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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the animal
- Inject analgesia 30 min before surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).
- Record the mouse body weight to adjust the dose of Rose Bengal to be injected (10 µL/g i.e., 100 µg/g).
- Place the mouse in the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops.
- Transfer the mouse into the stereotactic frame and place it in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. Then reduce and maintain the isoflurane concentration at 2%.
- Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 °C.
- Apply dexpanthenol eye ointment to both eyes and clean the skin and surrounding fur with a disinfectant agent.
2. Photothrombosis model
- Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.
- Remove the periosteum gently with cotton and identify the coronal sutures.
- Put on the protective glasses, switch on the 561 nm laser and mark the bregma +3 mm left.
- Switch off the laser, attach a sticker with a 4 mm diameter hole placed at the marked coordinates mentioned above.
- Inject the mouse with Bengal Rose (10 µL/g), intraperitoneally. Place the laser beam at 4-5 cm from the skull, switch on the 561 nm laser and illuminate the skull for 20 min.
- Apply two drops of 0.9% saline on the skull to rehydrate, suture the wound, and place the animal in a recovery chamber at 37 °C to recover from anesthesia. After 1 h, return the mice to their cages in a temperature-controlled room.
- Inject analgesia every 12 h for 3 days after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).