Myelin pathology in white matter tracts following chronic intermittent ethanol exposure in mice

Background: In the vertebrate brain, myelin sheaths accelerate action potential propagation. Chronic alcohol exposure disrupts myelin gene/protein expression and reduces white matter integrity in humans. This study investigated the effects of chronic intermittent ethanol (CIE) exposure on myelin ultrastructure in three mouse brain white matter tracts.

Methods: Electron microscopy was used to assess myelin sheath ultrastructure in the corpus callosum (cc), stria medullaris (sm), and anterior commissure (ac) of two C57BL/6J male mice exposed to six nonconsecutive weeks of CIE vapor inhalation (n=1) or air only (n=1). Each CIE week involved four cycles of 16-hour intoxication followed by 8-hour withdrawal and a final 72-hour withdrawal. Each 16-h period of ethanol vapor exposure was primed with an i.p. injection of ethanol (1.5 g/kg) to initiate intoxication and pyrazole (1 mmol/kg) to normalize ethanol clearance rate between individual mice. The air-exposed control mouse received pyrazole only. During vapor exposure, the CIE mouse had an average blood alcohol level of 199.0 ± 15.1 mg/dL. Mice were perfused five days after the final withdrawal and immersed in fixative for 3 weeks. Brain punches containing the cc, ac, and sm were processed for electron microscopy imaging at 5300x magnification, and axons >0.3 μm in diameter were analyzed for myelination status and pathologies using Fiji software.

Results: CIE exposure induced significant myelin pathology across all examined white matter tracts including splitting myelin, uncompacted myelin, degenerating axons, and electron-dense bodies. Degenerating axons were the most frequent pathology in the cc of the CIE mouse, with a mean relative frequency of 1.00 ± 0.00 compared to 0.21 ± 0.01 in the air-exposed mouse. Splitting myelin was elevated in the ac and sm of the CIE mouse, with relative frequencies of 1.00 ± 0.00 vs. 0.23 ± 0.02 (ac) and 0.21 ± 0.01(sm) in the air-exposed mouse. Electron-dense bodies were observed in both groups but were more frequent in the CIE mouse.

Conclusions: These findings demonstrate that CIE exposure disrupts myelin ultrastructure, with degenerating axons as the most frequent pathology. This effect may stem from intoxication, withdrawal, or both. Despite the small sample size, this study highlights the tract-specific impact of ethanol exposure on white matter integrity. Further research should explore whether these findings are consistent across larger cohorts, sexes, and additional brain regions to clarify the mechanisms underlying ethanol-induced myelin damage.