Fig. 1
Schematic drawing of the tandem stenosis. ApoE−/− mice at 6–8 weeks of age are fed with Western diet containing 22 % fat and 0.15 % cholesterol and tap water ad libitum. At 12–14 weeks of age, including 6 weeks of Western diet, the animals are anesthetized for TS surgery. The fur is removed around the surgical site and mice are placed on a 37 °C heater mat to prevent hypothermia. Vascular stenoses are created by two sutures on the common carotid artery using a 6-0 blue braided polyester fiber. The distal point is 1 mm apart from the carotid artery bifurcation. The proximal point is 3 mm apart from the distal point. The stenosis diameter is defined by the needle applied as a space holder
2 Materials
Prepare all solutions using medical grade saline or autoclaved phosphate-buffered saline (PBS).
1.
PBS: 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM monopotassium phosphate, pH 7.2.
2.
HFD: Mouse chow supplemented with 22 % fat and 0.15 % cholesterol (e.g., SF00-219, Specialty Feeds, Western Australia).
3.
Anesthetic: Mixture of ketamine (80–100 mg/kg), xylazine (16–20 mg/kg), atropine (0.96–1.2 mg/kg).
4.
Post-anesthetic: Atipamezole HCl (e.g., antisedan, 0.2 mg/kg, s.c.).
5.
Pain relief: Bupivacaine (marcaine, 2 mg/kg).
6.
Surgical instruments and accessories: High-precision tweezers (e.g., 4*Dumont Tweezers: T0545-811 Tweezers, style 5/45, Inox 08, high precision, polished), straight Castroviejo scissors (e.g., 1*TS1092 Castroviejo scissors, 420SS, straight, 100 mm), straight dissecting scissors (e.g., 1*TS1044 dissecting scissors, 420SS, straight, 115 mm), 150 μm diameter needles (e.g., Ethicon 8-0, Virgin Silk blue: W1782), 450-μm diameter needles (e.g., Terumo 26 G × ½″: NN*2613R), suture (e.g., 6-0 TI.CRON coated, braided polyester blue, 0.7 metric, Kendall), dissecting microscope, heater mat, depilatory hair removal cream, gauze, cotton tips, 1 mL syringe (Terumo), 30 G needle (Terumo).
3 Methods
1.
Use 6–8-week-old male ApoE−/− mice that weigh around 20–23 g, and maintain them on a high-fat diet and tap water ad libitum for 6 weeks prior to the surgery.
2.
At the day of surgery, weigh mice on a scale and record weight.
3.
It is recommended that at least semi-sterile conditions are maintained during the whole surgical procedure. Wear a surgical gown, gloves, mask, and hair cap. Sterilize surgical instruments using ultra-heat beads or autoclave the surgical instruments before use.
4.
Administer bupivacaine (2 mg/kg) 10–15 min prior to anesthesia. Anesthetize mice by i.p. administration of a mixture of ketamine, xylazine and atropine in saline according at a volume of 0.1 ml per 10 g of body weight, using a 30 G needle (see Note 1 ).
5.
Monitor the depth of anesthesia by the response to toe pinch. Movement or an increased breathing rate indicates inadequate anesthesia. In this situation, a top-up dose (half of initial dose) of anesthetic mixture should be given and it should be waited for at least 5–10 min without further stimulation. If the animal still responds to toe pinch, consider the option of using gas anesthetics.
6.
After anesthesia has taken effect, lay the animals down in the dorsal to ventral position on a 37 °C heat mat, disinfect the surgical area with 70 % ethanol, and clear the neck region of fur using hair removal cream.
7.
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A small incision (~1 cm) of the skin is made directly on top of the left common carotid artery region. It is highly recommended that from this step onwards, the procedure is performed under a dissecting microscope to help increase accuracy and avoid unnecessary bleeding.
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