In this study, 70 adult wistar rats (35 male rats and 35 female rats) weighing 180 - 220 grams were used. The rats had free access to food and water and were housed at standard temperature (22 ± 2°C) and humidity (40 — 50%), and a 12 h light–dark cycle. The male and female rats were each divided into five groups: intact, sham, exercised sham, neuropathy, and exercised neuropathy, with seven rats in each group. Because of their inability to perform the tests, three male rats and four female rats were excluded from the study.

All experimental protocols of this study were approved by the research committees of the Semnan University of Medical Sciences (IR.SEMUMS.REC.1399.298) and were conducted according to the national health institute’s guidelines for the use and care of laboratory animals. All of the behavioral experiments were performed between 9 and 12 a.m to minimize diurnal variations.

Neuropathic pain was induced via chronic constriction injury (CCI) as described by Bennett and Xie13. Each rat was anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg); then, the right thigh was shaved and a 2-cm incision was made at the sciatic nerve. The exposed sciatic nerve was ligated with four moveable 4/0 catgut chromic sutures. The ligations were placed 1 mm apart. Then, the incision was closed with 4/0 silk suture. Rats in the sham group underwent the same surgery without nerve ligation. The rats were placed in individual cages until they reached full consciousness and recovered.

Moderate-intensity swimming exercise was performed as described by Jose14. Animals were exercised for 4 weeks (5 days a week for 20 minutes daily). During swimming, a weight equal to 3% of the animal’s body weight was hung on its tail. A plastic cylinder (60 cm tall and 30 cm in diameter) filled with tap water (36 ± 1° C) was used. To accommodate the exercise program, the animals swam for 5, 10, and 20 minutes a day during the week before the experimental treatment began, and the animals that were unable to complete the program were excluded from the study (three male rats and four female rats).

Mechanical allodynia and thermal hyperalgesia (paw withdrawal threshold in response to mechanical and thermal stimulation), were evaluated via Von-Frey filament and plantar test devices, respectively, on the 30^th day after surgery. To adapt to the experimental conditions, the animals were transferred to the lab 30 minutes before the experiments were performed.

Biochemical tests included malondialdehyde (MDA) and ferric-reducing ability of plasma (FRAP) assays in blood serum. A spectrophotometer was used to read the wavelength results.

To prepare the serum, blood samples were taken from rats’ hearts and centrifuged for 10 minutes at 2,000 RPM. The prepared serum was kept at -80° C until the biochemical tests were performed.

One-way analyses of variance and two-way analyses of variance were used to analyze the data. Tukey’s and Bonferroni posthoc tests were used on these results, respectively. The data were analyzed with GraphPad Prism version 8.0 software (GraphPad, San Diego, CA, USA). All data are presented as mean ± SEM and p < 0.05 was considered statistically significant. The sample size in behavioral tests was 6–7 rats per group and 4–5 rats per group for biochemical experiments. Experiments were performed according to the following timeline.

Figure 1

Abbreviations: Exe: exercise, CCI: chronic constriction injury. *P < 0.05, **P < 0.01

In this study, we showed that swimming exercise improves neuropathic pain in both sexes and operates as an antioxidant in male rats.

We showed that CCI decreased the paw withdrawal threshold and paw withdrawal latency in both sexes compared to the respective sham groups. Consistent with our results, Cardenas et al. reported in 2021 that cuff compression injury to the sciatic nerve led to mechanical allodynia and thermal hyperalgesia in male and female mice18. Further, Dominguez et al. reported that sciatic injury led to mechanical allodynia and thermal hyperalgesia in rats of both sexes19.

Most of the available information is the result of research on male animals, and sex and gender differences are less frequently considered20. Various physiological differences have been identified between male and female animals, including nervous responses, cardiovascular responses, respiratory responses, and hormones21. In addition, sex and gender are known to play a role in the pathology of chronic pain22. In this study, we observed that thermal hyperalgesia following CCI was significantly greater in female rats than in males. Similarly, LaCroix-Fralish et al. showed that when mechanical allodynia and thermal hyperalgesia are measured, female rats are more sensitive than ovariectomized rats and male rats23. Further, Meyer et al. reported that females are more sensitive than males to thermal stimulation after polyneuropathy24. Boullon et al. showed that the response threshold to skin irritation caused by acetone in female neuropathic rats is significantly lower than that of male neuropathic rats25. Biological factors play an important role in the different responses of the two sexes to painful stimuli. Estrogen receptors occur in different central and peripheral regions associated with pain26. Estrogen also increases pain sensitivity by stimulating the expression of NMDAR1 (N-methyl-D-aspartate acid receptor in the spinal dorsal horn ganglion, reducing the response threshold to noxious stimuli in females27. Moreover, brain imaging studies have shown that the activity of pain-inhibitory regions of the brain (the rostral ventrolateral medulla) is reduced in women who take contraceptives28.

Although various medicinal methods have been developed to stop neuropathic pain, not only have none of them been completely effective but they exert many side effects on patients. Given the many adverse effects of pharmacological medication, adjuvant non-pharmacologic therapies play a prominent role in patients’ pain management and reduced drug consumption.

In this study, we used swimming exercise as a non-pharmacological approach to improve CCI-induced neuropathic pain. Our results showed that exercise reduces mechanical allodynia and thermal hyperalgesia in both sexes. Several studies have investigated the hypoalgesic effect of exercise and reported that exercise improves sensory and motor performance after nerve injury29. Furthermore, we have previously shown that swimming exercise reduced neuropathic pain after infraorbital nerve injury in both sexes12. Similarly, Sumizono et al. reported the hypoalgesic effect of treadmill exercise in rats with injured sciatic nerves30.

Evidence has suggested the role of oxidative stress in neuropathic pain30, 31. ROS production after nerve injury reportedly leads to oxidative stress and endoneurial lipid peroxidation32, 33. In this study, CCI did not increase the MDA level in male or female neuropathic rats compared to the equivalent sham groups. Tang et al. reported similar results, indicating that ischemic sciatic nerve lesions cause no change in MDA levels in male and female mice34. Conversely, Yuceli et al. reported that MDA levels noticeably increased in male rats after sciatic nerve ischemia via femoral artery clamping35. Furthermore, contrary to our results, Etienne et al. reported increased MDA levels in male and female diabetic neuropathy patients36 but found no significant difference between the sexes. Oxidative stress parameters change in response to physical activity in a time-dependent manner37, so the time of measurement will affect the determined value. The inconsistency between our results and other mentioned studies may be due to either a difference in the evaluated sample (e.g., serum in our study, but sciatic tissue in others), a difference in the time of measuring malonaldehyde (e.g., in our study, one month after the intervention, but in Yuceli et al., one day after the intervention), a difference in the type of intervention (e.g., in our study, pressure injury on the nerve, but in other studies, ischemia reperfusion), or a difference in the study subject (rats in our experiment, but humans in Etienne et al.).

According to our results, exercise significantly (p < 0.01) reduced MDA levels in the male neuropathic rats but not in the female neuropathic rats. Jiankang38 and Shirvani39 separately showed that physical training decreases MDA levels compared to a control group.

Physical training reportedly increases parasympathetic nervous system activity40. Increased parasympathetic tone has an anti-inflammatory effect by suppressing cytokine release41. In this study, financial limitations prevented us from evaluating inflammatory mediators such as cytokines, but in a previous study42, we showed the anti-inflammatory effect of exercise in CCI-treated male rats. As pain is one of the signs of inflammation, the observed hypoalgesia in this study may be due to increased parasympathetic activity and the resulting inflammation suppression. However, a close and direct relationship between inflammation and oxidative stress also exists43; therefore, the reduction of inflammatory factors through the attenuation of oxidative stress may reduce neuropathic pain.

We observed that the FRAP levels of neuropathic rats of both sexes were not significantly different from the sham groups. In addition, the FRAP levels of male neuropathic rats were not significantly different from those of female neuropathic rats. Heidari et al. observed that FRAP levels were significantly lower in female diabetic neuropathic patients compared to female diabetic patients without neuropathic pain44. Furthermore, Etienne et al. reported that FRAP level was significantly lower in male and female patients compared to their control groups, but there was no significant sex difference36. The difference between our results and those of the researchers mentioned above may reflect aspects such as the time of FRAP measurement or the examined subjects. In this study, we examined the serum level of FRAP at 4 weeks post-surgery, when the level of this parameter may have returned to control levels, as previously we showed that the FRAP level was significantly reduced 3 weeks after sciatic nerve CCI in male rats42. In addition, the difference between our results and Etienne et al.’s and Heidari et al.’s may be due to the difference in the examined species. Importantly, as in malondialdehyde, the total antioxidant capacity may also change over time.

Our result showed that exercise led to a significant increase in the FRAP levels of male neuropathic rats but not female neuropathic rats. These results align with Rytz et al., who showed that aerobic exercise prominently increased FRAP levels in men with metabolic syndrome but not in women with the same problem45. Jolien Hendrix also reported that repeated exercise increases total antioxidant capacity and exerts hypoalgesia in male rats46. Previously, we showed that 3 weeks of treadmill exercise increased FRAP levels in CCI-treated male rats42. Human and animal studies show that sex hormones, especially estrogen, exert antioxidant and neuroprotective effects, and estrogen’s role is more pronounced than testosterone’s47.

In addition to reproduction, estrogen plays a role in immune system performance through receptors on immune cells, such as lymphocytes, monocytes, and macrophages48. Estrogen prevents cytokine release by inhibiting microglia and astrocytes49. Meanwhile, as mentioned earlier, there is a close and direct relationship between inflammation and oxidative stress50, so cytokines can stimulate oxidative stress and vice versa51. Therefore, estrogen may prevent oxidative stress by inhibiting the release of inflammatory agents. The levels of vitamin E and glutathione peroxidase enzyme activity are high in female rats9, and these levels may have prevented oxidative stress and, therefore, limited changes in malondialdehyde and FRAP levels. Confirming this possibility, other studies have shown that the mitochondrial DNA damage caused by oxidative stress products is significantly reduced in female rats compared to male rats52. Several studies have indicated the direct protective effect of estrogen against oxidative stress damage in the heart and liver53, 54. In vitro reports have shown that the protective effect of estrogen and progesterone may be mediated through antioxidant properties or genomic effects55. According to the mentioned studies, which have shown that the amount and activity of antioxidant enzymes are higher in female than in male organisms, the stronger antioxidant potential in females may prevent oxidative stress or neutralize it in the initial stages. Therefore, the protective and antioxidant properties of estrogen may have prevented the changes in malondialdehyde and FRAP levels that were observed in male rats during this study.

Reportedly, in castrated male rats, MDA and the antioxidant power of plasma significantly increased and decreased, respectively, and either continuous exercise or testosterone therapy significantly reversed these trends toward control levels56. Moderate-intensity exercise has been shown to not only increase testosterone levels57 but also potentiate the effects of testosterone58. According to the literature, it is possible that in our study, exercise increased the level of testosterone in male neuropathic rats, thereby decreasing the level of malondialdehyde and increasing the FRAP level. This mechanism should be studied further to increase the reliability of the conclusions. Although financial constraints prevented us from using sex hormone antagonists or measuring testosterone levels in the rat sample, doing so would permit us to ascertain whether the reducing oxidative stress response effects of exercise could be attributed to sex hormones. This is one of the limitations of this study.

In this study, we did not detect oxidative stress in neuropathic rats of either sex but affirmed that exercise improves oxidative stress parameters in male rats. There may be an association between male sex hormones and oxidative stress suppression by exercise that does not manifest in females. The potential antioxidative properties of female sex hormones do not appear to be affected by exercise.

CCI: Chronic Constriction Injury, FRAP: Ferric-reducing ability of plasma, MDA: Malondialdehyde, NMDAR: N-methyl-D-aspartate acid receptor, ROS: Reactive Oxygen Species, RPM: Revolutions Per Minute

The authors of this article express their gratitude to Mr. Hossein Ali Safakhah for technical assistances. The authors wish to thank the Deputy of Research and Technology of Semnan University of Medical Sciences, Semnan, Iran for offering the grants (grant number: IR.SEMUMS.REC.1398.137) for present study.

Ali Ghanbari: Conceptualization, Methodology, Supervision, Reviewing and Editing, Sahar Ghasemi: Methodology, Writing- Original draft preparation, Data curation, Ahmad Reza Bandegi: Software, Visualization, Investigation, Reviewing, Ali Rashidy-pour: Software, Validation, Reviewing. All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors read and approved the final manuscript.

This study was supoorted by Deputy of Research and Technology of Semnan University of Medical Sciences, Semnan, Iran (permit number: IR.SEMUMS.REC.1398.137).

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Study involving animals complied with all relevant national regulations and institutional policies (permit number: IR.SEMUMS.REC.1398.137) for the care and use of animals.

Not applicable.

The authors declare that they have no competing interests.