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.
...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.