2.7. Creating Transient Middle Cerebral Artery Occlusion (t-MCAO) Model Rats by Filamentous Occlusion

For filamentous occlusion, 4.0 monofilament nylon thread (Ethicon, Johnson and Johnson, Somerville, NJ, USA) was used. The 4.0 monofilament nylon thread was cut to a length of 3.5 cm, roughly 2–3 mm sections at the ends were coated with a mixture of Provil^® novo Base and Provil^® novo Catalyst and were placed in an incubator for 24 h. Once the monofilament was completely dried, the ends were further coated with JM Silicone adhesive. The diameter of the portion forming the occlusion was adjusted to 0.39 mm by comparison with a 27G needle under a microscope. The monofilament was again placed in an incubator for 24 h to dry it completely.

Next, 300 g male SD rats were anesthetized with pentobarbital (50 mg/kg) via intraperitoneal injection. The rats were fixed on a cork board with the animal’s body in a face up position. The neck of the animal was wiped with ethanol for disinfection, and the hair was shaved with hair clippers. Next, 0.2 mL of 0.5% bupivacaine was administered to the neck by subcutaneous injection. An incision was made in the neck and the common carotid artery (CCA) was located and isolated with tweezers. Following this, the branching into the external carotid artery (ECA) and internal carotid artery (ICA) was located and isolated. Then, the superior thyroid artery (STA) and occipital artery (OA), which are found to branch off near the ECA, were cauterized via bipolar electrocautery (SX-2001, Tagawa Electricity Research Laboratories, Ltd., Chiba, Japan). Furthermore, after locating the pterygopalatine artery (PPA) branch point on the ICA, the PPA was cauterized. Then, the ECA was sutured, and artery clamps were placed on the CCA followed by the ICA. A half incision was made in the sutured ECA, and the prepared nylon suture was inserted. The artery clamp fixed on the ICA was removed, and the middle cerebral artery of the rat was occluded by further inserting the nylon suture up to the 20–23 mm mark from its end. The artery clamp fixed on the CCA was removed, and after confirming the absence of hemorrhaging, the cut-open neck was stitched back together.

After the middle cerebral artery occlusion caused ischemia for 2 h, the nylon suture was removed, reopening the blood vessel and creating a transient ischemia-reperfusion injury model rat. After again cutting open the neck where it was sewed shut, the occluded portion up to the incised part of the ECA where the nylon suture was inserted was extracted, and artery clamps were fixed onto the ICA and CCA in that order. After the nylon suture was completely extracted and the ECA was completely sutured, the artery clamps were removed, thus reopening blood flow in the rat’s middle cerebral artery. The surgical incision was again sewn shut and sterilized, and reperfusion was allowed to occur for 22 h.