TREM2 KO amyloid pathology study

EXPECTED OUTCOMES

## Expected Outcomes ### Primary Outcomes 1. **Increased amyloid burden in TREM2-/- mice:** TREM2 KO mice are expected to show 30–60% greater plaque coverage vs TREM2+/+ 5xFAD mice at 6 months based on published literature (Wang et al. 2015; Jay et al. 2015) 2. **Increased plaque number and size:** TREM2 KO expected to increase diffuse/immature plaque count; compaction (Thioflavin-S) may be reduced 3. **Accelerated temporal progression:** Differences expected to be larger at 9 months than 6 months ### Secondary Outcomes - TREM2 KO mice may show increased soluble Aβ42/Aβ40 ratio in brain lysates - Reduced microglial clustering around plaques in TREM2-/- (assessed by IBA1 co-staining) - No gross phenotypic differences between groups expected ### Negative/Null Result Interpretation - If no difference observed, may indicate TREM2 role is pathway-specific or age-dependent - Null result at 6 months should prompt re-analysis at 9+ months

SUCCESS CRITERIA

## Success Criteria ### Primary - [ ] ≥30% increase in cortical Aβ % area coverage in 5xFAD/TREM2-/- vs 5xFAD/TREM2+/+ (p < 0.05) - [ ] ≥30% increase in hippocampal Aβ % area coverage (p < 0.05) - [ ] Effect reproduces in both 6-month and 9-month cohorts ### Secondary - [ ] Thioflavin-S+ plaque density quantified in both groups - [ ] IBA1 microglial density around plaques quantified - [ ] All controls pass QC (positive shows >10% area, negative shows <0.5% area) ### Technical Quality Gates - [ ] ≥8/10 animals per group yield analyzable histology sections - [ ] Genotyping confirmed for 100% of animals included in analysis - [ ] Antibody staining variability (CV) < 15% across sections from same animal - [ ] Blinded analysis performed (analyst unaware of genotype during quantification)

PROTOCOL

## Protocol: TREM2 KO Amyloid Pathology Validation Study ### Study Design Parallel-group comparison of 5xFAD transgenic mice with and without TREM2 knockout (5xFAD/TREM2-/- vs 5xFAD/TREM2+/+) to assess amyloid burden. ### Animals and Genotyping 1. Use 5xFAD/TREM2-/- double mutant mice (n=10 per group) and 5xFAD/TREM2+/+ controls (n=10 per group) 2. Confirm genotype by PCR from tail biopsies at weaning (3 weeks) 3. Harvest tissue at 6 months (peak amyloid deposition) and 9 months (severe pathology) 4. Equal sex distribution per group (5M/5F) ### Tissue Collection and Preparation 1. Perfuse mice transcardially with ice-cold PBS followed by 4% paraformaldehyde 2. Dissect and post-fix brains in 4% PFA overnight at 4°C 3. Cryoprotect in 30% sucrose/PBS for 48 hours 4. Section coronally at 30 µm using a cryostat; collect serial sections through hippocampus and cortex 5. Store sections at -20°C in cryoprotectant until use ### Amyloid Burden Quantification **Immunohistochemistry (IHC):** 1. Block sections in 5% normal goat serum / 0.3% Triton X-100 / PBS for 1 hour 2. Incubate with anti-Aβ antibody (clone 6E10, 1:1000) overnight at 4°C 3. Apply biotinylated secondary antibody (1:500) for 2 hours RT 4. Develop using ABC kit and DAB chromogen 5. Counterstain with hematoxylin **Thioflavin-S Staining (compact plaque assessment):** 1. Incubate sections in 0.5% Thioflavin-S in 50% ethanol for 8 min 2. Differentiate in 70% ethanol (2x 5 min) 3. Mount with aqueous mounting medium ### Image Analysis 1. Acquire whole-cortex and hippocampus images at 10x using a slide scanner 2. Use FIJI/ImageJ to quantify % area covered by Aβ immunoreactivity (threshold-based) 3. Quantify plaque number, average plaque size, and total plaque area per ROI 4. Analyze 5 sections per animal, 3 sections per region per animal ### Controls - **Positive control:** 12-month 5xFAD/TREM2+/+ (high amyloid expected) - **Negative control:** Wild-type age-matched littermates (no amyloid expected) - **Antibody control:** Sections stained with secondary antibody only (no primary) - **Technical replicates:** 3 IHC runs per antibody ### Statistical Analysis 1. Compare % amyloid area between genotypes using two-tailed Student's t-test 2. Two-way ANOVA for age × genotype interaction 3. Significance threshold: p < 0.05 (Bonferroni corrected for multiple comparisons) 4. Effect size (Cohen's d) reported for all significant comparisons

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