Male mdx mice and wild-type C57BL/6 mice, kindly provided by University of São Paulo (USP), Brazil, were used in this study. The animals were 28 days old, and they weighed 18–23 g. The animals were kept in the Vivarium of the Experimental Neuroscience Laboratory (Lanex), in Unisul, Palhoça, Santa Catarina, Brazil. Each box contained five animals, in a 12-h light-dark cycle (6 am to 6 pm), with food and water provided ad libitum. The temperature of the environment was maintained at 22 ± 2°C. The study was conducted at Lanex and at the Laboratory of Biochemistry and Molecular Biology of Unisul, Palhoça, Santa Catarina, Brazil, during 2016 and 2017. Approval for conducting this study was obtained from the Animal Use Ethics Committee (CEUA) of Unisul under the number 16.003.4.01.IV.
2.2 Sample calculation
To calculate the number of animals required for each experimental group, we have to take: n= [(zα/2 + zβ) 2.s2]/(¯ x—µ)2. Where:
- zα/2 = z value on normal curve according to α value;
- zβ = beta error;
- s2 = variance;
- (X—µ)2 = estimated maximum difference between the sample mean (X) and the true population mean (μ). It is the error margin or maximum estimation error.
Therefore, the following values are assigned to calculate the number of animals:
- The alpha value will be set at 0.05. Hence, the zα/2 value, based on the table of z-values for two-tailed distribution, is 1.96.
- The beta value will be set at 0.20. Hence, the z beta value, based on the table of z-values (single-tailed distribution), is 0.84.
- The value of the standard deviation (s) is on average 30% of the means’ value (based on experimental data from our laboratory).
The difference between the groups’ means is at least 40% (based on experimental data from our laboratory). Biological experiments have an embedded error of about 10 –15% (resulting from individual variations, surgical procedure errors, dosage errors, etc.).
Assigning the values to the above formula, we have:
n= [(1,96 + 0,84) 2. 302]/(0,4—0,1) 2 = 7,84 animals
It is therefore understood that a minimum of eight animals should be used in each experimental group to ensure that the experiments’ conclusions are valid, within an acceptable risk of not observing differences where they exist and not seeing differences where they do not exist. This study used 10 animals per group to guarantee data safety and reliability.
2.3. Experimental design
As illustrated in Fig. 1. The animals were divided into the following four groups, each one consisting of eight animals: (1) non-exercised wild-type, (2) exercised wild-type, (3) non-exercised mdx, and (4) exercised mdx. Groups 2 and 4 were subjected to the low intensity aerobic exercise protocol of the swimming type for 4 weeks. After 24 hours of the last day of training, the tests for assessing aversive memory and habituation were conducted. Afterwards, the animals were sedated and submitted to the assisted painless death procedure, and the following structures were removed for determining lipid peroxidation, protein carbonylation, and thiol grouping: the gastrocnemius and quadriceps muscles, diaphragm, prefrontal cortex, cerebellum, hippocampus, striatum, and cerebral cortex.
2.4 Aerobic exercise protocol of swimming type
The groups of exercised animals were submitted to a protocol of swimming aerobic exercise in a plastic container adapted for this purpose (170 x 100 mm), with 35 liters of water at 28 to 30 ºC degrees, divided into eight lanes. The protocol consisted on four consecutive weeks of exercise, four times a week, with daily 15-minute sessions in the first week, 20 minutes in the second week, 30 minutes in the third and fourth weeks (adapted from ). It was used 1 ml of baby’s shampoo all over the container to decrease the surface tension of the water (adapted from ). After the protocol, the animals were gently dried. The groups of non-exercised animals did not perform any type of physical exercise, remaining in their housing boxes during the entire study period.
The determination of the exercise intensity was performed on the fourth week of protocol in the wt e mdx animals. Blood samples were collected before the test, on the 10th and 30th minutes of exercise for subsequent analysis of lactate concentration. The criterion for considering intensity was the increase in concentration of no more than 1 mmol/L between the 10th and 30th minutes of physical exercise. The blood collection was performed in an alcohol (70%) sanitized place. After this procedure, the distal portion of the animal’s tail was slightly sectioned with surgical scissors and 25 μl of a drop of blood was inserted in the lactate collection tape, and through a portable lactimeter, the blood lactate level was measured. Before each test, the equipment was calibrated according to the manufacturer’s instructions .
2.5. Aversive memory test
This test consists of an acrylic box whose floor is formed by parallel metal bars. A platform 7 cm wide and 2.5 cm long is placed near the left wall of the appliance. At the training session, the animals are placed on the platform, and the time taken by the animal to go down the platform with the four legs is measured, in seconds. This time is called latency. Immediately after going down the platform, the animal receives a 0.2 mA shock for two seconds. In the test session, the animal is placed on the platform again, and the time it takes to go down (latency) is measured; however, the shock is not administered. The test is also finalized if the animal does not go down within three minutes .
2.6. Habituation test
This test was performed in a 40 x 60 cm open field, delimited by four 50 cm high walls – three made of wood and one of transparent glass. The open field floor is divided into 16 equal squares, marked by black lines. In the training session, the animals were carefully placed in the square of the rear left corner of the appliance, from which they freely explored the environment for five minutes. Immediately afterward, the animals returned to the housing box. The test session was held 24 hours after the training, in which the training procedure is repeated. The number of four-legged crossings (crossings: motor activity) through the black lines and the number of times the animals rested on their back legs (rearings: exploratory activity) were evaluated in both sessions .
2.7. Oxidative stress measures
2.7.1. Measurement of thiobarbituric acid reactive substances (TBARS)
This method is used to evaluate the oxidation state of hydroperoxides in biological systems. Damage to membrane lipids is determined by the formation of lipoxidation by-products (such as MDA or malondialdehyde), which are reactive substances to thiobarbituric acid heating, formed during peroxidation in membrane systems. MDA reacts with thiobarbituric acid (TBA), generating a pinkish-colored product, read in a 535 nm microplate reader. The technique consists of the following: first of all, the dilution value was calculated so that in the TBARS reaction tube there are 100 µl of tissue protein in 500 µl of BHT buffer. Afterwards, 500 µl of the 0.67% TBA solution were added. The tubes were placed in a dry bath at 96 °C degrees for 30 minutes. To stop the reaction, the samples were placed on ice for five minutes. Finally, 200 µl of the reaction were placed in 96-well microplates and read in the microplate reader at 535 nm.
2.7.2. Measurement of oxidative damage in proteins
This method was used for protein oxidation dosing. It is based on the principle that several ROS attack protein residues, such as amino acids to produce products with the carbonyl group, which can be measured though the reaction with 2,4-dinitrophenylhydrazine. The carbonyl content is determined by a microplate reader at 370 nm, as described by Levine et al. . First, the tissue was homogenized in 1 ml BHT buffer.
The samples were centrifuged for 15 min at 4 °C at 14,000 rpm. 200 µl of the sample were separated for the blank and 200 µl for the test. 100 µl of 20% trichloroacetic acid (TCA) were placed in all eppendorfs. They were centrifuged for five minutes at 14,000 rpm. The supernatant was discarded. The pellet was redissolved in 100 µl of 0.2 molar NaOH. 100 µl dinitrophenylhydrazine 2.4 (DNTP) were placed in the sample and were left to rest for one hour. 100 µl 20% TCA were placed on all eppendorfs, which were centrifuged for three minutes. The supernatant was discarded. The pellet was washed three times with 500 µl ethanol and ethyl acetate (1: 1). For each wash, it was centrifuged for three min at 14,000 rpm and the supernatant discarded. After discarding the last wash, 1 ml of 3% sodium hydroxide (NaOH) was placed on all eppendorfs. The samples were taken to water bath at 60 °C for 30 minutes and read in the microplate reader at 370 nm.
2.8. Thiol groups
Sulfhydryl radicals represent all groups of thiols found in proteins such as albumin and low molecular weight compounds, such as glutathione. These groups can be oxidized when oxidative stress is high. The determination of total sulfhydryl groups, protein-linked sulfhydryl groups, sulfhydryl groups in low molecular weight compounds (free sulfhydryl) can be performed by using Ellman's reagent (2,2-dinitro-5,5-dithiobenzoic acid - DTNB). The thiol groups react with DTNB forming a light-absorbing complex at 412 nm. The technique consists of adding 10% TCA to the same sample volume (1: 1 dilution). White was prepared, containing 100 µl TCA by adding 100 µl PBS. It was centrifuged for 15 minutes at 3,000 rpm (temperature 4 °C degrees); the supernatant was collected, and 30 µl DTNB (1.7mM) and 300 µl hydrochloric acid (TRIS-HCl) were added to 75 µl of this supernatant. It was left to react for 30 minutes and transferred to a 96-well plate. The samples were read in a 412 nm microplate reader.
2.9. Protein Dosages
The proteins were determined by the BCA method and the bovine serum albumin was used as standard. The method is based on the reaction of the copper with the proteins in basic medium. The samples were analyzed by a plate reader at 562 nm.