POSSIBLE MECHANISM OF REFERRED PAIN IN THE PERINEUM AND PELVIS ASSOCIATED WITH THE PROSTATE IN RATS

0022-5347/05/1746-2405/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 174, 2405–2408, December 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000180421.90260.65

POSSIBLE MECHANISM OF REFERRED PAIN IN THE PERINEUM AND PELVIS ASSOCIATED WITH THE PROSTATE IN RATS YONG CHEN, BO SONG,* XI-YU JIN, EN-QING XIONG

AND

JIAN-HUA ZHANG

From the Departments of Urology, Southwest Hospital, Third Military Medical University (BS, X-YJ, E-QX) and Fuling Central Hospital (YC, J-HZ), Chongqing, China

ABSTRACT

Purpose: Since persistent pain in the perineum and pelvic floor associated with chronic prostatitis /chronic pelvic pain syndrome has been hypothesized to be referred pain, it might also be explained by neural mechanisms. Materials and Methods: Dual retrograde fluorescent labeling and immunohistochemistry were identified as methods with which to investigate the neurogenic aspect of this status. The dual distribution of dorsal root ganglia (DRG) cells was determined after double retrograde fluorescent staining of the prostate and pelvic floor, and the prostate and perineum somatic nerves. Calcitonin gene-related peptide (CGRP) and substance P (SP) in dual labeled cells were determined by immunohistochemistry, giving possible insight into the cause of pelvic pain. Results: Fluorescent double labeled cells were found in the lumbar and sacral DRG, while double labeled cells were distributed predominantly in L6 to S1 and L1 to L2 segment DRG in groups 1 and 2, respectively. On immunohistochemistry some of them were confirmed to contain CGRP and SP. Thus, there are crossover pathways between the prostate and pelvic floor. Conclusions: The findings that we present confirm that the peripheral process of DRG cells dichotomizes to the prostate, sphincter and somatic parties simultaneously. Some of these cells contain CGRP and SP, which indicate that referred pain in the perineum and pelvic floor may be caused by an axon reflex in the peripheral process of DRG neurons. KEY WORDS: prostate; ganglia, spinal; pain; prostatitis; rats, Wistar

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is frequently noted in the urological clinic.1 Patients with bacterial prostatitis represent a small minority of the large number with prostatitis syndromes. On the contrary, no demonstrable infection processes are observed in the prostate of most patients with CPPS. CP/CPPS is a challenge for the patient and urologist since it is often difficult to design treatment strategies for improving symptoms. The pain symptom appears to be the most prominent manifestation of chronic prostatitis. Moreover, pain was the only symptom that significantly decreases quality of life and function status in these individuals.2 Since chronic pain occurs in regions of the body other than the prostate, it is appropriate to consider this symptom as being referred from the prostate. However, little attention has been given to the possible neurological basis for referred pain in CP/CPPS.3 To understand prostatitis properly and find better treatments for chronic prostatitis its pathogenesis must be better elucidated. Regardless of its etiology a different perspective on the causative agents of chronic prostatitis may lead to new and more effective treatment modalities. It appeared reasonable to investigate the possible neurological basis for the symptoms associated with this condition. However, little is known concerning the relationship between prostatitis and the pelvic floor. We describe the results of double labeling fluorescence microscopic study and immunohistochemistry of the prostate, pelvic floor and peripheral nerves innervating the pelvic floor. We specifically looked at the relationship between the prostate and pelvic floor. MATERIALS AND METHODS

Healthy adult male Wistar rats weighing 180 to 280 gm were used in the study. Rats were anesthetized with intra-

peritoneal injection of pentobarbital (50 mg/kg), immobilized with gallamine triethiodide (20 mg/kg intravenously) and ventilated artificially via a tracheal cannula. The jugular vein was catheterized for intravenous administration of supplemental anesthesia and other drugs. Additional pentobarbital and gallamine triethiodide were injected via the catheter when necessary. Rectal temperature was maintained at 37.0C to 38.0C using an automatically regulated heating pad. The rats were placed supine. The prostate was exposed by a lower abdominal incision. The peripheral nerves originating from the spinal nerves of the T13 through L6 segments were exposed retroperitoneally, namely the iliohypogastric, ilioinguinal, genitofemoral, femoral and lateral femoral cutaneous nerves. The anal sphincter was exposed by a radiate incision around the anus. A total of 27 rats were used in this study, randomly divided into 3 groups. There were 12 rats in each experimental group and 3 in the control group. In experimental group 1 for fluorescence microscopic examination of the dorsal root ganglia (DRG) innervating the prostate and anal sphincter 10 ␮l 1% propidium iodide (PI) were injected into the right lateral lobe of the prostate. At 24 hours later 10 ␮l 1% propidium bisbenzimide (Bb) was injected into the right anal sphincter. In experimental group 2 for fluorescence microscopic examination of the DRG to the prostate and the nerves innervating the perineum 10 ␮l 1% PI were injected into the right lateral lobe of the prostate in the same way. At survival 24 hours later 3 ␮l Bb were injected into the right iliohypogastric, ilioinguinal, genitofemoral, femoral and lateral femoral cutaneous nerves. At 24 hours after Bb injection the animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with normal saline, followed by 250 to 500 ml of fixative (4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4). The bilateral DRG of T3/L1 to S4 were dissected and

Submitted for publication March 16, 2005. * Correspondence: Department of Urology, Southwest Hospital, Chongqing, China, 40008. 2405

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maintained overnight in 30% sucrose in the same buffer before they were coronally sectioned at 20 um with a freezing microtome. The slides were observed under a fluorescence microscope (Nikon, Tokyo, Japan) using appropriate WU filter combinations and photographs were taken. In the control group PI was injected intravenously. Six hours later Bb was injected intravenously and 3 hours later perfusion was performed. Spinal ganglia from the lumbosacral area were obtained to serve as controls to exclude fluorescence in the spinal ganglia neuron via blood flow. Meanwhile, in the experimental groups some contralateral spinal ganglia were obtained randomly to serve as controls. Observers were blinded to the treatment status of the animals evaluated. The positions of double labeling cells were then judged using a drawing easel. Slides that had fluorescence double labeling DRG cells were dried for 24 hours overnight at 37C. Nickel ammonium sulfate intensified immunocytochemistry was done with slight modification.4 Section were washed 3 times with 0.1 mol/l sodium phosphate buffer (PBS) (pH 7.4) for 5 minutes each and soaked in 3% H2O2 in PBS for 30 minutes to inactive endogenous peroxidase, followed by 3 washes. Sections were incubated in 0.4% Triton X-100 in PBS and then transferred to commercially available rabbit calcitonin gene-related peptide (CGRP) and rabbit substance P (SP) incubated for 2 hours at 37C. They were then left for 3 nights at 4C in horseradish peroxidase labeled streptavidin in bovine serum albumin-PBS-Triton X-100 for 1 hour at room temperature. After 4 washes in PBS sections were incubated in a chromogen solution containing 0.05% 3 3-diaminobenzidine (Sigma Chemical Co., St. Louis, Missouri), 2.5% nickel ammonium sulfate and 0.001% H2O2 in 0.1 mol/l acetic acid buffer (pH 6.0) for about 10 minutes at room temperature. A negative control slide was included at each staining batch by substituting PBS for the primary antibody. The reaction product appeared blue-black. Finally, sections were dehydrated, cleared in xylene, coverslipped with DePex (BDH, Poole, United Kingdom) and observed with an Olympus light microscope. Photographs were obtained. According to neighbors and figures cells were judged that were not only fluorescence double labeled, but also immunopositive.

FIG. 1. Three kinds of labeled cells in lumbosacral DRG cells. Red arrow indicates double labeled cell. Green arrow indicates Bb single labeled cell. Yellow arrow indicates PI single labeled cell. Reduced from ⫻400.

FIG. 2. Distribution of double labeled cells in prostate and anal sphincter in experimental group 1.

RESULTS

In the double labeling fluorescence microscope study 3 kinds of labeled neuron cells were observed in the DRG of the right L1 to S3. They were PI labeled cells, Bb labeled cells and PI/Bb double labeled cells. Red and blue fluorescence was observed in PI single labeled cell cytoplasm and Bb single labeled cell nuclei, respectively. Blue fluorescence in nuclei and red fluorescence in cytoplasm were observed in PI/Bb double labeled cells (fig. 1). Double labeled cells were distributed predominantly in L6 to S1 and L1 to L2 segment DRG in groups 1and 2, respectively (figs. 2 and 3). On immunohistochemistry using nickel intensified immunostaining CGRP and SP immunopositive materials were only detected in the cell cytoplasm. When comparing photographs of the 2 groups, some parts of double labeled cells contained CGRP and SP (fig. 4). DISCUSSION

Pain is the chief complaint in 61% of patients with CP/ CPPS and the secondary complaint in the remainder.2 Pain localized in the pelvic, suprapubic perineal, scrotal and low back areas is characteristics of CP. A low correspondence between symptoms and medical findings have been found. Since chronic pain occurs in regions of the body other than the prostate, it is appropriate to consider this symptom as being referred from the prostate. Thus, it was considered to be referred pain. However, much current research has con-

FIG. 3. Distribution of double labeled cells in prostate and lumbosacral nerves in experimental group 2.

tinued to concentrate on the microbiology of the prostate without considering this disease as a pain syndrome. The mechanism of referred pain caused by prostatitis remains unclear, although some studies have described these phenomena.3 A different perspective on the causative agents of CP/CPPS may lead to new and more effective treatment modalities. The mechanism of referred pain can be explained by an antidromic axon reflex via dichotomizing afferent neurons, viscerosomatic convergence in the spinal cord (afferentafferent interactions via the spinal cord) or a sympathetic reflex. Probably several mechanism act together to determine the sensory and trophic phenomena that appear in the area or referred pain.5– 8 Although the mechanism of referred pain is complex, it is believed that the first stage may be the DRG. The reasons are 1) DRG neurons have dichotomizing dendrites projecting to different sites,7 2) an axon reflex exists in the structure of

REFERRED PAIN IN PERINEUM AND PELVIS ASSOCIATED WITH PROSTATE

FIG. 4. Strongly stained CGRP and SP in cytoplasm of double labeled DRG cells (arrow) on same slide. Reduced from ⫻400.

branching projection, namely when 1 impulse from 1 branch transmit to the center, 1 part of the impulse may relay to the peripheral antidromically at the crotch, 3) sensory terminals can release some mediums, such as CGRP and SP, 9⫺11 and 4) neurotransmitters released by nerve terminals may induce neurogenic inflammation.12 Branching and/or collateral projections of DRG neurons have been confirmed by fluorescent dye retrograde double labeling.13⫺15 Fluorescent tracers are most likely incorporated into terminals by nonspecific endocytosis, followed by retrograde axonal transported in axons. The combination of immunohistochemistry with retrograde tracing permits the visualization of neurotransmitters in the same neuron that is labeled for its projection to a particular field. Because of their emission characteristics, retrograde dye and immunostaining can be simultaneously observed in the same neuron. Do the lumbosacral DRG project to the prostate, pelvic floor and peripheral innervating pelvis? After fluorescent dyes (PI and Bb) were injected into the prostate, pelvic floor and nerves in this study double labeled neurons were observed in lumbosacral DRG. There were no double labeled cells in other ganglia, which indicated that no focal leakage diffusion to peripheral tissues or labeling via blood flow occurred and also showed that 2 kinds of fluorescence were retrograde transported to cytoplasm or nuclei via 2 branches projected from their neurons to the prostate and referred areas. Thus, we could consider that these results were reliable. It is postulated that lumbosacral DRG project to the prostate, pelvic floor and perineum. Sensations from the prostate are conveyed within the parasympathetic system with a partial contribution from the sympathetic thoracolumbar system. Our results show that double labeled cells mainly localized in segments L1 to L2 and L6 to S2, and they were similar to sympathetic and parasympathetic segments in rats. The sympathetic preganglionic neurons are in L1 to L2 and the parasympathetic preganglionic neurons are in L6 to S1.12, 16 It was confirmed by fluorescent dye retrograde double labeling that lumbosacral DRG neurons have dichotomizing dendrites projecting to the prostate and pelvic floor, and the prostate and peripheral nerves innervating the perineum. Moreover, it was confirmed by immunohistochemistry that the part of neurons with dichotomized fiber contains CGRP and SP. This study provides evidence that some lumbosacral DRG neurons innervate the prostate and pelvic floor. This supports the notion that there are crossover pathways between the prostate and pelvic floor. With regard to the functional consequence of our findings we assume that crossover pathways between the prostate and pelvic floor may be involved in pain sensation transmission and the pelvic pain referred to as CP/CPPS may result from a DRG axon reflex.13 When pain impulses from the prostate were relayed to the nerve center, 1 part of the impulses was relayed to the peripheral along another branch at the crotch. When antidromic impulses arrive in the pelvic floor and perineum, some mediums, such as CGRP and SP, were released from the terminals of primary afferent neu-

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rons. Neurogenic inflammation then occurs. Wesselmann considered that neurogenic inflammation might have an important role in this persistent pain status.12 When these impulses from the pelvic floor were relayed to the nerve center along topical sensory terminals, pelvic referred pain was evoked. Recent studies suggest that CP/CPPS symptoms may be due to or associated with pelvic floor muscle dysfunction.17, 18 Since pain impulses resulting from pelvic floor muscle dysfunction might be relayed to nerve center through this crossover pathway, patients could not distinguish pain resulting from the prostate or pelvic floor subjectively. Therefore, it is considered that pain associated with CP/CPPS might be closely related to pelvic floor muscle dysfunction. However, it may be confirmed by further studies. Since there are anatomical differences in the lower urinary tract and nervous system in rats and humans, there are definite limitations to our results in human chronic prostatitis. However, these concepts could be applied to chronic persistent pain involving CP/CPPS. Applying the concept of the axon reflex and neurogenic inflammation to CP/CPPS might open an avenue for the development of novel therapies targeted toward different levels of the neural axis. To clarify the exact role of this collateral innervation in this persistent pain status electrophysiological study is necessary.19 The relationship between the prostate and pelvic floor may be corroborated in future studies.

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