*4.5. Immunohistochemical Staining*

After completion of the 18F fluorodeoxyglucose (FDG), rats were anesthetized with sodium pentobarbital (100 mg·kg<sup>−</sup>1, intraperitoneally). Rats were perfused with saline solution, followed by 4% paraformaldehyde (PFA). The brain was anatomized from the skull, post-fixed in PFA overnight, and stored in a 30% sucrose in PBS until it subsided. The brain was embedded and serially sectioned on a cryostat (Leica Microsystem Co., Ltd., Wetzlar, Germany) at 30 μm thickness in the coronal plane and the sections were collected in phosphate buffered saline (PBS). The primary antibodies against the following specific antigens were used: ChAT and AchE. The primary antibody was diluted from the concentrate with the blocking solution (0.2% phosphate buffered saline with tween 20 (PBST), 2% blocking serum in PBST). The primary antibody was infused into the brain sections for 72 h at 4 ◦C. After several rinses in PBST, the sections were incubated with secondary biotinylated antibodies against rabbit immunoglobulin G (IgG) or sheet IgG (Vector Laboratories, Burlingame, CA, USA) for

2 h. After washing with PBST, the sections were incubated with an appropriate biotinylated secondary antibody and processed with an avidin–biotin complex kit (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). The staining was conducted using 0.05% 3,3 -Diaminobenzidine (DAB) in the presence of 0.003% H2O2 in 0.1 M PB. After rinsing with 0.1 M PB, the stained tissue sections were mounted on the slide. The images were captured using a DP2-BSW imaging system (Olympus, CA, USA) and processed using Adobe Photoshop Cells. Those testing positive for ChAT and AChE were counted on a grid that was placed on CA1 and CA3 in the hippocampus area. The number of cells was counted at a magnification of 100×, using a rectangular microscopic grid measuring 200 <sup>×</sup> 200 <sup>μ</sup>m2. The cells were counted in 3 sections per rat within the hippocampus.

#### *4.6. Image Processing and Analysis*

After completion of the behavioral test, animals underwent prior fasting over 12–15 h and bedding was changed during the fasting period to avoid ingestion of bedding. The animals were treated with 500 mCi/100 g 18F-FDG via tail intravenous insjsmsjection, and the animals inhaled 2% isoflurane in 100% oxygen (Forane solution; ChoongWae Pharma, Korea) until the positron emission tomography (PET) scan. A transverse resolution of <1.8 mm was used at the center [48]. The emission data were acquired at an energy window setting (350–650 keV, 30 min). The acquired data in the emission list mode were arranged into three-dimensional (3D) sinograms and reconstructed using 3D Reprojection (3DRP) methods. To distinguish the cerebral glucose metabolism, between TMT + Bean-PS and TMT group datasets, a voxel-based statistical analysis was performed using SPM. In summary, the area of the brain was masked using the rectangular method. The PET data were spatially reconstructed onto a rat brain template, smoothed using a 3 mm isotropic Gaussian kernel, and counted. A Statistical Parametric Mapping 8 program was used in the voxel-wise t-test between the TMT + Bean-PS and TMT group datasets (*p* < 0.05, K > 50).
