Phase 1: Primary Cell Isolation and Culture -- Days 1-10
Human trabecular meshwork cells isolated from donor eyes (age 45-75 years, <48h post-mortem) obtained through eye banks with IRB approval. Tissue dissected under sterile conditions and cells cultured in DMEM with 10% FBS, 1% P/S. Validate TM cell identity by immunocytochemistry for myocilin, α-SMA, and fibronectin. Use cells between passages 2-5. Power calculation: n=15 cells per condition for 80% power to detect 30% change in current amplitude.
...Phase 1: Primary Cell Isolation and Culture -- Days 1-10
Human trabecular meshwork cells isolated from donor eyes (age 45-75 years, <48h post-mortem) obtained through eye banks with IRB approval. Tissue dissected under sterile conditions and cells cultured in DMEM with 10% FBS, 1% P/S. Validate TM cell identity by immunocytochemistry for myocilin, α-SMA, and fibronectin. Use cells between passages 2-5. Power calculation: n=15 cells per condition for 80% power to detect 30% change in current amplitude.
Phase 2: Chronic Dexamethasone Treatment -- Days 11-18
Seed cells on glass coverslips in 35mm dishes. Chronic DEX treatment protocol: Control (vehicle, 0.1% ethanol), DEX 100 nM, DEX 1 μM for 72-96 hours. Replace medium and drugs every 48 hours. Assess cell viability by calcein-AM staining (>95% viability required). Document morphological changes by phase-contrast microscopy.
Phase 3: Patch-Clamp Setup and Calibration -- Days 19-20
Patch-clamp amplifier (Axopatch 200B) and digitizer (Digidata 1550) calibrated. Borosilicate pipettes pulled to 3-5 MΩ resistance. External solution: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4, 300 mOsm. Internal solution: 140 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 3 mM Na2ATP, pH 7.2, 290 mOsm.
Phase 4: Whole-Cell Current Recordings -- Days 21-28
Whole-cell patch-clamp recordings at room temperature. Establish gigaseals (>2 GΩ) and break into whole-cell mode. Series resistance <15 MΩ with >70% compensation. Measure resting membrane potential immediately after break-in. Record baseline K+ currents using voltage steps (-80 to +60 mV, 10 mV increments, 200 ms duration).
Phase 5: ML-402 Response Testing -- Days 21-28
After stable baseline recording, apply ML-402 (TREK-1 agonist, 30 μM) via perfusion system. Record current responses for 5-10 minutes to assess activation kinetics and steady-state amplitude. Measure ML-402-evoked current as difference between peak and baseline. Test reversibility with washout. Include vehicle control applications (0.1% DMSO).
Phase 6: Data Analysis and Statistical Testing -- Days 29-32
Data analysis using pClamp 11 and GraphPad Prism. Measure: resting membrane potential, input resistance, ML-402-evoked current amplitude and kinetics. Current density calculated (pA/pF) using cell capacitance. Statistics: unpaired t-tests for between-group comparisons, paired t-tests for before/after drug applications. One-way ANOVA with Tukey's post-hoc for multiple group comparisons. Significance threshold p<0.05.