Background & Aims
Multiple sclerosis (MS) is a debilitating autoimmune disease characterized by inflammatory myelin lesions in the central nervous system, with over 50% of people with MS (pwMS) citing pain as their primary symptom. Current pain management for MS rely on off-label use of drugs and underlying mechanisms contributing to pain in MS remain unclear. Disruptions in circadian rhythms are often reported in pwMS, with long-term shift workers and those living at extreme north/south latitudes being at an increased risk for disease. Furthermore, pwMS often report a diurnal rhythm to disease-related symptoms, including fatigue and pain. We therefore sought to investigate the role of circadian rhythms in MS-related chronic pain using the experimental autoimmune encephalomyelitis (EAE) model.
Methods
C57BL/6J female mice (7-12 weeks old) were induced with EAE using myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freud’s adjuvant (CFA); sham mice received saline instead of MOG35-55. Pain outcomes were assessed at 7, 10, 14, 21 and 28 days post-immunization (dpi) using the von Frey, Hargreaves and acetone assay for mechanical, thermal and cold sensitivity respectively. Pain assays were carried out at multiple timepoints throughout the day (ZT2, 8, 14, and 20; where ZT0 corresponds to lights-on and ZT12 to lights-off). For mice tested only at peak disease, mice were behaviour-tested at ZT2 and 8, given an indomethacin injection at ZT11, and tested again at ZT14. Flow cytometry, qRT-PCR and immunofluorescence were used to characterize immune cell infiltration, neuroinflammation and cell morphology in the lumbar spinal cord. Box-and-hull analysis were used to assess circadian morphological changes in myeloid cells, including circulating monocytes and CNS-resident microglia.
Results
The results show that mechanical hypersensitivity oscillates in a circadian pattern, with EAE mice experiencing greatest sensitivity to mechanical stimuli at ZT8. At peak of disease, pro-inflammatory cytokines IL-1?, TNF-?, IFN-?, IL-6, and CCL2 exhibit a pronounced circadian pattern, with the lowest expression of the cytokines at ZT8. Spinal cord infiltration by activated monocytes and macrophages shows a circadian pattern with its trough at ZT8. Myeloid cell morphology at peak disease also exhibits a circadian rhythm with higher levels of ramification at ZT8 compared to other timepoints. Given the reciprocating circadian oscillations in mechanical hypersensitivity and neuroinflammation, their potential interactions were further validated. After indomethacin injection, EAE mice exhibited increased mechanical hypersensitivity at ZT14, similar to ZT8. Correspondingly, indomethacin injection reduced the inflammatory cytokine expression levels and increased myeloid cell ramifications.
Conclusions
Our data shows, for the first time, that neuropathic pain follows a circadian pattern in EAE and that this response may be regulated by circadian rhythms of neuroimmune interactions. The peak of EAE mimics the relapse episodes in MS, where neuropathic pain worsens, and non-steroid anti-inflammatory drugs are often used to reduce inflammation. We observed a novel mechanism that neuroinflammation masks neuropathic pain and this masking effect could be regulated by circadian rhythms. Thus, interactions between the nervous, immune, and circadian systems merit further investigation to determine whether modulating circadian rhythmicity may provide novel therapeutic targets for pain treatment. Further research may give rise to chronotherapy including light therapy and timed-release treatments, providing economic and effective relief for neuropathic pain.
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