Male adult Wistar rats between 9 to 10 weeks old and weighing 225 to 250 g were purchased from Cumhuriyet University Experimental Animal Center (Sivas, Turkey). The experimental protocols were approved by the Animal Ethics Committee of Cumhuriyet University (Ethic no: 2019/181). The rats were housed under 12-h/12_h light/dark conditions (lights on at 7:00 a.m.) in a room with controlled temperature (23 ± 2°C) and a humidity of 50–55%. Animals had free access to food and water and were used after a minimum of 7 days acclimatization to the housing conditions. All experiments were carried out blindly between 9:30 (a.m.) and 4:30 (p.m.). The guidelines in the "Manual for the Care and Use of Laboratory Rats" were followed in the housing and administration of the rats (Institute of Laboratory Animal Resources, Commission on Life Sciences 2011).
Acetylsalicylic acid (ASA, Aspirin; 50 mg/kg) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and administered intraperitoneally (i.p.) after dissolving in saline. Morphine HCl (10 mg/kg; i.p.) was obtained from Cumhuriyet University Research Hospital (Sivas, Turkey) and dissolved in sterile 0.9% saline. After purchasing streptozotocin (STZ) from Sigma-Aldrich, a fresh solution was produced by dissolving in citrate buffer (pH 4.5) and injected at a dose of 65 mg/kg (i.p.) to induce diabetic neuropathy. Selection of drug doses was based on data obtained from pre-tests and previous studies [29, 30].
To induce morphine tolerance, morphine was administered at a cumulative dose for three days in rats . The treatment schedule consisted of twice daily doses of morphine administered at 30 mg/kg (i.p.) (a.m.) and 45 mg/kg (p.m.) on day 1, 60 and 90 mg/kg on day 2, and 120 mg/kg twice on day 3. The development of tolerance to morphine in rats was evaluated by antinociceptive tests by administering a test dose of morphine (5 mg/kg) on the 4th day. If administration of a test dose of morphine produces a significant reduction in antinociceptive effect in rats, it is defined as morphine tolerance. The experimental protocol and study timeline are shown in Fig. 1.
Initially, tail flick (TF) and hot plate (HP) thermal antinociceptive tests were performed for each rat to determine basal latency times. In the next step, post-drug latency times for each rat were measured by TF and HP tests at 15, 30, 60, 90 and 120 minutes after administration of the test dose of morphine. Saline group rats were injected with the same dose of saline twice a day for three days as in the drug groups.
Thermal antinociceptive tests, TF test (May TF 0703 TF Unit; Commat, Ankara, Turkey) and HP test (May AHP 0603, Analgesic Hot Plate; Commat) were used to measure antinociceptive behaviors in rats. In the thermal TF test, after the drug was injected into the rats, the radiant heat source was applied to the 2 to 2.5 cm distal part of the rat tail and the tail flick latency (TFL) times were recorded in seconds. The radiant heat source was calibrated so that the basal TFL occurs in a mean of 2.5 ± 0.5 seconds in the nociceptive test. Rats with baseline TFL less than 2.0 seconds or greater than 3.0 seconds were excluded from subsequent antinociceptive tests. The test cut-off latency was programmed to be 20 seconds to avoid tissue injury in the rat tail. Responses to the TF test are often associated with central pain mechanisms [32, 33].
In the thermal HP test, rats were placed in a test device with a plate at an average temperature of 54 ± 0.6°C. Paw licking responses to avoid heat or delay in the rat's jumping behavior were considered an index of the pain threshold. In this antinociceptive test, the cut-off time was programmed as 30 s to prevent injury to the rat paw. It is stated that antinociceptive responses in HP test result from a combination of peripheral and central mechanisms of pain .
Induction of diabetes
To induce diabetic neuropathy, a single dose (65 mg/kg) streptozotocin (STZ) prepared in 0.1 mol/l sodium citrate buffer (pH 4.5) was injected intraperitoneally into diabetes-induced rats . One week later, blood glucose levels were measured with GolDeal Gluco Prober (Aurum Biomedical Tecnology, Hsinchu City, Taiwan) in rats that developed diabetic neuropathy. A few drops of blood from the animal's tail vein were poured onto the instrument strip. In the next step, blood glucose levels were read from the instrument monitor. Animals with a blood glucose level of ≥ 250 mg/dl were considered diabetic. After diabetes induction, the effect of aspirin on morphine antinociception and tolerance was evaluated in diabetic rats.
Homogenate preparation of the dorsal root ganglia
Control and diabetic group rats were sacrificed by cervical dislocation and rapidly surgically removed dorsal root ganglia (DRGs) at L3 to S4 spinal levels. DRG tissue samples in phosphate buffered saline solution (pH 7.4) were homogenized with a mechanical homogenizer device (Analytik Jena speedmill plus, Jena, Germany). Then, the tissues were centrifuged at 4°C and 4000 rpm for 10 minutes. Supernatants formed by centrifugation were stored at -80°C for predetermined biochemical analysis. Bradford protein assay kit was used to analyze total protein levels in tissue samples (Merck KGaA, Darmstadt, Germany) .
Measurement of Bax, caspase-3 and Bcl-2 levels
All commercial rat enzyme-linked immunosorbent assay (ELISA) kits (YL Biont, Shanghai, China) were used to measure Bax, caspase-3 and Bcl-2 levels from DRG tissue samples. Measurements were performed in accordance with the manufacturer's application guide. In summary, DRG tissue samples and standard were added to the plate and incubated at 36.5°C for approximately 60 minutes. Staining solutions were added to the washed tissues and incubated for approximately 15 minutes. After addition of the stop solution, the reading was performed at 450 nm. Standard curves were drawn to determine the value of the samples, and the coefficients of variation within and between plates were found to be less than 10%.
Detection of apoptotic cells
After the antinociceptive tests were applied to the rats, anesthesia was provided with ketamine injection. To isolate the dorsal root ganglia (DRG), rats were sacrificed and DRGs of the lumbosacral spinal cord at the L3-S4 level were isolated. The resulting tissues were quickly transferred to a glass petri dish containing a stable fixative solution (4% paraformaldehyde in 0.1 M phosphate-buffered saline, PBS) at 4°C . Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was used to evaluate apoptosis in DRG tissue. The TUNEL method is based on detecting DNA fragmentation by labeling the 3′-hydroxyl ends of double-stranded DNA breaks produced during apoptosis.
In situ cell death detection kit (fluorescein) was used to assess neuronal apoptosis (Roche Diagnostics GmbH, Mannheim, Germany). The procedures were performed in accordance with the kit manufacturer's application manual. First, DRG tissues were fixed in 10% neutral buffered formalin and then washed for histological examination. In the next stage, the preparations were passed through increasing concentrations of alcohol (70%, 80%, 90% and 100%) and triple xylene series and embedded in paraffin. 3–4 µm sagittal and horizontal serial sections were taken from paraffin-embedded neuronal tissue samples with a microtome (Leica SM 2000R, Heidelberg, Germany). Tissue sections left on slides were kept in the oven for 12 hours and exposed to chemical deparaffinization in xylol twice. To increase the water content of the tissue, ethanol, which was reduced 2 times, was first passed through distilled water and then through PBS. Then, DRG tissues were incubated in proteinase K solution in a dark and humid environment at 21–37°C for 30 minutes. In the next step, neuronal tissues were exposed to PBS 2 times. After applying 50 µl of kit label solution to the negative controls, the enzyme solution was mixed with the label solution and incubated. Before applying the resulting mixture to positive controls, for detection of DNA breaks, in DNase 1 recombinant solution (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 3000 U/ml to 3U/ml in 1 mg/ml BSA) at 20–25°C was suspended for 15 minutes. TUNEL mixture solution was applied to the tissues exposed to PBS twice and incubated at 37°C for 60 minutes in a dark and humid environment. Each neuronal tissue sample was washed 3 times with PBS-Triton-X-100. Detection of apoptotic cells was examined semi-quantitatively under fluorescence microscopy (Olympus BX51). Photographs of DRG sections were recorded using a microscope (Leica DM2500, Nussloch, Germany). A semi-quantitative scoring system was used to evaluate apoptotic cells visible by TUNEL staining. DRG cells that appeared bright and green in nuclei due to chromatin aggregation were considered apoptotic cells. Apoptotic cell counts were expressed as a percentage and graded. The grading is as follows: 1, light staining and affects only ≤ 5% of neurons; 2, staining affecting 5–10% of neurons; 3, staining affecting 10–15% of neurons; 3, staining affecting 15–20% of neurons; 5, staining affecting ≥ 20% of neurons .
Samples obtained from DRG sections for hematoxylin-eosin (H&E) staining were first deparaffinized in xylol for 10 minutes. Tissues were then exposed to 95%, 80%, and 70% alcohol for 10 minutes, respectively. Neuronal tissue samples were stained with hematoxylin for 5–6 minutes and then washed with water. Washed tissue samples were immersed in acid alcohol to turn pale blue within a few seconds. In the next step, neuronal tissues were exposed to the eosin solution for 3–4 minutes. In order to remove excess eosin in the preparation, the tissues were passed through 70%, 80% and 95% absolute alcohol, respectively, and treated with xylol 3 times for 10 minutes. Synthetic resin was used to bond the preparations extracted from xylol and then the preparation was left to dry.
The latency times measured by the hot plate test and the tail flick test were calculated as a percentage of the maximum possible effect (% MPE) with the following equation:
% MPE = [(test latency-baseline) / (cutoff-baseline)]×100.
The results are presented as the mean ± standard error of the mean (S.E.M.). Normal distribution was assessed in accordance with the Shapiro Wilk’s test. A paired t-test statistical analysis was used for the means of the two groups. Behavioral and biochemical data were analyzed via two-way repeated-measures analysis of variance (ANOVA) and multiple comparisons determined by Tukey test using SPSS computer program (version 22.0 for windows Chicago, IL, USA). In all groups, p < 0.05 was considered statistically significant.