**3. Results**

#### *3.1. E*ff*ect of Epicatechin Treatment on Spatial Memory in Rats Injected with Aß25–35 in the Hippocampus of Rats*

The animals of each group performed the spatial training test in the WM, where the latency time to find the escape platform was quantified. The behavior of the animals of all the experimental groups to find the platform with respect to the days of training showed a progressive decrease in the latency time. The comparative analysis indicates that the group administered with Aβ25–35 showed a significantly longer latency time compared to the control group and the group administered only with EC on the di fferent days that the training test was performed (one-way Analysis of Variance (ANOVA), *p* < 0.05). On the other hand, the group administered with EC + Aβ25–35 showed a latency time to find the platform lower in comparison with the group administered only with Aβ25–35 with a statistically significant di fference that was observed from day 2 of training until the end of the test (One-way ANOVA, *p* < 0.05) (Figure 1A).

For the memory test, carried out two days after the learning test (Figure 1B), it is shown that the latency time at the first crossing of the target quadrant was significantly higher in the group treated with Aβ25–35 with respect to the other three experimental groups, respectively. Particularly, when comparing the group of Aβ25–35 with respect to the group EC + Aβ25–35, it is shown that the administration of EC significantly lowered the latency time at the first crossing of the white quadrant, suggesting a diminishing of cognitive damage caused by the neurotoxicity of the peptide into the Hp (one-way ANOVA, *p* < 0.05) (Figure 1A).

**Figure 1.** The administration of EC prevents the deterioration in spatial memory that induces the injection of Aβ25–35 in the rat Hp. The spatial training test and spatial memory test of rats administered with saline solution, epicatechin, <sup>A</sup>β25–35, and epicatechin (EC) + Aβ25–35 were performed in the water maze. The parameters to be quantified were the latency time to find the escape platform (**A**) and latency of the first crossing in the objective quadrant (**B**). The values shown represent the standard error (SE) mean one-way ANOVA (\*\* *p* < 0.01) comparing all groups with respect to the vehicle group. (# *p* < 0.05) compared the Aβ25–35 group versus EC + Aβ25–35 group.

#### *3.2. E*ff*ect of the Administration of Epicatechin on the Oxidative Response Induced by A*β*25–35 in the Hippocampus of Rats*

The lipid lipoperoxidation data obtained from the Hp are shown in Figure 2A. The group injected with Aβ25–35 showed a significant fourfold increase compared to the basal levels of peroxidation in the control group. Treatment with EC + Aβ25–35 demonstrated its antioxidant effect by significantly decreasing (65%) the lipoperoxidation levels with respect to the group injected intrahippocampally with Aβ25–35 (one-way ANOVA with significance of *p* < 0.05), while the group treated only with EC did not show any considerable changes in lipid peroxidation when compared to the control group. The amount of ROS by 2,7-dichlorodihydrofluorescein found in the Hp is shown in Figure 2B. The statistical analysis reveals that the group injected with Aβ25–35 presented a significant increase of 59% regarding the control group. Likewise, when analyzing the data obtained from the hippocampi of the group treated with EC + Aβ25–35, there was a significant decrease of 28% in the levels of ROS in relation to the group injected only with Aβ25–35 (one-way ANOVA with a significance of *p* < 0.05). Regarding the group only injected with EC, the data obtained did not show a significant difference when compared with the control group. Figure 2C shows the enzymatic activity of Zn-SOD in the Hp. The Aβ25–35 group presented a decrement of 38% in SOD activity in relation to the vehicle group. In the same way, the SOD activity in the EC + Aβ25–35 group decreased by 25% with respect to the Aβ25–35 group (one-way ANOVA with a significance of *p* < 0.05), while between the control group and EC-treated group there were no differences. The Mn-SOD activity is shown in Figure 2D. The Aβ25–35 group and control group differed significantly by 35%. Likewise, when comparing the Mn-SOD activity in the EC + Aβ25–35 group in relation to the Aβ25–35 group, there was a significant change of 21% (one-way ANOVA with a significance of *p* < 0.05), while the EC group showed no differences regarding the control group.

**Figure 2.** Effect of epicatechin on oxidative stress in the Hp of rats injected with Aβ25–35. (**A**) Reactive Oxygen Species (ROS) assay; (**B**) lipid peroxidation assay; (**C**) Zn-Superoxide activity assay; (**D**) Mn-Superoxide activity assay. The mean ± SE is plotted. Data were analyzed with one-way ANOVA and post-test Bonferroni test. (\* *p* < 0.05; \*\* *p* < 0.01; and \*\*\* *p* < 0.001) comparing all groups with respect to the vehicle group. (# *p* < 0.05 and ## *p* < 0.01) compared the Aβ25–35 group versus EC + Aβ25–35 group.

#### *3.3. E*ff*ect of Epicatechin on the Production of IL-1ß and TNF-*α *in the Hippocampus of Rats Injected with Aß25–35*

The concentrations of IL-1β and TNF-α were determined from a supernatant of the hippocampal tissues of each of the experimental groups (Figure 3). The concentration of IL-1β clearly shows that the intrahippocampal injection of Aβ25–35 was significantly higher compared to the control group. The EC treatment prevented the increase of the IL-1β in the EC + Aβ25–35 group (one-way ANOVA with a significance of *p* < 0.05), but the EC-treatment-only group did not show changes in relation to the control group. Similar behavior was observed in the TNF-α level in the Hp of the Aβ25–35 group, which was significantly higher compared to the rest of the experimental groups, and EC treatment prevented the exacerbation of the cytokine level after Aβ25–35 injection; however, TNF-α did not have changes in the EC group (one-way ANOVA with a significance of *p* < 0.05).

**Figure 3.** The epicatechin treatment decreases the concentration of proinflammatory cytokines in Hp of rats with Aβ25–35. (**A**) IL-1β in Hp and (**B**) TNF-α in Hp. The mean ± SE is plotted. Data were analyzed with one-way ANOVA and post-test Bonferroni test (\* *p* < 0.05) comparing all groups with respect to the vehicle group. # *p* < 0.05 compared the Aβ25–35 group versus EC + Aβ25–35 group.

#### *3.4. The Administration of Epicatechin Changes the Immunoreactivity of HSP-60, HSP-70, and HSP-90 in the Hippocampus of Rats Injected with Aß25–35*

To understand the reactivity of HSP in response to the toxicity of Aβ25–35 and the EC treatment in rat hippocampi, immunofluorescence was performed to identify the HSP-60, HSP-70, and HSP-90 in the Hp-CA1 region in each experimental group. The photomicrographs are shown in Figure 4A. Qualitative analysis indicates that the group injected with Aβ25–35 generated a greater immunoreactivity in the HSP (green color) in the CA1 region of the Hp compared to the control group. Particularly, HSP-60 showed a more intense label compared to HSP-90 and HSP-70. The treatment with EC in animals injected with Aβ25–35 caused a decrease in the immunoreactivity of the three chaperone proteins (green color). Specifically, the HSP-70 immunoreactivity decreased in greater proportion with respect to HSP-90 and HSP-60 in the CA1 region of the Hp.

The statistical analysis of the number of cells reactive to HSP-60, HSP-70, and HSP-90 (Figure 4B–D) indicates that the group with Aβ25–35 shows a significant increase in the number of immunoreactivity cells of 400%, 83%, and 200% for HSP-60, HSP-70, and HSP-90, respectively, in the CA1 region of the Hp, whereas the treatment with EC plus Aβ25–35 caused a significant reduction of 17%, 72%, and 58% for HSP-60, HSP-70, and HSP-90, respectively, in the CA1 region of the Hp (one-way ANOVA with significance of *p* < 0.05).

**Figure 4.** Effect of epicatechin on immunoreactivity of HSPs in CA1 subfields of the Hp of rats injected with Aβ25–35. In (**A**) we observed the immunoreactivity (green color) for HSP-60, HSP-70, and HSP-90 in the CA1 subfield of the Hp of rats with different treatments: control, Aβ25–35, EC, and EC + Aβ25–35. The epicatechin treatment decreases the number of immunopositive cells to HSP-60 (**B**) HSP-70 (**C**) and HSP-90 (**D**) in the Hp of rats injected with Aβ25–35 (**A**). The mean ± SE is plotted. Data were analyzed with one-way ANOVA and post-test Bonferroni test (\* *p* < 0.05 and \*\*\* *p* < 0.001) comparing all groups with respect to the vehicle group. (# *p* < 0.05 and ## *p* < 0.01) compared the Aβ25–35 versus EC + Aβ25–35 groups.

#### *3.5. The Administration of Epicatechin Reduces the Immunoreactivity of Caspase-3 in the Hippocampus of Rats Injected with Aß25–35*

We performed an immunohistochemistry for caspase-3 and hematoxylin and eosin staining to investigate whether EC reduces neuronal death. The photomicrographs are shown in Figure 5A. The images reveal that Aβ25–35 induces a greater immunoreactivity to caspase-3 (green color) in the cells of the CA1 region of the Hp, compared to the control group, while for the EC + Aβ25–35 group, there exists a lower marker to caspase-3, with respect to the group injected only with Aβ25–35. The number of immunoreactive cells to caspase-3 indicates that Aβ25–35 increases the reactivity to caspase-3 by 40% with respect to the control group. However, treatment with EC in animals with Aβ25–35 reduces the immunoreactivity by 45% in the cells of the CA1 region of the Hp (Figure 5B) (one-way ANOVA with a significance of *p* < 0.05). Hematoxylin and eosin staining show damage to the neurons through cytoplasmic changes of eosinophilia, in addition to the observed pyknosis, karyorrhexis, and karyolysis. This was more severe in the CA1 region of the Hp of the group treated with Aβ25–35 than in the EC + Aβ25–35 group. These indicators were not observed in the control and EC group (Figure 5B). Figure 5A shows the viable cell number in the CA1 subfield of the Hp. The number of viable neurons in the Aβ25–35 group was less compared with the control group (54%), whereas the number of viable cells in the EC + Aβ25–35 group was greater with respect to the Aβ25–35 group (65%) (one-way ANOVA with a significance of *p* < 0.05). The group treated only with EC showed no changes

with respect to the control group. This indicates that EC decreases the neuronal death that Aβ25–35 induces in rat hippocampi.

**Figure 5.** The epicatechin decreased the immunoreactivity of caspase-3 and the number of damaged cells in CA1 subfields of the Hp rats of injected with Aβ25–35. In (**A**) we observed the immunoreactivity for caspase-3 (green color) and the cells stained with H & E of the CA1 subfield of the Hp of rats control, Aβ25–35, EC, and EC + Aβ25–35 groups. The epicatechin treatment decreases the number of immunoreactive cells to caspase-3 (**B**) and a number of damaged cells in the CA1 Hp of rats injected with Aβ25–35 (**C**). The mean ± SE is plotted. Data were analyzed with one-way ANOVA and post-test Bonferroni test (\* *p* < 0.05 and \*\*\* *p* < 0.001) comparing all groups with respect to the vehicle group. # *p* < 0.05 compared the Aβ25–35 and EC + Aβ25–35 groups.
