The plate was then placed in a shaker/CO2-incubator to equilibrate for 18 hrs at 37C. neuropathic pain and provided confidence over the possibilities to treat neuropathic pain with CCR2 antagonists. Results We provided evidence that dorsal root ganglia (DRG) cells harvested from CCI animals responded to stimulation by CCL2 with a concentration-dependent calcium rise involving PLC-dependent internal stores. This response was associated with an increase in evoked neuronal action potentials suggesting these cells were sensitive to CCR2 signalling. Importantly, treatment with AZ889 abolished CCL2-evoked excitation confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. We next showed that in vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in CCI rats. Additionally, Nomegestrol acetate application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord. Furthermore, Nomegestrol acetate AZ889 abolished NMDA-dependent wind-up of spinal withdrawal reflex pathway in neuropathic animals giving insight into the spinal mechanism underlying the analgesic properties of AZ889. Conclusions Overall, this study strengthens the important role of CCR2 in neuropathic pain and highlights feasibility that interfering on this mechanism at the spinal level with a selective antagonist can provide new analgesia opportunities. Background Neuropathic pain treatment is often refractory to available therapies and its underlying mechanisms remain poorly understood. This pathological state reflects abnormal sensory processes caused by a variety of cellular changes that result in abnormal CDC25B hyperexcitability, hyperactivity and spontaneous activity in the pain circuitry [1]. Many molecular events have been implicated for their contribution to neuropathic Nomegestrol acetate pain. The CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1/MCP-1) and its CC chemokine receptor (CCR2) are of interest as they have recently been shown to be overexpressed in glial and neuronal cells following injury to the nervous system and may contribute to the neuroinflammatory processes associated with the development and maintenance of neuropathic pain [2-14]. In addition, mice lacking CCR2 Nomegestrol acetate receptors failed to show mechanical allodynia in the partial nerve injury model [2] while antagonists of CCR2 reversed nociceptive responses in the spinal nerve ligation and varicella zoster animal model [15] as well as in the focal demyelination of the sciatic nerve model of neuropathic pain [4]. Although a growing body of evidence suggests that interventions aiming to block CCR2/CCL2 signalling may alleviate neuropathic pain, little is known about the actual cellular site of action of this effect. So far, there appears to be a disagreement on the site of action of CCR2 antagonists producing analgesia since studies have provided evidence that peripheral and central nervous system (CNS) mechanisms may be involved. Some studies have suggested that both resident and infiltrating spinal microglia activated by CCR2 [14] contributed to enhanced neuronal excitation [16] during the development of nerve Nomegestrol acetate injury induced neuropathy. Others suggested that CCR2 antagonists can inhibit activation of the sciatic nerve and DRG neurons which supports a peripherally-mediated analgesia mechanism [17]. Finally, DRG neurons activated by CCL2 could perhaps contribute to both centrally- and peripherally-mediated pathophysiology [18-20]. Here we provide further details on the cellular and pharmacological mechanisms of CCL2/CCR2 signalling in a model of neuropathic pain through the integration of cellular imaging, electrophysiology as well as the use of AZ889, a competitive CCR2 blocker. In addition, the behavioural evaluation of AZ889 in the CCI model of neuropathic pain was supplemented via critical pharmacokinetic measures of drug exposure that strengthened the in vitro to in vivo translation of pharmacological properties. Results Identification of the potent CCR2 antagonist Chemokine receptors are known to modulate intracellular calcium concentration [21]. A library of designed molecules was screened on HEK cells expressing the receptor using a calcium flux-assay (FLIPR). Compounds capable of blocking the intracellular calcium rise evoked by mouse CCL2 in HEK293 s cells (Gqi5) stably expressing the rat CCR2 receptor were selected and further profiled by performing dose-response curves. Mouse and rat CCL2 (mCCL2 and rCCL2) purchased from R&D Systems gave similar median effective concentrations (EC50) values (Additional file 1 Figure S1) and mCCL2 was selected to conduct the screening assay. Cells were pre-incubated (30 min) with a given concentration of compound (from 0.04 to 1 1 M) and CCR2 calcium-mediated activation was evoked by the addition.
