Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

プロトコル

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 animal

1. Inject analgesia 30 min before surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).
2. Record the mouse body weight to adjust the dose of Rose Bengal to be injected (10 µL/g i.e., 100 µg/g).
3. Place the mouse in the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops.
4. Transfer the mouse into the stereotactic frame and place it in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. Then reduce and maintain the isoflurane concentration at 2%.
5. Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 °C.
6. Apply dexpanthenol eye ointment to both eyes and clean the skin and surrounding fur with a disinfectant agent.

2. Photothrombosis model

1. Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.
2. Remove the periosteum gently with cotton and identify the coronal sutures.
3. Put on the protective glasses, switch on the 561 nm laser and mark the bregma +3 mm left.
4. Switch off the laser, attach a sticker with a 4 mm diameter hole placed at the marked coordinates mentioned above.
5. Inject the mouse with Bengal Rose (10 µL/g), intraperitoneally. Place the laser beam at 4-5 cm from the skull, switch on the 561 nm laser and illuminate the skull for 20 min.
6. Apply two drops of 0.9% saline on the skull to rehydrate, suture the wound, and place the animal in a recovery chamber at 37 °C to recover from anesthesia. After 1 h, return the mice to their cages in a temperature-controlled room.
7. Inject analgesia every 12 h for 3 days after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).

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資料

Name	Company	Catalog Number	Comments
561 nm wavelenght laser	Solna	Cobolt HS-03
C57Bl/6J mice	Charles River	664
Collimeter	Thorlabs	F240APC-A
Cotons	NOBA Verbondmitel Danz	974116
Ethanol 70%	CLN Chemikalien Laborbedorf	521005
Fine Scissors	FST	15000-00
Forceps	FST	11616-15
Heating blanket	FHC DC Temperature Controller	40-90-8D
Isoflurane	Abbot	B506
Protective glasses	Laser 2000	NIR-ZS2-38
Rose Bengal	Sigma Aldrich	198250-5G
Saline solution	Braun	131321
Stereotactic frame	Stoelting	51500U
Anesthesia system for isoflurane	Drager
Phosphate Buffered Saline PH: 7.4	Apotheke Innestadt Uni Munchen	P32799