EPSILON TOXIN–INDUCED DEMYELINATION IN THE MOUSE CORPUS CALLOSUM: IN VITRO AND IN VIVO EVIDENCE SUPPORTING A LINK TO MULTIPLE SCLEROSIS

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, can cross the blood–brain barrier and exert cytotoxic effects. In vitro studies have shown that ETX binds to myelin structures in mice and other species, including humans, inducing demyelination in mouse cerebellar organotypic cultures. Together with the detection of ETX antibodies in the sera of patients with multiple sclerosis (MS), these findings suggest a potential link between ETX and MS. The myelin and lymphocyte (MAL) protein is considered its putative receptor.
This study aimed to investigate the demyelinating effects of ETX in the mouse corpus callosum using in vitro and in vivo models. For in vitro analyses, fresh adult mouse brain slices were incubated with 150 nM ETX or its non-toxic prototoxin (pETX) for different period of time, followed by fixation and immunohistochemical analysis.
For in vivo experiments, mice received intraperitoneal injections of ETX or pETX (300 ng/kg) for three consecutive days, followed by a 15-day resting period. This cycle was repeated one to three times, resulting in exposure periods of up to three months. Locomotor activity was assessed at the end of the experiment, after which brains were collected for immunohistochemistry.
In vitro, MAG immunolabeling revealed morphological alterations at the oligodendrocyte–neuron interface. In vivo, ETX-treated mice exhibited locomotor impairment and a reduction in the myelin markers, along with increased astrocytic and microglial markers indicative of neuroinflammation. These findings demonstrate that ETX induces oligodendrocyte damage and demyelination, supporting its potential involvement in MS pathogenesis.