$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
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 material and instruments
- Connect the heat blanket to maintain the temperature of the operation area and the mouse body temperature during anesthesia at 37 °C.
- Autoclave scissors and forceps, prepare 70% ethanol solution and keep available dexpanthenol eye ointment, several pieces of cotton, and 5-0 coated braided polyester suture ready for use. Prepare a 1 mL syringe with 0.9% saline solution (without needle) to keep the animal's incision site hydrated. Prepare the anesthesia gas (100% O2 + isoflurane).
- Prepare a holder for the laser Doppler probe by cutting the tip of a 10 µL pipet tip (3-5 mm length).|
NOTE: All instruments are sterilized using a hot bead sterilizer. Surfaces are also disinfected before and after surgery with a microbial disinfectant spray. Prior to surgery, the areas surrounding the head and chest of mice are disinfected with a wound disinfection spray.
2. Preparation of the laser Doppler
- Inject analgesia to the mouse 30 min before the surgery (4 mg/kg Carprofen and 0,1 mg/kg Buprenorphine, intraperitoneally).
- Anesthetize the mouse by placing it in the induction chamber with an isoflurane flow rate of 4% until the cessation of spontaneous body movement and vibrissae.
- Place the mouse in a prone position in the operation area with its nose in the anesthesia mask. Maintain isoflurane concentration at 4% for another minute, then reduce it and keep it at 2%.
- Set the associated feedback-controlled heating pad for maintaining the mouse body temperature at 37 °C, and gently insert the rectal probe to monitor the temperature throughout the surgical procedures.
- Apply dexpanthenol eye ointment on both eyes.
- Disinfect the skin and hair surrounding the left eye and ear with 70% ethanol.
- Cut the scalp between the left ear and the eye (1 cm long) to expose the skull bone.
- Cut and retire the temporal muscle to visualize the MCA beneath the skull.
- Fix with glue the outer part of the tip holding the laser Doppler probe/fiber on top of the left MCA with glue. Then, glue the skin to close the wound around the tip holder. Apply 2-3 drops of hardener glue to speed up the process. Make sure that the laser Doppler fiber is not glued and can be easily removed from the tip holder at any time.
3.Transient MCAo model (occlusion)
- Turn the mouse into the supine position. Put the snout into the anaesthesia cone and fix the paws with tape.
- Disinfect the skin and hair surrounding the chest and make a 2-cm-long midline incision in the neck.
- Use forceps to pull the skin and the submandibular glands apart. Use retractors to hold the sternomastoid muscle, expose the surgical field and find the left common carotid artery (CCA). Dissect the CCA free from connective tissue and surrounding nerves (without harming the vagal nerve) and perform a transient ligation before the bifurcation.
- Dissect the external carotid artery (ECA) and tie a permanent knot at the most distal visible part. Place another suture under the ECA, close to the bifurcation, and prepare a loose knot to be used later.
- Dissect the internal carotid artery (ICA) and place a microvascular clip on it, 5 mm over the bifurcation. Make sure not to damage the vagal nerve.
- Cut a small hole into the ECA between the tight and the loose ligations; be careful not to cut the entire ECA.
- Introduce the filament and advance it towards the CCA. Tighten the loose ligation in the ECA around the lumen to shortly secure the filament in that position and avoid bleeding when removing the microvascular clip.
- Remove the microvascular clip and insert the filament through the ICA until the origin of the MCA is reached by detecting a sharp reduction (>80%) in the cerebral blood flow as measured by the laser Doppler. Fix the filament in this position by further tightening the knot around the ECA.
NOTE: When the filament goes toward the appropriate direction, it advances smoothly, and no resistance should be observed.
- Record laser Doppler values before and after filament insertion.
- Remove the retractor and relocate the sternomastoid muscle and the submandibular glands before suturing the wound. Remove the laser Doppler probe and place the animal in a recovery chamber at 37 °C for 1 h (until filament removal).