🧫 Experiment Protocol
Falsificationproposed
SUMMARY
# s:**
- ALOX15 overexpression in healthy astrocytes should be protective if the hypothesis is correct
- Measure both pro- and anti-inflammatory ALOX15
## Background and Rationale
This falsification experiment directly tests the hypothesis that ALOX15 (15-lipoxygenase) functions as a neuroprotective enzyme in astrocytes by overexpressing this enzyme in healthy astrocytic cultures and measuring resulting inflammatory profiles. The study utilizes lentiviral-mediated overexpression of ALOX15 in pri
METHODOLOGY NOTES
**Phase 1: Mouse Model Preparation (Weeks 1-2)**
• Obtain ALOX15 null mice (Alox15^-/-) and wild-type C57BL/6 controls (n=60 per group)
• Acclimatize mice for 1 week in controlled environment (12h light/dark cycle, 22±2°C)
• Perform baseline behavioral assessments using open field and rotarod tests
• Collect baseline blood samples for inflammatory marker analysis
**Phase 2: Viral Vector Preparation (Week 3)**
• Prepare AAV9-GFAP-ALOX15 viral vectors (titer: 1×10^12 genome copies/mL)
• Prepare AAV9-GFAP-GFP control vectors at matching titer
• Validate vector integrity and infectivity in primary astrocyte cultures
• Confirm astrocyte-specific expression using GFAP co-staining
**Phase 3: Stereotactic Injection (Week 4)**
• Anesthetize mice with isoflurane (2-3%)
• Perform bilateral hippocampal injections (coordinates: AP -2.0mm, ML ±1.5mm, DV -1.5mm)
• Inject 2μL of viral vector per site at 0.5μL/min rate
• Allow 3-week recovery period for optimal transgene expression
**Phase 4: Neuroi