Intestinal Mucosal Alterations Parallel Central Demyelination and Remyelination: Insights into the Gut-Brain Axis in the Cuprizone Model of Multiple Sclerosis

Carolina Ferreira^1,2,3,4Filipa Carvalho^1,2,3,4Pedro Vieira^1,2,3,4,5André Alves^1,2,3,4Filipe Palavra^1,3,4,6Jani Almeida^2,3,4,7Vera Alves^2,3,8Ezequiel R. Coscueta⁹Patrícia Dias Pereira^10,11Maria Manuela Estevez Pintado⁹Helena Sá^12,13,2,3Miguel Castelo-Branco^14,15,4Flávio Reis^1,2,3,4Sofia Viana^1,2,3,4,5*

• ¹Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
• ²Universidade de Coimbra Instituto de Investigacao Clinica e Biomedica de Coimbra, Coimbra, Portugal
• ³Universidade de Coimbra Centro de Inovacao em Biomedicina e Biotecnologia, Coimbra, Portugal
• ⁴Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal, coimbra, Portugal
• ⁵Instituto Politecnico de Coimbra Escola Superior de Tecnologia da Saude de Coimbra, Coimbra, Portugal
• ⁶Centre for Child Development - Neuropediatrics Unit, Hospital Pediátrico, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal, coimbra, Portugal
• ⁷Institute of Immunology, Faculty of Medicine (FMUC), University of Coimbra, Coimbra, Portugal, coimbra, Portugal
• ⁸7Institute of Immunology, Faculty of Medicine (FMUC), University of Coimbra, coimbra, Portugal
• ⁹Universidade Catolica Portuguesa Centro de Biotecnologia e Quimica Fina, Porto, Portugal
• ¹⁰Universidade do Porto Instituto de Ciencias Biomedicas Abel Salazar, Porto, Portugal
• ¹¹REQUIMTE LAQV Porto, Porto, Portugal
• ¹²Institute of Immunology, Faculty of Medicine (FMUC), University of Coimbra, coimbra, Portugal
• ¹³Nephrology Department, Centro Hospitalar e Universitário de Coimbra, ULS Coimbra, Coimbra, Portugal
• ¹⁴Universidade de Coimbra Instituto de Ciencias Nucleares Aplicadas a Saude, Coimbra, Portugal
• ¹⁵Institute of Physiology, Faculty of Medicine, University of Coimbra, coimbra, Portugal

Abstract Background: The gut-brain axis has been increasingly recognized as a critical factor in Multiple Sclerosis (MS) pathophysiology. While its role in demyelination is well documented, gut-brain axis involvement during remyelination remains largely unexplored. Methods: Using the cuprizone (CPZ) model, which induces reversible demyelination and spontaneous remyelination upon toxin withdrawal, we investigated gut and brain changes during both disease stages in C57BL/6 mice. Animals were administered 0.2% cuprizone for 5 weeks to induce demyelination, followed by a 2-week recovery phase. Intestinal changes were evaluated through 1) gut microbiota profiling and metabolite production (short-chain fatty acids (SCFAs), indoxyl sulfate), 2) structural and barrier integrity via histology, mucus staining, and tight junction markers (ZO-1, occludin, claudin-5), 3) mucosal immunity through M1/M2 macrophage profiling and Th17/Treg ratios, and 4) expression of inflammatory and oxidative stress markers. Differences in brain demyelination/remyelination, gliosis and related molecular changes were determined using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). Results: The demyelination peak was characterized by reduced abundance of SCFA-producing genus Akkermansia and Dubosiella, increased intestinal permeability at the level of the mucus layer and tight junction networks, and shifts in mucosal immunity toward a pro-inflammatory state characterized by M1 macrophages and Th17 cell expansion together with elevated levels of inflammatory cytokines (IL-17, IL-1β) and changes in oxidative stress-related enzymes (iNOS, HO-1, SOD1/2), all of which were partially reversed during the remyelination phase. Centrally, cuprizone-induced demyelination/remyelination and gliosis showed region-specific patterns. Neuroinflammation peaked during demyelination (TNF-α, IL-1β, IL-6, IL-17) and only partially resolved, suggesting that a balanced inflammatory response may aid remyelination. Conclusion: Our findings reveal that cuprizone-induced intestinal dysfunctions temporally parallel central nervous system (CNS) lesion dynamics, disclosing temporal coordination of both compartments and highlighting gut-brain axis impact on both disease stages.