Background: Development of new therapeutic approaches for the treatment of multiple sclerosis is hampered by the cost, time and ethical implications of the in vivo models available. Development of a medium-throughput in vivo screens would significantly accelerate drug development and provide useful dose-protection information.
Objectives: Develop a medium-throughput screen to allow the generation of dose-response curves for myelin protection over a range of drug candidate concentrations. Both immune-mediated and non-immune-mediated pathways will be modelled.
Methods: Following humane killing, live mouse brain sections are maintained under physiological conditions and myelin stained with the vital dye FluoroMyelin Red. Sections are then hemi-sected, transferred to neighbouring well-plates and exposed to either cuprizone or lipopolysaccharide (LPS). One of each pair of hemi-sections was exposed to a test agent, the other to vehicle control: agents were applied to the sections simultaneous to the period of myelin challenge. After the period of challenge, sections were PFA fixed and prepared for laser-scanning confocal imaging where the entire corpus callosum was image-tiled. Myelin staining was quantified in each hemi-section and intensity between vehicle and test conditions examined blind via paired statistical analysis. The injury produced by the myelin challenge was also examined via transmission electron microscopy.
Results: In vitro exposure to either LPS or cuprizone produced a significant loss of myelin staining from the corpus callosum. For the cuprizone model, ultrastructural analysis revealed an increase in myelinated axon g-ratio and myelin blebbing consistent with early features of demyelination. There was no significant mitochondrial swelling or pathology in glial soma. One novel drug treatment (ultra-low dose combined AMPA and NMDA receptor block) was identified using the assay and subsequently confirmed using a standard EAE model.
>Four slices were generated from each mouse brain, yielding 4 pairs of hemi-sections. The optic nerves were also suitable for this assay and yielded similar results, increasing the potential number of assays per mouse. The variability in the data required 5 hemi-section pairs for a meaningful level of sensitivity to drug protection, which can be taken from dissection to analysis in one day.
Conclusions: A rapid screen for protection against early myelin pathology has the potential to accelerate drug testing in multiple sclerosis.