heat stimuli in perineal area compared with healthy volunteers, and activation of afferent C fibers has been thought to underlie the pain in the patients.24,25 These points provide some clinical correlation for our testicular pain model. Although our model had only acute responses with testicular damage or inflammation, which may be different from the clinical situation of patients, the present study suggested that 947
nociceptive signals from testes may contribute to a part of bladder overactivity in patients with CP/CPPS.
CONCLUSIONS This study provides evidence that dilute acetic acid injection into the testes produces inflammation, prostaglandin synthesis, and activation of afferent C fibers, which lead to both pain behavior and bladder overactivity. This study suggests neural crosstalk between the testes and the bladder. This may provide a simple reproducible model to test novel therapies for CP/CPPS symptoms as well understanding the pathophysiological processes underlying bladder overactivity in CP/CPPS patients. Acknowledgments. The authors thank Dr. Karl B. Thor for the review of the manuscript and his valuable advices. Authors also thank Drs. Matthew O. Fraser, Velu Karicheti, and Ed. C. Burgard for valuable discussions. References 1. Teichman JM, Parsons CL. Contemporary clinical presentation of interstitial cystitis. Urology. 2007;69:S41-S47. 2. Tillisch K, Labus JS, Naliboff BD, et al. Characterization of the alternating bowel habit subtype in patients with irritable bowel syndrome. Am J Gastroenterol. 2005;100:896-904. 3. Alagiri M, Chottiner S, Ratner V, et al. Interstitial cystitis: unexplained associations with other chronic disease and pain syndromes. Urology. 1997;49:52-57. 4. Christianson JA, Liang R, Ustinova EE, et al. Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain. 2007;128:235-243. 5. Pezzone MA, Liang R, Fraser MO. A model of neural cross-talk and irritation in the pelvis: implications for the overlap of chronic pelvic pain disorders. Gastroenterology. 2005;128:1953-1964. 6. Litwin MS, McNaughton-Collins M, Fowler FJ Jr, et al. The National Institutes of Health chronic prostatitis symptom index: development and validation of a new outcome measure. Chronic Prostatitis Collaborative Research Network. J Urol. 1999;162:369375. 7. Schaeffer AJ, Landis JR, Knauss JS, et al. Demographic and clinical characteristics of men with chronic prostatitis: the national institutes of health chronic prostatitis cohort study. J Urol. 2002;168: 593-598. 8. Krieger JN, Egan KJ, Ross SO, et al. Chronic pelvic pains represent the most prominent urogenital symptoms of “chronic prostatitis”. Urology. 1996;48:715-721; discussion: 721-722. 9. Rudick CN, Schaeffer AJ, Thumbikat P. Experimental autoimmune prostatitis induces chronic pelvic pain. Am J Physiol Regul Integr Comp Physiol. 2008;294:R1268-R1275.
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10. Cheng CL, Ma CP, de Groat WC. Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res. 1995; 678:40-48. 11. Giamberardino MA, Valente R, de Bigontina P, et al. Artificial ureteral calculosis in rats: behavioural characterization of visceral pain episodes and their relationship with referred lumbar muscle hyperalgesia. Pain. 1995;61:459-469. 12. Laird JM, Martinez-Caro L, Garcia-Nicas E, et al. A new model of visceral pain and referred hyperalgesia in the mouse. Pain. 2001; 92:335-342. 13. Ikeda Y, Ueno A, Naraba H, et al. Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci. 2001;69:2911-2919. 14. Silverman JD, Kruger L. Lectin and neuropeptide labeling of separate populations of dorsal root ganglion neurons and associated “nociceptor” thin axons in rat testis and cornea whole-mount preparations. Somatosens Res. 1988;5:259-267. 15. Sarioglu-Buke A, Erdem S, Gedikoglu G, et al. Capsaicin effectively prevents apoptosis in the contralateral testis after ipsilateral testicular torsion. BJU Int. 2001;88:787-789. 16. Niemegeers CJ, Van Bruggen JA, Janssen PA. Suprofen, a potent antagonist of acetic acid-induced writhing in rats. Arzneimittelforschung. 1975;25:1505-1509. 17. Qin C, Foreman RD. Viscerovisceral convergence of urinary bladder and colorectal inputs to lumbosacral spinal neurons in rats. Neuroreport. 2004;15:467-471. 18. Ustinova EE, Gutkin DW, Pezzone MA. Sensitization of pelvic nerve afferents and mast cell infiltration in the urinary bladder following chronic colonic irritation is mediated by neuropeptides. Am J Physiol Renal Physiol. 2007;292:F123-F130. 19. Malykhina AP, Qin C, Foreman RD, et al. Colonic inflammation increases Na⫹ currents in bladder sensory neurons. Neuroreport. 2004;15:2601-2605. 20. Vera PL, Meyer-Siegler KL. Inflammation of the rat prostate evokes release of macrophage migration inhibitory factor in the bladder: evidence for a viscerovisceral reflex. J Urol. 2004;172:2440-2445. 21. Ishigooka M, Zermann DH, Doggweiler R, et al. Similarity of distributions of spinal c-Fos and plasma extravasation after acute chemical irritation of the bladder and the prostate. J Urol. 2000; 164:1751-1756. 22. Manzo J, Garcia LI, Camacho MA, et al. Influence of testosterone on the electrical properties of scrotal nerves at the cutaneous and spinal levels in the male rat. J Peripher Nerv Syst. 2003;8:75-81. 23. Takahashi Y, Morinaga T, Nakamura S, et al. Neural connection between the ventral portion of the lumbar intervertebral disc and the groin skin. J Neurosurg. 1996;85:323-328. 24. Lee JC, Yang CC, Kromm BG, et al. Neurophysiologic testing in chronic pelvic pain syndrome: a pilot study. Urology. 2001;58:246250. 25. Turini D, Beneforti P, Spinelli M, et al. Heat/burning sensation induced by topical application of capsaicin on perineal cutaneous area: new approach in diagnosis and treatment of chronic prostatitis/chronic pelvic pain syndrome? Urology. 2006;67:910-913.
UROLOGY 75 (4), 2010
METHODS
RESULTS
CONCLUSIONS
To develop an animal model of testicular pain to examine the hypothesis that neural crosstalk between testicular nociceptors and bladder reflex pathways may underlie bladder overactivity. In chronic pelvic pain disorders, neural crosstalk is thought to underlie referred pain and functional interaction in pelvic organs, and patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) suffer from pain in multiple organs, including the testes and perineum, as well as increased urinary frequency. In male Wistar rats, acetic acid was injected into the testes, and behaviors and bladder functions with conscious cystometry were examined. The effects of indomethacin and capsaicin pretreatment on both behaviors and bladder functional changes induced by acetic acid injection were examined. The weight of the testes and bladder after the testicular injection were measured. Injection of acetic acid (1% and 3%) induced pain behaviors and bladder overactivity proportional to the concentration. Indomethacin reduced, and capsaicin pretreatment almost completely abolished, both pain behavior and bladder overactivity induced by acetic acid injection. Administration of acetic acid increased testis weight and blanched the tissue, but no apparent changes were observed in the bladder. Injection of dilute acetic acid into the testes produces a reproducible testicular pain model involving testicular inflammation and activation of primary afferent C fibers and suggests a neural pathway for interaction between testicular pain and bladder overactivity. This study may provide a simple method to evaluate testicular pain, related bladder overactivity, and insight into the pathophysiology of bladder overactivity in patients with CP/CPPS. UROLOGY 75: 943–948, 2010. © 2010 Elsevier Inc.
C
hronic urogenital pain is a severe problem in urology. Chronic pelvic pain (CPP) disorders are present in multiple pelvic organs and may represent viscerovisceral referred pain. Interstitial cystitis, one of the CPP disorders, is characterized by suprapubic pain and increased urinary frequency and urgency.1 Irritable bowel syndrome is also encompassed in CPP disorders, and the patients suffer from unexplained lower abdominal pain, discomfort, and bloating in association with altered patterns of defecation, which ranges from diarrhea to constipation.2 Many patients present with overlapping symptoms of both interstitial cystitis and irritable bowel syndrome.3 Recent experiments suggest that this comorbidity is due to cross-organ sensitization (ie, crosstalk) between bladder and colon primary afferent fibers.4,5 For example, Pezzone et al demonstrated in rats
Financial support for this study was provided by Astellas Pharma Inc. From the Pharmacology Research Labs, Astellas Pharma Inc, Ibaraki, Japan; and Clinical Pharmacology, Development, Astellas Pharma Inc, Tokyo, Japan Reprint requests: Katsuro Yoshioka, 21, Miyukigaoka, Tsukuba-shi, Ibaraki 3058585, Japan. E-mail: [email protected] Submitted: June 7, 2009, accepted (with revisions): August 14, 2009
© 2010 Elsevier Inc. All Rights Reserved
that abdominal wall electromyography to colorectal distention (a measure of colonic nociception) increased after acute bladder irritation, whereas in contrast, urinary frequency (a measure of bladder nociception) increased as a result of colonic irritation. This cross-organ sensitization can be mediated by the convergence of separate populations of afferent fibers to second-order neurons or dichotomizing afferent fibers that individually innervate both colon and bladder.4 In CPP disorders, neural crosstalk may underlie pain and functional changes in multiple pelvic organs. Chronic prostatitis/chronic pelvic pain syndrome (CP/ CPPS), also one of the CPP disorders, is characterized by pain in multiple organs such as perineum, penis, and lower abdomen with discomfort during voiding or ejaculation. In particular, 40%-50% of patients with CP/CPPS are reported to have pain or discomfort in the testes.6,7 In addition, CP/CPPS is often associated with increased urinary frequency.8 It has been speculated that the symptoms arise from abnormal activation of afferent nerves in the pelvis, neurogenic inflammation of pelvic nerves, pelvic floor spasticity, pelvic organ ischemia, and/or au0090-4295/10/$34.00 doi:10.1016/j.urology.2009.08.043
943
toimmune reactions. However, the etiology remains uncertain, and there is no effective treatment. The present study developed an animal model of testicular pain to examine the hypothesis that neural crosstalk between testicular nociceptors and bladder reflex pathways may underlie bladder overactivity. An earlier model of CP/CPPS was developed in mice by injection of rat prostate gland extract into the mouse prostate, which evoked prostatic inflammation and referred pain to the lower abdominal area.9 However, to our knowledge, there have been no reports regarding animal models of testicular pain and ones about the crosstalk between testis and bladder. The testes can be stimulated by external triggers without surgery or anesthesia, which allows behavioral analysis to evaluate noxious responses. First, the present study examined whether the injection of dilute acetic acid into the testes of rats was able to induce pain behaviors. Second, we examined whether the injection of acetic acid into the testes induced bladder overactivity, suggesting existence of crosstalk between the testes and the bladder as we hypothesized earlier. We also examined the effects of an analgesic-, indomethacin-, and capsaicininduced C-fiber desensitization on the behaviors and bladder functional changes induced by acetic acid injection. Finally, we also measured the weight of the testis and bladder after the injection of acetic acid to assess inflammation.
MATERIAL AND METHODS Animals Male Wistar rats (Charles River, Japan, Kanagawa, Japan) were used. For behavioral tests, rats weighing approximately 100 g were used, and for cystometry and tissue weight measurements, rats weighing 180-340 g were used, respectively. All rats were fasted the evening before experiments. All animal experimental procedures were approved by the Committee for Animal Experiments of Astellas Pharma, Inc., and followed the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.
Testis Injection A 27-gauge needle and 1-mL syringe was used to administer 0.3%, 1%, or 3% aqueous solution of acetic acid or distilled water (DW) into the center of both the left and right testes (1 mL/kg each) of the rats that were under conscious, but restrained, conditions. In preliminary studies, a dye (Evans blue, Wako Pure Chemical Industries, Osaka, Japan) was injected into the testis along with acetic acid or DW. Successful testicular injections (as opposed to extratesticular scrotal injection) produced a characteristic swelling of the testis observed by palpation. Thus, in subsequent procedures, injection into the testis was confirmed by palpating the swollen testis. If both testes were not swollen, the rat was excluded from this study.
diately after the testicular injection. The following behavior was scored as a pain response: contraction of oblique musculature with inward flexion of the hind limb, stretching of the body, and flattening of the lower abdomen against the floor. In the time-course experiment, the number of pain responses during the period of 5-20 minutes, 35-50 minutes, and 65-80 minutes, after the injection of acetic acid (0.3%, 1%, 3%) or DW were counted (n ⫽ 5 each). In the evaluation of indomethacin and capsaicin pretreatment, only the number of responses from 5-20 minutes after 1% acetic acid injection was counted (vehicle: n ⫽ 9; indomethacin: n ⫽ 8; vehicle pretreatment: n ⫽ 5; capsaicin pretreatment: n ⫽ 5). Indomethacin (5 mg/kg) or its vehicle (0.1% Na2CO3) was orally administered 60 minutes before the injection.
Cystometry Rats were anesthetized with diethyl ether. The bladder was exposed through an abdominal midline incision, and a PE-50 catheter (Becton Dickinson & Company, Tokyo, Japan) was implanted into the bladder through the bladder dome, which was permanently sutured with silk thread. The catheter was tunneled subcutaneously and externalized at the back of the neck. Water was removed overnight to minimize urine production influencing bladder capacity measurement, and the next day conscious cystometry was performed by placing individual rats in Bollman cages (KN-326, Natsume, Tokyo, Japan). The bladder catheter was connected to a three-way connector, which led to an infusion pump (TE-331, Terumo, Tokyo, Japan) and a pressure transducer (DX-100, Becton Dickinson, & Company, Tokyo, Japan). Physiological saline was continuously infused into the bladder at a rate of 4 mL/h at room temperature while the intravesical pressure was continuously measured. Before testicular injection, cystometry was performed for 2 to 3 hours and after the voiding pattern stabilized, control values were recorded for an additional 1-1.5 hours. Immediately after the last void during the control period, the rat was removed from the cage. Acetic acid or DW was then injected into the testes. Immediately after the injection, each rat was placed back in the Bollman cage and cystometry was repeated. Bladder capacity (average volume of infusion between each micturition) was recorded during each 30-minute period after injection, and expressed as a percentage of the control capacity. In the timecourse experiment, cystometry was recorded for 3 hours after the injection of acetic acid or DW (n ⫽ 6-8). In the evaluation of indomethacin and capsaicin-pretreatment, cystometry was recorded for 2 hours after the injection of 1% acetic acid (vehicle: n ⫽ 7; indomethacin: n ⫽ 8; vehicle-pretreatment: n ⫽ 5; capsaicin-pretreatment: n ⫽ 6). Indomethacin (5 mg/kg p.o.) or its vehicle was administered 15 minutes before the testicular injection.
Measurement of Tissue Weight For the measurement of testis weight, rats were killed by exsanguination 0.5, 1, or 4 hours after the testicular injection of 1% acetic acid or DW (n ⫽ 3-4). After laparotomy, testes were harvested, weighed, visually examined, and palpated. Bladders were also harvested, weighed, and visually inspected 0.5 and 1 hours after the testicular injection of 1% acetic acid or DW.
Behavioral Test
Capsaicin Pretreatment
Rats were placed individually in a clear, round, plastic container (diameter 24.5 cm, height 30 cm) for observation imme-
To eliminate primary afferent C-fiber function, rats were pretreated with capsaicin. Capsaicin (125 mg/kg) or its vehicle
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(10% ethanol, 10% Tween 80, and 80% saline) was subcutaneously administered over a 2-day period 4-7 days before the experiments. Rats received 25 mg/kg of capsaicin on the first day, and 50 mg/kg twice on the second day with an interval of 10 hours. Desensitization of C fibers was confirmed by an eye wipe test before the experiments.10 Briefly, a drop of 20 g/mL capsaicin solution was instilled into the eye and rats that showed defensive wiping movements were excluded.
Compounds Acetic acid was purchased from Kanto Chemical (Tokyo, Japan) and diluted with DW. Indomethacin was purchased from Sigma Aldrich, Japan (Tokyo, Japan) and dissolved in 0.1% Na2CO3 solution (5 mg/3 mL). Capsaicin was purchased from Sigma Aldrich, Japan (Tokyo, Japan) and dissolved in 10% ethanol, 10% Tween 80%, and 80% saline.
Data Analysis Results are presented as means ⫾ standard error of means. Data were statistically analyzed using an SAS software (version 8.2, SAS Institute, Tokyo, Japan). The differences between 2 groups were analyzed with Student t test except for the differences in bladder capacity in indomethacin- and capsaicin-treated groups from their vehicle groups, which were analyzed with two-way analysis of variance. The differences in bladder capacity in acetic acid (0.3%, 1%, 3%) groups from DW group for each time point were analyzed with one-way analysis of variance or Dunnett multiple comparison test. P ⱕ.05 was considered significant.
Figure 1. Number of pain behaviors (mean ⫾ standard error of mean, n ⫽ 5) at various time periods (15 minutes each) after injection of 1% or 3% acetic acid.
RESULTS Testicular Acetic Acid Injection-induced Pain Behaviors Rats injected with 1% or 3% acetic acid showed characteristic behaviors such as contraction of oblique musculature with inward flexion of the hind limbs, stretching of the body, and flattening of the lower abdomen against the floor. These pain behaviors appeared from 5 minutes and gradually diminished to near zero within 80 minutes, and were proportional to the concentration of acetic acid (Fig. 1). Injections of DW and 0.3% acetic acid produced no responses (data not shown). Testicular Acetic Acid Injection-induced Decreases in Bladder Capacity Rats injected with 1% or 3% acetic acid showed significant decreases in bladder capacity immediately after the injection and gradually recovered across the next 1.5 hour. The decrease in bladder capacity was proportional to the acetic acid concentration at 1% and 3%, but rats injected with DW or 0.3% acetic acid did not show significant changes in bladder capacity (Fig. 2). Control bladder capacities were not significantly different among the 4 groups (DW: 1.3 ⫾ 0.2, 0.3%: 1.5 ⫾ 0.2, 1%: 1.5 ⫾ 0.2, 3%: 1.5 ⫾ 0.1 mL, P ⫽ .77), and maximum micturition pressure did not change significantly after DW or acetic acid injection (data not shown). Figure 2B shows the typical charts of intravesical pressure before and after the injection of DW (top trace) or 1% acetic acid (bottom trace). UROLOGY 75 (4), 2010
Figure 2. Time course of the bladder capacity changes after injection of DW or acetic acid (0.3%, 1%, 3%) into the testes. (A) Changes in bladder capacity in animals injected with DW and 0.3%, 1%, and 3% acetic acid (n ⫽ 6-8). Vertical lines indicate the standard error of mean. * P ⬍.05, ** P ⬍.01 vs DW group at the same time point by Dunnett multiple comparison test. (B) Physiograph tracings of bladder pressure during continuous infusion cystometry showing an example of the increase in bladder contraction frequency (because of a decrease in the bladder capacity) resulting from injection (at asterisk) of DW (top trace) or 1% acetic acid (bottom trace).
Indomethacin and Capsaicin Pretreatment Reduced Effects of Acetic Acid Injection Into Testes Indomethacin (5 mg/kg p.o.) significantly decreased the pain behaviors induced by 1% acetic acid injection (Fig. 945
Figure 3. Data are expressed as the mean ⫾ standard error of mean. (A) Number of pain responses from 5 to 20 minutes after 1% acetic acid injection into the testes of the vehicle-treated (n ⫽ 9) or indomethacin-treated (n ⫽ 8) animals. (B) Bladder capacity after testicular 1% acetic acid injection in vehicle-treated (n ⫽ 7) or indomethacin-treated (n ⫽ 8) animals across time. (C) Number of pain responses in vehicle-pretreated (n ⫽ 5) and capsaicin-pretreated (n ⫽ 5) groups (Capsaicin-pretreated group had zero responses). (D) Bladder capacity of vehicle-pretreated (n ⫽ 5) and capsaicinpretreated (n ⫽ 6) animals across time. (A,C) * P ⬍.05, ** P ⬍.01 vs vehicle group by Student t test. (B,D) * P ⬍.05 vs vehicle group as assessed by two-way analysis of variance.
3A). Indomethacin also significantly reduced the decrease in bladder capacity induced by 1% acetic acid injection (Fig. 3B). There was no significant difference between control values of bladder capacity in either group (vehicle: 1.3 ⫾ 0.1, indomethacin: 1.4 ⫾ 0.1 mL, P ⫽ .37). Capsaicin pretreatment significantly and completely inhibited the pain behaviors induced by 1% acetic acid (Fig. 3C). Capsaicin pretreatment also significantly and almost completely inhibited the decrease in bladder capacity induced by 1% acetic acid (Fig. 3D). There was no significant difference between control values of bladder capacity in either group (vehicle: 1.4 ⫾ 0.3, capsaicin: 1.2 ⫾ 0.1 mL, P ⫽ .37).
Acetic Acid Injection Into Testes Increased Testis Weight At every time point (0.5, 1, 4 hours), testes injected with 1% acetic acid showed evidence of tissue damage that was apparent by whitening and hardening of the tissue compared with testes injected with DW. In addition, the weight of the testis were significantly increased in the acetic acid group at 1 and 4 hours (Fig. 4), whereas the body weights of each group were nearly the same (DW: 0.5 hour 196 ⫾ 6 g, 1 hour 191 ⫾ 3 g, 4 hours 195 ⫾ 3 g; 1% acetic acid: 0.5 hour 195 ⫾ 5 g, 1 hour 946
Figure 4. Mean testicular weight (⫾ standard error of mean) at various time points after injections of DW or 1% acetic acid into the testes (n ⫽ 3-4). * P ⬍.05 vs DW group at the same time point by Student t test.
190 ⫾ 4 g, 4 hours 190 ⫾ 4 g). In contrast to the testes, the visual appearance and the tissue weight of the bladder remained unchanged by acetic acid injection into the testes (DW: 0.5 hour 64.5 ⫾ 3.3 mg, 1 hour 73.0 ⫾ 6.6 mg; 1% acetic acid: 0.5 hour 68.0 ⫾ 4.7 mg, 1 hour 65.2 ⫾ 7.5 mg). UROLOGY 75 (4), 2010
COMMENT In the present study, injection of acetic acid (1% and 3%) into the testes of conscious rats produced characteristic pain behaviors such as contraction of oblique musculature with inward flexion of the hind limb, stretching of the body, and flattening of the lower abdomen against the floor. Similar behaviors have been reported in other pain models such as the artificial ureteral calculosis model, the visceral pain model induced by intracolonic administration of mustard oil or capsaicin, and writhing tests.11-13 The behaviors induced by 1% acetic acid were reduced significantly by the analgesic, indomethacin, which indicates that the behaviors may reflect inflammatory pain and prostaglandins may contribute to the behaviors. In addition, the behaviors were completely eliminated by capsaicin pretreatment, indicating that these behaviors were mediated by primary afferent C fibers, which have been previously described in the testes.14,15 The fact that the testes had obvious tissue damage and increased weight after 1% acetic acid injection suggests that inflammation occurred during the observation period. Taken together, it appears that acetic acid injection into testes produces inflammation with an increase in prostaglandin synthesis and activation of capsaicin-sensitive afferent C fibers, which leads to the pain behaviors. Writhing tests with intraperitoneal injection of acetic acid in rodents have been widely used to evaluate analgesics for visceral pain, and our model is similar to the writhing tests in terms of acetic acid stimulation and inducing similar spontaneous pain behaviors at some level. Indomethacin (5 mg/kg p.o.) and capsaicin pretreatment, which was effective in the present study, was also shown to inhibit writhing behaviors induced by intraperitoneal acetic acid.13,16 However, an important difference between these models is that intraperitoneal injection of acetic acid produces global visceral pain, whereas the intratesticular injection of acetic acid is localized to the testes, as evidenced by the injection of dye, and the fact that intraperitoneal injection of 1% acetic acid at the same volume as our model (total 2 mL/kg) did not evoke pain behaviors in a preliminary study (data not shown) also supports the pain behaviors in our model as those reflecting testicular pain. Injection of acetic acid (1% and 3%) into the testes decreased bladder capacity without direct stimulation of the bladder under conscious conditions. Measurement of bladder capacity is commonly used for measuring bladder sensitivity, and a reduction in bladder capacity is considered an indicator of the clinical condition of bladder overactivity. A reduction in bladder capacity without any effects on maximum micturition pressure suggests an effect on the afferent limb of the bladder reflex rather than the efferent limb. This suggestion is supported by the fact that capsaicin pretreatment nearly abolished the bladder overactivity and further suggests that testicular primary afferent C fibers mediate the bladder overactivity. The fact that neither bladder weight nor visual appearance of UROLOGY 75 (4), 2010
the bladder was changed also supports modification of the afferent or sensory limb of the bladder reflex resulting from testicular pain. The modification of bladder activity by testicular pain suggests that neural crosstalk may exist between the testes and the bladder. Qin and Foreman17 showed that about a third of L6-S2 spinal neurons of rats received convergent inputs of afferent nerves from both bladder and colon. Ustinova et al18 implicated afferent C fibers in mediating crosstalk between colon and bladder as capsaicin pretreatment abolished bladder overactivity after trinitrobenzene sulfonic acid-induced colitis in rats. Consequently, the crosstalk between colon and bladder can be explained at least partially by the convergence of C-fiber afferent nerves in the spinal cord. Contribution of afferent C-fibers to neural crosstalk is also supported by the report that colonic inflammation by dextran sulfate sodium enhanced the response of bladder sensory neurons to capsaicin by 60%.19 Neural crosstalk has also been reported between prostate and bladder in a study in which Vera and MeyerSiegler20 reported that intraprostatic formalin injection produced immediate bladder overactivity and increased inflammatory mediators in L6-S1 spinal cord and bladder. Prostatic and bladder crosstalk were also reported by Ishigooka et al21 who showed that injections of chemical irritants into the bladder or prostate produced convergent expression of c-Fos within second-order interneurons in the L6-S1 spinal cord. Scrotal afferent nerves of rats have been reported to enter the spinal cord through L5-S1 dorsal roots, and cutaneous plasma extravasation in the groin results from application of capsaicin to the L5-L6 intervertebral disk, indicating overlaps between bladder and groin dermatomes at the level of second-order neurons.22,23 The present study suggests that there is also convergence of testicular C fibers and bladder afferent fibers onto second-order interneurons in the lumbosacral spinal cord. This convergence is further supported by the parallel reduction in both pain behavior and bladder overactivity by indomethacin and capsaicin. However, there is a slight discrepancy between the duration of the pain behavior and bladder overactivity after testicular injection with the pain behavior disappearing at 1 hour, whereas bladder overactivity remained for at least 3 hours. This may be due to differences in endogenous control of pain perception vs visceral nociception or possibly secondary inflammatory changes in the bladder. Patients with CP/CPPS are reported to have higher sensitivity to capsaicin and heat stimuli in perineal area compared with healthy volunteers, and activation of afferent C fibers has been thought to underlie the pain in the patients.24,25 These points provide some clinical correlation for our testicular pain model. Although our model had only acute responses with testicular damage or inflammation, which may be different from the clinical situation of patients, the present study suggested that 947
nociceptive signals from testes may contribute to a part of bladder overactivity in patients with CP/CPPS.
CONCLUSIONS This study provides evidence that dilute acetic acid injection into the testes produces inflammation, prostaglandin synthesis, and activation of afferent C fibers, which lead to both pain behavior and bladder overactivity. This study suggests neural crosstalk between the testes and the bladder. This may provide a simple reproducible model to test novel therapies for CP/CPPS symptoms as well understanding the pathophysiological processes underlying bladder overactivity in CP/CPPS patients. Acknowledgments. The authors thank Dr. Karl B. Thor for the review of the manuscript and his valuable advices. Authors also thank Drs. Matthew O. Fraser, Velu Karicheti, and Ed. C. Burgard for valuable discussions. References 1. Teichman JM, Parsons CL. Contemporary clinical presentation of interstitial cystitis. Urology. 2007;69:S41-S47. 2. Tillisch K, Labus JS, Naliboff BD, et al. Characterization of the alternating bowel habit subtype in patients with irritable bowel syndrome. Am J Gastroenterol. 2005;100:896-904. 3. Alagiri M, Chottiner S, Ratner V, et al. Interstitial cystitis: unexplained associations with other chronic disease and pain syndromes. Urology. 1997;49:52-57. 4. Christianson JA, Liang R, Ustinova EE, et al. Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain. 2007;128:235-243. 5. Pezzone MA, Liang R, Fraser MO. A model of neural cross-talk and irritation in the pelvis: implications for the overlap of chronic pelvic pain disorders. Gastroenterology. 2005;128:1953-1964. 6. Litwin MS, McNaughton-Collins M, Fowler FJ Jr, et al. The National Institutes of Health chronic prostatitis symptom index: development and validation of a new outcome measure. Chronic Prostatitis Collaborative Research Network. J Urol. 1999;162:369375. 7. Schaeffer AJ, Landis JR, Knauss JS, et al. Demographic and clinical characteristics of men with chronic prostatitis: the national institutes of health chronic prostatitis cohort study. J Urol. 2002;168: 593-598. 8. Krieger JN, Egan KJ, Ross SO, et al. Chronic pelvic pains represent the most prominent urogenital symptoms of “chronic prostatitis”. Urology. 1996;48:715-721; discussion: 721-722. 9. Rudick CN, Schaeffer AJ, Thumbikat P. Experimental autoimmune prostatitis induces chronic pelvic pain. Am J Physiol Regul Integr Comp Physiol. 2008;294:R1268-R1275.
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