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Objective Cancer pain, especially bone cancer pain, affects the life quality of cancer patients, and current treatments for this pain are limited.The present study was deSigned to investigate the time course and the role of neuroinflammation including glial activation, ERK activation, proinflammatory cytokines and Toll-like receptor 4 (TLR4) expressions in the spinal cord in bone cancer pain (BCIP) on a modified rat model.Methods The mechanical allodynia was tested by yon Frey hairs in rats inoculated with Walker 256 mammary gland carcinoma cells.Pharmacological approaches as well as Western Blotting and fluorescent immunohistochemistry staining were applied to assess the roles played by spinal neuroinflammation in bone cancer pain.Results (1) Glial activation and proinflammatory cytokine expression: After intra-tibial inoculation of syngeneic Walker 256 mammary gland carcinoma cells, the rats showed significant bilateral mechanical allodynia.A robust activation of both astrocyte and microglia was observed in bilateral spinal dorsal horn at 8 and 16 days after unilateral Walker 256 inoculation, characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglia marker), and elevated level of IL-1β, IL-6 and TNF-α mRNA, in both sides of spinal dorsal horn.On day 12 after the Walker 256 inoculation, single spinal administration of fluorocitrate (inhibitor of glial metabolism) or minocycline (inhibitor of microglia) by lumbar puncture has significant anti-allodynic effects.(2) ERK activation and its role in cancer pain: Down stream, the activation of spinal extracellular signal-regulated kinase ERK1/2 contributes to the development of hyperalgesia and allodynia in neuropathic and inflammatory pain.However, little is known about the possible role of spinal ERK in bone cancer pain.The present study demonstrated in addition that spinal ERK activation also plays a crucial role in bone cancer pain.Intratibia inoculation of carcinoma cells induced a robust and persistent (>21 D) activation of ERK in the L2-L3 and L4-L5 spinal dorsal horn.The pERK1/2-immunoreactivity was observed in both Iba-1-expressing microglia and GFAP-expressing astrocytes.A single intrathecal injection of the selective MEK (ERK kinase) inhibitors PD98059 (10 μg) on day 12 and U0126 (1 μg, 3 μg) on day 14, respectively attenuated the mechanical allodynia tested by Von Frey hair.(3) Upstream—involvement of TLR4 receptor: The expression of TLR4 mRNA was elevated bilaterally in spinal cord at 8, 16 and 22 days after the Walker 256 inoculation.Intrathecal injection of nonstereoselective TLR4 receptor antagonist naloxone significantly blocked the increased expression of inflammatory cytokines and bilateral allodynia induced by bone cancer.Conclusions These results demonstrated that the bilateral sustained activation of spinal astrocyte as well as microglia cells critically contributes to the bilateral mechanical allodynia induced by unilateral bone cancer in rats.Spinal TLR4 might play an important role in the sustained activation of microglias and hence the robust and sustained spinal neuroinflammation in BCIP in rats.ERK activation in spinal microglia and astrocytes is required for the induction and maintenance of mechanical allodynia in bone cancer induced pain.