TREK-1 effects on spontaneous OHT in rats using telemetry

PRIMARY OUTCOME

intraocular pressure reduction

EXPECTED OUTCOMES

## Primary Outcomes **IOP Reduction**: ML-402 treatment reduces 24-hour mean IOP by ≥25% vs. vehicle controls in SOHT rats (p < 0.01, repeated measures ANOVA). Peak IOP during dark cycle shows ≥30% reduction. Expected baseline IOP in SOHT rats: 28-32 mmHg vs. 18-22 mmHg in naive controls. **Optic Nerve Protection**: ML-402-treated eyes show optic nerve degeneration score ≤1.5 (scale 0-5) vs. ≥3.0 in vehicle-treated SOHT eyes (p < 0.01). Retinal ganglion cell density maintained at ≥85% of naive control levels. ## Secondary Outcomes **TREK-1 Pathway Activation**: TM tissue from ML-402 eyes shows increased TREK-1 expression (≥1.8-fold vs. vehicle, p < 0.05) and reduced fibronectin deposition (-35%, p < 0.05), consistent with TM remodeling toward improved outflow.

SUCCESS CRITERIA

## Primary Success Criteria **IOP Lowering**: ML-402 must produce ≥20% reduction in mean IOP (averaged across 24h, 5-day recording period) vs. vehicle control SOHT rats. Reduction must be statistically significant (p < 0.05) and sustained for ≥4 weeks post-first injection. **Safety Profile**: ML-402 treatment must not produce significant corneal opacity, anterior chamber inflammation (>2+ cells), or systemic toxicity (body weight loss < 10% vs. pre-treatment baseline) in ≥90% of treated animals. ## Secondary Success Criteria **Dose-Response**: Lower dose ML-402 (1 μg/μL) produces intermediate IOP reduction (15-25%), confirming dose-response relationship and biological specificity. **Mechanistic Confirmation**: TREK-1 expression in TM must correlate inversely with IOP values across all groups (Pearson's r ≤ -0.65), supporting mechanism of action.

PROTOCOL

# TREK-1 Effects on Spontaneous Ocular Hypertension in Rats Using Telemetry Protocol ## Phase 1: Rat Model and Telemetry Implantation (Days 1-21) **Animal Group**: Use adult male Brown Norway rats (300-350g, 12 weeks old, n=18 total). Randomize into 3 groups: (a) naive control (n=6), (b) spontaneous ocular hypertension (SOHT) + vehicle (n=6), (c) SOHT + ML-402 treatment (n=6). Confirm baseline IOP via TonoLab (5 measurements per eye, averaged). **Telemetry Probe Implantation**: Under isoflurane anesthesia (3-5% induction, 1.5-2% maintenance), implant telemetry probes (DSI #CA-F1-40) into the anterior chamber of the right eye via temporal limbal incision. Secure with 10-0 nylon suture. Tunnel subcutaneous leads to a dorsoscapular pocket for signal transmission. Allow 14-day recovery. **IOP Telemetry Data Collection**: Continuous IOP recording (5 min every 30 min, 24 hours/day) for 5 consecutive days at baseline (pre-treatment), during treatment weeks 2-4, and at endpoint. Extract hourly means, diurnal patterns (light/dark cycle), and peak IOP values. ## Phase 2: ML-402 Treatment Protocol (Days 22-42) **Drug Preparation**: ML-402 (Tocris #6376) dissolved in 20% hydroxypropyl-β-cyclodextrin (HPβCD) vehicle. Fresh preparation weekly. Store at 4°C protected from light. **Dosing Regimen**: ML-402 administered via intraocular injection (2 μL, 5 μg/μL concentration, bilateral) at days 0, 14, and 28. Control groups receive equal volume of vehicle (20% HPβCD). Treatment-blinded operators perform injections. **Monitoring Protocol**: Measure bodyweight weekly. Monitor for signs of ocular inflammation (redness, corneal clarity, anterior chamber cells) via slit-lamp biomicroscopy at each injection timepoint and weekly. ## Phase 3: Histological and Molecular Analysis (Days 43-56) **Optic Nerve Assessment**: At endpoint (day 42), perfuse rats with 4% PFA. Dissect optic nerves, embed in paraffin, and section (3 μm). Stain with paraphenylenediamine (PPD) to assess axonal degeneration (Brüett-Gordon scoring: 0-5 scale). Score by masked evaluator. **Trabecular Meshwork Analysis**: Collect superior trabecular meshwork (TM) tissue at termination. Process for: (a) RNA sequencing (NovaSeq, 30M reads/sample) to assess TREK-1 pathway modulation, (b) western blot for TREK-1, p-ERK1/2, and fibronectin (FN1) as marker of TM remodeling. **Aqueous Humor Outflow**: Perform anterior chamber perfusion with fluorescent dextran ( FITC-dextran, 70 kDa) to measure outflow facility (μL/min/mmHg) in ex vivo eyes at termination.

LINKED HYPOTHESES

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[TREK-1 effects on spontaneous OHT in rats using telemetry](http://scidex.ai/artifact/exp-3762f9a4-78dd-46b0-bd08-87caf6bf3934)

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