Positioning Laser Doppler Probe: A Technique to Implant Probe Over Middle Cerebral Artery for Blood Flow Measurement in Murine Model

프로토콜

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation and positioning of the laser Doppler

1. 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. Inject analgesia to the mouse 30 min before the surgery (4 mg/kg Carprofen and 0,1 mg/kg Buprenorphine, intraperitoneally).
3. 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.
4. 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%.
5. 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.
6. Apply dexpanthenol eye ointment on both eyes.
7. Disinfect the skin and hair surrounding the left eye and ear with 70% ethanol.
8. Cut the scalp between the left ear and the eye (1 cm long) to expose the skull bone.
9. Cut and retire the temporal muscle to visualize the MCA beneath the skull.
10. 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.

자료

Name	Company	Catalog Number	Comments
45° ramp	H&S Kunststofftechnik		height: 18 cm
5 mL Syringe	Braun
Anesthesia system for isoflurane	Drager
Bepanthen pomade	Bayer
C57Bl/6J mice	Charles River	000664
Ethanol 70%	CLN Chemikalien Laborbedorf	521005
Ethanol 96%	CLN Chemikalien Laborbedorf	522078
Ethanol 99%	CLN Chemikalien Laborbedorf	ETO-5000-99-1
Filaments	Doccol	602112PK5Re
Fine 45 angled forceps	FST	11251-35
Fine forceps	FST	11252-23
Fine Scissors	FST	14094-11
Glue	Orechseln	BSI-112
Hardener Glue	Drechseln & Mehr	BSI-151
Heating blanket	FHC DC Temperature Controller
Isoflurane	Abbot	B506
Laser Doppler	Perimed	PF 5010 LDPM, Periflux System 5000
Laser Doppler probe	Perimed	91-00123