7-Nitroindazole, an nNOS inhibitor, reduces migraine-like nociception, demyelination, and anxiety-like behavior in a mouse model of relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is a complex neuroinflammatory disease often associated with migraine and anxiety, both of which impair quality of life. MS pathology involves intense inflammatory and oxidative processes, including increased nitric oxide (NO) production. However, the role of NO in MS-related migraine symptoms remains unclear. This study evaluated whether repeated administration of 7-nitroindazole (7-NI), a selective neuronal nitric oxide synthase (nNOS) inhibitor, could alleviate migraine-like nociception, anxiety-like behavior, and neuroinflammatory biomarkers in a relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) mouse model. RR-EAE was induced in female C57BL/6 mice (20–30 g) using myelin oligodendrocyte glycoprotein (MOG35-55) and Quillaja saponin as an adjuvant. Mice received daily intragastric 7-NI (120 mg/kg) from day 20–35 post-induction. Disease progression, mechanical/spontaneous allodynia, and anxiety-like behavior were assessed. At the end of the protocol, oxidative and inflammatory biomarkers were analyzed. 7-NI treatment significantly reduced disease severity and nociception, exerted an anxiolytic effect, and improved myelin quality parameters. It inhibited the increase of oxidative and nitrosative markers (NOx, H 2 O 2 ) in the brainstem, trigeminal ganglion, and plasma. Treatment also prevented plasma calcitonin gene-related peptide elevation and increased anti-inflammatory cytokines (IL-4, IL-10), suggesting positive modulation of neuroinflammation in RR-EAE. These findings highlight the therapeutic potential of 7-NI in MS; however, further studies are required to confirm its safety and efficacy in different populations and chronic disease contexts. • 7-NI reduced migraine-like pain and anxiety in the RR-EAE mouse model. • 7-NI increased IL-4 and IL-10 while preventing plasma CGRP elevation. • IL-4 and IL-10 increased, while plasma CGRP elevation was prevented. • 7-NI shows therapeutic potential for MS-related pain and inflammation.