Activation of Src Family Kinases in Spinal Microglia Contributes to Formalin-Induced Persistent Pain State Through p38 Pathway


      Protein tyrosine phosphorylation has been implicated in normal and pathological functions such as cell proliferation, migration and differentiation. Recently, some studies have shown that Src family kinases (SFKs) were involved in neurological disorders and neuropathic pain states in which microglial activation plays a role. In the formalin test, we have reported that microglia undergo at least 2 distinct stages of activation on the basis of signaling events regarding p38 mitogen-activated protein kinases (MAPK). Here, we investigated the involvement of SFKs signaling in a formalin pain animal model and the association with p38 MAPK. Our results showed that SFKs were activated in the spinal microglia beginning 1 day after peripheral formalin injection lasting for 7 days. Pretreatment with SFK specific inhibitor PP2 could not inhibit formalin-induced spontaneous pain behaviors. However, PP2 inhibited formalin injury, induced persistent mechanical hyperalgesia, and reversed microglial phospho-p38 expression as well using immunohistostaining and Western blot at day 3 and 7 after injection. Our results suggested that the activation of the Src/p38MAPK signaling cascade in spinal microglia contributed to late stage persistent mechanical hyperalgesia evoked by formalin injection into the paw.


      This study presents unique properties of spinal microglial activation in a pain animal model. This finding could potentially help clinicians to further understand the contributions of spinal microglia to acute and persistent pain state.

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        • Callera G.E.
        • Touyz R.M.
        • Tostes R.C.
        • Yogi A.
        • He Y.
        • Malkinson S.
        • Schiffrin E.L.
        Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src.
        Hypertension. 2005; 45: 773-779
        • Choi J.S.
        • Kim H.Y.
        • Chung J.W.
        • Chun M.H.
        • Kim S.Y.
        • Yoon S.H.
        • Lee M.Y.
        Activation of Src tyrosine kinase in microglia in the rat hippocampus following transient forebrain ischemia.
        Neurosci Lett. 2005; 380: 1-5
        • Coyle D.E.
        Partial peripheral nerve injury leads to activation of astroglia and microglia which parallels the development of allodynic behavior.
        Glia. 1998; 23: 75-83
        • Eriksson N.P.
        • Persson J.K.
        • Svensson M.
        • Arvidsson J.
        • Molander C.
        • Aldskogius H.
        A quantitative analysis of the microglial cell reaction in central primary sensory projection territories following peripheral nerve injury in the adult rat.
        Exp Brain Res. 1993; 96: 19-27
        • Fu K.Y.
        • Light A.R.
        • Matsushima G.K.
        • Maixner W.
        Microglial reactions after subcutaneous formalin injection into the rat hind paw.
        Brain Res. 1999; 825: 59-67
        • Fu K.Y.
        • Light A.R.
        • Maixner W.
        Long-lasting inflammation and long-term hyperalgesia after subcutaneous formalin injection into the rat hindpaw.
        J Pain. 2001; 2: 2-11
        • Fu K.Y.
        • Tan Y.H.
        • Sung B.
        • Mao J.
        Peripheral formalin injection induces unique spinal cord microglial phenotypic changes.
        Neurosci Lett. 2009; 449: 234-239
        • Graeber M.B.
        • Streit W.J.
        • Kreutzberg G.W.
        Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells.
        J Neurosci Res. 1988; 21: 18-24
        • Guo W.
        • Wei F.
        • Zou S.
        • Robbins M.T.
        • Sugiyo S.
        • Ikeda T.
        • Tu J.C.
        • Worley P.F.
        • Dubner R.
        • Ren K.
        Group I metabotropic glutamate receptor NMDA receptor coupling and signaling cascade mediate spinal dorsal horn NMDA receptor 2B tyrosine phosphorylation associated with inflammatory hyperalgesia.
        J Neurosci. 2004; 24: 9161-9173
        • Hanisch U.K.
        • Kettenmann H.
        Microglia: Active sensor and versatile effector cells in the normal and pathologic brain.
        Nat Neurosci. 2007; 10: 1387-1394
        • Hashizume H.
        • DeLeo J.A.
        • Colburn R.W.
        • Weinstein J.N.
        Spinal glial activation and cytokine expression after lumbar root injury in the rat.
        Spine (Phila Pa 1976). 2000; 25: 1206-1217
        • Hashizume H.
        • Rutkowski M.D.
        • Weinstein J.N.
        • DeLeo J.A.
        Central administration of methotrexate reduces mechanical allodynia in an animal model of radiculopathy/sciatica.
        Pain. 2000; 87: 159-169
        • Honore P.
        • Rogers S.D.
        • Schwei M.J.
        • Salak-Johnson J.L.
        • Luger N.M.
        • Sabino M.C.
        • Clohisy D.R.
        • Mantyh P.W.
        Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons.
        Neuroscience. 2000; 98: 585-598
        • Hu P.
        • Bembrick A.L.
        • Keay K.A.
        • McLachlan E.M.
        Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve.
        Brain Behav Immun. 2007; 21: 599-616
        • Igwe O.J.
        c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia.
        Eur J Neurosci. 2003; 18: 1719-1730
        • Ji R.R.
        • Suter M.R.
        p38 MAPK, microglial signaling, and neuropathic pain.
        Mol Pain. 2007; 3: 33
        • Ji R.R.
        • Gereau RWt
        • Malcangio M.
        • Strichartz G.R.
        MAP kinase and pain.
        Brain Res Rev. 2009; 60: 135-148
        • Kalia L.V.
        • Gingrich J.R.
        • Salter M.W.
        Src in synaptic transmission and plasticity.
        Oncogene. 2004; 23: 8007-8016
        • Katsura H.
        • Obata K.
        • Mizushima T.
        • Sakurai J.
        • Kobayashi K.
        • Yamanaka H.
        • Dai Y.
        • Fukuoka T.
        • Sakagami M.
        • Noguchi K.
        Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury.
        J Neurosci. 2006; 26: 8680-8690
        • Kim L.C.
        • Song L.
        • Haura E.B.
        Src kinases as therapeutic targets for cancer.
        Nat Rev Clin Oncol. 2009; 6: 587-595
        • Krady J.K.
        • Basu A.
        • Levison S.W.
        • Milner R.J.
        Differential expression of protein tyrosine kinase genes during microglial activation.
        Glia. 2002; 40: 11-24
        • Li K.
        • Lin T.
        • Cao Y.
        • Light A.R.
        • Fu K.Y.
        Peripheral formalin injury induces 2 stages of microglial activation in the spinal cord.
        J Pain. 2010; 11: 1056-1065
        • Lin T.
        • Li K.
        • Zhang F.Y.
        • Zhang Z.K.
        • Light A.R.
        • Fu K.Y.
        Dissociation of spinal microglia morphological activation and peripheral inflammation in inflammatory pain models.
        J Neuroimmunol. 2007; 192: 40-48
        • Lowell C.A.
        Src-family and Syk kinases in activating and inhibitory pathways in innate immune cells: Signaling cross talk.
        Cold Spring Harb Perspect Biol. 2011; 3
        • Molander C.
        • Hongpaisan J.
        • Svensson M.
        • Aldskogius H.
        Glial cell reactions in the spinal cord after sensory nerve stimulation are associated with axonal injury.
        Brain Res. 1997; 747: 122-129
        • Page T.H.
        • Smolinska M.
        • Gillespie J.
        • Urbaniak A.M.
        • Foxwell B.M.
        Tyrosine kinases and inflammatory signalling.
        Curr Mol Med. 2009; 9: 69-85
        • Ren K.
        An improved method for assessing mechanical allodynia in the rat.
        Physiol Behav. 1999; 67: 711-716
        • Scholz J.
        • Abele A.
        • Marian C.
        • Haussler A.
        • Herbert T.A.
        • Woolf C.J.
        • Tegeder I.
        Low-dose methotrexate reduces peripheral nerve injury-evoked spinal microglial activation and neuropathic pain behavior in rats.
        Pain. 2008; 138: 130-142
        • Storkson R.V.
        • Kjorsvik A.
        • Tjolsen A.
        • Hole K.
        Lumbar catheterization of the spinal subarachnoid space in the rat.
        J Neurosci Methods. 1996; 65: 167-172
        • Streit W.J.
        • Graeber M.B.
        • Kreutzberg G.W.
        Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system.
        J Neuroimmunol. 1989; 21: 117-123
        • Thomas S.M.
        • Brugge J.S.
        Cellular functions regulated by Src family kinases.
        Annu Rev Cell Dev Biol. 1997; 13: 513-609
        • Tsuda M.
        • Tozaki-Saitoh H.
        • Masuda T.
        • Toyomitsu E.
        • Tezuka T.
        • Yamamoto T.
        • Inoue K.
        Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after peripheral nerve injury.
        Glia. 2008; 56: 50-58
        • Tsuda M.
        • Ueno H.
        • Kataoka A.
        • Tozaki-Saitoh H.
        • Inoue K.
        Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling.
        Glia. 2008; 56: 378-386
        • Watanabe T.
        • Tsuda M.
        • Tanaka S.
        • Ohba Y.
        • Kawaguchi H.
        • Majima T.
        • Sawa H.
        • Minami A.
        Adaptor protein Crk induces Src-dependent activation of p38 MAPK in regulation of synovial sarcoma cell proliferation.
        Mol Cancer Res. 2009; 7: 1582-1592
        • Zhang F.Y.
        • Wan Y.
        • Zhang Z.K.
        • Light A.R.
        • Fu K.Y.
        Peripheral formalin injection induces long-lasting increases in cyclooxygenase 1 expression by microglia in the spinal cord.
        J Pain. 2007; 8: 110-117