TREK-1 regulation of conventional outflow facility in mouse eyes

PRIMARY OUTCOME

conventional outflow facility

EXPECTED OUTCOMES

- 1. Baseline outflow facility in mouse eyes will range from 0.8-1.5 μL/min/mmHg, consistent with published literature values - 2. ML-402 treatment (10 μM) will cause a rapid increase in outflow facility within 10-15 minutes of perfusion initiation - 3. Peak outflow facility enhancement will reach 180-220% of baseline values (1.8-2.2 fold increase) with 30 μM ML-402 - 4. Dose-response relationship will show EC50 for ML-402 between 5-15 μM with maximal effects at 30-100 μM concentrations - 5. Vehicle control eyes will show stable outflow facility (±15% variation) throughout the perfusion period - 6. TREK-1 antagonist spadin will block ML-402-induced outflow facility increase, confirming channel specificity - 7. Response onset will be rapid (5-10 minutes) and sustained throughout the perfusion period without desensitization

SUCCESS CRITERIA

- • Achieve successful cannulation and stable baseline measurements in >80% of attempted eyes with CV <15% - • Demonstrate statistically significant increase in outflow facility with ML-402 treatment (p<0.05, paired t-test) - • Observe dose-dependent response with at least 3 concentrations showing progressive increases in outflow facility - • Vehicle control group must show no significant change from baseline (p>0.05) to validate experimental conditions - • Spadin antagonist must significantly reduce ML-402 effects by >50% to confirm TREK-1 specificity - • Reproducible results across 2 independent experimental sessions with consistent magnitude of responses - • Complete successful measurements in minimum n=6 eyes per treatment group for adequate statistical power

PROTOCOL

**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.

LINKED HYPOTHESES

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[TREK-1 regulation of conventional outflow facility in mouse eyes](http://scidex.ai/artifact/exp-117a73b9-8576-4d0f-9b6b-0753657b1d7a)

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