Inducing Ischemic Stroke via Middle Cerebral Artery Occlusion Using a Thread Bolt

Protocol

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

1. Establishment of a unilateral middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats

NOTE: During the operation, use the microforceps gently to prevent breakage of the blood vessel. Avoid damage to the nerves and other blood vessels in the neck of the rat when the vessel is isolated. Care must be taken to present appropriate aseptic technique for all survival surgical procedures. The technique illustrated later in the video should be practiced through the entire procedure.

1. Throughout the surgery, maintain the body temperature of the rats at 37.0 ± 0.5 °C in a small animal thermostat. Prepare four 6 cm 5-0 sutures.
2. Set the oxygen flow rate of a small animal anesthesia machine (with a waste gas treatment device) at 0.4−0.6 L/min and the concentration of isoflurane to be 5%. Place the rat in the anesthesia machine.
3. After the animal has fainted, place the rat on a surgical fixing table. Connect the mouth of the rat to the mask of the anesthesia machine (the oxygen flow rate remains unchanged; adjust the concentration of isoflurane to be 3%). Confirm that the animal has entered deep anesthesia by observing a lack of extremity tension, corneal reflexes, and pain.
4. Fix its limbs to lie on the operating table with paper bandages (or other tools).
5. Remove the neck coat with an electric shaver and sterilize with 75% alcohol (iodophor is better). Fix the mouth of the rat with a hook.
6. Cut 2−3 cm along the central longitudinal shape of the neck with ophthalmic scissors.
7. Separate the common carotid artery. Separate the subcutaneous muscle with ophthalmic forceps. Use a homemade retractor to fully expose the field of vision. After separating the anterior muscle of the trachea with ophthalmic forceps, separate along the right sternocleidomastoid tendon until the common carotid artery is visible.
8. Isolate the common carotid artery, the external carotid artery, and the internal carotid artery with ophthalmic forceps. Ligate the common carotid artery (hard knot), external carotid artery far from the heart end (hard knot), and internal carotid artery (loose knot) with 5-0 sutures. Line the external carotid artery near the heart end with 5-0 sutures.
9. Insert a thread bolt. Cut a small opening in the external carotid artery using ophthalmic scissors and gently insert a thread bolt. Ligate the suture of the external carotid artery that has been in a loose knot and cut off the external carotid artery.
   1. Loosen the loose knot of the internal carotid artery and continue inserting the thread bolt to the beginning of the middle cerebral artery (suture marked). Then, cut off the exposed thread bolt.
10. After the ischemic time is reached (2-3 h), fix the fracture of the external carotid artery with one microforceps and gently pull out the thread bolt with another microforceps. When the front end of the thread bolt is completely withdrawn from the internal carotid artery, ligate the external carotid artery that was lined with 5-0 sutures and then pull out the thread bolt completely.
11. Loosen the common carotid artery, and daub ~50,000 U of penicillin sodium powder on the surface of the wound to prevent infection. Suture subcutaneous muscles and skin with 4 sutures.
12. Give ~0.2 mL of sterile saline to the rat orally using a 1 mL syringe (SQ-PEN injection is better) to prevent postoperative water shortage after placing the rat back in the cage.

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Materials

Name	Company	Catalog Number	Comments
Anatomical Microscope	Leica (Germany)	S8	Microscopic operating instrument
Electric Shaver	Shanghai Yuyan Scientific Instruments Co., Ltd.	3000#	Removal of hair from the neck of rats
Image Pro Plus Software	Media Cybernetics Inc.	6	Analyze the infarct volume
Isoflurane	RWD Life Science	217170702	Anesthetic gas
Microforceps	Shanghai Jinzhong Medical Devices Co., Ltd.	10 cm	Vascular micromanipulation
Microshear	Shanghai Jinzhong Medical Devices Co., Ltd.	10 cm	Vascular micromanipulation
Ophthalmic Forceps	Shanghai Jinzhong Medical Devices Co., Ltd.	10 cm	Auxiliary skin and muscle anatomy
Ophthalmic Scissors	Shanghai Jinzhong Medical Devices Co., Ltd.	10 cm	Using for cutting the skin of neck
Small Animal Anaesthesia Machine	Shanghai Yuyan Scientific Instruments Co., Ltd.	ABM3000	A gas anesthetic machine
Small Animal Thermostat	Beijing Damida Technology Co., Ltd.	DM.7-YLS-20A	To maintain animal body temperature constant during operation
Surgical Scissors	Shanghai Medical device Co., Ltd.	16 cm	Using for decapitate and brain removing
Suture	Shanghai Jinhuan Medical Devices Co., Ltd.	4-0 / 5-0	Using for skin and muscle sutures / Using for vascular ligations
Thread Bolt	Beijing Cinontech Co. Ltd.	2636/2838/3040/3043-A4	Blockage of the middle cerebral artery in rats