Astaxanthin (multiple mechanisms; 3,3′-Dihydroxy-β-carotene-4,4′-dione; C₄₀H₅₂O₄) and gabapentin (GABA synthetic enzyme modulator; 1-(Aminomethyl)-cyclohexaneacetic acid; C₉H₁₇NO₂) were purchased from Sigma–Aldrich (Sigma Chemical Co., St. Louis, MO, United States). Naloxone [μ-opioid receptor antagonist; (5α)-4,5-Epoxy-3,14-dihydroxy-17-(2-propen-1-yl) morphinan-6-one; C₁₉H₂₁NO₄; Caspian Tamin Pharmaceutical Company, Iran], flumazenil [GABA_A receptor antagonist; 5,6-dihydro-4H-imidazo(1,5-a)(1,4)benzodiazepine; C₁₅H₁₄FN₃O₃; Hameln, Germany], ketamine [NMDA receptor antagonist; 2-(2-Chlorophenyl)-2-(methylamino) cyclohexanone, 2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone hydrochloride; Alfasan IBV, Turkey], and xylazine [α2 adrenergic receptor agonist; 2-(2,6-Dimethylphenylamino)-5,6-dihydro-4H-thiazine; C₁₂H₁₆N₂S; Alfasan IBV, Turkey] were analytical grade reagents purchased from commercial sources.

In this research, 54 adult male Wistar rats (weighing 230–250 g) were selected from the reproductive colony of Kermanshah University of Medical Sciences. They were kept in a suitable situation with access to food and water with a 12-h light/dark cycle, a temperature of 25 ± 2°C, and also at optimum humidity. Also, all the protocols were confirmed by the Ethics Committee in Medical Research of Kermanshah University of Medical Sciences (Ethical code: IR.KUM.REC.1400.431). In the current study lowest numbers of animals and surgeries were done in a clean situation (Soleimani et al., 2019; Zhang et al., 2021).

To evaluate the effect of AST on neuropathic pain, the CCI was induced according to the Bennett method (Bennett and Xie, 1988). For this purpose, rats were anesthetized with 50 mg/kg ketamine and 5 mg/kg xylazine. Following disinfection, incision of muscles and skin, and appearing the sciatic nerve on the left side, four loos chromic catgut (4.0) were tied on the sciatic nerve exactly near the trifurcation of the sciatic. Finally, after neuropathy induction, the animals were transferred to the cage for recovery (Amin et al., 2016; Soleimani et al., 2019).

In total, 54 rats were divided into nine groups of six, including, the sham group (undergoing surgery without CCI and receiving DMSO 5%), CCI group (undergoing CCI and receiving DMSO 5%), gabapentin (positive control group, 100 mg/kg in DMSO 5%), AST-treated groups (5 and 10 mg/kg in DMSO 5%), flumazenil (FLU) (0.5 mg/kg), naloxone (NAL) (0.1 mg/kg), AST (10 mg/kg) + flumazenil (0.5 mg/kg) (AST + FLU), and AST (10 mg/kg) + naloxone (0.1 mg/kg) (AST + NAL) (Supplementary Figures 1, 2). All groups received intraperitoneal doses on days 1, 3, 5, 7, 10, and 14, about 1 h prior to assessing behavioral tests. Animals were anesthetized on days 7 and 14 with ketamine/xylazine (100/10 mg/kg), and blood samples were obtained from the retro orbital sinus to evaluate the activities of MMP-2 and MMP-9. Besides, glutathione, catalase, and nitrite levels were measured in serum samples. Finally, the sciatic nerve was separated for histopathological analysis (Soleimani et al., 2019). Blood sampling from retro orbital sinus by an expert is a valuable method without animal expiration after sampling. This method allows serial sampling even in different days and researchers can continue performing experiments on the animals until the end of study timeline (e.g., in our study sampling was done on day 7 and rats were studied until day 14 for behavioural analysis and re-sampling). This strategy also minimizes the number of rats as per the National Institutes of Health Guidelines on the care and use of laboratory animals. Once required blood sampling is done, the capillary tube is removed and wiped with sterile cotton. Bleeding can be stopped using gentle finger pressure. Then, rats should be housed individually in large cages and monitored, until the possible bleeding is stopped. Ad libitum intake of water helps replace the lost blood (Sharma et al., 2014; Fakhri et al., 2021b).

Behavioral tests were done on days 1, 3, 5, 7, 10, and 14 after surgery. Open field tests were used for the evaluation of motor activity. In addition, acetone drop and hot plate tests were done to assess cold allodynia and heat hyperalgesia, respectively. All behavioral tests were carried out as per the mentioned order at 30-min intervals.

To evaluate serum antioxidant potential, catalase and glutathione activities were measured on days 7 and 14. Serum levels of nitrite were also measured to assess oxidative capacity.

The gelatin zymography method was used to evaluate the pattern of gelatinases in the serum. Briefly, serum samples with a total protein content of 100 μg using the Bradford method were loaded on sodium dodecyl sulfate-polyacrylamide gels copolymerized with 0.1% gelatin. Then, electrophoresis was performed at a constant voltage of 150 V using a mini-gel slab device Mini Protean III (Bio-Rad, Hercules, CA). The gel was washed in the renaturation buffer for 1 h on a shaker. The renaturation buffer contained 2.5% Triton X-100 (in 50 mM Tris-HCl). After that, the gel was incubated at 37°C in the incubation buffer for 18 h. The incubation buffer comprises 0.02% NaN₃, 10 mM CaCl₂, and 0.15 NaCl in Tris-HCl (50 mM). Respectively, Gel staining was done with Coomassie blue, followed by destaining in acetic acid (5%) and methanol (7%). The formed clear bands indicate gelatinolytic activity. ImageJ software (National Institute of Health, United States) was used to quantify the relative intensity of the bands in compare to the sham group (Fakhri et al., 2021b; Fakhri et al., 2022).

In this research, rats were anesthetized and sacrificed with a mixture of intraperitoneal xylazine (20 mg/kg) and ketamine (100 mg/kg). Samples from each group were selected on the 7th and 14th day. The close parts of the ligated sciatic nerves were excised and fixed with a 10% formalin solution (Muthuraman et al., 2008). After that, tissues were kept in paraffin. Using hematoxylin and eosin (H&E), consecutive sections of the injury site were stained (Sudoh et al., 2004), and then the sections were qualitatively examined by experts to investigate axonal degeneration under a light microscope (×400) in accordance with routine histological techniques.

The obtained results were assessed with Prism version 8.0 software and a Two-way ANOVA statistical test and Bonferroni post hoc for behavioral analysis. One-way ANOVA statistical test with Tukey post hoc test were employed for biochemical analysis. The area under the curve (AUC) was calculated using the linear trapezoidal method. All the data are presented as mean ± SEM and significant level was set at p < 0.05.

The results of the acetone drop test indicated that the paw withdrawal reflex in the CCI group had a considerable increase compared to the sham group (p < 0.01) (Figure 1A). Moreover, the results indicated that treatment with AST in both doses increased the tolerance threshold of cold stimulus after CCI, with the better result seen in the group receiving AST at the dose 10 mg/kg. This group showed a remarkable decrease in cold allodynia compared to the CCI group on days 7 (p < 0.01), 10 and 14 (p < 0.001) (Figure 1A). Also, in this group the area under the curve of cold allodynia shad a remarkable decrease compared to the CCI group (p < 0.05) (Figure 1B).

On the other hand, the paw withdrawal reflex in response to the cold stimulus in the rats receiving flumazenil and naloxone alone was not considerably different from the CCI group (Figure 1C). Nevertheless, the results showed that the intraperitoneal injection of naloxone half an hour before AST (10 mg/kg) remarkably reduced the analgesic activity of AST on day 14 (p < 0.01), which led to an increase in cold allodynia in this group (Figure 1C). In addition, in this group, the area under the curve of cold allodynia also indicated a considerable increase compared to the group treated with AST (10 mg/kg) (p < 0.01) (Figure 1D).

The data of the hot plate test (Figure 2A) indicated that the time delay of paw withdrawal (PWL) by rats, as the time to reach the pain threshold by heat, was the same in the sham group animals and they were unresponsive. In the CCI group, rats strongly reacted to heat and exhibited a considerable decline in PWL time compared to the first group. The significant differences were seen on the first day with p < 0.01 and from days 3–14 with p < 0.001. Likewise, the data indicated that treatment with AST caused an increase in PWL delay following CCI, and the better result was related to the group receiving AST at the dose of 10 mg/kg. PWL delay showed a considerable increase in this group compared to the CCI group on days 7 and 14 (p < 0.01) and on day 10 (p < 0.001) (Figure 2A). In addition, the area under the PWL curve in the groups treated with AST was significantly increased compared to the CCI group, and this increase was observed in AST 5 mg/kg (p < 0.05) and AST 10 mg/kg (p < 0.001) groups (Figure 2B). In this study, in rats receiving flumazenil and naloxone, PWL delay did not differ compared to the CCI group (Figure 2C). Besides, the data showed that the area under the PWL curve in the AST + FLU (p < 0.05) and AST + NAL (p < 0.01) was significantly decreased compared to the AST group (10 mg/kg) indicating a significant increase in thermal hyperalgesia after blocking the opioid and benzodiazepine receptors (Figure 2D).

The data of the open field test showed that all the three components of locomotor activity were the same in the sham group animals and they were unresponsive (Figure 3). The numbers of rearing (Figure 3A), crossing (Figure 3C) and grooming (Figure 3E) in animals of the CCI group were greatly decreased compared to the sham group (p < 0.001).

Results also showed that AST improved all the three motor function parameters compared to the CCI group (Figure 3) (p < 0.01). The area under the curve also illustrated the reduction of these three movement parameters following the CCI model (p < 0.001), along with the beneficial effect of AST in those parameters (Figures 3B, D, F) (p < 0.01).

In the groups receiving flumazenil and naloxone alone, the locomotor activity of rats in all the three components including rearing (Figure 4A), crossing (Figure 4C) and grooming (Figure 4E) did not show any remarkable difference compared to those in the CCI group. However, these parameters were partially reduced in the groups receiving AST + NAL and AST + FLU compared to the AST group (10 mg/kg), which was significant in crossing (p < 0.05). The area under the curve clearly showed such changes (Figures 4B, D, F).

In addition to the aforementioned Figures, a Supplementary Figure also provided to refuse the role of solvent volume on neuropathic pain and motor dysfunction (Sham, sham received v2, CCI, CCI received v2) (Supplementary Figures 1, 2). Since unnecessarily use of different rats is against the National Institutes of Health Guidelines on the care and use of laboratory animals nothing role of solvent volume extrapolated to biochemical and histological analysis.

The serum level of glutathione in CCI group compared to the sham group considerably declined on days 7 and 14, which was related to the antioxidant activity of glutathione (p < 0.01) (Figure 5A). The intraperitoneal injection of AST at both doses, especially the dose of 10 mg/kg increased the level of glutathione compared to the CCI group (p < 0.001) (Figure 5A). Administration of naloxone and flumazenil alone did not change serum glutathione levels as compared to the CCI group. However, in both AST + NAL and AST + FLU groups, serum levels of glutathione were shown to be significantly decreased compared to the AST group (10 mg/kg) (p < 0.001) (Figure 5B).

The data showed (Figure 6A) that the serum level of catalase in the CCI group was significantly decreased compared to the sham group (p < 0.001). Besides, the serum levels of catalase at both doses of AST, especially the dose of 10 mg/kg (p < 0.05), increased significantly compared to the CCI group. Injection of flumazenil and naloxone alone did not affect the serum levels of catalase in rats. The results of flumazenil and naloxone injections on catalase serum levels were similar to the CCI group. The injection of these two together with AST prevented the positive effect of AST on catalase level (p < 0.05) (Figure 6B).

Our findings demonstrated a significant increase in serum nitrite levels in the CCI group compared to the sham group (p < 0.05). In addition, AST administration was shown to reduce this increased pattern of nitrite following CCI similar to the positive control group (GBP group) (p < 0.001) (Figure 7A). The results of flumazenil and naloxone injections on nitrite serum levels were similar to the CCI group. Their injection with AST did not cause considerable changes in the serum levels of nitrite when compared to the group receiving 10 mg/kg AST on days 7 and 14 (Figure 7B).

The data showed an increase in the levels of MMP-9 (Figure 8) and MMP-2 (Figure 9) activity following CCI compared to the sham group (p < 0.001). Both doses of AST could reverse such increased patterns and had effects close to the effects of GBP (positive control) (p < 0.01). Injection of flumazenil and naloxone half an hour before AST administration reduced the positive effects of AST on MMP-2 and MMP-9 activity levels (p < 0.01).

In this study, we utilized H&E staining to assess tissue damage resulting from the CCI model and the impact of AST on it. The sciatic sections of the sham group exhibited well-organized myelin sheaths. Conversely, the stained nerve sections of the CCI group displayed axonal swelling, disrupted myelin sheaths, hemorrhage between cells, and wide separation between nerve fibers. However, CCI rats treated with AST (10 mg/kg) demonstrated a significant improvement in myelin sheath integrity and Schwann cells. These positive effects of AST significantly decreased in the AST + FLU (0.5 mg/kg) and AST + NAL (0.1 mg/kg) groups (Figure 10).