Animals:
The institutional review board approved the animal experiments of the research. The research was conducted with 45 male conventional rats whose mean age was 21.4 ± 4.2 (range 17–25) months, and mean weight was 306.5 ± 29.3 g (range 275–338). The rats were kept in wire cages at 60% humidity and room temperature (25 °C) and the cages were cleaned every day to maintain hygienic conditions throughout the research. The environment was illuminated in a 12:12 light/dark cycle to imitate the daylight and 100% air exchange was carried out 10 times in 1. A pellet diet and refined water were provided to the animals. Food restriction and water restriction, respectively, 24 and 6 h before the injection were applied. The rats were separated into 3 groups, with 15 rats in each group. The 1st group (control group) was administered a topical saline injection, the 2nd group was administered a TXA injection, and the 3rd group was administered an IV TXA injection from the tail area. Following the completion of the research, all of the rats were euthanized using the cervical dislocation method.
The National Institutes of Health Guiding Principles has been taken as a guide regarding the care and use of animals used in this experimental study.
Surgical Procedure:
First, the rats were anesthetized with 100 mg/kg intramuscular sodium ketamine hydrochloride (Alphamine, Egevet, 100 mg/mL) and 20 mg/kg intramuscular xylazine (Alfazyne, Egevet, 2%) using a 30 G needle. The therapeutic dose (10 mg/kg) was selected for the dose of TXA to be used topically and IV, and diluted accordingly[15,17]. The same amount of saline solution was also prepared to ensure standardization. After general anesthesia, arthrotomy was performed in the right hip of all of the rats via the dorsal gluteal approach. After arthrotomy, a topical saline injection was given in the hip joint of the 1st group and topical TXA injection was given to the hip joint of the 2nd group. In the 3rd group, hip arthrotomy was performed only to ensure standardization, and the joint capsules and skin and subcutaneous tissues of all of the rats were closed after IV TXA was administered through the tail vein. After these procedures, we waited for 4 weeks for the effects of TXA to occur. At the end of the 4 weeks, all of the rats were euthanized and their right hips were removed as a whole joint and sent for histological analysis.
Histological Analyses for Cartilage and Joint Capsule:
Tissues were fixed in 10% neutral buffered formalin and decalcified by immersion in formic acid solution. After decalcification, the samples were dehydrated in ethanol series, cleared in xylene, and embedded in paraffin. Cross-sections measuring 5 µm thick were cut with a sliding microtome (Leica Microsystems, Wetzlar, Germany) and stained with hematoxylin & eosin (H&E) and toluidine blue. After the muscle layer, the sections were obtained by randomly taking at least 45 sections of 5 microns, from the beginning to the end, for each femoral head.
All of the femoral head samples were evaluated in this way. Histological evaluation was performed using a scoring system with minor modifications as previously described[18]. The amount of remaining articular cartilage was graded as: less than 25% cartilage remaining, grade 4+; 25% to 50% cartilage remaining, grade 3+; 50% to 75% cartilage remaining, grade 2+; 75% to 99% cartilage remaining, grade 1+; and cartilage intact, grade 0. In each slide, cartilage degeneration in the femoral head of selected areas were evaluated under a light microscope (objectives of 10X, 20X, and 40X). The stained sections were examined under the light microscope (Olympus BX43, Hamburg, Germany) and photographed under objectives of 10X, 20X, and 40X.
For each femoral head analysis (control or experimental groups), great care was taken to provide 3D realistic results in terms of both scoring and the measurements. Scoring was made by 3 independent researchers by closing the preparation groups (without knowing the groups by the researchers) and evaluated without bias. During the joint capsule measurements after scoring, the names of the groups were known by the researchers, and the results of the study were recorded to avoid any inaccuracies between the groups
Histological Analyses for Sciatic Nerve:
All of the procedures were conducted according to institutional guidelines.
After fixation in 10% buffered formol fixation for at least 48 h, the portion of each sciatic nerve tissue was divided into multiple parts and embedded in paraffin, and included as many parts of the peripheral nerve as possible. Sections with a thickness of 4 µm were cut, mounted on slides (Dako, Türkiye), and deparaffinized with xylenes and a series of graded ethanol solutions. Each section was stained with H&E and toluidine blue to identify inflammation, intact axon, degenerate axon, and degradation of myelin respectively routine histochemical procedures were performed for the immunofluorescence experiment (see below)[19].
Immunofluorescence was performed as described in[20], with the following modifications. Antigen retrieval was performed in a microwave oven with citrate buffer, and the tissues were then blocked in 0.5% bovine serum albumin. Using mouse monoclonal Occludin (sc-133256, 1:50, Santa cruz, ABD), mouse monoclonal MAG (sc-166849, 1:100, Santa cruz, ABD) and mouse monoclonal MBP (sc-271524, 1:100, Santa cruz, ABD) antibodies were detected. Primary antibodies were incubated with the tissues overnight at 4 °C. Negative control sections were treated with an isotype of Mouse IgG and rabbit IgG antibodies. After incubation with primary antibodies, the tissue sections were washed twice with phosphate buffered saline for 5 min each time, and then, Texas red-conjugated secondary antibodies (st026, abm, US) were added and incubated for 60 min at room temperature. The preparations were mounted on a fluoroshield with DAPI medium (ab104139, Abcam, US). H-SCORE analyses were used for the immunohistochemistry evaluation as previously described[21].
Statistical Analysis:
Statistical analyses were performed using one-way ANOVA followed by the Dunnett test and the Mann-Whitney U test using Sigma Plot 12 (Jandel Scientific Corp., San Rafael, CA). The statistical significance of the data was defined as P < 0.01. All of the data were presented as the mean ± standard error (SEM) of 3 independent experiments.