2.1. Animals
The male Sprague-Dawley rats (250–280 g) were purchased from the Animal Laboratory Center of Shiraz University of Medical Sciences, Shiraz, Iran. The animals were kept under standard conditions at room temperature (22 ± 2°C), with normal humidity and 12 h light-dark cycles. The rats had free access to standard food and water. All animal procedures were performed under the standard rules established by the Animal Care and Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1401.009).
2.2. Experimental design
Fifty-five male Sprague-Dawley rats were randomly divided into the following five groups: 1- sham group, laminectomy was performed; 2- SCI group, the spinal cord clip compression model was done; 3- SCI-MP group, the rats were treated intraperitoneally (IP) with MP (30 mg/kg) immediately after SCI [15]; 4- SCI-GFRS group, the rat received GFRS (200 µl, IP) for six consecutive days; and 5- SCI-MP + GFRS group, the rats were treated with MP (30 mg/kg, IP) immediately after SCI, then received GFRS (200 µl, IP) for six consecutive days. Six rats from each group were used for stereological and histological estimation, and five for lipid peroxidation and antioxidant status measurements.
2.3. Spinal cord surgery procedure
Before induction of SCI model, the rats were administrated with antibiotics (ceftriaxone, 50 mg/kg), and then were anesthetized intraperitoneally with ketamine (80 mg/kg; Pasteur, Romania) and xylazine (10 mg/kg). The animals were placed on a heating pad at 36°C during surgery, and were subjected to laminectomy at the thoracic level to expose the spinal cord. An aneurysm mini clip (1 N; 103 gms) held in an applicator in the open position, it was rapidly released from the applicator and applied vertically onto the intact T10 level of exposed spinal cord. The clip was then rapidly (1 minute) released from the applicator to produce acute impact compression injury (Fig. 2) [16, 17]. Acute extradural clip compression injury is a reliable model for producing acute experimental spinal cord injury. It should be noted that the rat thoracic cord clip compression model is a reproducible, clinically relevant spinal cord injury model [16]. After surgery, rats were given diclofenac (25 mg/kg) for pain relief. Then, the rats were kept in separate cages and underwent urinary bladder massage at least twice a day until the recovery of spontaneous bladder function [18].
2.4. Growth factor-rich serum preparation
Blood was collected from healthy male Sprague-Dawley rats by cardiac punctures under anesthesia. Every 14 ml of blood was added to a tube containing 0.7 ml of 3.8% sodium citrate, then centrifuged at 360 g for 10 min. The plasma was separated above the buffy coat to prevent mixed-up white blood cells. Whole blood cells and the obtained plasma fraction (PRP) were analyzed with a hematologic analyzer (XP-300, Sysmex, Kobe, Japan), and the platelet yield was adjusted to above 60%. To activate the release of biological factors from platelets, 10% calcium chloride (50 µl) was added to every 1 ml of plasma. To complete the clotting and retracting process, the plasma was incubated for 20 min at 37 ˚C and centrifuged at 1000 g and 4 ˚C for 20 min. The obtained supernatant, known as growth factor-rich serum, was aliquoted and stored at -80 ˚C [19].
2.5. Behavioral analysis
2.5.1. Basso-Beattie-Bresnahan test
The 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale was used to assess the locomotor behavior recovery after SCI. The BBB scores were documented by observers who were blind to the treatment [20].
2.5.2. Rotarod test
The rats were pre-trained on the rotarod apparatus prior to compression injury at a speed of 5 rpm for at least 5 min on three consecutive days. On the day before SCI induction, the rats were tested by accelerating the speed from 5 to 20 rpm over 5 min. Then, 28 days after SCI, motor performance was examined with the same test conditions [21, 22].
2.6. Stereological analysis
2.6.1. Tissue preparation
The animals were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) on day 28 after SCI. Then, the spinal cords (including the epicenter of injury) were removed (approximately 6 mm) and kept in 10% neutral buffered formalin for 24 h. Subsequently, they were transferred to 30% sucrose in 0.1 M PBS (pH 7.4) for 72 h at 4 ℃, flash-frozen in isopentane (2-methlybutane), and stored at 80°C. Afterward, spinal cord tissues were embedded in optimal cutting temperature compound (OCT, Tissue-Tek) and cut into 40 µm serial transverse sections on a cryostat. Fifteen sections were prepared from each sample (at an interval of 400 µm) and stained with Cresyl Violet (Nissl staining).
2.6.2. Estimation of volumes
The total volumes of the spinal cord, spared white matter, spared gray matter, and lesion were estimated using the Cavalieri method [23]. The slides were labeled with a specific codes and the analysis processes were blinded. A point grid was superimposed on the image of each section on the monitor using stereology software designed by Shiraz University of Medical Sciences, Shiraz, Iran. The total volumes were determined by counting the number of points hitting these areas in the sampled sections, and multiplying the sum of points (∑P) by the area around each point (a/p, 90000 µm2) and the interval between the sampled sections (d). The formula is presented below:
V = ∑P × (a/p) × d
2.6.3. Estimation of cell numbers
A computer linked to a light microscope (Nikon E200, Japan) with an oil immersion lens (100×, numerical aperture = 1.4) was employed to estimate the total number of neurons and other cells in the spared gray matter (the non-neuronal cells which are morphologically different from neuronal cells, characterized by smaller nuclei). According to the optical disector method, the unbiased counting frame with acceptance (upper and right) and forbidden (lower and left) borders was superimposed on the images of the brain sections viewed on the monitor. Then, the microscopic fields were scanned and sampled by moving the microscope stage at equal distances in X and Y directions to ensure systematic uniform random sampling [24, 25]. The nucleus of cells that came into focus within the sampling box (h × a/f) was selected if it was located totally or partially inside the counting frame and did not touch the left and bottom borders of the frame. The total number of cells in the spared gray matter was estimated by multiplying the numerical density (Nv) by the volume of spared gray matter:
Nv = [∑Q/∑P×(a/f)×h]× [t/BA]
where ΣQ is the total number of cells in the spared gray matter coming into focus when scanning the height of the disector; ΣP is the total number of counting frames in most fields; h is the height of the disector; a/f (2500 µm2) is the frame area; t is the mean section thickness calculated in every sampled field using the microcator; BA is the block advance of the microtome set at 40 µm.
2.7. Histological evaluation
The Nissl-stained sections were examined under a light microscope (Olympus BX51, Tokyo, Japan) by a pathologist who was blinded to the experimental groups. The groups were graded 0 to 3 (absent, mild, moderate, severe) to assess necrosis, inflammation, and hemorrhage parameters. The number of cavities in the sections was also evaluated [26, 27].
2.8. Biochemical analyses
On day 28, the animals were sacrificed after deep anesthesia with ketamine (80 mg/kg) and xylazine (10 mg/kg). Then, the spinal cords (including the epicenter of injury) were immediately removed and weighted. The specimens of spinal cord (each specimen: 0.025 g) homogenized in 1 mL of 0.1 M phosphate buffer solution (PBS; pH 7.4). The homogenates were centrifuged at 3000 rpm for 20 min at 4°C; then, the supernatants were aliquoted and stored at -80 ˚C until assessment.
2.8.1. Malondialdehyde assay
The supernatant from each homogenized sample was used for the malondialdehyde (MDA) assay by thiobarbituric acid reactive substances (TBARS) test. Briefly, the solution containing 0.25 N hydrochloric acid (HCl, Sigma, Germany), 20% trichloroacetic acid (TCA, Sigma, Germany), and 0.8% thiobarbituric acid (TBA, Sigma, Germany) was added to the supernatants and standards (1,1,3,3-tetra ethoxy propane, Sigma, Germany). Then, they were incubated at 87 ◦C for 1 h and centrifuged at 12,000 rpm for 5 min and finally read with a microplate reader (Biotek, USA) at the absorbance wavelength 532 nm [28].
2.8.2. Glutathione assay
The glutathione (GSH) concentration was measured by glutathione colorimetric assay kit (ZellBio GmbH, Ulm, Germany). In this assay, DTNB (5,5′- dithio-bis-[2-nitrobenzoic acid]) reacts with reduced glutathione to form a yellow product that absorbs at 412 nm. The optical density, determined at 412 nm, was directly proportional to glutathione concentration in the samples.
2.8.3. Superoxide dismutase and catalase activity
The superoxide dismutase (SOD) and catalase (CAT) enzyme activities were determined by using Assay Kits (ZellBio GmbH, Ulm, Germany) according to the manufacturer’s instructions.
2.8.4. Protein assay
The assessment of protein content of the supernatants was done according to the Bradford method using bovine serum albumin (BSA) as standard [29].
2.9. Statistical analysis
Quantitative results are presented as mean ± standard deviation (SD). GraphPad Prism software version 6.0 (Graphpad Software, La Jolla, CA, USA) was used for statistical analysis. After assessing the data normality of distribution, statistical significance for the normal distribution results were evaluated using ANOVA followed by post-hoc Tukey, and to analyze data lacking normal distribution was used Kruskal-Wallis test. P-values below 0.05 were considered statistically significant.