Phase 1: Animal Preparation and Ethical Approval -- Days 1-3
C57BL/6 mice (8-12 weeks, both sexes) maintained on 12h light/dark cycle. IACUC approval obtained. Pre-experiment health screening including ophthalmoscopy to exclude baseline ocular abnormalities. Randomization to treatment groups using computer-generated sequences. Power calculation: n=8 eyes per group for 80% power to detect 50% change in outflow facility with α=0.05.
...Phase 1: Animal Preparation and Ethical Approval -- Days 1-3
C57BL/6 mice (8-12 weeks, both sexes) maintained on 12h light/dark cycle. IACUC approval obtained. Pre-experiment health screening including ophthalmoscopy to exclude baseline ocular abnormalities. Randomization to treatment groups using computer-generated sequences. Power calculation: n=8 eyes per group for 80% power to detect 50% change in outflow facility with α=0.05.
Phase 2: iPerfusion System Setup and Calibration -- Days 4-5
Epithelial Instruments iPerfusion system calibrated according to manufacturer specifications. Pressure transducers zeroed and calibrated with known pressures. Flow rate accuracy verified using gravimetric method. Prepare perfusion medium: Dulbecco's PBS with 5.5 mM glucose, 1 mM pyruvate, pH 7.4, osmolality 290-300 mOsm, sterile filtered.
Phase 3: Baseline Outflow Facility Measurements -- Days 6-8
Mice anesthetized with isoflurane (2-3% induction, 1-2% maintenance). Eyes enucleated within 5 minutes post-euthanasia. Anterior chamber cannulated with 33-gauge needle connected to iPerfusion system. Perfusion initiated at 8.2 mmHg for 45-60 minutes to establish stable baseline. Real-time monitoring of flow rate and pressure. Calculate outflow facility (C) using: C = Flow rate / (Perfusion pressure - Episcleral venous pressure).
Phase 4: ML-402 Treatment and Response Measurement -- Days 6-8
After stable baseline (coefficient of variation <15% for 15 minutes), switch perfusion to medium containing ML-402 TREK-1 agonist. Treatment concentrations: Vehicle control (0.1% DMSO), ML-402 10 μM, ML-402 30 μM. Continue perfusion for 60-90 minutes. Record flow rate every 30 seconds. Calculate fold-change in outflow facility relative to baseline.
Phase 5: Dose-Response Analysis and Controls -- Days 9-12
Conduct dose-response experiments with ML-402 concentrations: 1, 3, 10, 30, 100 μM. Include appropriate controls: DMSO vehicle, inactive analog compound, and TREK-1 antagonist (spadin, 1 μM) to confirm specificity. Each eye serves as its own control with before/after measurements.
Phase 6: Data Analysis and Validation -- Days 13-15
Statistical analysis using mixed-effects models to account for paired measurements. Primary endpoint: fold-change in outflow facility from baseline. Secondary endpoints: time to maximum response, duration of effect. Outlier detection using Grubbs' test. Significance testing with repeated measures ANOVA followed by Dunnett's post-hoc test comparing to vehicle control.