To evaluate the effect of botulinum neurotoxin A (BoNT/A) on prevention of chronic migraine (CM) in mice and explore the potential mechanism.

Twenty-four male C57BL/6 mice were randomly divided into control group, nitroglycerin (NTG) group, and BoNT/A+NTG group (n=8). Mice in the latter two groups were intraperitoneally injected with 10 mg/kg NTG on the 1^st, 3^rd, 5^th, 7^th and 9^th d of experiments to establish CM models. Mice in the BoNT/A+NTG group were injected with 0.18 U/100 μL BoNT/A one h before the first injection of NTG. Mice in the control group were injected with the same dose of normal saline. Basal mechanical withdrawal threshold (MWT) and evoked MWT 2 h after NTG in the facial and hindpaw regions on the 1^st, 3^rd, 5^th, 7^th and 9^th d of experiments were evaluated by von Frey filament test. The motor function of mice 2 h after NTG injection was tested by rotarod test on the 1^st, 3^rd, 5^th, 7^th and 9^th d of experiments. On 9^th d of experiments, the mice were sacrified; the calcitonin gene-related peptide (CGRP), synaptosomal-associated protein 25 (SNAP25), glial fibrillary acidic protein (GFAP) and TRP channel protein expressions in the trigeminal ganglia (TG) and trigeminal nucleus caudalis (TNC), and NOD-like receptor protein 3 (NLRP3) inflammatory factor pathway-related protein expressions in TNC were detected by Western blotting; real-time quantitative PCR (RT-qPCR) was used to detect the NLRP3 inflammatory factor pathway-related mRNA expressions in TNC. The CGRP expression in TNC was detected by immunofluorescent staining.

(1) As compared with the control group, the NTG group had significantly decreased basal facial MWT on the 7^th and 9^th d of experiments (P<0.05); as compared with the NTG group, the BoNT/A+NTG group had significantly increased basal facial MWT on the 7^th and 9^th d of experiments (P<0.05). As compared with the control group, the NTG group had significantly decreased evoked facial MWT on the 5^th and 9^th d of experiments (P<0.05); as compared with the NTG group, the BoNT/A+NTG group had significantly increased evoked facial MWT on the 5^th and 9^th d of experiments (P<0.05). As compared with the control group, the NTG group had significantly decreased basal and evoked MWT in the hindpaw regions on the 3^rd, 5^th, 7^th and 9^th d of experiments (P<0.05); as compared with the NTG group, the BoNT/A+NTG group had significantly increased basal and evoked MWT in the hindpaw regions on the 3^rd, 5^th, 7^th and 9^th d of experiments (P<0.05). (2) There was no significant difference in running time on rotarod among the three groups (P>0.05). (3)Western blotting results showed that as compared with those in the control group, the CGRP and SNAP25 protein expressions were significantly increased in TG of the NTG group (P<0.05); and those in the BoNT/A+NTG group were significantly decreased as compared with those in the NTG group (P<0.05). As compared with those in the control group, the CGRP and NLRP3 protein expressions were significantly increased in TNC of NTG group (P<0.05); and those in the BoNT/A+NTG group were significantly decreased as compared with those in the NTG group (P<0.05). (4)RT-qPCR results showed that as compared with that in the control group, the IL-1β mRNA expression in TNC of the NTG group was significantly increased (P<0.05), and that in the BoNT/A prevention group was statistically decreased as compared with that in the NTG group (P<0.05). (5) Immunofluorescent staining results showed that as compared with that in the control group, the CGRP expression in TNC of the NTG group was significantly increased, and that in the BoNT/A+NTG group was significantly decreased as compared with that in the NTG group (P<0.05).

BoNT/A can reduce the SNAP25 expression in TG, reduce the CGRP release in TG and TNC, and prevent CM onset; BoNT/A can regulate NLRP3 level in TNC.

Chronic migraine; Botulinum neurotoxin A; Calcitonin gene-related peptide; NOD-like receptor protein 3