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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. Establishment of infarction in the pons
- Preheat the heating pad immediately before anesthesia.
- Attach the skull drill to the holder on the stereotaxic frame.
- Intraperitoneally inject the rats with 50 mg/kg ketamine and 5 mg/kg xylazine. Check for the lack of toe-pinch response.
- Mount the rat onto the stereotaxic frame in the prone position. Position the ear bars above the ear canal to secure the head. Ensure that the skull is kept horizontal to avoid any skewing of the injection.
- Maintain anesthesia by isoflurane (100% oxygen, 2.5% isoflurane) via a stereotaxic nose cone attachment for rats with inlet and outlet ports. Use a heating pad to maintain the temperature at 37 °C and monitor it throughout the procedure.
- Use eye ointment to prevent corneal drying. Use forceps to pinch the paws to ensure no pain response slightly.
- Shave the hair of the skull with a micro-shaver. Circularly apply chlorhexidine surgical scrub starting at the surgical incision site and rotating outward.
- Make a 3 cm midline incision with a scalpel from the line of the bilateral lateral canthus to 0.5 cm behind the posterior fontanelle, which a surgical marker pen should mark.
- Use a cotton swab to remove blood.
- A piece of surgical tape should be placed on each side of the skin flap to expose the scalp (Figure 1).
- Gently remove the connective tissues from the skull bone with a cotton swab dipped in 0.9% NaCl. If not removed, the connective tissues will get caught in the drill.
- Identify the bregma. Choose the central point of the bregma as the origin point and mark it using a fine-tip black surgical marker pen.
- Place a drill at 6.0 mm AP, 2.0 mm ML (range from 0.5–3.0 mm, Figure 2A).
- Perform craniotomy (1 mm diameter) using an automatic drill. Proceed carefully because this point is close to the venous sinus.
- Remove the drill from the stereotaxic frame.
- Place a 22 G probe with an insulated sheath in the stereotaxic frame (Figure 3A). The probe tip should be placed 2 mm above the proximal end of the sheath (Figure 3A, B; Figure 2B).
- Ensure that the sheath enters the brain 7 mm (7 mm DV, Figure 2B; Figure 1C).
- Advance the probe along the sheath (Figure 1D) until the tip of the probe is 9 mm below the surface of the brain (Figure 2D).
- Connect the electrodes to an electrical stimulator (Figure 3C). Connect the anode to the probe as shown in Figure 1D. Connect the cathode to the rats (usually to the ear of the rats).
- Turn the electrical stimulator on and set up the following parameters: single pulse width = 4,050 ms; voltage = 50 V; and current = 4 mA (Figure 3C). During electrical stimulation, the rat will exhibit trembling. In this study, the device was not turned on for control group rats used for the behavioral tests, MRI, and TTC.
- Leave the probe in position for 5 min after stimulation.
- Remove the probe from the brain (Figure 1F).
- Use bone cement to cover the craniotomy. Allow the cement to dry before wound suturing.
- Suture the wound with 4-0 polyamide suture filaments. After three or four stiches, tie 2-1-1 standard surgical knots.
- Inject the rats with penicillin (0.25 mL, 80 IU diluted in 4 mL of saline) intraperitoneally to prevent infection.
- Inject the rats subcutaneously with meloxicam at a dose of 2 mg/kg and then repeat it every 24 h.
- Monitor the rats every 15 min until fully awake and return them to the cage with a heating pad. Provide free access to food and water until sacrifice.
NOTE: All procedures should follow the aseptic surgical principles. Before the surgery, put on a scrub top, surgical mask, and sterile gloves after a surgical scrub of the hands. Always maintain sterile suture material within the sterile field.
