Inducing Thrombotic Stroke Through Hypoxia and Ischemia in a Mouse Model

Protocol

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

1. Setup

1. Prepare the surgical bed on a warming pad connected to a heat pump at 37 °C for at least 15 minutes before the surgery. Place a neck roll using the barrel of a 3 ml syringe on the surgical bed. Prepare the anesthesia gas with 2% isoflurane in medical air.
2. Prepare autoclaved forceps, scissors, micro needle holders, a hemostat, cotton swabs, and sutures. Also, prepare tissue glue and eye ointment.
3. Set up the hypoxia system and temperature controllers with a heating lamp and rectal probe. Prepare hypoxia gas with 2% isoflurane in 7.5% O₂ balanced by 92.5% N₂.
4. One hour before surgery, mice are analgesized by subcutaneous injection of a slow-release Meloxicam (4.0 mg/kg).

2. Transient Cerebral Hypoxia-ischemia (Figure 1B)

1. Anesthetize 10-13 week-old male C57BL/6 mice weighing 22 to 30 g in the anesthesia induction chamber with 3% isoflurane until the animal is unresponsive to foot squeeze, and then remove the hair on the right neck using an electronic shaver.
2. Place mice on the surgical bed connected with 2% isoflurane in medical air at a flow rate of 2 L/min. Secure forelimbs stretched out along neck roll at sides using medical tape.
3. Clean the surgical site for incision with betadine followed by alcohol and then cotton swabs.
4. Under a dissecting microscope, make a 0.5 cm right-cervical incision using straight forceps and micro scissors about 0.2 cm lateral from the midline skin.
5. Use a pair of finely serrated forceps to pull apart the fascia and tissue to expose the right common carotid artery (RCCA). Carefully separate the RCCA from the vagal nerve using a pair of fine smooth forceps.
6. Live knot two precut 5-0 silk sutures (releasable) on the RCCA, and then sew up the skin using 4-0 Nylon monofilament sutures (Figure 1C).
7. Apply eye ointment on both eyes to prevent dryness.
8. Quickly transfer the mice to the hypoxia system and put their noses and mouths in a face mask with 2% isoflurane in 7.5% O₂ at a flow rate of   0.5-1 L/min for 30 min.
   1. During hypoxia, use temperature controllers with heating lamps to control the rectal temperature at 37.5 ± 0.5 °C. Monitor the respiratory rate at 80-120 breaths/min. Maintaining the body temperature above 37 °C during hypoxia is important to create consistent brain infarction. Low respiratory rate usually happens after 20 minutes of hypoxia. Remove the face mask and allow normal air supply if the respiratory rate drops below 40. This takes 1-2 minutes and does not count toward the 30-minute hypoxia duration

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النتائج

Figure 1: Procedure of transient cerebral hypoxia-ischemia (tHI) insult in adult mice. (A) Virchow's triad that propels thrombosis includes stasis of blood flow, endothelial injury, and hypercoagulability of the blood. (B) A schematic diagram of the tHI stroke procedure. Two releasable knots were tied onto the right common carotid artery (CCA), and followed by delivery of 7.5% oxygen via a nose cone for 30 min, while the mouse rectal temperature was maintained at 37-38 °C. After the transient systemic hypoxia, the CCA ligation was released by pulling out one end of the releasable suture knots. MCA, middle cerebral artery; ICA, internal carotid artery; ECA, external carotid artery; CCA, common carotid artery. (C) Surgical procedures for transient right CCA occlusion. 1. Two precut sutures (#1 and #2) were placed under an isolated right CAA. 2. Two releasable knots were made. 3. The incision line was closed up by suture #3. Make sure that the ends of suture #1 and #2 were approachable outside the incision line. 4. Carefully pull suture #1 and #2 from outside to release the CCA. When performed gently, this procedure will not cause a laceration of the CCA.

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Materials

Name	Company	Catalog Number	Comments
Adult male mice	Charles River	C57BL/6	10~13 weeks old (22~30 g)
Mobile Laboratory Animal Anesthesia System	VetEquip	901807	Anesthesia
Medical air (Compressed) air tank	Airgas	UN1002	Anesthesia
Isoflurane	Piramal Healthcare	NDC 66794-013-25	Anesthesia
Multi-Station Lab Animal Anesthesia System	Surgivet	V703501	Hypoxia system
7.5% O2 balanced by 92.5% N2 tank	Airgas	UN1956	Hypoxia system
Temperature Controller with heating lamp	Cole Parmer	EW-89000-10	Temperature controllers
Rectal probe	Cole Parmer	NCI-00141PG	Temperature controllers
Dissecting microscope	Olympus	SZ40	Surgical setup
Heat pump with warming pad	Gaymar	TP700	Surgical setup
Fine curved forceps (serrated)	FST	11370-31	Surgical instrument
Fine curved forceps (smooth)	FST	11373-12	Surgical instrument
micro scissors	FST	15000-03	Surgical instrument
micro needle holders	FST	12060-01	Surgical instrument
Halsted-Mosquito hemostats	FST	13008-12	Surgical instrument
5-0 silk suture	Harvard Apparatus	624143	Surgical supplies
4-0 Nylon monofilament suture	LOOK	766B	Surgical supplies
Tissue glue	Abbott Laboratories	NC9855218	Surgical supplies
Puralube Vet ointment	Fisher	NC0138063	Eye dryness prevention