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Activated spinal astrocytes contribute to the later phase of carrageenan-induced prostatitis pain.

ABSTRACT: BACKGROUND:Prostatodynia is the main symptom of chronic prostatitis and the main reason that patients go to the hospital for treatment. Although a variety of factors, including inflammatory immune response, nervous system sensitization, and psychological factors, have been shown to play important roles in the induction and development of chronic pain in prostatitis, the underlying cause of chronic prostatodynia maintenance in prostatitis patients remains unclear. METHODS:A mouse model of chronic prostatitis induced by carrageenan injection was used. The von Frey test was used to measure pain behavior. The microglial and astrocyte activations were immunohistochemically demonstrated with antibodies against Iba1 and GFAP. The expression of cytokine or signaling pathway was detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. RESULTS:In this study, we provide several lines of evidence to demonstrate that activated spinal astrocytes contribute to the later phase (5 weeks after carrageenan injection) of carrageenan-induced prostatitis pain. First, activation of spinal astrocytes but not microglia was found in the spinal cord dorsal horn at 5 weeks. Second, intrathecal injection of the astroglial toxin L-2-Aminoadipate acid (L-AA) but not microglial inhibitor minocycline reduced mechanical allodynia at 5 weeks. Third, chronic prostatitis induced a profound and persistent upregulation of connexin-43 hemichannels in spinal astrocytes, and spinal injection of the connexin-43 inhibitor carbenoxolone (CBX) effectively reduced pain symptoms. Fourth, increased expression and release of chemokine C-X-C motif ligand 1 (CXCL1) in the spinal dorsal horn and intrathecal injection of a CXCL1 neutralizing antibody or the CXCR2 (a major receptor of CXCL1) antagonist SB225002 significantly reduced mechanical allodynia at 5 weeks. CONCLUSIONS:In this study, we found that a novel mechanism of activated spinal astrocytes plays a crucial role in maintaining chronic prostatitis-induced persistent pain via connexin-43-regulated CXCL1 production and secretion.

SUBMITTER: Deng GC

PROVIDER: S-EPMC6814979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Accumulating evidence suggests that spinal cord astrocytes play an important role in neuropathic pain sensitization by releasing astrocytic mediators (e.g. cytokines, chemokines and growth factors). However, it remains unclear how astrocytes control the release of astrocytic mediators and sustain late-phase neuropathic pain. Astrocytic connexin-43 (now known as GJ1) has been implicated in gap junction and hemichannel communication of cytosolic contents through the glial syncytia and to the extracellular space, respectively. Connexin-43 also plays an essential role in facilitating the development of neuropathic pain, yet the mechanism for this contribution remains unknown. In this study, we investigated whether nerve injury could upregulate connexin-43 to sustain late-phase neuropathic pain by releasing chemokine from spinal astrocytes. Chronic constriction injury elicited a persistent upregulation of connexin-43 in spinal astrocytes for >3 weeks. Spinal (intrathecal) injection of carbenoxolone (a non-selective hemichannel blocker) and selective connexin-43 blockers (connexin-43 mimetic peptides (43)Gap26 and (37,43)Gap27), as well as astroglial toxin but not microglial inhibitors, given 3 weeks after nerve injury, effectively reduced mechanical allodynia, a cardinal feature of late-phase neuropathic pain. In cultured astrocytes, TNF-α elicited marked release of the chemokine CXCL1, and the release was blocked by carbenoxolone, Gap26/Gap27, and connexin-43 small interfering RNA. TNF-α also increased connexin-43 expression and hemichannel activity, but not gap junction communication in astrocyte cultures prepared from cortices and spinal cords. Spinal injection of TNF-α-activated astrocytes was sufficient to induce persistent mechanical allodynia, and this allodynia was suppressed by CXCL1 neutralization, CXCL1 receptor (CXCR2) antagonist, and pretreatment of astrocytes with connexin-43 small interfering RNA. Furthermore, nerve injury persistently increased excitatory synaptic transmission (spontaneous excitatory postsynaptic currents) in spinal lamina IIo nociceptive synapses in the late phase, and this increase was suppressed by carbenoxolone and Gap27, and recapitulated by CXCL1. Together, our findings demonstrate a novel mechanism of astrocytic connexin-43 to enhance spinal cord synaptic transmission and maintain neuropathic pain in the late-phase via releasing chemokines.

Project description:It is now widely accepted that the glial cells of the central nervous system (CNS) are key players in many processes, especially when they are activated via neuron-glia or glia-glia interactions. In turn, many of the glia-derived pro-inflammatory cytokines contribute to central sensitization during inflammation or nerve injury-evoked pathological pain conditions. The prototype of pro-inflammatory cytokines is interleukin-1beta (IL-1β) which has widespread functions in inflammatory processes. Our earlier findings showed that in the spinal cord (besides neurons) astrocytes express the ligand binding interleukin-1 receptor type 1 (IL-1R1) subunit of the IL-1 receptor in the spinal dorsal horn in the chronic phase of inflammatory pain. Interestingly, spinal astrocytes are also the main source of the IL-1β itself which in turn acts on its neuronal and astrocytic IL-1R1 leading to cell-type specific responses. In the initial experiments we measured the IL-1β concentration in the spinal cord of C57BL/6 mice during the course of complete Freund adjuvant (CFA)-induced inflammatory pain and observed a peak of IL-1β level at the time of highest mechanical sensitivity. In order to further study astrocytic activation, primary astrocyte cultures from spinal cords of C57BL/6 wild type and IL-1R1 deficient mice were exposed to IL-1β in concentrations corresponding to the spinal levels in the CFA-induced pain model. By using cytokine array method we observed significant increase in the expressional level of three cytokines: interleukin-6 (IL-6), granulocyte-macrophage colony stimulating factor (GM-CSF) and chemokine (C-C motif) ligand 5 (CCL5 or RANTES). We also observed that the secretion of the three cytokines is mediated by the NFkB signaling pathway. Our data completes the picture of the IL-1β-triggered cytokine cascade in spinal astrocytes, which may lead to enhanced activation of the local cells (neurons and glia as well) and can lead to the prolonged maintenance of chronic pain. All these cytokines and the NFkB pathway can be possible targets of pain therapy.

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Activated spinal astrocytes contribute to the later phase of carrageenan-induced prostatitis pain.

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