Purinergic receptors and microglial activation in pain

Previous studies in our laboratory have shown that long-term increases in pain-related behavior were correlated with the activation of spinal microglia following formalin injection. In vitro studies in collaboration with our lab have demonstrated that microglia are activated by ATP via P2Y receptors that are blocked by suramin. The present studies examine whether intrathecal delivery of suramin blocks microglia activation and long-term hyperalgesia induced by formalin injection. Suramin delivery began one day prior to the formalin injection and lasted for 7 days. Rats were observed using a modified hotplate test before and at different times following formalin injection. The spinal cord was surveyed for changes in microglia labeling as revealed by OX-42 staining at different times after formalin injection. Suramin decreased both the hyperalgesic sensitivity to the thermal stimuli and microglial activation induced by formalin injection as compared to the saline-treated group. This suggests that ATP is one potential mediator that activates spinal cord microglia and enhances pain-related behavior in the formalin model. To further identify what subtype purinergic receptors are responsible for the activation of microglia, the profile of P2Y receptor subtype expression in glial cells was characterized. Primary cultures of microglia in a ramified state do not express P2Y₁, P2Y₄ and P2Y ₆. In rat spinal cord, P2Y1 colocalized with astrocytes and endothelial cells and not with microglia and neurons. P2Y4 colocalized with astrocytes and there was no neuronal or microglial colocalization. Combing pharmacology data with the RT-PCR and imunohistochemistry, these findings suggest microglia do not express P2Y1, P2Y4 and P2Y6. It is likely that Microglia express P2Y12 and P2Y13. Since P2Y₁₂ and P2Y₁₃ are best activated by ADP, ATP's action at low concentrations on microglia is likely mediated by ADP acting on P2Y₁₂ and/or P2Y₁₃. The additional study was also performed to examine ATP's effect on the production of pro-inflammatory cytokines in mouse microglia primary culture. ATP induced IL-6 release from primary microglia culture in a concentration dependent manner. IL-1 beta and TNF-alpha were not detected after incubation with ATP for 6 hours and 24 hours. Therefore, ATP in physiological concentrations can release at least one proinflammatory cytokine.