*3.2. Preparation of Ischemic Stroke Rat Models*

#### 3.2.1. Animals

Nine-week-old male Sprague Dawley (SD) rats (290–300 g; KOATECH, Gyeonggi-do, Korea) were handled in accordance with the animal welfare guidelines issued by the Korean National Institute of Health and the Korean Academy of Medical Sciences for the care and use of laboratory animals.

## 3.2.2. Middle Cerebral Artery Occlusion

We applied a modified surgical procedure of the standard method [26,27]. Briefly, rats were anesthetized with isoflurane in a mixture of 30% oxygen and 70% nitrous oxide. Using an operative microscope, a 3-0 nylon suture was inserted into the internal carotid artery (ICA) through the external carotid artery (ECA). After 90 min of occlusion, the suture was withdrawn for 7 days for reperfusion. The animals were randomly divided into three groups (*n* = 12 per group): group I, sham-operation (sham), group II, MCAo/reperfusion-induced ischemic group with saline treatment (control), group III, MCAo/reperfusion-induced ischemic group with 10 μM Ech A treatment.
