Animals and experimental design
Male Wistar rats of approximately two months old and weight of 250 – 350 g were used. The animals were conditioned in controlled room temperature at 22 ºC with light/dark cycles of 12 h and kept in appropriate boxes with capacity for 5 animals, covered with shaving, which was switched every two days. They received water and food ad libitum. The maximum concern was deliberated with the purpose of minimizing the suffering of the animals and reducing the number of animals to be used in the research.
The animals were divided into six groups (n = 10) and were divided into control groups: group naïve, group submitted to the intra-hippocampal infusion of saline, group submitted to the hippocampus infusion of LPS; and groups which practiced muscular strength exercises: a group which performed only muscular strength exercises (CT), a group which performed muscular strength exercises and was submitted to the infra-hippocampal infusion of saline (exercise + saline) (n = 10), and a group that performed muscular strength exercise and was submitted to the intra-hippocampal infusion of LPS (exercise + LPS) (n = 10).
The animals were submitted to eight weeks of training three times a week. After the final day of training, all animals performed the muscular strength testing by gripping through the Grip Strength equipment. Then, the surgery was performed to induce acute neuroinflammation through LPS, which was infused in the CA1 region of the hippocampus. The post-operative period was five days, followed by the behavioral tests in the following sequence: open field, object recognition, social recognition, plus maze and hot plate. At last, the animals were euthanized. Then, the heart of each animal was removed to access its mass, whereas the hippocampus, and the prefrontal cortex were removed to analyze the activity of antioxidant enzymes. As per the Additional file 2: Figure S1.
Ethics approval
All the experiments were according to the rules of the Ethics Committee in the Use of Animals—Federal University of Pampa (CEUA/UNIPAMPA) (report #046/2017). the study is reported in accordance with ARRIVE guidelines.
Strength exercise protocol
Muscular strength training was performed using a personalized vertical scale made of wood and iron (1.1 × 0.18 m, 2 cm grip, an inclination of 80°) with a wooden box (20 × 20 × 20 cm) put in the upper part of the stairs. Initially, the animals were familiarized with the exercise, performing four climbing attempts per day for three days. Once they were able to climb to the wooden box, they could rest inside the wooden box for 120 s. The strength training started a week after the familiarization. In the first week, the load corresponding to 50% of the rat's body mass was fixed at the base of the animal's tail. In the second week of training, the maximum load test was performed to determine a load of exercise for each animal individually. It was added 30 g to each repetition of the next climb until the rat was not able to finish the exercise. The heavier load transported successfully was considered the maximum load. The maximum load was determined on the first day of each week of training. The training sessions consisted of eight climbs in the stairs with two repetitions for each load of 50%, 75%, 90%, and 100% of the individual result of the maximum load, resulting in 8 climbs with a rest interval of 1 min between the repetitions, with three training sessions per week (one day for the maximum load and two days for the training sessions), during eight weeks [17].
Grip strength meter
After the final day of training, all animals performed the muscular strength test by gripping through Grip Strength equipment. The grip gauge was positioned horizontally facing the equipment. The animals were put in the metal grid and, next, were pulled backward in the horizontal plane by the tail. The forces applied to the grid through the animal's legs were measured in grams. Immediately before losing the adherence, the maximum tension was recorded. Three tests were performed, and as a result, a mean value was obtained [18].
LPS administration
The animals were submitted to stereotaxic surgery. Subsequently, the intra-hippocampal bilateral administration (CA1 region) administration of vehicle (saline solution) or LPS (from Escherichia coli 055:B5; Sigma) dissolved in PBS in the concentration of 10 mg/ml and administrated in the dose of 40 μg/side, through the infusion of 4 μL/side during 10 min. The stereotaxic surgery coordinates were adapted from the Paxinos & Watson Anatomic Atlas, being as follows: antero-posterior (AP) = − 4.2 mm; mid-lateral (MD) = ± 3.0 mm; dorsum-ventral (DV) = − 3.0 mm; lateral tilting (LT) = 0° starting from the Bregma point. The LPS infusion was performed using a Hamilton (5 µl) syringe. The procedures were performed with previously anesthetized animals with 75 mg/kg of ketamine and 10 mg/kg of xylazine through the intra-peritoneal route. After the surgery, the rats were put in housing boxes under smooth heating to avoid hypothermia.
Object recognition memory test
From the first to the fourth day of the experiment of such task, regarding the habituation period, the animal was put in the left superior corners of the device, a 50 cm × 50 cm × 50 cm box, made of compensated, acrylic transparent polyvinyl chloride, and then left it there for 5 min, without a single object inside the box, so that the animal could get used to the environment.
During the training session, one day after the last day of the habituation day, the animal was once again put in the box with two equal objects, A and B, located right at the center of the box. The animal was left there for 5 min to explore the environment freely. The exploration time of each object was clocked for posterior evaluation. After 3 h, the short-term memory was tested. The animal was replaced in the box with object A and object C (replacing the previous object B) in 5 min for free exploration. The long-term memory duration was tested 24 h after the previous test, in which the animal was once again placed inside the box, with objects A and D (different from objects A, B, and C), and the animal had 5 min for free exploration [19].
Social recognition memory test
This task is an adaptation of the social interaction test proposed by Kaidanovich-Beilin et al., [20]. The task was performed in 3 days. First, the animals were placed in a habituation field (the same size and characteristics described in the previously described object recognition task) with two small cages for 20 min for free exploration. On the following day, the training was performed with a juvenile rat in one of the cages for one hour of free exploration. At the same time, one cage was left empty. After 24 h, the test was performed when the same rat of the training (that is, the rat that became familiar) and a new rat were put for exploration for 5 min. The time exploring the new rat and the familiar rat was registered. Exploring the animal was defined as smelling or touching the cages with the nose or front legs [21].
Open-field test
To evaluate the locomotor ability of the animals, they were put for 5 min in the open-field arena. The apparatus has the same size and characteristics previously described in the object recognition task. The experiments were held in a low sound room under low-intensity lighting. Each rat was placed in the center of the open field and the number of squares crossed and rearing were registered [22].
Elevated plus-maze
To evaluate the state of anxiety of the animals, they were put in an elevated crossway maze (ECM), and the number of entries and the time spent in the open and close arms were registered during a 5-min session [23].
Hot plate
To evaluate the nociceptive response and the sensitivity of the animal's legs, each of them was placed in a device with a warm metal sheet of paper (55 ± 0,5 °C) and the time until the animal showed reaction to the thermal stimulation by raising or licking one's leg was determined [24].
Euthanasia
The animals were sacrificed by physical method, through decapitation by guillotine. The entire procedure was based on the guidelines written by the national council for control of animal experimentation (CONCEA), which aims to establish procedures that evoke minor pain and suffering to the animal.
Heart mass
After all the behavioral tasks, the animals were euthanized, and then the collection of the hippocampus, prefrontal cortex, and heart mass were performed. For the heat mass collection, the open chest procedure was performed, and blood was drawn by cardiac puncture, followed by the removal of the heart. After such removal, the heart was washed with saline solution (0.9%) to remove the excess blood. The heart mass was immediately weighed using an analytic scale. The cardiac hypertrophy index was calculated by the reason between heart mass (mg) and body mass (g).
Histological analysis
After euthanasia, the hippocampus was removed and then fixed in 10% formaldehyde, dehydrated, and embedded in paraffin. Then, Sects. (5 mm) were stained with hematoxylin and eosin (H&E) and analyzed under a microscope. The physical dissection method was used for quantitative analysis of dark neurons recognized by hyper basophilia density and morphological features [25, 26]. All dark neurons present in 400 × 400 µm squares were quantified using ImageJ® software version 1.52c (National Institutes of Health, Bethesda, MD, USA) and visually confirmed using an optical microscope (L3000B, Labor Import®, 400 × magnification). For each rat, three squares were analyzed for the CA1 and dentate gyrus (DG) regions and two squares for the CA3 region. The mean of these values was used as the final value in dark neurons/mm2.
Preparation of the homogenate and protein analysis
Tissue samples of the hippocampus and prefrontal cortex were weighed and homogenized with 200 mM phosphate buffer. This homogenate was used for the quantification of the reduced glutathione (GSH) and lipoperoxides (LOOH) amounts. Further, the homogenate obtained was centrifuged at 10,000 rpm for 20 min. The precipitate was used to the determination of myeloperoxidase enzymes (MPO), whereas the supernatant was used to verify the dismutase superoxide (SOD), catalase (CAT) and S-transferase glutathione (GST) activities.
The protein concentration was measured with a spectrophotometer at 590 nm using Bradford reagent, and bovine albumin (0.012 – 0.100 mg/mL) was used for the standard curve.
Determination of MPO activity
The obtained precipitate (according to the previous description) was re-suspended with 500 µL of 80 mM potassium phosphate buffer with 0.5% of hexadecyltrimethylammonium bromide. After the homogenization, the samples were once again centrifuged at 12,000 rpm, 20 min at 4 °C of temperature in a high-speed refrigerated microcentrifuge. Aliquots of 60 μl of the supernatant of each sample was added to 200 μL of a reactional solution (100 μL of 80 mM phosphate buffer, 85 μL of 22 mM phosphate buffer, and 15 μL of 0.017% H2O2) in triplicate using a 96-well plate. The reaction was started with the addition of 20 μl of tetramethylbenzidine and incubated for 3 min at 37ºC. After, the reaction was interrupted by adding 30 μL of 1,46 M sodium acetate (pH = 3.0) in each well. The absorbance was read at 620 nm, and the results were expressed as units of optical density (OD)/mg of protein, according to Bradley et al. [27].
Quantification of the GSH and LOOH levels
As described previously [28, 29], 50 µl of the homogenate was added to 40 µL of 12,5% of trichloroacetic. Next, the material was centrifuged at 9000 rpm during 15 min. After the centrifuge, 20 μL of the supernatant was added to 270 μL of TRIS buffer (pH 8.9) and 10 μL of 5,5'-dithio-bis-[2-nitrobenzoic acid (DTNB). The absorbance was measured after 5 min at 415 nm, and the obtained values were interpolated in a standard deviation curve of GSH (1.25–10.00 μg/mL) and expressed as μg/g of tissue.
Determination of SOD, CAT, and GST activities
The obtained supernatant (according to the previous description were used to verify the SOD activity, and it was based on the capacity of the SOD in inhibiting the auto-oxidation of the pyrogallol. In a conic tube, the following were added: 442,5 μL of Tris -EDTA buffer and 20 μL of the sample. After agitation, 25 μLof 1 mM pyrogallol was added and incubated. Then, they were centrifuged, and 300 μL of the supernatant was pipetted in a microplate for the reading in the spectrophotometer at 205 nm. The results were compared with the control group being such value equal to 100%. The amount of protein that inhibits the reaction in 50% (IC 50) equals one unit (U) of SOD. The results were expressed in U of SOD/mg of protein [30].
The supernatant was also used to verify the participation of the catalase enzyme (CAT). In a 96-well microplate 10 μL of the sample was placed, added to 290 go of the reaction broth (5 mM Tris/EDTA buffer, pH 8.0, plus 30% H2O2 and ultra-pure water) [31]. After, the decrease in the absorbance was monitored at 240 nm. The results were expressed in µmol of H2O2 consumed/min/g of tissue.
The supernatant was used to verify the activity of the glutathione S-transferase enzyme. In this assay, 50 μL of the supernatant and 250 μL of the reaction broth (0,1 M phosphate buffer, 3 mM 1-Chloro-2,4-dinitrobenzene and 3 mM GSH) were added in a 96-well microplate. Then, the increase in the absorbance was monitored at 340 nm, and the results were expressed in μmol/min/mg of protein [32].
Data analysis
The latency and the time percentage of the behavioral experiments and the mean numbers obtained in the biochemical experiments were compared between the different groups through the parametric Student test (two groups) or ANOVA (more than two groups) with the post-hoc Newman-Keuls test for multiple comparisons, or Dunnett’s test for the comparisons with the control group. P values < 0.05 were considered statistically significant.