Background

Multiple sclerosis (MS) is an autoimmune disorder with over 8 million individuals diagnosed [1]. Current treatments rely on immunosuppressive disease modifying therapies; however, these therapies have limited therapeutic efficacy and significant side effects [2–4].

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of MS extensively used in the field to investigate disease pathophysiology and advance therapeutic discovery and curative strategies [5–9]. EAE mimics key MS pathophysiology and successfully recapitulates the hallmarks of the disease—demyelination, neuroinflammation, gliosis, and axonal damage [6,9–12]. Moreover, it also replicates the two prominent symptoms of MS—neuropathic pain and motor deficits [13–15]. Most often, active EAE induction in C57BL/6 mice requires immunization with the myelin oligodendrocyte glycoprotein (MOG_35-55) peptide emulsified in complete Freund’s adjuvant (CFA) and heat-killed Mycobacterium tuberculosis, alongside co-administration of pertussis toxin (PTX) [6,7,16].

PTX is a bacterial exotoxin that facilitates disease induction by artificially disrupting the blood–brain barrier (BBB), enabling CNS infiltration of peripheral immune cells [17,18]. However, apart from its synthetic effect on immune cell infiltration in the CNS, it also introduces several confounding variables. PTX disrupts G protein-coupled receptor (GPCR) signaling through ADP-ribosylation, complicating studies that seek to understand GPCR function in neuroinflammation and immune regulation, which is relevant given the growing recognition of GPCRs in MS pathogenesis [19–22].

PTX’s broad immunomodulatory properties extend beyond adjuvant effects. It alters the immune landscape through suppression of regulatory T cells and skews T helper responses toward pathogenic Th1 and Th17 phenotypes, which artificially exacerbates the pro-inflammatory effect of EAE pathology [23–26]. Additionally, it has also been shown to mask sex differences that are prevalent in MS pathology [27]. Together, these factors limit the translational relevance of PTX-based EAE models and warrant the use of alternative protocols that better preserve physiological immune responses.

Recent work from our lab demonstrates that EAE can be reliably induced in C57BL/6 mice without PTX, using optimized antigen and adjuvant conditions [27–31]. This non-PTX EAE model recapitulates key clinical and pathological features of MS, including motor deficits, neuropathic pain, demyelination, neuroinflammation, gliosis, and axonal loss, without relying on artificial BBB disruption. Importantly, omitting PTX reveals biologically meaningful sex-specific differences in disease onset, immune activation, and neurodegeneration that are often masked in traditional PTX-dependent EAE models [27,31].

Increase in myelin and axonal pathology, immune cell infiltration, and glial activation in the brain and lumbar spinal cord have been assessed in our model using immunohistochemistry, western blotting, and flow cytometry [27,29,30]. Moreover, in our non-PTX EAE model, females develop paresis significantly earlier than males; however, immunopathological analyses revealed heightened glial and immune activation in EAE males, mirroring the clinical trajectory of MS. Specifically, male EAE animals exhibited significantly greater CD45⁺ leukocyte and peripheral macrophage infiltration in the lumbar spinal cord relative to female EAE mice [27].

Given the increasing interest in modeling MS pathophysiology in a more biologically and translationally relevant manner, the non-PTX EAE model offers a powerful platform for mechanistic studies, therapeutic testing, and the investigation of sex as a biological variable. In this paper, we describe a detailed and reproducible protocol for non-PTX EAE induction in C57BL/6 mice, along with phenotypic readouts and histopathological assessments that validate the model’s utility in capturing the heterogeneity of MS.