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P2X7 receptor mediates activation of microglial cells in prostate of chemically irritated rats

Abstract

Purpose

Evidence shows that adenosine triphosphate (ATP) is involved in the transmission of multiple chronic pain via P2X7 receptor. This study was to investigate the P2X7 and microglial cells in the chronic prostatitis pain.

Materials and Methods

Rats were divided into control group and chronic prostatitis group (n = 24 per group). A chronic prostatitis animal model was established by injecting complete Freund's adjuvant (CFA) to the prostate of rats, and the thermal withdrawal latency (TWL) was detected on days 0, 4, 12 and 24 (n = 6 at each time point in each group). Animals were sacrificed and the pathological examination of the prostate, detection of mRNA expression of P2X7 and ionized calcium binding adaptor molecule 1 (IBA-1) and measurement of content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the dorsal horn of L5-S2 spinal cord were performed on days 0, 4, 12 and 24. In addition, the content of TNF-α and IL-1β in the dorsal horn of L5-S2 spinal cord was measured after intrathecal injection of inhibitors of microglial cells and/or P2X7 for 5 days.

Results

The chronic prostatitis was confirmed by pathological examination. The expression of P2X7 and IBA-1 and the content of TNF-α and IL-1β in rats with chronic prostatitis were significantly higher than those in the control group. On day 4, the expressions of pro-inflammatory cytokines became to increase, reaching a maximal level on day 12 and started to reduce on day 24, but remained higher than that in the control group. Following suppression of microglial cells and P2X7 receptor, the secretion of TNF-α and IL-1β was markedly reduced.

Conclusion

In chronic prostatitis pain, the microglial cells and P2X7 receptor are activated resulting in the increased expression of TNF-α and IL-1β in the L5-S2 spinal cord, which might attribute to the maintenance and intensification of pain in chronic prostatitis.

Prostatitis; Cells; Receptors, Purinergic P2X7; Tumor Necrosis Factor-alpha; Interleukin-1


INTRODUCTION

Chronic prostatitis, a common urological condition in young and middle-age men, is caused by multiple etiological factors. Pain is a major presentation of chronic prostatitis (11. Luzzi GA: Chronic prostatitis and chronic pelvic pain in men: aetiology, diagnosis and management. J Eur Acad Dermatol Venereol. 2002; 16: 253-6.). Previous studies focused on the pathological changes in the prostate, while the pathways related to neurotransmission and the regulatory mechanisms of chronic prostatitis pain have not been studied. Recent studies have identified the chronic prostatitis pain as a visceral referred pain, which is usually accompanied by the dysfunction of pelvic floor muscles. The prostate is innervated largely by the pelvic nerves arising from the L5-S2 spinal cord (22. Ishigooka M, Nakada T, Hashimoto T, Iijima Y, Yaguchi H: Spinal substance P immunoreactivity is enhanced by acute chemical stimulation of the rat prostate. Urology. 2002; 59: 139-4.,33. Chen Y, Song B, Jin XY, Xiong EQ, Zhang JH: Possible mechanism of referred pain in the perineum and pelvis associated with the prostate in rats. J Urol. 2005; 174: 2405-8.).

It has also been shown that the transmission and regulation of pain are associated with not only the neurons but the microglia and astrocytes (44. Beggs S, Salter MW: Microglia-neuronal signalling in neuropathic pain hypersensitivity 2.0. Curr Opin Neurobiol. 2010; 20: 474-80.,55. Watkins LR, Milligan ED, Maier SF: Glial activation: a driving force for pathological pain. Trends Neurosci. 2001; 24: 450-5.). Studies also demonstrated that astrocytes and microglias may secrete pro-inflammatory cytokines such as tumor necrosis factors (TNF), interleukin-1 (IL-1), nerve growth factor (NGF), and nitric oxide (NO), which may lead to the neuronal injury and chronic pain (66. Madiai F, Hussain SR, Goettl VM, Burry RW, Stephens RL Jr, Hackshaw KV: Upregulation of FGF-2 in reactive spinal cord astrocytes following unilateral lumbar spinal nerve ligation. Exp Brain Res. 2003; 148: 366-76.,77. Kucher BM, Neary JT: Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes. J Neurochem. 2005; 92: 525-35.). Especially, the microglias are widely distributed in the central nervous system (CNS). The detrimental stimulation of CNS (such as trauma, ischemia and infection) may activate microglias. Under this condition, their morphology, the receptor expression on these cells and their function alter; these cells are ameboid; the markers for activation increase on these cells (88. DeLeo JA, Tanga FY, Tawfik VL: Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist. 2004; 10: 40-52.). There is evidence showing that the microglias in the posterior horn of spinal cord are significantly activated after damage to peripheral nerves (99. Beggs S, Salter MW: Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury. Brain Behav Immun. 2007; 21: 624-33.). This suggests that the activation of microglias in the spinal cord is related to the occurrence and transmission of neuropathic pain. However, the role of microglias in chronic prostatitis pain is still poorly understood, and molecules activated after injury on these cells and the exact mechanisms are unclear.

There are a lot of P2X7 receptors of adenosine triphosphate (ATP). ATP is a type of pain-causing neurotransmitter, and its receptors can be classified as P2X receptors and P2Y receptors. P2X7 receptor is a special subtype of purinergic receptor P2X family and an ATP-gated non-selective cation channel. P2X7 receptor contains 595 amino acids and three P2X7 receptors form homologous polymers generally. P2X7 receptor is a dual functional receptor. Under pathological conditions, P2X7 receptor is involved in the transmission of pain. It was reported that microglias may be activated by the P2X7 receptor, which is up-regulated in various types of chronic pain (1010. Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y: Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci. 2004; 24: 1-7.). Chessell et al. found P2X7 knockout mice failed to present with hyperalgesia to heat and mechanical stimulation after nerve injury (1111. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, et al.: Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain. 2005; 114: 386-96.). However, whether the microglias and P2X7 receptor in the posterior horn of L5-S2 spinal cord are activated to regulate the chronic prostatitis pain remains unclear. Hence, the present study was to investigate the role of microglias and P2X7 receptor in the chronic prostatitis pain and the possible therapeutic strategies for chronic pelvic pain syndrome.

MATERIALS AND METHODS

Animals

The specific pathogen free (SPF) rats weighing 200 ± 25 g were purchased from the Experimental Animal Center of the Third Military Medical University and randomized into experiment group and control group. All rats were intraperitoneally anesthetized with 1% pentobarbital and then the prostate was exposed through a ventral midline incision (1 cm). For rats in the experiment group, injection with complete Freund's adjuvant (CFA; Sigma-Aldrich, Sigma) was done once at bilateral ventral lobes (10µL for each). For rats in the control group, 20µL of normal saline was injected (Nackley et al. (1212. Nackley AG, Suplita RL 2nd, Hohmann AG: A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience. 2003; 117: 659-70.), Butler et al. (1313. Butler SH, Godefroy F, Besson JM, Weil-Fugazza J: A limited arthritic model for chronic pain studies in the rat. Pain. 1992; 48: 73-81.) and Zhou et al. (1414. Zhou ZS, Song Bo, Lu GS: The research on the relationship between chronic prostatitis pain and spinal astrocytes activation. J Third Milit Med Univ 2005; 27: 1853-4.)). Then, the wound was closed. Rats were sacrificed on days 0, 4, 12 and 24 d after injection (n = 6 at each time point in each group) and pathological examination and detection of the mRNA expression of P2X7 and ionized calcium binding adaptor molecule 1 (IBA-1) and the content of TNF-α and IL-1β in spinal cord were performed. All procedures were performed in accordance with the guidelines for animal care and use of National Institute of Health, and this study was approved by the Ethics Committee of our Hospital.

Prostatic Inflammation Model Identification

Rats were intraperitoneally anesthetized with 10% chloral hydrate (0.3 mL/100 g) on days 0, 4, 12 and 24 after CFA injection, and transcardially perfused with 200 mL of 0.9% saline and then with 0.01 mol/L phosphate buffer (about 300 mL) containing 4% paraformaldehyde at 48° C Subsequently, the left and right prostatic tissues were collected, fixed in 4% paraformaldehyde at 4° C overnight, embedded in paraffin, cut into sections, stained, and finally examined under a microscope (1515. Wu AN, Xiong EQ, Song B: Secretory alterations of urethral glands in complete Freund's adjuvant-induced prostatitis in rats. J Third Milit Med Univ 2009; 31: 2266-8.).

Detection of heat pain threshold

Detection of mRNA expression of P2X7 and IBA-1 in L5-S2 posterior horn

Animals were sacrificed by decapitation. The posterior horn of L5-S2 spinal cord was carefully collected on ice under a microscope, and stored at -70° C for use. Total RNA was extracted from the L5-S2 spinal cord using the RNAgents Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer's instructions. The concentration and purity of total RNA were determined by spectrophotometric analysis at A260 and A280 (1.8-2.0). The quality of RNA was determined by methanal agarose gel electrophoresis following ethidium bromide staining. Total RNA (2µg) was subjected to reverse transcription using the Reverse Transcription System (Jikang, Shanghai, China) with random primer oligo(DT)18 (0.5µg). The reaction conditions were as follows: 70° C for 5 min, 37° C for 60 min. and 70° C for 10 min., and products were then stored at -70° C.

The resulting cDNA (20 ng) was used as templates for real-time fluorescence quantitative (FQ) PCR using a SYBR green PCR core reagent kit (Applied Biosystems, Foster City, CA) in DNA Engine OPTIONTm2 (MJ RESEARCH, USA). The primers were designed using the Geneworks software package as follows: P2X7: 5′-GACAAACAAAGTCACCCGGAT-3′ (forward) and 5′-CGCTCACCAAAGCAAAGCTAAT-3′ (reverse); IBA-1: 5′-TTGATCTGAATGGCAATGGA-3′ (forward) and 5′-CCTCC AATTAGGGCAACTCA-3′ (reverse). The PCR conditions were as follows: reverse transcription at 50° C for 30 min., Hot Start Taq (1.25 unit/sample) activation for 15 min at 95° C, 40 cycles of denaturation at 94° C for 15 s, annealing at 56° C for 30 s, and extension at 72° C for 30 s. The SYBR Green fluorescence was acquired by a final extension at 79° C. The melting curve analysis was performed after each reaction. GAPDH (5′-TTTAACTCTGGTAAAGTGGATATTGTTG-3′ [forward] and 5′-ATTTCCATTGATGACAAGCTTCC-3′) served as an internal control (Table-1). The expression of target genes could be calculated according to the amplification standard curve and regression equation of GAPDH automatically by the DNA Engine OPTIONTm2. The expression of target genes was normalized to that of GAPDH as the relative expression. Average was obtained from 6 animals in each group.

Table 1
- Primers and conditions for RT-PCR.

Contents of TNF-α and IL-1β in L5-S2 posterior horn

L5-S2 posterior horn was homogenized in 0.5 mL of ice-cold lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% TritonX 100, 0.5% sodium deoxycholate, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1% sodium dodecyl sulfate (SDS), 10 mM NaF and 1 mM vitriolu acid sodium. Homogenates were centrifuged at 1800 rpm for 10 min. and the supernatant was collected and stored at -70° C. The contents of IL-1β and TNF-α were detected using the commercially available ELISA kits according to the manufacturer's instructions. Briefly, 50µg) was subjected to reverse transcription using the L of biotinylated antibody was added to 100µg) was subjected to reverse transcription using the L of samples in an anti-rat TNF-α or IL-1β pre-coated plate (Santa Cruz Biotechnology, Inc., Santa Cruz) followed by incubation for 3 h at room temperature. After washing three times, a prepared 100µg) was subjected to reverse transcription using the L of strepta vidin horseradish peroxidase solution and 3, 3′, 5, 5′-tetramethylbenzidin substrate solution were added followed by incubation. Absorbance was measured at 492 nm in a microplate reader (Salzburger Labortechnik, Salzburg, Austria). A standard curve was delineated to determine the content of TNF-α and IL-1β. The sensitivity of this assay is > 10 pg/mL. Average was obtained from 6 animals in each group.

Intrathecal injection of agonist or antagonist of P2X7 and microglial cells

The second part of the experiment involved intrathecal cannulation according to the procedures described by Yaksh and Rudy (1717. Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav. 1976; 17: 1031-6.). In brief, rats were intraperitoneally anesthetized with pentobarbital, a cannula (PE-10 tubing) was inserted through the cisterna magna at 6 cm to the L1 spinal cord via the spinal subarachnoid space. A recovery period of 7 d was allowed, and rats showing no motor impairment following surgery were used for further experiments. The prostatitis was induced as mentioned above. From day 7, intrathecal injection of drug was done for consecutive 5 days. Rats were divided into 5 groups (n = 6 per group) and treated as follows: 1) P2X7 receptor agonist: 2′-3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP; 100µmol/L); 2) P2X7 receptor antagonist: oxidized ATP (oATP; 100µmol/L); 3) inhibitor of microglial cells: minocycline (50µg); 4) P2X7 receptor agonist + inhibitor of microglial cells; 5) in the control group, injection was done with artificial cerebrospinal fluid (ACSF; pH5.5) of equal volume. Six rats were included in each group. Drugs were injected at a volume of 15µL and flushing was done with 5µL of ACSF. Injections were finished within 5 min. The content of TNF-α and IL-1β in the spinal cord was determined.

Statistical analysis

All data were expressed as means ± standard error (SEM) and statistical analysis was performed with SPSS version 13.0 for Windows. When F-test showed significance, means were compared with the LSD test of post hoc analysis (Dunnett's t test). Analysis of variance (ANOVA) was conducted for comparisons of data among different groups. A value of P < 0.05 was considered statistically significant.

RESULTS

Pathological changes in prostate

Pathological examination showed thAT CFA treated prostates presented with degeneration, necrosis and exfoliation of mucosal cells in the prostate gland. Infiltration of large amounts of lymphocytes and monocytes was noted in the interstitium, and some lymphocytes aggregated in cluster. In the control group, the mucosal epithelial cells were regularly arranged, and the infiltration of leukocytes was not observed in the interstitium (Figure-1).

Figure 1
Pathological changes of the prostate in different groups. In the control group (A) and inflammation 0 day group (B), the mucosal epithelium maintained an orderly organization, without leukocyte infiltration in interstitial tissues. In contrast, in the inflammation 4, 12, 24 days groups (C,D,E), degeneration, necrosis, and exfoliation of mucosal cells in the prostate gland were observed. Interstitial substance was infiltrated with large amounts of lymphocytes and monocytes.

Detection of pain sensation

The TWL of CFA treated rats was 14.73 ± 0.93s, 12.15 ± 0.99s and 12.45 ± 1.19 respectively at 4 d, 12 d and 24 d after injection, respectively, which were significantly shorter than that in the control group (P < 0.01). This suggests that hyperalgia was induced following CFA injection at the prostate and the rat chronic prostatitis model was successfully established (Figure-2).

Figure 2
Thermal withdrawal latency (s) in two groups.

*P < 0.01 vs. control group; #P < 0.01 vs. 0 d.


mRNA expression of P2X7 and IBA-1 in posterior horn

The mRNA expression of P2X7 and IBA-1 was significantly increased in the posterior horn of L5-S2 spinal cord in the experiment group on days 4, 12 and 24 as compared to the control group at the corresponding time points and to the experiment group at baseline (P < 0.01). The P2X7 expression reached a maximal level on day 12 (P < 0.05) (Figures 3A and 3B).

Figure 3
mRNA expression of P2X7 (A) and IBA-1 (B) in dorsal horn and contents of TNF-α (C) and IL-1β (D) (ng/mL) in dorsal horn at different time points in two groups.

*P < 0.01 vs. control group; #P <0.01 vs. 0 d; &P <0.05 vs. 4 d and 24 d.


Content of TNF-α and IL-1β in L5-S2 posterior horn

The content of TNF-α and IL-1β in the posterior horn of L5-S2 spinal cord was significantly increased in the experiment group on days 4, 12 and 24, as compared to the control group at corresponding time points (P < 0.01) and to the experiment group at baseline (P < 0.01). The content of TNF-α and IL-1β reached a peak on day 12 (P < 0.05) (Figures 3C and 3D).

Content of TNF-α and IL-1β following inhibition of microglial cells and/or P2X7

In the experiment group, following injection of minocycline and oATP, the content of TNF-α and IL-1β was markedly reduced (P < 0.01). However, the P2X7 agonist (BzATP) could promote the secretion of TNF-α and IL-1β (P < 0.01 and < 0.05, respectively), and minocycline inhibited the bioeffects of BzATP (P < 0.05). In the control group, intrathecal injection of ASCF had no influence on the contents of TNF-α and IL-1β in the spinal cord (Figure-4).

Figure 4
Contents of IL-1β and TNF-α in dorsal horn after injection of different agonist or antagonist (ng/mL).

*P < 0.05 vs. CSF; #P < 0.05 vs. BzATP.


DISCUSSION

Chronic prostatitis is a common urological disease in young and middle-age men, and patients with chronic prostatitis account for 25-35% of inpatients at the urological clinic. Of all chronic prostatitis of different types, chronic nonbacterial prostatitis (IIIa) or chronic prostatitis/chronic pelvic pain syndrome (IIIb) is the most common and accounts for about 95% of chronic prostatitis (1818. Millán-Rodríguez F, Palou J, Bujons-Tur A, Musquera-Felip M, Sevilla-Cecilia C, Serrallach-Orejas M, et al.: Acute bacterial prostatitis: two different sub-categories according to a previous manipulation of the lower urinary tract. World J Urol. 2006; 24: 45-50.,1919. Zhou Z, Hong L, Shen X, Rao X, Jin X, Lu G, et al.: Detection of nanobacteria infection in type III prostatitis. Urology. 2008; 71: 1091-5. Erratum in: Urology. 2008 ;72: 723.). Generally, chronic prostatitis is characterized by refractory pelvic or perineal pain without evidence of urinary tract infection, which is usually accompanied by bladder and urethra dysfunction. Chronic prostatitis is a major reason for hospital visit. The etiology of chronic prostatitis and the pathogenesis of pain in chronic prostatitis are still poorly understood. In recent years, studies have shown that nanobacteria infection might be a major cause of chronic nonbacterial prostatitis (2020. Shen X, Ming A, Li X, Zhou Z, Song B: Nanobacteria: a possible etiology for type III prostatitis. J Urol. 2010; 184: 364-9.). Currently, the antibiotic therapy achieves unfavorable efficacy for patients with chronic prostatitis, and effective strategies have not been developed for these patients to date. The diagnosis and treatment of chronic prostatitis have been a challenge in urology. In addition, the long-lasting pain may result in physical and psychological disorders. Thus, the investigation of the etiology of chronic prostatitis and the pathogenesis of pain in chronic prostatitis are crucial for the accurate diagnosis and development of effective strategies for the treatment of chronic prostatitis.

Pain appears to be a most prominent manifestation of chronic prostatitis. However, the diagnosis and treatment of pain in chronic prostatitis are still challenging because of the complicated pathogenesis of chronic prostatitis pain (2121. Baxter C, Bolus R, Mayer E: Choice and outcomes of alternative therapies in patients with interstitial cystitis (IC) and chronic pelvic pain (CPP). J Urol. 2010; 183: 580.). Patients with chronic prostatitis often experience pains not only at prostate, but at the sites adjacent to or tissues outside the prostate which are found to be also controlled by the L5-S2 spinal cord. Moreover, some patients feel pain even after prostatitis disappears. Hence, the pain in the chronic prostatitis is often characteristic of “extra-territorial” and “mirror” image pain. Increasing evidence demonstrates that there are abnormalities in the cell-mediated neurological regulation and the transmitters in the L5-S2 spinal cord in chronic prostatitis (2222. Zhang H, Liu L, Lu G, Chen Z, Fang Q, Yang Z, et al.: Chemical irritation of the prostate sensitizes P(2)X(3) receptor-mediated responses in rat dorsal root ganglion neurons. Neurourol Urodyn. 2011; 30: 612-8.,2323. Baranowski AP: Chronic pelvic pain. Best Pract Res Clin Gastroenterol. 2009; 23: 593-610.).

Accumulating studies have revealed that the pathological pain is due to not only neuronal dysfunction, but the activation of astrocytes and microglias (2424. Watkins LR, Milligan ED, Maier SF: Glial activation: a driving force for pathological pain. Trends Neurosci. 2001; 24: 450-5.), especially in the chronic exaggerated and continuing pain. Microglias and astrocytes are regarded as “immune cells” in the nervous system, and can secrete some pro-inflammatory cytokines such as IL-1β, TNF-α, NGF, NO, prostaglandin and bradykinin following activation, leading to the exaggeration and persistence of pain by acting on other glial cells and neurons (2525. Li HL, Qin LY, Wan Y: Astrocyte: a new star in pain research. Sheng Li Ke Xue Jin Zhan. 2003; 34: 45-8.,2626. Aronica E, Gorter JA, Rozemuller AJ, Yankaya B, Troost D: Activation of metabotropic glutamate receptor 3 enhances interleukin (IL)-1beta-stimulated release of IL-6 in cultured human astrocytes. Neuroscience. 2005; 130: 927-33.). Therefore, microglial cells play important roles in the pathogenesis of pathological pain (2727. Mingam R, De Smedt V, Amédée T, Bluthé RM, Kelley KW, Dantzer R, et al.: In vitro and in vivo evidence for a role of the P2X7 receptor in the release of IL-1 beta in the murine brain. Brain Behav Immun. 2008; 22: 234-44.,2828. Barger SW, Goodwin ME, Porter MM, Beggs ML: Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation. J Neurochem. 2007; 101: 1205-13.). In rats with sciatic inflammation, intrathecal injection of minocycline, an inhibitor of microglial cell activation, was found to inhibit the abnormal mechanical pain with low threshold (2929. Ledeboer A, Sloane EM, Milligan ED, Frank MG, Mahony JH, Maier SF, et al.: Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain. 2005; 115: 71-83.).

In the pathogenesis of pathological pain, P2X7 plays an important role in the secretion of pro-inflamamtory cytokines mediated by microglial cell activation (2727. Mingam R, De Smedt V, Amédée T, Bluthé RM, Kelley KW, Dantzer R, et al.: In vitro and in vivo evidence for a role of the P2X7 receptor in the release of IL-1 beta in the murine brain. Brain Behav Immun. 2008; 22: 234-44.). P2X7 is an ATP receptor, a transmitter and modulator in the nervous system. P2X7 is a special subtype of purinergic receptor P2X family. In rats with inflammatory pain, visceral pain and neuropathic pain, focal or intraperitoneal injection of antagonist of P2X7 (oATP or A-740003) was found to inhibit the mechanical hyperalgesia, allodynia and hypersensitivity (3030. Fulgenzi A, Ticozzi P, Gabel CA, Dell'Antonio G, Quattrini A, Franzone JS, et al.: Periodate oxidized ATP (oATP) reduces hyperalgesia in mice: involvement of P2X7 receptors and implications for therapy. Int J Immunopathol Pharmacol. 2008; 21: 61-71.,3131. Honore P, Donnelly-Roberts D, Namovic MT, Hsieh G, Zhu CZ, Mikusa JP, et al.: A-740003 [N-(1-{[(cyanoimino) (5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a novel and selective P2X7 receptor antagonist, dose-dependently reduces neuropathic pain in the rat. J Pharmacol Exp Ther. 2006; 319: 1376-85.). In the P2X7 receptor deficiency mice, the neuropathic hypersensitivity to mechanical or heat stimulation was absent following nerve injury (1111. Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, et al.: Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain. 2005; 114: 386-96.). However, under physiological conditions, P2X7 receptor is not activated. Under the pathological conditions, P2X7 receptor is activated and involved in the pain transduction. Our findings also revealed that P2X7 receptor activation significantly increased the secretion of pro-inflammatory cytokines in animal inflammation model. Following activation, P2X7 receptor involves in the pain transduction, which is associated with the calcium related signal transduction (3232. Fumagalli M, Brambilla R, D'Ambrosi N, Volonté C, Matteoli M, Verderio C, et al.: Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors. Glia. 2003; 43: 218-03.,3333. Wang CM, Chang YY, Kuo JS, Sun SH: Activation of P2X(7) receptors induced [(3)H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl(-)/HCO(3) (-)-dependent mechanism. Glia. 2002; 37: 8-18.).

In the present study, our results indicated that, in rats with chronic prostatitis pain, the expression of P2X7 and IBA-1 was elevated in the posterior horn of L5-S2 spinal cord, and the excretion of TNF-α and IL-1β was also up-regulated. However, after inhibition of P2X7 receptor and/or microglial cells, the secretion of TNF-α and IL-1β was dramatically reduced suggesting that P2X7 receptor mediates the microglial cell activation in rat with prostate prostatitis leading to the increased secretion of pro-inflammatory cytokines. It has been well established that TNF-α and IL-1β are responsive to inflammatory stimuli and cytotoxicity towards neurons, and they can induce chronic inflammation and pain (3434. Stalder AK, Carson MJ, Pagenstecher A, Asensio VC, Kincaid C, Benedict M, et al.: Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor. Am J Pathol. 1998; 153: 767-83.). These findings suggest that there is neurogenic inflammation in the L5-S2 spinal cord as a result of microglial cell activation via the P2X7 receptor in rats with chronic prostatitis pain. These observations demonstrate that P2X7 mediated microglial cells activation in the L5-S2 spinal cord may take part in the regulation of chronic pain and might lead to the persistence and exaggeration of prostatitis pain. Hence, identification of new neurotransmission pathways and the mechanisms underlying the regulation of chronic prostatitis pain may be helpful to find novel therapeutic targets for chronic prostatitis pain. Currently, oral or intravenous non-steroidal anti-inflammatory drugs have been used to treat spinal cord inflammation. However, the focal drug concentration is at a low level leading to unfavorable efficacy. In Traditional Chinese Medicine, the surface projection of L5-S2 spinal cord is also known as Shenshu point and acupuncture of Shenshu point has been used in the treatment of chronic prostatitis pain (3535. Chen ZX: Observation on therapeutic effect of warm needle moxibustion on chronic non-bacterial prostatitis. Zhongguo Zhen Jiu. 2009; 29: 275-8.). To date, we have applied “water-needle therapy” for chronic prostatitis pain on the basis of our previous findings, in which the acupuncture of acupoint at L5-S2 spinal cord was performed followed by focal injection of B12, B1, hydrocortisone and Chinese herbs. This treatment achieves favorable efficacy, but is still in its infancy stage.

Of note, our findings can not explain the whole molecular mechanisms underlying the activation of microglial cells in chronic prostatitis because there are other receptors (such as P2X4, P2Y12 and Toll like receptor) related to the neuropathic pain (3636. Kim D, Kim MA, Cho IH, Kim MS, Lee S, Jo EK, et al.: A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity. J Biol Chem. 2007; 282: 14975-83.,3737. Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K: P2Y12 receptors in spinal microglia are required for neuropathic pain after peripheral nerve injury. J Neurosci. 2008; 28: 4949-56.). Studies have shown that the P2X7 receptor is related to the P2X4 receptor in structure and function, and there is interaction between P2X7 and P2X4 in the microglial cell mediated pain (3838. Casas-Pruneda G, Reyes JP, Pérez-Flores G, Pérez-Cornejo P, Arreola J: Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia. J Physiol. 2009; 587: 2887-901.). In addition, the activation of Toll-like receptor 4 in the dorsal horn and the release of IL-1β are dependent on the activation of P2X7 receptor, and inhibitors of P2X7 receptor (oxidized ATP, A-438079) may suppress the hyperalgesia to heat and mechanical stimulation following intrathecal injection of LPS (a agonist of Toll-like 4 receptor) (3939. Clark AK, Staniland AA, Marchand F, Kaan TK, McMahon SB, Malcangio M: P2X7-dependent release of interleukin-1beta and nociception in the spinal cord following lipopolysaccharide. J Neurosci. 2010; 30: 573-82.). These findings demonstrate that the P2X7 receptor on the microglial cells can interact with the above molecules, which then aggregates the neuropathic pain However, the specific mechanisms of their interactions require further studies.

CONCLUSIONS

The chronic prostatitis is related to the activation of P2X7 and microglial cells and the high expression of TNF-α and IL-1β in the dorsal horn of L5-S2 spinal cord. Moreover, TNF-α and IL-1β expression in the L5-S2 spinal cord can be inhibited by inhibitors of P2X7 receptor and microglial cells. These findings indicate that chronic pelvic pain syndrome may cause secondary inflammation in the L5-S2 spinal cord by activating the microglial cells via P2X7 receptor, a phenomenon probably associated with the persistence and intensification of chronic prostatitis pain.

ABBREVIATIONS

ACSF = artificial cerebrospinal fluid

ANOVA = analysis of variance

ATP = adenosine triphosphate

BzATP = 2′-3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate

CFA = complete Freund's adjuvant

CNS = central nervous system

IBA-1 = ionized calcium binding adaptor molecule 1

IL-1 = interleukin-1

NGF = nerve growth factor

NO = nitric oxide

oATP = oxidized ATP

PMSF = phenylmethanesulfonyl fluoride

SDS = sodium dodecyl sulfate

SEM = standard error

SPF = specific pathogen free

TNF-α = tumor necrosis factor-α

TWL = thermal withdrawal latency

CONFLICT OF INTEREST

None declared.

This study was supported by the National Natural Science Foundation (NO.81100537).

REFERENCES

  • 1
    Luzzi GA: Chronic prostatitis and chronic pelvic pain in men: aetiology, diagnosis and management. J Eur Acad Dermatol Venereol. 2002; 16: 253-6.
  • 2
    Ishigooka M, Nakada T, Hashimoto T, Iijima Y, Yaguchi H: Spinal substance P immunoreactivity is enhanced by acute chemical stimulation of the rat prostate. Urology. 2002; 59: 139-4.
  • 3
    Chen Y, Song B, Jin XY, Xiong EQ, Zhang JH: Possible mechanism of referred pain in the perineum and pelvis associated with the prostate in rats. J Urol. 2005; 174: 2405-8.
  • 4
    Beggs S, Salter MW: Microglia-neuronal signalling in neuropathic pain hypersensitivity 2.0. Curr Opin Neurobiol. 2010; 20: 474-80.
  • 5
    Watkins LR, Milligan ED, Maier SF: Glial activation: a driving force for pathological pain. Trends Neurosci. 2001; 24: 450-5.
  • 6
    Madiai F, Hussain SR, Goettl VM, Burry RW, Stephens RL Jr, Hackshaw KV: Upregulation of FGF-2 in reactive spinal cord astrocytes following unilateral lumbar spinal nerve ligation. Exp Brain Res. 2003; 148: 366-76.
  • 7
    Kucher BM, Neary JT: Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes. J Neurochem. 2005; 92: 525-35.
  • 8
    DeLeo JA, Tanga FY, Tawfik VL: Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist. 2004; 10: 40-52.
  • 9
    Beggs S, Salter MW: Stereological and somatotopic analysis of the spinal microglial response to peripheral nerve injury. Brain Behav Immun. 2007; 21: 624-33.
  • 10
    Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y: Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci. 2004; 24: 1-7.
  • 11
    Chessell IP, Hatcher JP, Bountra C, Michel AD, Hughes JP, Green P, et al.: Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain. 2005; 114: 386-96.
  • 12
    Nackley AG, Suplita RL 2nd, Hohmann AG: A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience. 2003; 117: 659-70.
  • 13
    Butler SH, Godefroy F, Besson JM, Weil-Fugazza J: A limited arthritic model for chronic pain studies in the rat. Pain. 1992; 48: 73-81.
  • 14
    Zhou ZS, Song Bo, Lu GS: The research on the relationship between chronic prostatitis pain and spinal astrocytes activation. J Third Milit Med Univ 2005; 27: 1853-4.
  • 15
    Wu AN, Xiong EQ, Song B: Secretory alterations of urethral glands in complete Freund's adjuvant-induced prostatitis in rats. J Third Milit Med Univ 2009; 31: 2266-8.
  • 16
    Cheng H Yang JP, Zhang YB: Spinal microglia activation intrathecal minocycline inhibited by and attenuation on thermal hyperalgesia in CFA-induced inflammatory rats. Suzhou Univ J Med Sci 2007; 27: 14-6.
  • 17
    Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav. 1976; 17: 1031-6.
  • 18
    Millán-Rodríguez F, Palou J, Bujons-Tur A, Musquera-Felip M, Sevilla-Cecilia C, Serrallach-Orejas M, et al.: Acute bacterial prostatitis: two different sub-categories according to a previous manipulation of the lower urinary tract. World J Urol. 2006; 24: 45-50.
  • 19
    Zhou Z, Hong L, Shen X, Rao X, Jin X, Lu G, et al.: Detection of nanobacteria infection in type III prostatitis. Urology. 2008; 71: 1091-5. Erratum in: Urology. 2008 ;72: 723.
  • 20
    Shen X, Ming A, Li X, Zhou Z, Song B: Nanobacteria: a possible etiology for type III prostatitis. J Urol. 2010; 184: 364-9.
  • 21
    Baxter C, Bolus R, Mayer E: Choice and outcomes of alternative therapies in patients with interstitial cystitis (IC) and chronic pelvic pain (CPP). J Urol. 2010; 183: 580.
  • 22
    Zhang H, Liu L, Lu G, Chen Z, Fang Q, Yang Z, et al.: Chemical irritation of the prostate sensitizes P(2)X(3) receptor-mediated responses in rat dorsal root ganglion neurons. Neurourol Urodyn. 2011; 30: 612-8.
  • 23
    Baranowski AP: Chronic pelvic pain. Best Pract Res Clin Gastroenterol. 2009; 23: 593-610.
  • 24
    Watkins LR, Milligan ED, Maier SF: Glial activation: a driving force for pathological pain. Trends Neurosci. 2001; 24: 450-5.
  • 25
    Li HL, Qin LY, Wan Y: Astrocyte: a new star in pain research. Sheng Li Ke Xue Jin Zhan. 2003; 34: 45-8.
  • 26
    Aronica E, Gorter JA, Rozemuller AJ, Yankaya B, Troost D: Activation of metabotropic glutamate receptor 3 enhances interleukin (IL)-1beta-stimulated release of IL-6 in cultured human astrocytes. Neuroscience. 2005; 130: 927-33.
  • 27
    Mingam R, De Smedt V, Amédée T, Bluthé RM, Kelley KW, Dantzer R, et al.: In vitro and in vivo evidence for a role of the P2X7 receptor in the release of IL-1 beta in the murine brain. Brain Behav Immun. 2008; 22: 234-44.
  • 28
    Barger SW, Goodwin ME, Porter MM, Beggs ML: Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation. J Neurochem. 2007; 101: 1205-13.
  • 29
    Ledeboer A, Sloane EM, Milligan ED, Frank MG, Mahony JH, Maier SF, et al.: Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain. 2005; 115: 71-83.
  • 30
    Fulgenzi A, Ticozzi P, Gabel CA, Dell'Antonio G, Quattrini A, Franzone JS, et al.: Periodate oxidized ATP (oATP) reduces hyperalgesia in mice: involvement of P2X7 receptors and implications for therapy. Int J Immunopathol Pharmacol. 2008; 21: 61-71.
  • 31
    Honore P, Donnelly-Roberts D, Namovic MT, Hsieh G, Zhu CZ, Mikusa JP, et al.: A-740003 [N-(1-{[(cyanoimino) (5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a novel and selective P2X7 receptor antagonist, dose-dependently reduces neuropathic pain in the rat. J Pharmacol Exp Ther. 2006; 319: 1376-85.
  • 32
    Fumagalli M, Brambilla R, D'Ambrosi N, Volonté C, Matteoli M, Verderio C, et al.: Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors. Glia. 2003; 43: 218-03.
  • 33
    Wang CM, Chang YY, Kuo JS, Sun SH: Activation of P2X(7) receptors induced [(3)H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl(-)/HCO(3) (-)-dependent mechanism. Glia. 2002; 37: 8-18.
  • 34
    Stalder AK, Carson MJ, Pagenstecher A, Asensio VC, Kincaid C, Benedict M, et al.: Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor. Am J Pathol. 1998; 153: 767-83.
  • 35
    Chen ZX: Observation on therapeutic effect of warm needle moxibustion on chronic non-bacterial prostatitis. Zhongguo Zhen Jiu. 2009; 29: 275-8.
  • 36
    Kim D, Kim MA, Cho IH, Kim MS, Lee S, Jo EK, et al.: A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity. J Biol Chem. 2007; 282: 14975-83.
  • 37
    Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K: P2Y12 receptors in spinal microglia are required for neuropathic pain after peripheral nerve injury. J Neurosci. 2008; 28: 4949-56.
  • 38
    Casas-Pruneda G, Reyes JP, Pérez-Flores G, Pérez-Cornejo P, Arreola J: Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia. J Physiol. 2009; 587: 2887-901.
  • 39
    Clark AK, Staniland AA, Marchand F, Kaan TK, McMahon SB, Malcangio M: P2X7-dependent release of interleukin-1beta and nociception in the spinal cord following lipopolysaccharide. J Neurosci. 2010; 30: 573-82.

Publication Dates

  • Publication in this collection
    Mar-Apr 2013

History

  • Received
    4 June 2012
  • Accepted
    17 Jan 2013
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