Background

Although there is strong evidence for an excitotoxic component to myelin injury in multiple sclerosis, functional protection against demyelination by glutamate receptor antagonists is controversial.

Objectives

To deploy a novel medium-throughput screen to generate dose-response curves for myelin protection by NMDA- and AMPA-type glutamate antagonists and probe for synergy between these drugs at low concentrations.

Methods

An in vitro super-acute myelin protection assay based upon isolated brain slice and optic nerve exposure to cuprizone was used to test the protective properties of several experimental and clinically approved AMPA and NMDA receptor antagonists. A standard EAE model of multiple sclerosis was used to examine one paradigm.

Results

Both AMPA and NMDA receptor antagonists protected central myelin from acute cuprizone-mediated injury in a concentration-dependant manner. When these two drug types were combined at concentrations 1-2 order lower than the minimally effective concentrations required when applied alone, significant protection was achieved. In the case of clinically approved drugs, an effective combined concentration was ~2 orders of magnitude below the normal treatment doses (200 nM perampamel + 500 nM memantine). One combined low-dose regiment (1 mg/Kg CP465022 + 2 mg/Kg QNZ-46) was tested in a standard EAE model of disease and produced significant levels of functional and clinical protection.

Conclusions

Super-low combinations of AMPA and NMDA antagonists were identified as a potential therapy using a novel myelin protection assay. The approach was confirmed in an in vivo model of disease. These very low doses of clinically approved medicines will have a low barrier to translation as they work at much lower doses to those used in current clinical practice in the treatment of non-demyelinating disorders.

Background

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

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

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

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

Conclusions: A rapid screen for protection against early myelin pathology has the potential to accelerate drug testing in multiple sclerosis.

Background

Although there is strong evidence for an excitotoxic component to myelin injury in multiple sclerosis, functional protection against demyelination by glutamate receptor antagonists is controversial.

Objectives

To deploy a novel medium-throughput screen to generate dose-response curves for myelin protection by NMDA- and AMPA-type glutamate antagonists and probe for synergy between these drugs at low concentrations.

Methods

An in vitro super-acute myelin protection assay based upon isolated brain slice and optic nerve exposure to cuprizone was used to test the protective properties of several experimental and clinically approved AMPA and NMDA receptor antagonists. A standard EAE model of multiple sclerosis was used to examine one paradigm.

Results

Both AMPA and NMDA receptor antagonists protected central myelin from acute cuprizone-mediated injury in a concentration-dependant manner. When these two drug types were combined at concentrations 1-2 order lower than the minimally effective concentrations required when applied alone, significant protection was achieved. In the case of clinically approved drugs, an effective combined concentration was ~2 orders of magnitude below the normal treatment doses (200 nM perampamel + 500 nM memantine). One combined low-dose regiment (1 mg/Kg CP465022 + 2 mg/Kg QNZ-46) was tested in a standard EAE model of disease and produced significant levels of functional and clinical protection.

Conclusions

Super-low combinations of AMPA and NMDA antagonists were identified as a potential therapy using a novel myelin protection assay. The approach was confirmed in an in vivo model of disease. These very low doses of clinically approved medicines will have a low barrier to translation as they work at much lower doses to those used in current clinical practice in the treatment of non-demyelinating disorders.

Background

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

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

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

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

Conclusions: A rapid screen for protection against early myelin pathology has the potential to accelerate drug testing in multiple sclerosis.