Physiological actions of a humanized P2X4 scFv on peripheral and central neurons in male mice with neuropathic pain.

Neuropathic pain remains a challenging clinical condition due to its resistance to conventional analgesics. The purinergic P2X4 receptor (P2X4R), an ATP-gated ion channel, is upregulated in sensory neurons and glial cells following nerve injury and is pivotal in chronic pain pathogenesis. This study evaluates the therapeutic potential of a novel humanized single-chain variable fragment antibody (hP2X4R scFv) targeting P2X4R in male mice models of neuropathic pain. Using spared nerve injury (SNI) and foramen rotundum inflammatory compression of the trigeminal infraorbital nerve (FRICT-ION) models, we demonstrate that a single intraperitoneal dose of hP2X4R scFv significantly reverses mechanical hypersensitivity for up to four weeks. Electrophysiological recordings from FosTRAP mice revealed that hP2X4R scFv reduced the excitability of Fos+ neurons in the spinal dorsal horn and ventrolateral periaqueductal gray (vlPAG), key regions involved in pain processing. In vitro, patch-clamp studies further showed that hP2X4R scFv selectively decreased action potential firing in larger diameter dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons from SNI and FRICT-ION mice, respectively, without affecting naïve neurons. These findings suggest that hP2X4R scFv modulates both central and peripheral neuronal excitability associated with chronic pain. The specificity and long-lasting efficacy of hP2X4R scFv highlights its promise as a non-opioid therapeutic candidate for neuropathic pain management.