Fifty male Wistar rats (200 + 50 g; obtained from the Laboratory Animal Center of Hamadan University of Medical Sciences, Hamadan, Iran) were randomly assigned to five groups and kept in a 12h light-dark cycle (lights on: 07:00–19:00 h) at a controlled temperature of 23 ±1 °C. They had free access to food and water. The used experiments were approved by the Veterinary Ethics Committee (Code of Ethics Committee: Grant Number: IR.UMSHA.REC.1394.397), the Hamadan University of Medical Science and were conducted following the Guidelines of the National Institutes of Health (NIH Publication 80–23, 1996). Also, the study was carried out in compliance with the ARRIVE guidelines.
Amyloid-beta- 1–42 (Aβ1–42)
Aβ powder (Sigma–Aldrich, USA) was dissolved in normal saline (PH 7.2) at 1 μg/μL, and we incubated the solution at 37°C for a week before its application. The drug (5μl) was injected via intracerebroventricular (ICV) injection to induce the AD .
Preparation of Vit C
Vit C (Sigma–Aldrich.-USA) was dissolved in distilled water, and 200 mg/kg of it was administered to the rats orally for 60 days.
Animals were habituated to the lab environment for seven days before the study and were randomly divided into five groups (n = 10 rats/group): 1- Control: receiving no intervention and treatment; 2- Sham: receiving 5 μL of Aβ solvent via ICV injection; 3- AD: receiving 5 μL of Aβ1-42 via ICV injection, 4- Vit C: receiving 200 mg/kg of Vit C by oral gavage daily for 2 months, and 5- AD+Vit C: receiving Vit C by oral gavage daily for 30 days followed by 5 μl of Aβ1-42 and Vit C (200 mg/kg) daily for 30 days (fig.1).
Aβ injections and surgery
The rats were anesthetized by ketamine and xylazine (100 and 10 mg/kg, respectively) and transferred to a stereotaxic apparatus (Stoelting Co., USA). Aβ injection was conducted using a 5-μL microsyringe (Hamilton Laboratory Products, USA). The bregma and lambda were determined after determining the distance between the two points, according to the atlas of Paxinos, and the coordinates of the brain ventricular region were adjusted . Aβ solution (5 ml) was injected into the ventricle at both sides of the brain at 5 μL / 20 min. For a better deposition of Aβ injection after 5 min, 2 min of rest was considered, and then injection was continued. After surgery, animals were kept in a warm box for one hour before returning to their cages. Animals were given two weeks to create the AD model. Then, 5–7 days of recovery time were considered after the surgery [30, 31], and then the treatment began.
Locomotor activity test
The present study was based on the measurement of motor activity measured by the video tracking system. In this regard, the animals were first subjected to the treatment period. Then, they were transferred to the video tracking box for half an hour, so that their activities could be analyzed. The output of this analysis included the calculation of two criteria of the physical locomotion of the animal, namely the distance (m) and speed (cm/sec) of movement.
Object recognition test
The tendency of rodents to search for new and old objects can be examined by examining the objects. In this study, rats were placed in a wooden chamber with a length and width of about 40 × 50 cm and a height of 40 cm, with opaque paint [32, 33]. The light of the chamber was also adjusted in a way that no shadow would form. The rats were tested in three phases. First, compatibility with empty compartment; second, familiarization phase with two identical objects; and third, the test phase (test day). The rats were trained and tested for two consecutive days [24, 34]. During 5 min, the animal’s exploration time was measured and recorded for the old object (cube) and the new object (circle) using a timer. The exploratory behavior was defined monitored by the rat's nose pointed toward and within a 2-cm radius of the object. The exploration ratio was calculated by dividing the time to search the new object over the total time exploring the old and the new objects .
Spatial memory assessment
A hippocampal-dependent spatial learning test for rodents , the MWM task, was used for examination of the spatial memory. The apparatus has a circular pool painted black with a diameter of 155 and a height of 60 cm, which was filled with water to a depth of 35 cm (22 ± 1 °C) and divided into four quadrants of the same size. A hidden (10 × 10 cm) Plexiglas platform was located 2 cm below the water surface at the eastern (target) quadrant center. The swim path of the rats was recorded using a video tracking system equipped with a CCD camera (Panasonic, Japan) to be analyzed later using video tracking. Visual cues were provided by large posters on the room wall. To be adapted to the environment, the rats were allowed to swim for 60 s in the tank without a platform an entire day prior to the start of the hidden platform training.
The previous procedure [36-38] was served as the basis for the training session, briefly involving one block of 4 daily trials for four days in a row. Each trial was started by placing the animals in one of the four quadrants. They were allowed to swim in the pool for 90 s in order to find the hidden platform and kept at one of the four quadrants. Animals that failed to find the platform within that period were provided with manual guidance from the researcher toward the platform. A 10-minute rest period was considered for rats between two consecutive trials.
For the assessment of the memory acquisition in the MWM test, swimming distance, and escape latency (the time required to reach the platform) were utilized. For calculation of the rats’ motor activity, swimming speed was employed. For the measurement of the path, by which the rats remembered the platform location (memory retention), the time spent in the target quadrant was utilized.
A day following the fourth session, the probe test was performed, where the platform was eliminated, and rats were given a 60 s free-swimming period prior to elimination. The animals were released in water, exactly opposite the location of the platform. A video tracking system (using Maze Router homemade software) was used for recording behavior . For later analysis, swimming speed and escape latency were recorded. After elevating the platform above the surface of the water, it was covered by aluminum foil in bright color and located elsewhere 30 min following the probe trial, and the rats were allowed to swim for 60 s and find the visible platform so that their visual ability could be examined. The experiments were conducted within 10 to 12 min.
Test of passive avoidance learning (PAL)
A passive avoidance apparatus (Shuttle box) consists of a light and a dark compartment. The rat was located in the light part facing away from the guillotine door that was elevated after 5 s. The door was shut once the rat entered the dark chamber, followed by the application of a 50-Hz square wave with a 1.2 mA constant current shock for 1.5 s. The rat was given a foot shock once it entered the dark compartment again. Once the rat stayed for 120 s on a row in the lighted part, training was stopped. The rat was located in the lighted chamber again in the retention test that was conducted 24 h following the acquisition trial, and the time spent in the dark part (TDC) and step-through latency (STLr) was recorded as a criterion for retention performance [40, 41]. The behavioral tests were conducted between 8 am and 11 am. Once the rat stayed in the lighted part for 120 s on a row, training was stopped. The number of entries into the dark part (i.e. the number of trials, NTa) was recorded. Then, 24 h following the PAL acquisition trial, the retention test was conducted. Similar to the PAL training, the rats were located in the lighted part, and the guillotine door was elevated 5 s later. The STLr and TDC were recorded for up to 300 s. The retention test was stopped once the rat did not enter the dark part within 300 s.
Surgery and electrophysiological recording
Initially, the animal was anesthetized by intraperitoneal urethane injection (1.5 g / kg). After anesthetization, the animal was placed in a stereotactic device and a warm-up pad was used to maintain its body temperature in a range of 36.5±5.5ºC. Small holes were created on the right or left side of the skull. Two stimulating electrodes and a 125-micron microelectrode made from Teflon-coated stainless steel were placed in the perforant path (AP = 8.1 mm = 3.6 mm, ML = 3.35 mm, and DV = 3-3.3 mm from the skull surface) and teeth scattering area (AP = 3.8 mm vs. Bergema, 2.4 mm = ML, 2.7-2.7 mm / 2 mm from the skull surface) of the right or left hemisphere (fig.2). The stabilized electrode was connected to the microwave electrolyte amplifier and the stimulating electrode to the stimulating system. In response to the stimulation of the perforant path, field evoked potentials were recorded from the teeth scattering area. The perforant path was stimulated by monophasic square pulses for 0.1 ms and at 10-second intervals. An excitation command was transmitted through the computer to the stimulator, and from there through the stimulus isolator and stabilizer, and the final stimulation was transmitted through the bipolar electrodes to the perforant path. The perforant path was stimulated with different intensities until the field potential peaked in response to a single wave of the stimuli. To this end, it was necessary to replace the stimulation electrode several times in order to obtain the best points for stimulation and recording. Among the intensities used, the severity of 50% of the maximum response was selected, and to generate LTP, the stimuli were filled. High-frequency stimulation (HFS) was applied to the perforant area with the same intensity, and the percentage of excitatory postsynaptic potential (EPSP) and the number of spikes (PS) were compared. In LTP recordings, the slope of EPSP and the amplitude of the PS were analyzed for 1 h. The exciting potential in the dentate gyrus (DG) region has two components: PS and fEPSP . The benchmark in this study was to measure the slope of fEPSP and the amplitude of PS. The PS amplitude was calculated as the mean voltage difference between the two peaks. FEPSP slope, the voltage variation at 80% of the middle point of the first positive wave (point A) to the peak of the first positive wave (point B) divided by the difference in time between those two points was calculated. Data were recorded and analyzed by a computer. At the end of the study, the animals were sacrificed using a high dose of ketamine.
Using GraphPad Prism (v. 5.0; GraphPad Software, Inc., La Jolla, CA) we conducted the Statistical analyses. One-way analysis of variance (ANOVA) followed by a Tukey test for multiple comparisons were used to analyze the data. All results were expressed as mean ±S.E.M. At all stages, a P <0.05 was considered significant.