Pannexin 1 (panx1) is a large-pore membrane channel expressed in many tissues of mammals, including neurons and glial cells. Panx1 channels are highly permeable to calcium and adenosine triphosphatase (ATP); on the other hand, they can be opened by ATP and glutamate, two crucial molecules for acute and chronic pain signaling in the spinal cord dorsal horn, thus suggesting that panx1 could be a key component for the generation of central sensitization during persistent pain. In this study, we examined the effect of three panx1 blockers, namely, 10panx peptide, carbenoxolone, and probenecid, on C-reflex wind-up activity and mechanical nociceptive behavior in a spared nerve injury neuropathic rat model involving sural nerve transection. In addition, the expression of panx1 protein in the dorsal horn of the ipsilateral lumbar spinal cord was measured in sural nerve-transected and sham-operated control rats. Sural nerve transection resulted in a lower threshold for C-reflex activation by electric stimulation of the injured hindpaw, together with persistent mechanical hypersensitivity to pressure stimuli applied to the paw. Intrathecal administration of the panx1 blockers significantly depressed the spinal C-reflex wind-up activity in both neuropathic and sham control rats, and decreased mechanical hyperalgesia in neuropathic rats without affecting the nociceptive threshold in sham animals. Western blotting showed that panx1 was similarly expressed in the dorsal horn of lumbar spinal cord from neuropathic and sham rats. The present results constitute the first evidence that panx1 channels play a significant role in the mechanisms underlying central sensitization in neuropathic pain.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.

A well-recognized molecular entity involved in pain-related neuroplasticity is the N-methyl-D-aspartate receptor (NMDAR), which is crucial for developing chronic pain. Likewise, the pannexin 1 (Panx1) channel has been described as necessary for initiating and maintaining neuropathic pain, driving nociceptive signals dependent on spinal NMDAR through several possible mechanisms. Through behavioral, pharmacological, and molecular approaches, our study in male rats has revealed several key findings: (1) neurons located in spinal cord laminae I and II express functional Panx1 channels in both neuropathic and sham rats. These channels can open (indicated by YOPRO-1 uptake) through the stimulation of NMDARs with intrathecal NMDA; (2) intrathecal NMDA leads to increased expression of pSrc and pPanx1 in dorsal horn neurons. This elevation exacerbates existing mechanical hyperalgesia in nerve-injured rats; (3) inhibition of Src with intrathecal PP2 or blockade of Panx1 with intrathecal ¹⁰Panx effectively mitigates NMDA-induced effects and reduces the spontaneous mechanical hyperalgesia of nerve-injured rats. Notably, while ¹⁰Panx successfully alleviates hyperalgesia, it does not alter pSrc expression; and (4) NMDA-stimulated YOPRO-1 uptake in neurons of laminae I-II of spinal cord slices were prevented by the NMDAR antagonist D-AP5, the Src inhibitor PP2 (but not PP3), as well as with the ¹⁰Panx and carbenoxolone. Therefore, NMDAR activation in dorsal horn neurons triggers an NMDAR-Src-Panx1 signaling pathway, where Panx1 acts as an enhancing effector in neuropathic pain. This implies that disrupting the NMDAR-Panx1 communication (eg, through Src inhibitors and/or Panx1 blockers) may offer a valuable strategy for managing some forms of chronic pain.