*2.3. ChAT and AchE Immunoreactivity*

The results of the evaluations of the ChAT-positive cells per section from the different hippocampal formations are shown in Figure 3. The ChAT activity in the hippocampus of the normal group was significantly higher than that of the TMT group. In particular, there were significant differences in the hippocampal CA1 (F2, 18 = 8.53, *p* < 0.01) and CA3 (F2, 18 = 22.94, *p* < 0.001).

**Figure 2.** Effect of Bean-PS on escape latency into the dark phase of the retention test during the passive avoidance task. Each value is expressed ± S.E.M. Normal group (*N* = 6); TMT group (*N* = 5); TMT-Bean-PS (*N* = 5).

The numbers of ChAT-positive cells in the TMT+Bean-PS group were higher than those in the TMT group, particularly in CA1 (*p* < 0.05). Immunoreactivity of ChAT in the TMT + Bean-PS group was significantly increased in CA1 (*p* < 0.05), as seen in Figure 3A,B. The TMT + Bean-PS group in the CA3 region showed no difference compared with that of the TMT group as seen in Figure 3A,B. The results of the evaluations of the acetylcholinesterase (AchE) immunoreactive cells per section from the different hippocampi are shown in Figure 4A,B. The AChE activity in the hippocampus of the TMT group was significantly lower than that of the normal group (*p* < 0.001). In particular, there were significant differences in both CA1 (F2, 15 = 12.07, *p* < 0.01) and CA3 (F2, 15 = 10.85, *p* < 0.01). However, the AChE reactivity in the TMT+Bean-PS group was higher than that in the TMT group, particularly in CA1 (*p* < 0.01) and CA3 (*p* < 0.01).

**A Figure 3.** *Cont*.

**Figure 3.** Effect of Bean-PS on the number of choline acetyltransferase (ChAT)-positive neurons in the hippocampus. Representative photographs and the number of positive neurons are indicated in (**A**) and (**B**). Black square represents region of CA1 and CA3 in the hippocampus and the scale bar represents 200 μm. Results are expressed as means ± S.E.M. \*\*\* *p* < 0.001, \*\* *p* < 0.01 vs. normal group, # *p* < 0.05 vs. TMT group. Normal group (*N* = 9); TMT group (*N* = 5); TMT-Bean-PS (*N* = 5).

**Figure 4.** *Cont*.

**Figure 4.** Effect of Bean-PS on the density of acetylcholinersterase (AchE)-immunostained nuclei in the hippocampus. Representative photographs and the density of AchE-immunostained nuclei are indicated in (**A**) and (**B**). Black square represents region of CA1 and CA3 in the hippocampus and the scale bar represents 200 μm. The values are presented as means ± S.E.M. \*\* *p* < 0.01 vs. normal group, ## *p* < 0.01 vs. TMT group, respectively. Normal group (*N* = 6); TMT group (*N* = 5); TMT-Bean-PS (*N* = 5).

### *2.4. Brain Glucose Metabolism*

Figure 5 and Table 1 show the results of 18F-fluorodeoxyglucose (FDG) uptake measurements from the different brain regions. The cerebral glucose activity of the TMT group was markedly reduced in the hippocampus and frontal lobe compared with that of the normal group in statistical parametric mapping (SPM) analysis of FDG-positron emission tomography (PET) (Figure 5A and Table 1A, *p* < 0.05). In addition, the activity of the TMT + Bean-PS group was significantly increased in the frontal lobe and hippocampus compared with the TMT group (Figure 5B and Table 1B, *p* < 0.05).

**Table 1.** The changes of Z values in the hippocampus and frontal lobe. The results of voxel-wise comparison between the TMT + Bean-PS and TMT group datasets are shown (Table 1A). In the statistical parametric mapping (SPM) analysis, the cerebral glucose uptake of TMT+Bean-PS datasets was significantly increased in the hippocampus and frontal lobe compared with that of the TMT group (Table 1B).


**Figure 5.** Brain regions showing regional glucose metabolism. (**A**) Brain regions where the regional 18F-fluodeoxyglucose (FDG) uptake in the TMT group (*N* = 3) was markedly lower than in the normal group (*N* = 3) (hippocampus and frontal lobe). (**B**) Brain region where reginal glucose uptake in the TMT + Bean-PS group (*N* = 3) was significantly higher than in the TMT group (frontal lobe and hippocampus). Green line: cross hair
