P2Y₁₂Receptors in Spinal Microglia Are Required for Neuropathic Pain after Peripheral Nerve Injury

Abstract

Extracellular nucleotides have been implicated as signaling molecules used by microglia to sense adverse physiological conditions, such as neuronal damage. They act through purinoceptors, especially the G-protein-coupled P2Y receptor P2Y 12 R. Emerging evidence has indicated that activated spinal microglia responding to nerve injury are key cellular intermediaries in the resulting highly debilitating chronic pain state, namely neuropathic pain. However, the role of microglial P2Y 12 Rs in neuropathic pain remains unknown. Here, we show that the level of P2Y 12 R mRNA expression was markedly increased in the spinal cord ipsilateral to the nerve injury and that this expression was highly restricted to ionized binding calcium adapter molecule 1-positive microglia. An increase in the immunofluorescence of P2Y 12 R protein in the ipsilateral spinal cord was also observed after nerve injury, and P2Y 12 R-positive cells were double labeled with the microglial marker OX-42. Blocking spinal P2Y 12 R by the intrathecal administration of its antagonist AR-C69931MX prevented the development of tactile allodynia (pain hypersensitivity to innocuous stimuli), a hallmark of neuropathic pain syndrome. Furthermore, mice lacking P2ry 12 (P2ry 12 ؊/؊ ) displayed impaired tactile allodynia after nerve injury without any change in basal mechanical sensitivity. Moreover, a single intrathecal administration of AR-C69931MX or oral administration of clopidogrel (a P2Y 12 R blocker clinically in use) to nerve-injured rats produced a striking alleviation of existing tactile allodynia. Together, our findings indicate that activation of P2Y 12 Rs in spinal microglia may be a critical event in the pathogenesis of neuropathic pain and suggest that blocking microglial P2Y 12 R might be a viable therapeutic strategy for treating neuropathic pain.

IRF8 is a transcriptional determinant for microglial motility

“…In fact, following peripheral nerve injury, reactive microglia engulf myelinated axons with their processes in the spinal dorsal horn in a manner that is dependent on P2Y 12 R signals [27]. Furthermore, microglial chemotaxis-related genes are upregulated in the spinal cord and are required for the generation of neuropathic pain [28][29][30][31]. However, whether microglial motility itself correlates with the degree of pain hypersensitivity remains unclear.…”

P2Y purinergic receptors: New targets for analgesic and antimigraine drugs

“…The UDP-sensitive P2Y 6 receptor subtype appears to be directly involved in the modulation of microglia phagocytosis [71], whereas a prominent role has been proposed for the P2Y 12 subtype in the development of allodynia following nerve injury. In the latter case, an ipsilateral and time-dependent up-regulation of microglial P2Y 12 was observed following nerve ligation, whereas the genetic ablation of P2Y 12 receptor expression or administration of P2Y 12 selective antagonists (i.e., intrathecal AR-C69931MX or oral Clopidogrel) prevented the development of tactile allodynia [72]. Moreover, intrathecal infusion of the P2Y 12 agonist 2-(methylthio)-ADP in naïve rats mimicked nerve injuryassociated p38 activation and pain behavior [73].…”

“…Nevertheless, when trying to identify new molecular druggable targets, the data can be puzzling. Concerning the purinergic system, total inhibition of pain and development of allodynia following nerve injury has been observed in P2X4-KO, and also in P2Y 12 -KO mice [68,72] (see above for details), indicating that the blockade of one or both of these receptors is sufficient to relieve neuropathic pain. So, based on these results what is the best purinergic target for drug development in pain in humans?…”

P2X4 purinoceptor signaling in chronic pain

“…In vivo imaging studies show that upon traumatic injury microglia rapidly extend processes by an ATP-dependent mechanism and converge on the site of injury [19,32,45,53]. Microglia express a range of P2 purinergic receptors: of the metabotropic P2YRs, microglia express P2Y1, 2, 4, 6, and 12Rs [7,25,36,61], with P2Y12R signaling mediating chemotaxis and tactile allodynia associated with nerve injury [44,69]. By contrast, microglial expression of the ionotropic P2XR is restricted to the P2X4 and P2X7R subtypes [16,28,36,75].…”