Optic nerve myelination analysis

PRIMARY OUTCOME

G-ratio of optic nerve axons

EXPECTED OUTCOMES

## Expected Outcomes ### Primary Outcomes 1. **Axon loss:** 15-30% reduction in total optic nerve axon count in Gabra1/Gabrg2 mutants vs WT 2. **Myelin thinning:** Decreased myelin sheath thickness, elevated G-ratio (axon diameter / fiber diameter ratio approaches 1.0) 3. **Demyelination:** Increased percentage of unmyelinated axons in mutants 4. **Ultrastructural defects:** Myelin lamellar loosening, vacuolization, or axon degeneration ### Secondary Outcomes - Correlation between genotype and optic nerve function (if ERG performed) - Age-dependent progression of pathology - Sex-specific differences in severity ### Null Result Interpretation - If no axon loss at 12 weeks, examine younger (4-8 weeks) and older (24 weeks) timepoints - May need to examine optic nerve crush injury response (regeneration capacity) - Verify genotype by sequencing to confirm mutation

SUCCESS CRITERIA

## Success Criteria ### Primary - [ ] ≥6 animals per genotype yield analyzable histology - [ ] Axon count ≥ 500 per optic nerve cross-section - [ ] G-ratio significantly different between genotypes (p < 0.05) - [ ] Myelin thickness ≥10% difference between groups ### Secondary - [ ] Electron microscopy quality: clear myelin lamellae at 20,000x - [ ] Correlation between genotype and pathology severity - [ ] Sex included as biological variable in analysis ### Technical Quality Gates - [ ] Consistent sectioning depth (2 mm from globe) across all samples - [ ] Imaging parameters identical across all specimens - [ ] Blinded analysis: analyst unaware of genotype during quantification - [ ] Random sampling for axon measurements (systematic random sampling)

PROTOCOL

## Protocol: Optic Nerve Myelination Analysis in Gabra1/Gabrg2 Mutant Mice ### Study Design Histological and ultrastructural analysis of optic nerve myelination in Gabra1+/-/Gabrg2+/- mice (model of epilepsy with optic atrophy) compared to WT littermates. ### Animals and Tissue Preparation 1. Gabra1+/-/Gabrg2+/- mutant mice (n=8 per genotype) and WT controls (n=8) 2. At 12 weeks (adult), perfuse transcardially with 4% PFA/PBS 3. Dissect optic nerves, post-fix in 2% glutaraldehyde/2% PFA overnight at 4°C 4. Osmium tetroxide post-fixation: 1% OsO4 in 0.1M cacodylate buffer for 2 hours 5. Dehydrate through graded ethanol series (50%, 70%, 90%, 100%) 6. Embed in Epon resin for semithin (0.5 µm) and ultrathin (70 nm) sectioning ### Light Microscopy (Semithin Sections) 1. Collect 0.5 µm transverse sections of optic nerve at 2 mm posterior to globe 2. Stain with 1% toluidine blue for myelinated axon visualization 3. Image at 100x oil immersion (Zeiss AxioImager) 4. Analyze using FIJI/ImageJ: - Total axon count per optic nerve cross-section - G-ratio calculation (axon diameter / fiber diameter) for ≥200 axons - Myelin thickness measurement ### Transmission Electron Microscopy (Ultrathin Sections) 1. Collect 70 nm sections on copper grids, contrast with uranyl acetate and lead citrate 2. Image at 5,000x for axon density, 20,000x for myelin ultrastructure 3. Assess: - Myelin sheath thickness (10 measurements per axon) - G-ratio (axon caliber / total fiber caliber) - Number of myelinated vs unmyelinated axons - Myelin compaction (major dense line resolution) ### Controls - **Positive control:** Age-matched WT C57BL/6J optic nerve (normal myelination) - **Negative control:** No primary antibody control for IHC - **Technical replicates:** 3 sections per animal, 3 animals per group initially ### Expected Outcomes 1. Reduced total axon number in Gabra1/Gabrg2 mutants (15-30% loss expected) 2. Decreased myelin thickness and altered G-ratio (higher G-ratio = thinner myelin) 3. Increased proportion of unmyelinated axons in mutants 4. Ultrastructural myelin abnormalities (loosening, vacuolization) ### Success Criteria - [ ] ≥6 animals per genotype with analyzable optic nerve sections - [ ] Axon density: count ≥ 500 axons per optic nerve cross-section - [ ] G-ratio: mean ± SD reported, significant difference if p < 0.05 - [ ] Myelin thickness: ≥10% difference between genotypes - [ ] Electron microscopy: clear myelin lamellae visible, no imaging artifacts - [ ] Blinded quantification throughout

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