Botulinum toxin type A attenuates trigeminal neuralgia-like pain by suppressing CGRP release and modulating NaV1.7-associated signaling

BACKGROUND: Trigeminal neuralgia (TN) is a disabling facial pain disorder, and current treatments remain insufficient for a substantial proportion of patients. Botulinum toxin type A (BoNT/A) has shown therapeutic benefit in TN, but its peripheral analgesic mechanisms remain incompletely understood. We aimed to determine whether BoNT/A alleviates TN-like pain by modulating the calcitonin gene-related peptide (CGRP)-extracellular signal-regulated kinase (ERK)-NaV1.7 pathway in trigeminal ganglion neurons. METHODS: TN-like pain was induced in mice by chronic constriction injury of the infraorbital nerve (CCI-ION). On postoperative day 14, BoNT/A (10 U) or saline was injected intradermally into the whisker pad. Evoked and spontaneous pain-like behaviors were assessed using von Frey testing, the Ugo Basile facial pain test, and spontaneous scratching recordings; anxiety-like behavior was evaluated using the elevated plus maze. Myelin pathology was examined by Luxol fast blue staining and transmission electron microscopy. CGRP, NaV1.7/NaV1.8, and ERK/MAPK signaling were examined in trigeminal ganglion neurons using immunofluorescence, Western blotting, RT-qPCR, ELISA, calcium-influx assays, and whole-cell patch-clamp recordings. Olcegepant and PD98059 were used to interrogate CGRP receptor- and ERK-dependent mechanisms. RESULTS: BoNT/A significantly attenuated CCI-ION-induced mechanical allodynia, increasing the facial withdrawal threshold from 0.11 to 1.29 g, and reducing spontaneous pain-like behaviors. BoNT/A also improved anxiety-like behavioral measures in CCI-ION mice. Histological and ultrastructural analyses shown partial amelioration of myelin pathology, accompanied by increased myelin basic protein expression and a lower g-ratio. Mechanistically, BoNT/A reduced CGRP-associated activity in TRPV1-positive trigeminal ganglion neurons, accompanied by lower serum CGRP levels and attenuated capsaicin-evoked calcium influx. These changes were associated with a preferential reduction in NaV1.7 membrane localization, without a comparable reduction in NaV1.8, and with a marked decrease in sodium current density, with peak sodium current reduced by 78.6%. Pharmacological inhibition of CGRP receptors or ERK phosphorylation reproduced the effects of BoNT/A on NaV1.7 membrane localization. CONCLUSIONS: These findings suggest that BoNT/A alleviates CCI-ION-induced TN-like pain, at least in part, by suppressing CGRP-dependent ERK activation and subsequent NaV1.7 membrane recruitment in trigeminal ganglion neurons. The CGRP-ERK-NaV1.7 pathway may represent a peripheral mechanism of BoNT/A-mediated analgesia and a potential target for neuropathic facial pain. CLINICAL TRIAL NUMBER: Not applicable.