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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. Craniectomy and controlled cortical impact
- Preparation of the controlled cortical impact device and surgical supplies.
- Load a 1 mL slip-tip syringe with 0.5 mL of sterile saline for wound irrigation. Attach a 25 G needle to the syringe to control irrigation.
- Prepare a dilute solution of cyclosporine A (CsA) in dimethyl sulfoxide (DMSO) to a final concentration of 1 mg/mL. Load a second 1 mL slip-tip syringe with 0.5 mL of CsA solution for immunosuppression. Attach a 25 G needle or larger to the CsA syringe.
- Attach the controlled cortical impact piston to an arm on a stereotaxic frame and set it to a 15 ° angle. Attach a 3 mm impactor probe to the piston.
- Set the velocity of impact to 1.5 m/s and the impact dwell time to 0.1 s to produce a mild cortical injury.
- Perform a unilateral craniectomy
- Place the mouse in an anesthesia induction chamber connected to an isoflurane vaporizer with a compressed oxygen source. Induce anesthesia with ~3% isoflurane at ~0.7 L/min oxygen. Check for the depth of anesthesia by the lack of response to the toe pinch.
- Shave the scalp using electric clippers and wipe away any loose fur.
- Place the mouse in a stereotaxic frame with an attached anesthetic delivery nose cone.
- Place a 37 °C warming pad on the stereotaxic frame under the mouse to maintain body temperature under anesthesia. Fix the head in place with ear bars and a bite bar and orient the head so that the skull frontal bone is horizontal. Maintain anesthesia at ~1.5%-2% isoflurane for the duration of the surgery.
- To perform preoperative care and aseptic surgical preparation, apply antibiotic ophthalmic ointment to the eyes using a sterile cotton swab. Apply an iodine-based solution to the shaved scalp area. Remove this with 70% ethanol. Cover the animal with a fenestrated surgical drape so that the top of the head is visible, but the eyes are covered.
- Make a midline incision (1.5-2 cm) on the scalp using a scalpel or scissors. Use sterile cotton swabs to clean the wound and to clear the fascia left of the midline at bregma.
- Use the impactor probe to identify the craniectomy site.
- Set the stereotaxic reference point (X = 0, Y = 0) to bregma. Adjust the probe laterally to 2 mm left of the midline. Outline a 5 mm diameter circle around the probe using a fine-tip surgically safe marker. Raise and rotate the impactor out of position.
- Use the high-speed rotary micromotor kit hand tool to make an open hole in the skull using a round-tip 0.6 mm or 0.8 mm burr drill bit at ~70%-80% maximum speed. Apply light pressure to the skull while drilling along the 5 mm circumferential outline to thin this border.
- Do not apply excess pressure while drilling. Vibration, compression, or accidental penetration can cause cortical injury. Instead, allow the speed of the drill bit to do the work.
- Do not drill in any given spot for too long to avoid excessive friction heating of the skull. Irrigate the craniectomy occasionally with sterile saline to remove debris and reduce the heating from the rotary tool.
- Pay close attention while drilling over the coronal suture line as these points are vulnerable to hemorrhage.
- Use a pair of fine tweezers to remove the skull flap when the craniectomy outline is sufficiently thinned. Grasp the flap medially, and gently lift and pull laterally with a radial motion.
- Do not damage the dura mater when lifting the flap; this can cause severe injury and hemorrhaging.
- Perform a mild controlled cortical impact injury
- Clean the impactor probe with a sterile alcohol prep pad. Move the impactor probe back into position over the exposed cortex. Lower the probe until it touches the dura mater surface. Mark this position as Z = 0.
- Withdraw the piston and move to Z = -1.0 mm. Discharge the piston to impact the cortex.