Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Transient Middle Cerebral Artery Occlusion (MCAO)

1. Surgery Procedure (Figure 1)

Transient middle cerebral artery occlusion (tMCAO) is performed on 2- to 3-month old male C57Bl/6 mice (22-28g). This protocol was approved by the IRCM bioethics committee animal care. Surgical tools were sterilized by autoclaving (121 °C at 15 psi for 60 min). Between each animal, they were sterilized using the hot bead sterilizer (15 sec). Surgery table and other equipment are sanitized using 70% ethanol.

Two hours before surgery, mice were analgesized with buprenorphine (0.03 mg/kg b.w. i.p.).

Figure 1A.

1. Deeply anaesthetize mice with isoflurane 5% and then maintain the anesthesia at 2.5% isoflurane. Body temperature of the mice is maintained constant during surgery with a heating pad.
2. Disinfect the fur and skin with 70% ethanol or Betadine. Since shaving produces hair fragment release, microabrasions and inflammation that may impact on stroke pathophysiology, we do not shave mouse fur.
3. Make a midline neck incision and gently pull apart the soft tissues.
4. Under a stereo microscope (Stemi 2000, Zeiss), a blunt dissection is performed to expose the trachea and retract the muscles to locate the carotid artery.
5. The left common carotid artery (CCA) is carefully dissected from surrounding tissue and the CCA is temporarily occluded by a temporary suture (1) using 5-0 silk suture cut into 20 mm segments. Great care should be exercised not to harm the vagal nerve.
6. Separate the bifurcation of the left internal common carotid artery (ICA) and external common carotid artery (ECA). A permanent suture (2) is placed around the ECA, as distally as possible, and another temporary suture slightly tight (3) is placed on the ECA distal to the bifurcation.
7. Clip the left ICA (4) using a reverse-action tweezers to avoid bleeding. Great care should be exercised not to harm the vagal nerve.
8. Cut a small hole into ECA (5) between permanent (2) and temporary (3) sutures.

Figure 1B.

9. Introduce a 12 mm-long 6-0 silicon-coated (about 9-10mm is coated silicon) monofilament suture (Doccol Corporation) into the ECA (6), completely cut the ECA distal to the permanent suture and invert the occluder into the ICA. The suture is tightly tied around the monofilament to prevent bleeding and the reverse-action tweezers are removed.
10. The occluder is introduced to occlude the origin of the MCA in the circle of Willis. Stop its insertion around 9-10 mm beyond the bifurcation of ECA and CCA. The occluder is blocked and cannot move anymore. Care must be exercised not to penetrate the pterygopalatine artery. The suture (3) on the ECA is tightly tied to fix the monofilament in position.
11. Close the skin with an autoclip wound closing system.
12. Inject 1 ml of saline solution subcutaneously and place mice under an infrared heating lamp during all the post-occlusion period (60 min).

2. Restoration of Middle Cerebral Artery Blood Flow

Before reanesthesia, the neuroscore can be checked to evaluate the success of the surgery.

1. Anesthetize the mice as previously described and remove the autoclips.
2. Slightly open the suture (3) onto the ECA to allow monofilament withdrawal and blood reperfusion.
3. Permanently tie off the temporary suture on the ECA to prevent blood loss. The monofilament is kept for reuse.
4. Remove the temporary suture (1) onto the CCA to allow blood recirculation.
5. Close the wound. The mice should receive another 1 ml saline solution subcutaneously.
6. Place the mice under the infrared heating lamp for 1 hr. After checking that the animal regains mobility, the mouse is returned to its cage and left under the heating pad and towels for 72 hr. Note that only half of the cage is placed on the heating pad to allow mice to choose their environment. Because post-surgical weight loss is generally observed, mashed food is placed in a Petri dish to encourage eating.
   Twelve hours post-surgery, mice received another dose of buprenorphine (0.03 mg/kg b.w. i.p).

3. Sham Operation

For sham operations, all procedures are identical except that the occluder is not inserted.

4. Neuroscore

Neurological deficits allow the evaluation of the success of tMCAO just after reperfusion and later the estimation of the degree of severity of the injury. Neurological deficits are scored as previously described ⁵ and performed at 1, 24, 48 and 72 hr post-reperfusion. An expanded six-point scale is used:

• 0: normal.
• 1: mild circling behavior with or without inconsistent rotation when picked up by the tail, <50% attempts to rotate to the contralateral side.
• 2: mild consistent circling, >50% attempts to rotate to the contralateral side.
• 3: consistent strong and immediate circling, the mouse holds a rotation position for more than 1-2 sec, with its nose almost reaching its tail.
• 4: severe rotation progressing into barreling, loss of walking or righting reflex.
• 5: comatose or moribund.

5. Cresyl Violet Staining (Figure 2)

Following PBS solution perfusion, brains are quickly frozen in isopentane and stored at -80 °C. Mouse brains may be also perfused with 4% paraformaldehyde (PFA) depending on the planned immunohistochemistry studies. Cryostat-cuts of coronal brain sections (17 μm) are performed. One section out of every 30 is collected on the same slide to have a representative cerebral injury. For volume quantification, 2 slides are stained by cresyl violet staining and an image analysis system (Scion Corporation, Frederick, MD) was used to evaluate the lesion. The injury volume was calculated in arbitrary units (pixels), and expressed as a percentage of the contralateral non-lesioned area for each section. Other slides are kept at -80 °C for immunohistochemistry studies. Alternatively, PFA-fixed brain can be cut at 30-50 μm using vibratome and the infarct volume measured as recommended by Han et al. (2009)⁶.