*4.2. Middle Cerebral Artery Occlusion and Reperfusion*

Rats were anesthetized by i.p. injection of 50 mg/kg sodium pentobarbital (Hanlim Pharmaceutical, Seoul, South Korea) and were placed on a heated mat to maintain body temperature of 37.0 ± 1.0 ◦C. The surgical and injection process is illustrated in Figure 8. The Koizumi method [50] of MCAO was used for inducing ischemic reperfusion injury. Cervical skin incision was made to expose the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The CCA was permanently ligated below the bifurcation to the ECA and ICA. The ECA was temporarily ligated while a small incision was made in the CCA to insert a silicone coated nylon filament (403723PK10Re, Doccol Corporation, Sharon, MA, USA) into the ICA until a mild resistance stopped the insertion. The length of the insertion was approximately 18-20 mm (Figure 8a). The rat was occluded for 90 min. and the filament was removed. The experimental group undergoing sham surgery did not have the filament inserted. Polyethylene tubing with an inner diameter of 0.28 mm and outer diameter of 0.61 mm (427401, Becton, Dickson and Company, Sparks, MD, USA) was inserted for injection of LE or vehicle (Figure 8b). Injections were made at 0.5 mL/min over 2 min. The polyethylene tubing was removed after the injection and the CCA above the incision was permanently ligated. The temporary ligation of the ECA was removed. A total of 374 rats were used for this study. The MCAO surgery was conducted on 310 rats and sham surgery was conducted on 64 rats. 84 rats that deceased during MCAO, reperfusion or intra-arterial injection were excluded from the experiment. None of Sham-operated rats deceased. 34 rats that underwent MCAO and reperfusion but did not show infarction due to surgical errors were also excluded from the study.

**Figure 8.** Illustration of middle cerebral artery occlusion and injection. (**a**) Insertion of nylon filament for MCAO. The CCA was ligated permanently while the ECA and ICA were temporarily ligated to stop blood flow. A small incision was made in the CCA for the filament insertion. The silicone-coated filament was inserted until it reached the MCA. The artery was occluded for 90 min. Then the filament was removed for reperfusion; (**b**) Insertion of polyethylene tubing for injection of LE or vehicle. The polyethylene tubing was inserted near to the MCA. After injection, the tubing was removed and the CCA near the bifurcation was permanently ligated. The temporary ligations on the ICA and ECA were removed. Abbreviations: CCA—common carotid artery, ECA—external carotid artery, ICA—internal carotid artery, MCA—middle cerebral artery.

#### *4.3. Drug Treatment*

LE (Intralipid™ 20%, Fresenius Kabi, Uppsala, Sweden) was dissolved in sterile 0.9% NaCl to a final concentration of 10% for the injection of the LE 10% group. Vehicle, 10% LE and 20% LE were intra-arterially administered during reperfusion after 90 min. of MCAO. Intra-peritoneal injection XAV939 (S1180, Selleck Chemicals, Houston, TX, USA) 40mg/kg or DMSO were injected for 2 days prior to the MCAO surgery and on the day of the surgery before MCAO.
