*2.1. Animals*

Adult male Wistar rats (200–250 g, *n* = 60) were obtained from the vivarium of the faculty of medicine of the Universidad Nacional Autonoma de Mexico (UNAM). Animals were individually housed in a temperature- and humidity-controlled environment in a 12:12 h light-dark cycle with free access to food and water. The procedures described in this study were carried out in accordance with current national and international regulations for the use and care of laboratory animals of the Mexican Council of Animal Care (NOM-062-ZOO-1999), Guide of the National Institute of Health for the Care and Use of Laboratories, as well as Animals and Ethics Committee of the UNAM (UNAM-FMED-104-2017), in addition to minimizing the number of animals used for experimental purposes, which ensured generating the minimum possible pain.

#### *2.2. Epicatechin Administration Protocol*

Four experimental groups were considered for the study (15 rats per group): (1) Control (animals were given only sterile water), (2) Aβ25–35 [100 μM], (3) Epicatechin (EC) (200 mg/kg/day × 4 days), and (4) EC + Aβ25–35. The oral administration of EC started 1 day before the surgical injection and continued for 3 days more (4 days total). The treatment doses employed were chosen or calculated based on previous reports [18]. The Aβ25–35 peptide [100 μM] was dissolved in sterile water and the solution incubated at 37 ◦C for 24 h. The animals were then anesthetized with ketamine-xylazine (0.1 mL/100 g, i.p.) and placed in a stereotaxic frame (Stoelting Co. Wood Dale, Illinois, USA). The stereotaxic coordinates used to produce a bilateral lesion in the Hp were A: –4.2 mm from bregma, L: –2.0 mm from the midline, V: –2.2 below dura) [17]. Injections of Aβ25–35 or Control (1 μL) per side were administered for 5 min with a Hamilton syringe. After surgery, the animals were returned to their cages to recover.

#### *2.3. Water Maze Spatial Task*

After the treatment, the spatial memory was evaluated in the water maze (WM) at the Laboratory of Excitatory Amino Acids, Instituto Nacional de Neurología, INNN (Mexico City; Mexico), which consisted of a circular water pool (140 cm diameter and 80 cm high) that was filled with water to a height of 42 cm at 23 ± 24 ◦C and dyed with 0.01% white titanium oxide (TiO2). Four quadrants (N, S, E, or W) were traced into the pool. A platform of 20 cm diameter and 40 cm high, located at a constant position in the middle of one quadrant, was submerged 2 cm below the water's surface. This procedure was repeated during 4 days of training. The animals (*n* = 15/group) were allowed to search for the platform for 90 s and were gently guided to it if they did not reach the target on their own. The time of 90 s was assigned as the maximum score. The parameter to be evaluated was the time to find the platform. The memory test was performed 2 days after training; in this test, the platform was removed from the pool. The test consisted of each of the animals performing a single try to find the place where the platform was. The parameter to be evaluated was the latency time at the first crossing, with the aim of evaluating if the animal memorized where the platform was located. The trials were recorded by a video camera mounted above the center of the pool (Sharp VL-WD450 U, SHARP Corporation, Osaka, Japan).

#### *2.4. Quantifying IL-1*β *and TNF-*α *Cytokines*

After the spatial memory test, the animals were decapitated (*n* = 8/group). The brain was removed from the cranial cavity of the animals. After, the Hp was extracted according to the protocol described by Diaz et al. [6] The hippocampal tissue was placed in a microcentrifuge tube with 1.5 mL of a phosphate bu ffer solution (PBS) (0.1 M; pH 7.4) at a temperature of 4 ◦C, to be liquefied for one minute at 100 rpm, with the help of a homogenizer. Afterward, homogenate hippocampal tissue was centrifuged at 12,500 rpm at 4 ◦C [19,20]. The supernatant was extracted from the centrifuged tissue and subsequently aliquoted in microtubes (200 mL) and stored at –70 ◦C. The supernatants were used for protein and proinflammatory cytokine measurements. The concentrations of IL-1β and TNF-α in the supernatants were quantified by an immunoassay procedure, as specified in the kit protocols (R&D Systems, Minneapolis, MN, USA). Samples were treated with a monoclonal antibody in precoated wells where the immobilized antibody bound to the protein. After washing away any unbound substances, an enzyme-linked specific antibody was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. Samples were read in a microplate reader at a wavelength of 450 nm where it was found that the intensity of the measured color was in proportion to the amount of each cytokine.

#### *2.5. Assay of Lipid Peroxidation*

The lipid-soluble fluorescent compounds were determined by means of the method established by Cuevas et al., and Perez Severiano et al. [17,21]. The supernatant obtained from the hippocampal tissue was mixed with chloroform-methanol (2:1) placed on ice and in a dark room. Subsequently, the chloroform phase was taken to quantify the fluorescence at an excitation of 370 nm and emission wavelengths of 430 nm on a Perkin Elmer LS50-B luminescence spectrometer (Waltham, MASS, USA). The fluorescent signal from the kit was adjusted to 140 fluorescence units (FU) with a standard quinine solution (0.001 mg/mL quinine in 0.05 M H2SO4). The results were expressed as relative FU (RFU) per mg of protein. [19].

#### *2.6. Assay of Reactive Oxygen Species*

We used 5 μL of the supernatant of the hippocampal tissue previously centrifuged, which was diluted in 9 volumes of TRIS and HEPES (40 mM). Subsequently, samples were incubated with 2'7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (5 μM) [22] for one hour at 37 ◦C under constant agitation. The fluorescence signals were determined at 488 nm excitation and emission wavelengths of 525 nm (Perkin Elmer LS50-B luminescence spectrometer, Waltham, MASS, USA). The results were plotted as the mean of the DFC nm formed by mg of protein per minute. [20].

#### *2.7. Determination of Superoxide Dismutase Activity*

To evaluate the activity of superoxide dismutase (SOD), 20μL of the supernatant of the hippocampal tissue previously centrifuged was used, which was incubated with reduced cytochrome-C solution (10 μM), sodium azide (NaN3, 10 μM), disodium ethylenediaminetetraacetic acid (EDTA, 10 mM), sodium bicarbonate (NaHCO3, 20 mM), xanthine (100 μM), and Triton X-100 with a pH of 10.2. To the reaction was added xanthine oxidase (0.1 mM EDTA) and the absorbance was determined at 550 nm. The activity of Zn-SOD was calculated as the total activity minus the activity measured in the presence of potassium cyanide (KCN, 1 mM) (Mn-SOD).

## *2.8. Histological Examination*

After the spatial memory test, the animals (*n* = 7 per group) were anesthetized with pentobarbital (40 mg/kg). Once the animal was in a state of hypnosis, the rib cage was opened and the pericardium was separated. Next, the vertex of the left ventricle was sectioned (with fine-tipped scissors) and a rigid cannula that was connected to the peristaltic pump was inserted, at a pressure of 80 mmHg that generated a continuous flow of 200 mL of PBS in the ascending aorta. The cannula was adjusted to the ventricle with the help of flat forceps. When the perfusion was started, the evacuation of the perfusion blood was facilitated by a cut in the right atrium. Once the perfusion with PBS was finished, perfusion was continued with 300 mL of 4% paraformaldehyde in a continuous flow and to the same pressure to ensure a better fixation of brain tissue. The brains were removed and postfixed in the same fixative solution for 48 h. The brain tissue was dehydrated and embedded in paraffin with the help of a histoquinet (Leica). Subsequently, the tissues were included in paraffin blocks and cut coronally (5 mm) with the help of a microtome (Leica), at the anterior temporal region level, in approximately 3.8 to 6.8 mm of bregma.
