Fig. 14.1
Preparation of the intraluminal suture. (a) The intraluminal suture for the middle cerebral artery occlusion model. (b) The tip of the 6-monofilament was rounded using a soldering iron. (c) The intraluminal suture for the intracranial internal carotid artery injury model; the tip was coated with epoxide resin
IICAI model: A 5–0 monofilament suture (Nesco suture GA05NA, Alfresa Corporation, Japan) is cut into 15–20 mm long pieces and coated with epoxide resin solution (#16323, Konishi, Japan) for a length of 6 mm and 300 μm in diameter from the tip (Fig. 14.1b). One way to make this suture is to first make a hole in a piece of paper using a 29-G insulin syringe (ss-05 M2913, Terumo, Japan). After placing epoxide resin solution around the 5–0 monofilament, the suture is passed through the hole and marked 10 mm from the tip using white correction fluid.
14.3 Surgeries
The protocols for the MCAo and IICAI models are the same from step 3 to step 16.
3.
Mice are anesthetized using isoflurane (4 %) in a chamber, and the fur on the head and the ventral neck region is shaved using a hair clipper (ER 803P, Panasonic, Japan). The level of isoflurane is then reduced to 1.7 %, and artificial tear ointment is applied to both eyes.
4.
The mouse is placed in the abdominal position on a heating pad attached to a mouse Rotary Brain Anchor-block (MBB-100, Unique medical, Japan). A probe is inserted into the rectum, and the body temperature is maintained between 36.5 and 37.5 °C using a thermostat (ATC 402, Unique Medical).
5.
After sterilization using 70 % ethanol, 0.5 % bupivacaine is injected subcutaneously along the prospective incision site. A midline incision is made on the head, and a fiber optic probe is glued on the right parietal bone 2-mm posterior and 4-mm lateral to the bregma (the right MCAo region) and connected to a laser Doppler flowmeter (Unique Medical).
6.
The mouse is rotated and placed in the supine position. It is important that the neck is dorsiflexed sufficiently to obtain a large enough operative field. After sterilization using 70 % ethanol and the subcutaneous injection of 0.5 % bupivacaine, a midline incision is made on the neck under a stereomicroscope (Fig. 14.2a). A sufficient operative field is obtained using a retractor, which is remolded from a 26-G needle (Fig. 14.2a, asterisk).
Fig. 14.2
Surgical procedures (protocols 6–11). (a) Protocol 6. The asterisk shows the retractor made from a 26-G needle. (b) Protocol 7. (c) Protocol 8. The asterisk shows the 6–0 silk suture around the CCA. (d) Protocol 9. (e) Protocol 10. The asterisk shows a 6–0 suture pulled in the direction of the rostrum. (f) Protocol 11. CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery
7.
The common carotid artery (CCA) is exposed carefully and is dissected free from the surrounding veins, nerves, and fascia (Fig. 14.2b).
8.
A 6–0 silk suture is placed loosely around the CCA and then pulled in the caudal direction to show the external and internal carotid arteries (Fig. 14.2c).
9.
The external carotid artery (ECA) and ICA are exposed carefully, and then dissected free from the surrounding veins, nerves, and fascia (Fig. 14.2d).
10.
11.
The proximal portion of the ECA is tied loosely using a 6–0 silk suture (Fig. 14.2f), and the surgeon then ensures that no blood is flowing in the ECA.
12.
A small incision is made in the portion between the distal and proximal sutures in the ECA using a 30-G needle (Dentronics, Japan) (Fig. 14.3a).
Fig. 14.3
Surgical procedures (protocols 12–14). (a) Protocol 12. (b) Protocol 13. The asterisk shows the loosened 6–0 suture in the proximal site of the ECA. (c) Protocol 14. (d) Protocol 14. The asterisk showed the tip of suture inserted into ICA. (e) Protocol 15. (f) Protocol 17. CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery
13.
14.
15.
16.
The inserted suture is moved into the origin of the MCA until the CBF falls down (Figs. 14.4 and 14.5). Generally, 8–9 mm from the tip is sufficient to occlude the MCA.
Fig. 14.5
Surgical procedures (protocols 15–17). (a) CBF is measured using laser Doppler in the right MCA area. (b) The intraluminal suture is advanced into the origin of the MCA. (c) The dotted area in the straight segment of the IICA will be damaged after IICAI. Arrowhead, the direction of blood flow. ACA, anterior cerebral artery; CCA, common carotid artery; ECA, external carotid artery; IICAI, intracranial internal carotid artery injury; ICA, internal carotid artery; MCA, middle cerebral artery; PA, pterygoid artery; PCA, posterior cerebral artery (Fig. 14.5 was published previously [12]). CBF was reduced compared with baseline (arrowhead in b). The suture is placed for 5 min, which dilates the artery. Slightly withdrawing the suture recovers CBF, and the suture is then advanced and withdrawn 10 times (asterisks in b)
17.
A piece of PE-50 catheter is placed between the CCA and the suture around the CCA, and the suture is then tied to stop the flow in the CCA (Fig. 14.3f). Several drops of sterile saline are placed into the operative field to moisten the CCA, ICA, and ECA.
MCAo Model
18.
Duration of ischemia is 40 min in our lab, but it should be adjusted to get sufficient infarct volume without causing high mortality. Typically, the CBF increases gradually 4–7 min after injury (# in Fig. 14.4a) and then drops dramatically and recovers to the level of the CBF immediately after MCAo (# in Fig. 14.4a). This increase-drop-recovery wave occurs several times.
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