Targeted Gene Therapy for Ischemic Stroke - Gene Delivery and Therapy for Neurological Disorders - page 197

10. After opening the dura with a sharp end of a bent 30-gauge
needle, further dissect the arachnoid membrane to free the trunk
of the MCA so that it can be slightly elevated above the sur-
face of the cortex, and then cauterize the artery using the elec-
trocautery device (Fig. 2b , see Note 3 ).

11. Close the incision site with surgical 6-0 monofi lament nylon
sutures.
12. Measure surface blood fl ow before and after the occlusion
using a Laser Doppler Flowmeter (Vasamedics, St Paul, MN,
USA) of the artery to make sure the blood fl ow drops to below
20 % of baseline after the occlusion of the artery.

3.3 Intravenous
Injection of AAV
Vectors (See Note 4)

1. Warm the tail by immersing it in warm water or using a heat-
ing pad or placing the animal under a heat lamp.
2. Inject the vectors using 26-gauge needle, and fi nish the injec-
tion of 100

ʼ l viral vectors in 15 s.

3.3.1 Tail Vein Injection
(Fig. 3a , See Note 5 )

1. Anesthetize mouse using 5 % isofl urane inhalation, maintain-
ing at 1 ~ 1.5 % isofl urane during the whole procedure.
2. Put the mouse in a supine position. Shave the fur on the front
neck and disinfect the skin.
3. Make a 1-1.5 cm incision between the chin and the sternum
on the right side.
4. Expose the external jugular vein and a part of the pectoral
muscle (anterior margin of the pectoral muscle is marked by
the disappearance of the external jugular vein beneath it).
5. Place the needle into the vein through the pectoral muscle
with the bevel facing toward you.
6. Inject AAV vectors (2 × 10 11 gcs) in 200

3.3.2 Jugular Vein
Injection

ʼ l in PBS using syringe

with 26-gauge needle.
7. Close the wound with sutures after the injection.

Fig. 3 Schematic illustrations of IV injection and LacZ gene expression mediated
by IV-injected AAV9-H9 vector. ( a ) AAV vector is IV-delivered. ( b ) Infarct area is
circled in brown dots . LacZ gene expression ( blue color ) is exclusively in the peri-
ischemic region. Scale bar: 1 mm