*2.3. Tissues Collections for Molecular and Morphological Analysis*

For biochemical studies, the mice (8–10/group) were anesthetized, sacrificed, and the brain sections were separated. Next, the brain tissue was homogenized in a protein extraction solution (PRO-PREPTM), according to the instructions (iNtRON Biotechnology, Inc., Sungnam, South Korea). After homogenization, the samples were centrifuged at 13,000 r.p.m. at 4 ◦C for 25 min. The supernatants were collected and stored at −80 ◦C.

For the morphological studies, the mice (seven to eight per group) were anesthetized and perfused transcardially with saline at a flow rate of 10 mL/min for 3 min, followed by perfusion with a 4% paraformaldehyde solution for 8 min using a peristaltic pump, as provided [23]. The brains were removed and fixed in 4% cold neutral buffer paraformaldehyde for 48 h, and cryoprotected by immersing into a 30% sucrose phosphate buffer for 48 h at 4 ◦C [23]. After that, the whole brain was frozen in an OCT (optimal cutting temperature compound) compound (Sakura, Torrance, CA, USA), and 14 μm sections were made in the coronal planes using a microtome (Leica cryostat CM 3050S, Nussloch, Germany). The sections were mounted on the probe-on plus charged slides (Fisher, Pittsburgh, PA, USA), and were stored at −70 ◦C for further analyses.
