operation steps:

1. Body temperature of the mice is maintained at 36.5°C ± 0.5°C during surgery with a heating plate. A feedback controlled heating pad, which warms according to the rectal temperature of the mouse, is highly recommended.
2. Disinfect the skin and surrounding fur with an appropriate agent (e.g. 70% ethyl alcohol) and dry it afterwards.
3. A midline neck incision is made and the soft tissues are pulled apart.
4. The left common carotid artery (LCCA) is carefully dissected free from the surrounding nerves (without harming the vagal nerve) and a ligature is made using 6.0/7.0 string. 5.0 string can also be used.
5. The left external carotid artery (LECA) is then separated and a second knot is made.
6. Next, the left internal carotid artery (LICA) is isolated and a knot is prepared with a 6.0 filament.
7. After obtaining good view of the left internal carotid artery (LICA) and the left pterygopalatine artery (LPA), both arteries are clipped, using a microvascular clip.
8. A small hole is cut in the LCCA before it bifurcates to the LECA and the LICA. A monofilament made of 8.0 nylon coated with silicon hardener mixture (see below) is then introduced into the LICA, until it stops at the clip. Attention has to be paid not to enter the occipital artery. (Figure 2)
9. The clipped arteries are opened while the filament is inserted into the LICA to occlude the origin of the LMCA in the circle of Willis.
10. The third knot on the LICA is closed to fix the filament in position. And use the laser speckle check the blood perfusion. (Figure 1B), if the blood perfusion has decline more than 70%, that means we had put the suture in the right place.
11. The mice receive saline 0.5 mL subcutaneously as volume replenishment. For pain relief, Lidocaine gel is topically applied in the wound.
12. If reperfusion is intended, mice stay for 30 – 90 min occlusion in a heated cage, the wound could be closed with a small suture clip. Afterwards, a second anaesthesia is performed, the third knot on the ICA is momentary opened and the filament withdrawn. And use the laser speckle check the blood perfusion. (Figure 1C)
13. The remaining sutures are shortened and the skin is adapted with a surgical suture.
14. All animals receive a second volume replenishment as described above.
15. Animals are put in a heated cage for two hours to control for body temperature. The animals must be checked daily after surgery for signs of discomfort. The mice could show some post surgical weight