Animals
All experiments performed as a part of this study involved adult male Balb/C mice weighing 25 to 30 g and adhered fully to the guidelines of the ARRIVE (Animal in Research: In Vivo Experiments) as well as the principles of the International Association for the Study of Pain (IASP) Research and Ethics Committee (Zimmermann, 1983). This study was carried out with the approval of Firat University Animal Experiments Local Ethics Committee (dated 16.01.2019, protocol number 2019/06 with the decision number 2019/09). The animals were housed for one week before starting the experiments to acclimatize them and were maintained under standard laboratory conditions on a 12-hour light/dark cycle at a constant temperature (23 ± 2 oC) and humidity (60 ± 5%) with food and water supplied ad libitum.
Behavioral tests were performed on all groups in a quiet room at the same time by the same researchers blind to the randomization, thus minimizing the influence of environmental factors and researcher effects. Tests and exercises were conducted between 9.00 am and 12.00 am and all treatments were delivered intraperitoneally (i.p) (Figure 1).
Healthy Mice
Healthy animals were randomly divided into five subgroups: control, morphine group (50 mg/kg), asprosin (1 µg/kg), asprosin (10 µg/kg), and asprosin (30 µg/kg) (n=7, each group).We first evaluated the effectiveness of asprosin (1, 10 and 30 µg/kg, i.p.) and morphine (10 mg/kg) on nociceptive behavior in healthy animals, which were individually acclimatized to the environment and test setup, respectively. The effective dose of asprosin used in this study was calculated as LogEC50 = 0.9281.
Streptozotocin-induced diabetes model in mice
The streptozotocin (STZ)-induced diabetic neuropathic pain group was randomly divided into three subgroups: STZ (diabetic control), STZ+asprosin (10 µg /kg), and STZ+gabapentin (50 mg/kg) (n=10, each group). Which were determined to be normoglycemic were administered STZ i.p. at a dose of 150 mg/kg. STZ was dissolved in 0.4 ml (0.1 M) Na+ citrate buffer (pH 4.5). The control group was injected i.p. with the solvent sodium citrate buffer. Three days later, blood glucose was measured with manual glucometer (Optima, Taiwan) in all groups (all measurements were made between 9.00 and 10.00 am). Only mice with glucose levels above 250 mg/dL in blood taken from the tail vein were retained. From this group, mice that lost ≥10% of body weight before starting the experiment, exhibited decreased activity or hair erection were also excluded. Behavioral tests were conducted three weeks after STZ administration to mice.
Induction of neuropathy: Chronic constriction nerve injury
The neuropathic pain group with sciatic nerve chronic constriction injury (CCI) was divided into three subgroups: CCI (sham control), CCI+asprosin (10 µg /kg), and CCI+gabapentin (50 mg/kg) (n=10, each group).Chronic construction of the sciatic nerve was performed using clamps as previously described (Salvat et al., 2018). First, mice were anesthetized with a ketamine/xylazine mixture (3 mL/kg body weight i.p.) and right sciatic nerve was exposed after dissection. Next, 2 mm PE-20 polyethylene tube (Harvard Apparatus, 59-8323, Les Ulis, France) was placed at around 5 mm distance from the trifurcation of the sciatic nerve, after which the wound was closed by suturing muscle (chromic catgut) and skin (3.0 silk). The sham groups were operated as described above, but without a polyethylene tube (cuff implantation). Animals with CCI were subjected to behavioral tests after 2 weeks of waiting for their wounds to heal.
Oxaliplatin-induced neuropathy
The neuropathic pain group after oxaliplatin (OXA) use was also divided into three subgroups, denoted as OXA (control), OXA+asprosin (10 µg /kg), and OXA+gabapentin (50 mg/kg) ( n=10, each group).OXA (Sigma) was prepared at a concentration of 3 mg/mL in a 5% dextrose solution according to the weight of each animal and was administered i.p. at a dose of 3 mg/kg twice a week for 4.5 weeks. The control group was injected with 5% dextrose solution. After 2 weeks of 4.5 weeks of OXA drug administration, animals were subjected to behavioral tests.
Nociceptive test procedures
Groups were determined by randomly selecting mice. Before commencing the behavioral tests, the animals assigned to all groups were numbered by marking their tails. Behavioral tests were performed on all groups in a quiet room between 9.00 am and 12.00 pm by the same researchers that were blind to randomization, thus minimizing the influence of environmental factors and researcher effects. Gabapentin (50 mg/kg, i.p.) was used as positive control.
Hot plate test: The hot plate test was performed by placing the animals on a warm floor within a clear plexiglass cylinder of 15 cm diameter and 22.5 cm height to prevent them from coming out. The hot plate analgesimeter table was set to 50 ± 0.5 °C. Pain behavior was determined as the time (in seconds, with a 40 s cut-off) that lapsed from placing the animal on the floor to its visible reaction to pain (quickly pulling, licking or contracting its extremities). At the baseline, pain threshold values were determined before any injection was given to the mice (the time of injection was accepted as 0 minutes), and then measurements were performed 30, 60, 120, and 180 minutes after the injection.
Cold plate test: Cold hyperalgesia was evaluated using a cold plate at the optimum temperature of 5 ± 0.2 °C. The sum of paw lifts and jumps during the 5-minute measurement interval (while excluding movement-related behaviors and coordinated movements of all four extremities) was considered a response to cold hyperalgesia. As with the cold plate test, the time of injection was accepted as 0 minutes, and further measurements were performed at 30, 60, 120, and 180 minutes after injection.
Von Frey test: Mechanical allodynia was assessed using von Frey filaments as previously described (Yalcin et al., 2014). Prior to testing, animals were acclimated to the environment and the test setup for 10 days. On the day of the test, animals were kept in the boxes for 15 minutes without any treatment, after which each von Frey filament was applied 5 times to the plantar surface of the right hind paw until it bent. The filament that produced the same response in at least three instances was defined as the result. These measurements were performed at baseline (i.e., before the mice were given any injections) and the time of injection was accepted as 0 minutes. The test was repeated one hour after injection. This procedure was applied to all groups.
Motor activity
Rotarod test was used to determine motor coordination. Prior to testing, mice were acclimated to the environment and the test protocol by placing them on the fixed bar for 5 minutes for two consecutive days and then on the rotating bar while increasing its speed from 4 rpm to 40 rpm. Following the acclimation period, mice were placed on accelerating rotating rods to determine the effect of asprosin on motor activity and the time taken for the animals to fall off the rod was recorded (cut-off time = 300 s). As with other tests, the baseline measurements were performed before the mice were given any injections and the time of injection was accepted as 0 minutes. The test was repeated one hour after the injection, as this interval corresponds to the highest pain threshold. This procedure was applied to all groups.
Asprosin measurements
Once all experimental protocols were completed, the animals were decapitated between 9.00 and 10.00 am and the blood serum were stored at -80 °C until required for analysis, which was performed using the mouse asprosin ELISA Kit (USCN catalog number: SEA332Mu) following the manufacturer’s protocol. The amount of asprosin antigen in the serum was determined using a microplate coated with the asprosin antibody included in the kit. The color change that occurs at the end of the experiment indicates the presence of antigen and the measuring range of the kit spans from 78 to 5000 pg/mL, while its sensitivity is ≤ 32 pg/mL, with the inter-assay and intra-assay %CV (coefficient of variation) values of ˂ 12% and ˂ 10%, respectively. The experimental protocol included in the ELISA kit was used to determine the serum asprosin level.
Drugs
Gabapentin, STZ and OXA were purchased from Sigma. Gabapentin was prepared by dissolving in saline, while the recombinant asprosin (Elabscience Company, U.S.A.) was dissolved in phosphate buffered saline (PBS). All drugs were freshly prepared on the day of the experiment.
Statistical analyses
SPSS Statistics 22.0 for Windows software package was used for all statistical analyses. Conformity to normal distribution was evaluated via the Shapiro Wilk test and oneway analysis of variance was performed to compare the values of quantitative variables between groups when there were three or more groups. When a significant difference was identified, multiple comparisons were conducted using Dunnet's test and Tukey test, with p < 0.05 indicating statistical significance.