Complete Protocol for Ang II-Induced Hypertension Model in C57BL/6 Mice

Complete Protocol for Ang II-Induced Hypertension Model in C57BL/6 Mice

Title: Tackling the Core Challenges in Ang II-Induced Hypertension Modeling: A Complete Solution from Precision Implantation to Dynamic Monitoring

Introduction

Hypertension and its complications represent a global health challenge. The Angiotensin II (Ang II)-induced hypertension model, a cornerstone preclinical model for studying the Renin-Angiotensin-Aldosterone System (RAAS) and related drug targets, is critical for research success. However, issues like a failure rate exceeding 50%, low post-operative survival, and erratic blood pressure data are common. The root causes often lie in the overlooked details of drug release accuracy and surgical trauma. This article provides a detailed, highly reproducible protocol for establishing an Ang II-induced hypertension model in C57BL/6 mice, directly addressing these pain points to enhance your experimental success.

I. Background

Angiotensin II (Ang II) is a potent vasoconstrictor within the RAAS. Chronic exposure to high concentrations of Ang II can trigger a series of pathophysiological changes, including but not limited to: hypertension, cardiac and vascular remodeling, oxidative stress, inflammatory responses, endothelial dysfunction, and renal injury. Therefore, continuous subcutaneous infusion of Ang II to establish a hypertensive animal model has become a classic method in cardiovascular research.

II. Experimental Preparation

Animals: Male C57BL/6 mice, SPF grade, 8-10 weeks old, weighing 25-30g. Acclimatize for at least one week before procedures.

Reagents: Angiotensin II (Ang II), Saline, Isoflurane, Povidone-iodine, 75% Ethanol, Depilatory Cream.

Equipment & Consumables:

Equipment: Animal Anesthesia Machine with Induction Chamber, Homeothermic Heating Pad, Surgical Platform, Instrument Sterilizer, Cold Light Source, Non-invasive Tail-cuff Blood Pressure System.

Consumables: Alzet® Osmotic Pump (Model 2004W or equivalent, rate 0.25 μL/h, duration 28 days), Fine Surgical Instruments (Scissors, Forceps, Hemostats), Wound Clips/Applier, Hair Clipper, 0.22μm Sterile Filter, Sterile Syringes.

III. Detailed Experimental Procedure

Step 1: Acclimatization & Baseline Measurement (Crucial!)

Purpose: To minimize stress during BP measurements and obtain reliable baseline data.

Procedure: For at least 5-7 days pre-surgery, handle mice daily and acclimatize them to the tail-cuff restraint. Measure and record stable systolic blood pressure (SBP) for at least 3 consecutive days as baseline.

Step 2: Precise Drug Preparation (Key to Success)

Vehicle: To maintain Ang II stability, use 0.01N Acetic Acid-Saline (pH≈5.0) as the vehicle. Add 0.57 mL of glacial acetic acid (17.5 M) to 1L of saline and mix. Saline alone can serve as a control.

Drug Concentration Calculation (Dose: 750 ng/kg/min, Pump: 2004W):

Principle: Calculate based on the maximum body weight in the experimental cohort to ensure sufficient dosing for all individuals.

Known: 2004W Pump Rate ≈ 0.25 μL/h, Pump Volume ≈ 200 μL, Duration 28 days.

Calculation:

Total Ang II required per hour per mouse = Max Body Weight (kg) × 750 (ng/kg/min) × 60 (min)

Ang II Stock Concentration = Hourly Total (ng) / Pump Rate (0.25 μL/h)

Total Ang II Required = Stock Concentration (ng/μL) × Pump Volume (μL) × Number of Mice (n)

Example: For a max weight of 30g (0.03 kg), Hourly Ang II = 0.03 × 750 × 60 = 1350 ng. Required Concentration = 1350 ng / 0.25 μL/h = 5400 ng/μL = 5.4 μg/μL. Total drug (for 10 mice) ≈ 5.4 μg/μL × 200 μL × 10 = 10.8 mg.

Preparation: Filter the prepared Ang II solution through a 0.22μm sterile filter, aliquot, and store at -20°C.

Step 3: Pump Filling & Priming (Precision Control)

Weigh Empty Pump: Record the initial weight of the empty pump (with flow moderator) (W1).

Aseptic Filling: Slowly inject the drug solution into the pump through the filling hole using a sterile syringe until a droplet appears at the outlet, ensuring no air bubbles.

Weigh Filled Pump: Weigh again (W2). The fill volume (W2 - W1) should be >90% of the nominal pump volume. If not, empty and refill.

In Vitro Activation (Priming): To ensure immediate and stable release post-implantation, submerge the filled pump in sterile saline at 37°C and incubate for 40-48 hours (for model 2004W). This step significantly improves initial delivery stability.

Step 4: Subcutaneous Implantation Surgery (Minimally Invasive Technique)

Anesthesia & Preparation: Anesthetize the mouse using isoflurane. Shave the fur between the scapulae and disinfect the skin alternately with iodine and alcohol.

Incision & Blunt Dissection: Make a ~1 cm transverse skin incision along the midline between the scapulae. Use blunt forceps to carefully create a subcutaneous pocket towards the animal's hindquarters, large enough to hold the pump.

Pump Implantation: Insert the pump into the pocket with the delivery portal facing the tail (away from the incision).

Closure: Close the skin incision using wound clips or sutures. Re-disinfect the area around the incision.

Step 5: Post-operative Care: The Critical 72 Hours (Enhancing Survival)

Warmth: Place the mouse singly in a cage with clean bedding on a 37°C heating pad or under a heat lamp until fully awake and mobile.

Supportive Care: Administer pre-warmed saline subcutaneously (e.g., 0.5-1 mL) if needed to prevent dehydration. Consider analgesics (e.g., Buprenorphine) for post-operative pain relief.

Monitoring: Closely monitor the mouse's behavior, food/water intake, and wound healing for at least 3 days.

Step 6: Blood Pressure Monitoring & Data Collection

Timing: Measure BP 2-3 times per week after surgery.

Environment: Measurements must be conducted in a quiet, warm (~30°C), dimly lit room. Pre-warm the mouse in a restraint cage under a heat lamp for 15-20 minutes to dilate tail arteries.

Measurement: Record 10-15 valid readings consecutively, discard outliers, and record the average. Perform measurements at the same time each day (e.g., morning) to minimize diurnal variation.

IV. Model Validation & Expected Results

Blood Pressure: Successful modeling typically results in a significant SBP increase within 3-7 days, reaching a plateau around week 2, with an elevation of 20-60 mmHg above baseline.

Endpoint Analyses (Optional):

Organ Weight: At termination, harvest organs (heart, aorta, kidneys). Calculate organ weight/tibia length or body weight ratios. Expected findings include cardiac hypertrophy.

Histology: H&E staining, Masson's Trichrome, etc., can reveal pathology like cardiomyocyte hypertrophy, vascular wall thickening, and collagen deposition (fibrosis).

Molecular Biology: Upregulation of genes related to hypertrophy, fibrosis, and inflammation (e.g., ANP, BNP, Collagen I/III, TNF-α) can be detected in heart, vessel, and kidney tissues.

Conclusion

By strictly adhering to this protocol, with particular emphasis on precise drug formulation, proper pump priming, meticulous minimally invasive surgery, and thorough post-operative care, researchers can effectively overcome the core challenges of poor reproducibility and low survival rates in Ang II-induced hypertension modeling, thereby establishing a stable and reliable animal model foundation for cardiovascular research.

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