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Methotrexate neurotoxicity: in vitro studies using cerebellar explants from rats
M R Gilbert1, B L Harding, S A Grossman
1Department of Neurology, Johns Hopkins Hospital, Baltimore, Maryland 21205.
Abstract:
The mechanism of methotrexate (MTX)-induced neurotoxicity was investigated using cerebellar explant cultures from fetal rats. After 3 weeks of growth, myelinated cultures were treated with MTX at 1 microM, lysolecithin at 1 mg/dl, or unaltered nutrient medium. Myelin sheaths devoid of axons were observed by histological and electron microscopic preparations after 2 weeks of MTX exposure. After 5 weeks, cultures were almost entirely devoid of myelin sheaths. Myelin basic protein in the media removed from the cultures showed an increase in concentration after 3 weeks of MTX exposure and was significantly greater than control after 5 weeks of exposure. 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity, a measure of oligodendroglial function, was not significantly different in the MTX group compared to controls. Lysolecithin-treated cultures showed widespread destruction and an early increase in myelin basic protein release into the medium. These data indicate that, in the cerebellar explant cultures, MTX is primarily a neuronal toxin, and the demyelination is a consequence of axonal loss and is not related to a change in oligodendroglial cell function. These findings provide new insight into the pathogenesis of MTX-induced neurotoxicity.
Insights
Methotrexate (MTX) causes neurotoxicity by damaging neurons, leading to secondary demyelination. This study found MTX-induced demyelination in rat cerebellar cultures is due to axonal loss, not direct harm to myelin-producing cells.
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Methotrexate (MTX) is a chemotherapy agent with known neurotoxic side effects.
- The precise mechanism underlying MTX-induced neurotoxicity, particularly demyelination, remains incompletely understood.
Purpose of the Study:
- To investigate the mechanism of methotrexate (MTX)-induced neurotoxicity in cerebellar explant cultures.
- To determine if MTX directly affects oligodendroglial function or causes demyelination secondary to neuronal damage.
Main Methods:
- Primary cerebellar explant cultures from fetal rats were established and myelinated.
- Cultures were treated with MTX (1 microM) or lysolecithin (1 mg/dl) for up to 5 weeks.
- Histological, electron microscopy, myelin basic protein (MBP) quantification, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity assays were performed.
Main Results:
- MTX treatment led to progressive loss of myelin sheaths, with axons becoming denuded after 2 weeks and cultures largely demyelinated by 5 weeks.
- Increased myelin basic protein was detected in the media of MTX-treated cultures, indicating myelin breakdown.
- Oligodendroglial function, assessed by CNPase activity, showed no significant difference between MTX-treated and control cultures.
- Lysolecithin, a known myelinotoxic agent, caused rapid demyelination and increased MBP release.
Conclusions:
- Methotrexate (MTX) acts primarily as a neuronal toxin in this model, causing axonal damage.
- Demyelination observed in MTX-treated cultures is a secondary consequence of axonal loss, not a direct effect on oligodendrocytes.
- These findings offer new insights into the pathogenesis of MTX-induced neurotoxicity, highlighting neuronal injury as the primary event.