MCAO Stroke Model Protocol for Mouse

MCAO Stroke Model Protocol for Mouse

Luke R. Lemmerman, Hallie N. Harris, W. David Arnold, Natalia Higuita-Castro, Daniel Gallego-Perez

Materials

• Surgical tools
  • Forceps
    • Straight fine tip forceps
    • Curved fine tip forceps (2)
    • Micro clip applying forceps
  • Scissors
    • Iris scissors
    • Tenotomy scissors
    • Vanna scissors
  • Other
    • Wound clip stapler
    • Wound clip remover
    • Needle holder
    • Curved microclips
• Bipolar forceps with system
  • Similar to Accurate Surgical and Scientific Instruments ref. no. AC1905 and ref. no. ASSI.BPNS12723)
• Laser doppler flowmetry system and probes
  • Similar to Moor instruments ref. no. DRT4 and ref. no. VP10M200ST with ref. no. P10D
• Silicone rubber-coated monofilament (MCA occluder)
  • Select per size of animal
• Slide warmer with Temperature Control
• Sutures
  • 6-0 spool black braided silk sutures
  • 6-0, P-10 braided absorbable sutures
  • 6-0, C-22 black braided silk sutures
• Isoflurane induction system
• Eye lubricant
• Electric razor
• Chlorhexidine
• 70% Ethanol
• Surgical tape
• Tissue adhesive

Protocol

Preparation:

1. All surgical instruments and supplies are sterilized by autoclave prior tosurgery.

1. Note: Between same day animalsurgeries, instruments are soaked in a 70% EtOH solution and then rinsed in sterile water prior to the next surgery. An aseptic surgical field is maintained around the animal throughout the procedure.

Surgery:

2. The mouse is anesthetized initially with 4-5% isoflurane in an induction chamber and then moved to nose cone and maintained at 1-2% isoflurane for the duration of the MCAO induction procedure.

3. After confirming anesthetic depth via lack of response to a toe pinch, eye lubricant is applied. Then the area between the right eye and right ear is prepped for incision. A small incision is made in the prepped area for the laser doppler flowmetry probe (LDF) to be placed against the skull.

4. The mouse is then placed in a supine position and the legs are restrained with surgical tape to the surgicalarea.

5. The neck is shaved and then repeatedly scrubbed three times with chlorhexidine and 70% EtOH (alternating).

6. A midline ventral neck incision is made. The 6-0 black braided suture is used to open the surgical area to have increased visualization.

7. The laser doppler filament is placed against the skull and the system is calibrated.

8. The underlying tissues (including hyoid bone) are blunt dissected to expose the common, external, and internal carotid arteries.

9. The superior thyroid artery is cauterized. The external carotid is permanently ligated at the proximal end with 6-0 black braided silk suture. A loose knot of 6-0 black braided silk suture is placed around the distal end of the external carotid. A microclip is placed such that it blocks blood supply from both the common carotid and internal carotid arteries.

10. Once the microclip is placed and blood flow has sufficiently stopped, a small incision is made in the external carotid artery and the MCA occluder is inserted. The loose knot at the distal end is tightened around the MCA occluder. The microclip clip is removed. The external carotid artery is then bisected at the original incision site. The MCA occluder is advanced through the internal carotid via the carotid bifurcation.

11. The filament is advanced until the LDF blood flow drops >40-70% of baseline

12. Following induction of ischemia, all instrumentation is removed, and the surgical wounds are closed with wound clips. The scalp incision is closed with tissue adhesive and the animal is allowed to recover in a new, sterile cage during the ischemic event (either 30, 45, 60, or 90 min of occlusion time).

13. The use of a slide warmer (under the cage) is used to help maintain body temperature during recovery.

14. Following 30, 45, 60, or 90 minutes of ischemia the animal is re-anesthetized with 1-5% isoflurane.

15. The midline neck incision is reopened and the MCA occluder is removed by gently pulling it out of the external carotid to allow for reperfusion of the MCA territory. The knot is tightened around the external carotid stump to ensure there is no blood loss.

16. The neck incision is then closed with 6-0 dissolvable suture in a single interrupted suture pattern.

17. The animal is allowed to recover, and a visual neurological assessment is performed.

1. Note: Typical neurological deficits include hemiparesis, weakness, and lethargy

Post-op Supportive Care:
 

18. During the recovery period the animal is closely monitored for the first four hours. Body temperature is maintained at 37°C with the use of a slide warmer. Warm sterile NaCl (0.5-1mL) is given via subcutaneous injection right after surgery. Mash (i.e., wetted food) and hydrogel are given in petri dishes on the cage floor to help assist the animal with feeding/fluid intake. Mash and hydrogel will be provided at least 48 hours post-surgery or until the animal is able to eat sufficiently from the normal cage bars. If an animal continues to have difficult rearing/eating normally, mash will be provided till the end of the study. Mash and hydrogel are changed out daily.

19. Most animals should have recovered sufficiently to eat and drink independently by six hours post-operation. Following recovery, animals will be returned to the housing facility where they will remain on supplemental heat (slide warmer set to 37°C) for up to 21 day following surgery or until animals are able to thermoregulate adequately on theirown.

20. Each animal will be closely monitored every day following surgery for complications due to the surgery until harvest date. If fluid intake is insufficient or if dehydration is suspected, another dose of warm sterile NaCl (0.5-1mL) will begiven.

1. Note: To assess dehydration a skin turgor test will be performed.