Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model

Protokoll

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

1. Surgery

1. Use female elderly Lister-Hooded rats at 16-18 months (250 to 400 g) and induce anesthesia with 5% isoflurane in 1.5 L/min O₂. Following induction of anesthesia, reduce the level of isoflurane and maintain it at an adequate but minimized depth for surgery (e.g., 1.5-2%). Deliver anesthesia to the animal by a face mask, and use a scavenging system to limit the surgeon's exposure to isoflurane.
   1. Perform all procedures up to, but not including craniotomy on sham animals, as this procedure can produce behavioral deficits. Consider experimental goals when designing middle cerebral artery occlusion (MCAO) experiments, and decide whether or not a sham group to control for craniotomy should be included.
2. Administer pain relief pre- or peri-operatively (Carprieve, 0.25 mg/kg, s. cut).
   1. Add 1 ml to 19 ml sterile 0.9% saline to make the stock solution and inject 0.6 ml per 300 g rat. The use of Carprieve is preferred over Carprofen because the former is stable at room temperature (RT).
3. Shave the fur on the ventral neck region and the temporal region on the right hemisphere to expose the skin. Disinfect the surgical sites using ethanol swabs.
   1. Perform this step away from the operating area to minimize the amount of fur at the incision site.
4. Place the rat on a corkboard covered with a sterile drape in the supine position on a heating pad. Apply lidocaine cream to the shaved regions of the head and neck. Insert a rectal probe to monitor and maintain the animal’s temperature between 36.5-37.5 °C with a homeothermic system.
   1. Put lacrilube ointment on the eyes to prevent drying. Give the rat an injection of atropine sulfate solution (0.05 ml of a 600 µg/ml solution, subcutaneously) to reduce tracheal secretions.
      NOTE: Researchers should consider measuring physiological variables such as blood gases and pressure.
5. Before beginning surgery, check the hindpaw pinch withdrawal and blink reflexes to confirm full anesthesia.
6. Under a dissecting microscope, make a 2 cm central midline incision on the exposed neck using a scalpel. Move the salivary glands gently lateral to the trachea on both sides.
7. Loop non-absorbable silk suture around the skin that overlies the common carotid artery on one side. Gently pull away the skin from the site using the silk suture and stick these down to the corkboard using surgical tape to reveal the common carotid artery. Carefully blunt dissect the arteries free from the surrounding fascia and the vagal nerves using fine forceps.
   1. Be careful not to damage the muscle or vagus nerves during this step, as this will impair feeding, swallowing, and breathing.
8. Once the common carotid artery is exposed, use a non-absorbable silk suture (5/0) to isolate the carotid, first separating the carotid from the vagus nerve by reverse dissecting with fine forceps and being careful not to make contact with the nerve. Once the vessel has been looped (but not tied) with the suture, tape the ends of the suture together using surgical tape to prevent this step from being undone. Remove the sutures holding back the overlying skin.
9. Repeat for the other side.
10. Place a sterile saline-soaked gauze into the wound to keep tissues moist during the remainder of the surgery. Loosely suture the skin and place an additional saline-soaked gauze over the area to prevent further dehydration.
11. Place rats in a lateral position and make an incision in the skin at the midpoint between the right orbit and the external auditory canal. Retract the skin using up to five elastic 3 mm hook retractors pinned to the corkboard and then blunt dissect the temporalis muscle to reveal the skull.
12. Place a thumb wheel-adjusted gravity-driven saline drip above the open site (flow rate approximately 2 ml/min) and set up an aspirator system to remove bone debris and to clear minor bleeding from the exposed site throughout this phase of the surgery.
    1. Place the saline delivery nozzle at the highest point of the skull near the ear, and the aspirator nozzle at the lowest point. Adjust the wheel during bleeds to supply more saline to the area to better visualize the source of any bleed for cautery of the vessel, reducing the amount of time the animals are under anesthesia.
13. Perform a craniotomy on the exposed region (approximately 5 mm x 5 mm) using a dental drill with a coarse 1.6 mm diamond coated drill burr at approximately 8,000 rpm, ensuring the application of circular and lateral pressure and not downward pressure whilst drilling the exposed area. Once the bone is thin enough that it looks completely transparent, remove using forceps.
14. Use a homemade dural hook, made by bending one tip of fine forceps approximately 180° to form an arc, to carefully open the dura, being cautious to avoid the large surface blood vessels, as they are delicate and easy to rupture.
    NOTE: The exposed area of the brain will reveal the middle cerebral artery (MCA); the desired segment measures approximately 2 mm in length (see Figure 1).
15. Coagulate the MCA from where the inferior cerebral vein crosses, to the point of artery bifurcation and then along the caudal branches of the MCA using a pair of diathermy forceps until fully occluded. Use angled Jeweler diathermy forceps with 0.25 mm pointed tips.
    1. When occluding the artery, alter the saline flow as necessary to keep this area cool, preventing the coagulating forceps from adhering to the blood vessel. When occluded, the blood vessel appears black and no sign of blood flow should be present; blood flow can be seen in partially occluded vessels.
    2. Cut the MCA at this point to confirm complete occlusion. Using microvascular scissors, cut beneath where the inferior cerebral vein crosses the MCA.
    3. Cover the exposed area with a saline-soaked gauze pad before continuing to the next step.
16. Turn the rat back to a supine position and reopen the loosely tied suture on the neck to re-expose the carotid arteries. Ligate the carotid artery on the same side as the occluded MCA (right) permanently by tying a knot in the silk suture around the carotid, whilst the left carotid artery is occluded transiently using a 13 mm stainless steel artery clip with 125 g pressure for 60 min. Loosely suture up the incision in the neck during this time and place a sterile saline-soaked gauze on top to prevent dehydration.
    1. Note that the animal's right-hand side will be on the surgeon's left-hand side, given the supine positioning.

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Ergebnisse

Figure 1. Surgical setup of the permanent distal middle cerebral artery occlusion model. The equipment used in the setup for the rat craniotomy is shown for the right hemisphere, and inset, the positioning of the aspirator, and saline drip around the craniectomy site. Also shown are the key features of the vasculature; the middle cerebral artery (red) and inferior cerebral vein (blue) are shown, and the shaded area indicates where coagulation of the artery occurs. Confirmation of the occlusion is performed by cutting the MCA below the inferior cerebral vein.

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Materialien

Name	Company	Catalog Number	Comments
Carprofen	Norbrook	Vm No; 02000/4229	give 0.25 mg/kg
Atropine Sulfate	AmTech	RXATRINJ-100
Alcohol swabs	UHS	20021
Lidocaine cream (Emla)	AstraZeneca	12901	Apply a pea sized drop to the shaved neck and temporal regions
Homeothermic Blanket System	Harvard Instruments	507222F
Forceps	Fine Science Tools	11019-12
Isoflurane	Abbott	B506
Silk sutures	Harvard Apparatus	723288
Cautery system	Eschmann
0.25 mm Jeweler caurtery forceps	Eschmann	8330349
fine Dumont forceps	Fine Science Tools	11251-10
Thumb driven saline drip system
Vacusafe aspirator system	INTEGRA BIOSCIENCES	158320
16 mm coarse diamond coated Steel burrs		K801 104 016
Handheld dental drill	NSK	NSKVMAXVRE (Handpiece NSKEX6B)
Vannas Spring Scissors	Fine Science Tools	15000-03
Microvascular scissors	World Precision Instruments	501790
4-0 Vicryl sutures	Ethicon
Vascular clip and applicator
Operating microscope	Zeiss
Compact Anaesthesia System Isoflurane K/F Single Gas	VetTech Solutions
Carbon Steel Scalpel blades No. 10	Swann-Morton	201
25g needles	Terumo	NN-2525R
Syringes (1 ml and 5 ml)	Terumo	SS+01T1 / SS*05SE1
Saline (Sodium Chloride 0.9%)	Fresenius Kabi	Pl 08828/0178
Cotton buds	Johnson and Johnson	5.00021E+12	sterilize before use
Gauze			sterilize before use