*2.2. Ech A Encourages A*ffi*rmative Behavioral Changes after Ischemic Stroke*

Functional assessments were applied to determine behavioral changes in the rat model for aggressive central nervous damage [16]. Two methods, namely, the cylinder test and swim test, were adopted and modified. The cylinder test evaluates the frequency of forelimb use and asymmetric movement in postural weight support [17]. In the cylinder test, cerebrally injured rats (control group) exhibited increased asymmetry use, especially practical use of the unaffected forelimb, when compared to the sham group. Furthermore, the 10 μM Ech A-treated group showed behavioral recovery with increased use of both forelimbs and decreased use of the unaffected forelimb, as compared to the control group (simultaneous use: sham 38.0%, control 26.0%, 10 μM Ech A 35.0%; unaffected use: sham 26.0%, control 39.0%, 10 μM Ech A 32.0%) (Figure 2A).

**Figure 1.** Echinochrome A (Ech A) alleviates the infarcted brain region of rat middle cerebral artery occlusion (MCAo). (**A**) Representative TTC staining of brain sections of the damaged area in the sham, control and 10 μM Ech A treatment groups of a rat MCAo/reperfusion model. (\* *p* < 0.05). (**B**) Quantification of size in the infarcted brain region from each experimental group. (**C**) Quantification of water content in infarcted brain region from each experimental group (\*\* *p* < 0.01).

Stroke results in the reduction of predominant activities such as mobility, climbing, and swimming [18]. Since the forced swim test (FST) is frequently used to confirm these activities, this method was applied to assess the antidepressant-like behavior in MCAo models [18]. In this test, while brain infarct rats showed increasing immobility, the behavioral recovered rats (after 10 μM Ech A treatment) show alleviated immobility time during the FST (sham: 33 s, control: 81 s, 10 μM Ech A: 51 s) (Figure 2B). Our results indicate that treatment with an appropriate amount of Ech A enhances the motor ability in brain ischemic disease.

**Figure 2.** Declined movements are restored after Ech A treatment following ischemic stroke. (**A**) Assessment of percent use of affected (left), unaffected (right), and simultaneous (both) forelimbs on the wall of the cylinder (\* *p* < 0.05). (**B**) Total amount of immobility time in the forced swim test (# *p* < 0.01 compared with the values of the sham group; \*\* *p* < 0.01 compared with the values of the control group).
