Animals and experimental design:
A total of 35 adult albino rats, with weights ranging from 230 to 250 g, were acquired from the Department of Pharmacology at Urmia University of Medical Sciences. The rats were housed in standard laboratory settings, which included a light-dark cycle of 12 hours each, a temperature-controlled environment ranging from 23 to 25 °C, and ad libitum access to food and drink. The experimental protocols adhered to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.
Randomly, five groups of rats were formed (7 animals per group): Group 1: Control group; in this group, rats did not have sciatic nerve crush injury induction and received normal saline and distilled water as vehicles daily for seven days. Group 2: Lesion group, a sciatic nerve crush injury was surgically induced in rats, and they received normal saline and distilled water as vehicles daily for seven days. Group 3: Crocin (CR) group: After sciatic nerve crush injury induction, rats received crocin (soluble in normal saline) i.p at a 50 mg/kg dose daily for seven days (Karami et al. 2013). Group 4: Azithromycin (AZ) group: After sciatic nerve crush injury induction, rats received azithromycin (soluble in distilled water) orally at 160 mg/kg daily for seven days (Kopper et al. 2019). Group 5: Crocin + Azithromycin (AZ+CR) Group: After inducing sciatic nerve crush injury, rats were given 50 mg/kg crocin and 160 mg/kg azithromycin daily for seven days.
Surgery:
The rats were impacted by intraperitoneal administration of ketamine (80 mg/kg) and xylazine (10 mg/kg) for the purpose of sedation. The surgical area was thereafter completely shaved and sterilized using betadine solution, spanning from the inferior aspect of the femoral head to the superior aspect of the knee joint. A small incision about 2–3 cm in length was made on the skin in the desired area. Subsequent to the separation of the skin from the underlying tissues and the displacement of the muscles, the sciatic nerve became discernible inside the depths of this region. Subsequently, a compression maneuver was performed on the sciatic nerve with medium surgical hemostatic forceps for a duration of 60 seconds at a location situated one centimeter proximal to the trifurcation site. The compression of the nerve was standardized across all rats by using a single pair of locking forceps. After compression, the nerve was repositioned to its original anatomical location, the incision margins were sutured, and the wound was thoroughly cleansed. The animals were provided with appropriate thermal conditions both during and after the treatment, until they regained consciousness. Following the rats' restoration of consciousness, they were then transferred to individual enclosures and subjected to standard environmental settings, including regulated light exposure, temperature, and humidity levels (Tamaddonfard et al. 2013a).
Sciatic functional index (SFI):
On the 14th, 28th, and 42nd postoperative days, SFI assessed functional recovery. For this experiment, the rat's hind feet were soaked in ink and permitted to walk through a corridor covered with white paper. The SFI was then determined using the following formula:
SFI = -38.8 [EPLNPL/NPL] + 109.5 [ETSNTS/NTS] + 13.3 [EITNIT/NIT] – 8.8
In this formula, PL indicated sole length, TS was the distance between toes 1 and 5, IT was the distance between toes 2 and 4, E was for the experimental side, and N was for the healthy side. All measurements were taken in millimeters. An SFI score of zero was considered normal leg movement, while a minus 100 indicated significant impairment (Nobakhti-Afshar et al. 2016).
Hot plate test:
According to previous research (Ma et al. 2013), the hot plate test was utilized to assess thermal hyperalgesia. The test was conducted on the 42nd day using digital hot plate equipment. During the experimental process, the temperature of the gadget was adjusted to 56 degrees Celsius, after which the rats were placed on the hot plate. The measurement of response time included recording the duration, in seconds, that the rats needed to withdraw their injured leg from the screen. The process was conducted three times for each animal, with a time interval of 10 minutes between each repetition. To minimize the risk of harm to the rat's foot, the duration of exposure on the hot plate did not exceed 12 seconds.
Mechanical withdrawal thresholds:
Von Frey filaments and a mesh apparatus were used to evaluate the responses of animals to mechanical stimulation in order to measure the presence of mechanical allodynia. The Von Frey hair consists of a handle and a slender polyethylene thread with different thicknesses, resulting in a range of forces exerted on the animal's feet, ranging from 8 to 300 grams. The mesh structure consists of a metallic net that is supported by four bases, allowing for unrestricted access from the lower side. On the 42nd postoperative day, the rats were positioned on the mesh substrate for the purpose of conducting this experiment. Subsequently, their injured hind paws were subjected to stimulation originating from the mid-plantar surface, using a variety of filaments that varied in pressure intensity, ranging from low to high. Each stimulation lasted for a duration of 6 to 8 seconds. The filament with the lowest pressure that elicited the animal's paw withdrawal reaction was defined as the animal's stimulation threshold (Cobianchi et al. 2014).
Biochemical analysis of malondialdehyde (MDA) and total antioxidant capacity (TAC):
On the 42nd day of the study, the animals were administered ketamine (80 mg/kg) and xylazine (10 mg/kg) to induce sedation. Subsequently, a total of 5 ml of blood was collected directly from their cardiac region and immediately transferred to test tubes that were stored on ice. The sample underwent centrifugation at a speed of 3000 revolutions per minute (rpm) for a duration of 10 minutes. Following the separation of the serum, it was then held at a temperature of -70°C until the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were assessed.
The MDA test was conducted in order to assess the extent of lipid peroxidation. The test used the thiobarbituric acid reaction technique, as described by Ohkawa et al. (Ohkawa et al. 1979). The present methodology involves the evaluation of the interaction between thiobarbituric acid and MDA via the use of colorimetric analysis. The resultant formation of a chromogenic compound exhibiting optimal light absorption at a wavelength of 535 nm is quantified. The MDA standard curve is generated by using tetraethoxy propane, and the concentration of MDA is then determined in mol/l units based on this curve.
The TAC test was conducted using a total antioxidant kit from Randox (Crumlin, County Antrim, UK). As a result, the compound known as 3-ethylbenzothiazoline-6-sulfanate (ABTS) is subjected to incubation alongside peroxidase and hydrogen peroxide (H2O2), resulting in the formation of the radical cation ABTS+. Ultimately, a consistent blue-green hue was detected at a wavelength of 600 nm (Ghyasi et al. 2019).
Histopathological evaluation:
Following the administration of sedatives to the animals and subsequent blood collection for biochemical analysis, a segment measuring 1 cm from the central portion of the sciatic nerve was taken inside the area of injury and then preserved using paraformaldehyde. Following a 48-hour period, the tissue underwent processing. Subsequently, 5-micron slices were generated from the tissue and subjected to staining with Luxol Fast Blue. For each tissue sample, 10 random fields of view were selected and histological evaluation (myelinated fibers diameter, myeline sheath diameter and number of myelinated fibers) was then conducted with a light microscope set at a magnification of 400x and image J software.
The Luxol Fast Blue staining technique was used to stain the myelin sheath in tissue slices of the sciatic nerve, resulting in a blue coloration. The morphometrics of the sciatic nerve, including the number of myelinated filaments, axon diameter, and myelin sheath thickness, were evaluated using Image J software in this study.
Immunohistochemical staining for apoptosis:
The tissue segment slides were subjected to a temperature of 60 °C for about 25 minutes using a hot air oven (Venticell, MMM, Einrichtungen, Germany). The tissue slices underwent deparaffinization in xylene on two occasions, with each instance lasting for a duration of 5 minutes. Subsequently, rehydration was carried out by subjecting the tissue slices to an alcohol gradient, starting with a concentration of 90% and gradually decreasing to 80%, 70%, and finally 50%. The antigen retrieval technique was conducted using a solution containing sodium citrate at a concentration of 10 mM. The immunohistochemical staining procedure was conducted according to the instructions provided by the manufacturers, Biocare and ScyTek, based in the United States. The endogenous peroxidase activity was suppressed by immersing the sample in a peroxidase-blocking solution containing 0.03% hydrogen peroxide with sodium acid for a duration of 5 minutes. The tissue slices underwent a gentle rinsing using phosphate buffered saline (PBS, pH 7.2) prior to being subjected to overnight incubation at -4 °C with primary antibodies targeting caspase-3 (1:500). Prior to immersion in a buffer solution, the sections were subjected to a gentle washing process using washing buffer (PBS, pH 7.2). Subsequently, the slides were placed into a chamber that had been humidified and contained an anti-microbial substance along with an appropriate amount of streptavidin-HRP (streptavidin chemically linked to horseradish peroxidase) in phosphate-buffered saline (PBS). The slides were incubated for 15 minutes. The tissue sections were cleaned in a washing buffer solution and then immersed in a buffer bath. The tissue slices were subjected to the addition of a DAB chromogen, which underwent incubation for a duration of 5 minutes. Subsequently, the sections were subjected to a counterstaining process using hematoxylin for a duration of 20 seconds. The sections were thereafter immersed in a solution of mild ammonia (0.037 ml) for a duration of 10 seconds, followed by rinsing with distilled water and subsequent covering. The quantification of caspase-3-positive cells inside a tissue sample was performed by counting the number of such cells present in a square millimeter area. This measurement was then used to compare and analyze the differences between the different experimental groups. In addition, software-based methods were used to investigate the cellular distribution (Mosadegh et al. 2017).
Statistical analysis:
SPSS version 26 software was utilized for statistical analysis. To evaluate the significant differences between groups, statistical analysis was performed using a one-way analysis of variance (ANOVA). A Bonferroni-Post hoc analysis was used to compare the groups. All data were presented in terms of mean standard error of the mean (SEM), with P < 0.05 regarded statistically significant.