Botulinum toxin type A (BoNT-A) induces a bilateral analgesic effect following unilateral injection in rodent bilateral or mirror pain models. This occurs either by indirect plasticity-related actions, or by the toxin’s direct central action in bilateral spinal circuits. Herein, we aimed to resolve this question by assessing the role of trans-synaptic toxin traffic in a bilateral inflammatory pain model. The analgesic effect of the toxin was examined in rats pre-treated with unilateral intraplantar BoNT-A (7 U/kg) and subsequently challenged with bilateral carrageenan-evoked hind-paw inflammation (2%, 50 µL/paw, 6 days post BoNT-A). Specific neutralizing antitoxin injected into the lumbar intrathecal space (2 IU, 24 h post BoNT-A), aimed at preventing the spinal trans-synaptic traffic of BoNT-A, abolished its bilateral analgesic effect. The toxin trans-synaptic effect was associated with reduced c-Fos neuronal activation and BoNT-A-mediated cleavage of synaptosomal-associated protein 25 (SNAP-25) in the bilateral dorsal horn. Here, we showed that, in bilaterally occurring pain, BoNT-A exerts a direct contralateral analgesic action extending beyond the level of the dorsal root ganglion sensory neuron that directly links the hindlimb injection site to the primary sensory region. This points to the crucial role of the toxin’s central trans-synaptic traffic, and its direct action at propriospinal nociceptive circuits in its pain-relieving efficacy.

We previously demonstrated that botulinum neurotoxin A (BoNT-A) exerts bilateral antinociceptive effects, involving trans-synaptic transport at the level of the lumbar spinal cord. However, the potential distribution of the toxin to supraspinal sites has not yet been investigated. In the present study, we examined the distribution of cleaved SNAP-25 (cl-SNAP-25), a marker of BoNT-A activity, in the rat brain following peripheral unilateral BoNT-A administration. Brain tissues from rats treated with BoNT-A (7 U/kg, into the hind paw) were analyzed using immunofluorescent tyramide signal amplification to detect cl-SNAP-25. To assess the contribution of trans-synaptic transport, a BoNT-A-neutralizing antitoxin (2 IU) was administered intrathecally 24 h after BoNT-A injection. Signal intensity was evaluated using a semi-quantitative immunohistochemical scoring method based on cl-SNAP-25-positive nerve fibers. Bilateral cl-SNAP-25 immunoreactivity was observed in multiple supraspinal regions, most prominently within the trigeminal complex and the facial and gracile nuclei. Signal intensity was significantly reduced by intrathecal antitoxin, indicating that trans-synaptic transport contributes to central BoNT-A distribution. Peripherally administered BoNT-A reaches distant supraspinal regions, possibly via neuronal retrograde and trans-synaptic transport. Further studies are warranted to clarify exact pathways and alternative distribution routes, determine the functional relevance of central BoNT-A presence, and assess its clinical implications.