Overview
This video describes the surgical procedure to immobilize the laser Doppler probe over the murine brain's middle cerebral artery, or MCA. The probe helps to measure the blood flow in the brain's microvasculature by measuring the Doppler shift caused due to the motion of red blood cells within the artery.
Protocol
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
- 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. - 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.
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Materials
| 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 | |
