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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 Rat
NOTE: We performed the experiments in male rats to avoid any hormonal changes.
- Anesthetize the animal using a mix of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg) injected intramuscularly; with this dose, the rat should be deeply anesthetized for around 1 h.
- Pinch the rat's toe; absence of a withdrawal reflex indicates that the animal is fully unconscious. If the rat responds to the pinch, give supplementary anesthesia at one third of the initial dose.
- Shave the scalp and disinfect the surgical area with povidone-iodine.
- Place the animal on a heating pad to maintain a temperature of 38 °C and stabilize the animal's head in a stereotaxic frame using two ear bars and a bite bar. Use caution to avoid piercing the tympanic membrane with the ear bars.
- Protect the eyes by applying a drop of ophthalmic gel or serum saline to each eye.
2. Location of the AC in the Temporal Bone of the Rat
- Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the surface of the cranium.
- Use a sterile cotton tip to gently remove any blood covering the surface of the skull to visualize bregma, lambda, and interaural 0 according to the Paxinos and Watson atlas of the rat brain.
- Make an incision in the temporal muscle near its dorsal insertion on the skull with a scalpel. Pull the muscle out using a needle and suture material and fix the suture material to the stereotactic frame; this will expose the temporal bone. If bleeding occurs, rinse with cold sterile saline.
- Place a sterile straight needle in the stereotactic micromanipulator, making sure that it is fully secured.
- Slowly lower the needle until it is right above the surface of the skull, so that the tip of the needle is set to interaural 0. Set this point as zero and determine coordinates from this point.
- Depending on the brain area of interest, vary the stereotactic coordinates. Determine these coordinates by utilizing the Paxinos and Watson atlas of the rat brain. Once the coordinates are determined, move the needle to match those coordinates.
- Target the AC using the coordinates of the following four points: A: anteroposterior (A/P) = -5.8 mm, mediolateral (M/L) = +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. Lower the needle to right above the temporal bone to visualize each of these four points. Using a marker pen, mark the points on the temporal bone and connect them in order to draw a rectangle; the rectangle will serve as a guide to open of a window in the bone (Figure 1).
3. Surgical Exposure of the AC
- Open the window using an electric drill and a small drill bit (0.6 mm Ø). Drill the perimeter of the rectangle at 8,000 rpm until the bone gives away. Cool the drilling surface by rinsing with cold sterile saline to prevent damage to subcortical structures. When the bone gives way, a drop in resistance can be detected. Be careful to not drill the brain.
- When the borders are loose, pull up the covering bone with fine forceps and store it in cold sterile saline.
4. Ablation of the AC
- Using a surgical microscope (10X), gently cut the meninges with a microsurgical knife and remove them using two fine-pointed forceps. If bleeding occurs, rinse with cold sterile saline.
- Gently aspirate the AC using a surgical suction device (pressure -0.24 bar) coupled to a sterile 20 G blunt tip needle. This point is critical and needs to be performed very carefully: aspirate only the six cortical layers and not the underlying white matter.
- Aspirate until the perforating arteries stop bleeding.
- When the aspiration is finished, cover the injured area with the extracted bone and apply an absorbable hemostatic gauze.
- Let the temporal muscle recover its original position, and then suture the skin using wound clips (9 mm). Apply antibiotic ointment. Continue to apply ointment to the wound twice daily for three days.
NOTE: Each application consists of a thin layer applied on the wound.
- Inject buprenorphine subcutaneously in the back of the rat (0.05 mg/kg) as an analgesic 1 h after the surgery, and then every 8 h during 72 h.
- Keep the animal on the heating pad until it wakes up and return it to its housing cage to recover.
