Nucleotide receptors in trigeminal satellite glial cells as new targets for the pharmacological control of migraine pain: in vitro and in vivo studies

Background and Purpose - The main aim of my PhD research project is to study pain transduction mechanisms in trigeminal ganglia (TG) in migraine, to understand why sensory trigeminal neurons become hyperactive in this pathological status, and how their activity can be pharmacologically modulated. We focused our studies on the role of the purinergic system in the neuron-to-glial cells communication within the TG, and its cross-talk with known pro-algogenic systems (such as bradykinin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine. Methods and Results – For the in vitro studies, we set up primary mixed neuron-glia or purified satellite glial cell (SGCs) cultures from theTG of C57BL6 mice. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging, and the extracellular concentrations of CGRP and PGE2 were measured by ELISA assays. Western blot experiments on P2Y1 and P2Y2 receptor subtypes were also performed. Concerning the role of metabotropic purinergic receptors, our data show that exposure of mixed-neuron glia cultures to BK induced the neuronal release of CGRP, which in turn significantly potentiated the ADP-responsive P2Y1 and the UTP-sensitive P2Y2 receptor subtypes on surrounding SGCs. The increased activity of P2 receptors was related to increased receptor protein expression. Interestingly, the anti-migraine drug sumatriptan fully inhibited both CGRP release and glial P2Y-receptor potentiation. Moreover, exposure to BK led to increased production of PGE2, an effect completely abolished by the COX-1 inhibitor acetylsalicylic acid (ASA), whichalso blocked neuronal CGRP release. Taken together, these results suggest that a complex cross-talk between neuronal and glial cells takes place in the TG, involving pain mediators and extracellular nucleotides. Modulation of this network by known anti-migraine drugs, such as triptans and COX inhibitors, suggests that it might play an important role in the development of migraine pain. In vivo studies were aimed at evaluating the pro- or anti-algogenic role of P2Y receptors through their selective inhibition. To this purpose, we set up a sub-chronic model of inflammatory trigeminal hypersensitivity, by injecting complete Freund adjuvant (CFA) into the temporomandibular joint (TMJ) of rats. CFA-injected animals showed ipsilateral mechanical allodynia and TMJ edema. Glial cell activation was evaluated in the spinal-trigeminal system by immunohistochemistry and western blotting analysis of GFAP protein expression, a typical marker of activated SGCs. A significant glial activation within the TG was observed starting from 24 hours up to 11 days after CFA injection, thus confirming that our model leads to TG sensitization. Moreover, upregulation of P2Y1 and P2Y2 protein expression was also observed, thus corroborating our in vitro data. Interestingly, the non-selective P2Y antagonist PPADS showed a strong analgesic effect on CFA-induced TG inflammation, which is comparable to ASA-mediated analgesia. Conclusions - These results suggest a possible the pro-algogenic role for P2Y receptors in the development of trigeminal sensitization and migraine pain, opening the future perspective of identifying innovative and more selective pharmacological approaches for the sake of those migraineurs who are insensitive to currently available drugs. References [1] G. Magni, S. Ceruti. P2Y purinergic receptors: new targets for analgesic and antimigraine drugs. Biochem Pharmacol, 85(4):466-77, 2013. [2] S. Ceruti, G. Villa, M. Fumagalli, L. Colombo, G. Magni, M. Zanardelli, E. Fabbretti, C. Verderio, A. M. J. M. van den Maagdenberg, A. Nistri, M. P. Abbracchio. CGRP−mediated enhancement of purinergic neuron/glia communication by the algogenic factor bradykinin in mouse trigeminal ganglia from wild type and R192Q Cav2.1 knock-in mice: implications for basic mechanisms of migraine pain. J Neuroscience, 31(10):3638 –3649, 2011 [3] G. Villa, S. Ceruti, M. Zanardelli, G. Magni, L. Jasmin, P. T. Ohara, M. P. Abbracchio.. Temporomandibular join inflammation activates glial and immune cells in both the trigeminal ganglia and the spinal trigeminal nucleus. Mol Pain, 6:89, 2010.