The effects of chronic nanoselenium treatment on sciatic nerve injury: behavioral and biochemical responses

Nanoselenium as a free radical scavenger suggested being a neuroprotective agent in some neuronal diseases. As the neuropathic pain could be a consequence of a defect in antioxidant defenses and changes in oxidative stress parameters, the present study was planned to investigate the effect of nanoselenium particles on pain-related behaviors and spinal antioxidant defense parameters in the sciatic nerve injury model. Adult male albino Wistar rats (n = 32) were randomly allocated to the four experimental groups: control group, neuropathy group with chronic constriction injury of the sciatic nerve (CCI), CCI + nanoselenium, CCI + vehicle. The CCI model was used to create neuropathic pain-related symptoms. Nanoselenium or vehicle was injected intraperitoneally for 14 days. The behavioral evaluation was carried out to assess the pain threshold by the radiant heat and von Frey tests. Malondialdehyde (MAD), superoxide dismutase (SOD) levels, and catalase activity in the spinal cord were evaluated to investigate the possible relation. Our data displayed that CCI triggered neuropathic pain-related behaviors in rats. Chronic treatment with nanoselenium meaningfully improved pain threshold (P < 0.001; F = 37.86, F = 29. 82) and decreased the level of MDA (P < 0.01; F = 33.16) and increased the SOD level (P < 0.001; F = 13.43) and catalase activity (P < 0. 05; F = 10.17) in the spinal cord of CCI rats. Chronic nanoselenium treatment can improve pain-related behavior and is associated with a reduction in MDA level and increasing in SOD level and catalase activity in the spinal cord of the CCI rats. Nanoselenium provides a therapeutic alternative for the treatment of neuropathic pain by alteration in lipid peroxidation and antioxidant defense system factors.


Introduction
Neuropathic pain affects approximately 6% of the adult population worldwide, usually becomes a chronic and unsuitable condition, and impacted a patient's quality of life. It is a common consequence of peripheral neuropathy with sensory symptoms such as spontaneous pain, allodynia, and hyperalgesia, which also is associated with cognitive and emotional disorders. Common analgesics have not effectively treated or alleviated neuropathic pain because of the heterogeneous etiology and the multifactorial mechanisms underlying this disorder [1].
Recent documents determined that oxidative stress and inflammation contributed to the formation and development of neuropathic pain. Oxidative stress and inflammation result in peripheral and central sensitization that is involved in neuropathic pain. It can originate cellular dysfunction, DNA damage, and apoptosis, whereas antioxidants, in contrast, defend the cells from oxidative damage. The mammalian nervous system has a high content of phospholipids and axonal mitochondria, but their neuronal antioxidant defense is weak. It is especially susceptible to free radicals, including oxygen and nitrogen reactive species. Therefore, neuropathic pain could be a consequence of the imbalance between reactive oxygen species and endogen antioxidants [2].
Peripheral nerve injury causes increased production of free radicals which belong to reactive oxygen species at the injury site that inflamed tissue, stimulate nociceptive, and promote hyperalgesia and allodynia, not only at the lesion site but also in the central level of the nervous system. In this regard, it demonstrated that decreasing antioxidant protection, increasing oxidant generation, and loss of the capacity of repair from oxidative damage could lead to a neuropathic pain situation [2,3].
Selenium is a necessary micronutrient that has a crucial function in the active site of many antioxidant enzymes such as glutathione peroxidase and thioredoxin reductase in mammalian cells. Selenium-containing selenoproteins are involved in antioxidant defense and anti-inflammatory effects. Selenium supplementations are known to be neuroprotective agents in spinal cord injury, epilepsy, and pain. It has been shown that the neuroprotective effect of selenium is applied by the regulation of scavenging enzymes, Ca 2+ ion channels and inhibition of apoptosis [4]. Furthermore, selenium is concerned as a neuroprotective agent in peripheral pain through the ability to modulate signal transduction, molecular pathways, antioxidant potential, and regulation of the TRPM2 and TRPV1 channels [5,6].
Although recently selenium compounds have been highlighted as a pharmacologically active synthetic molecule, there are limiting factors concerning its use as bioavailability and toxicity. Since the materials at the nanometer dimension revealed novel and different properties, many studies have researched different methods of producing less toxic and more effective nanoparticles of selenium. Recent studies confirmed the efficacy of nanoselenium particles with lower toxicity and acceptable bioavailability [9]. Nanoscale selenium has a varied range of biomedical uses. Its effect on the reduction of oxidative stress is determined [10,11].
Moreover, its use as an antioxidant with reduced risk of toxicity, the present study assessed the outcome of chronic administration of nanoselenium on pain-related behavior and antioxidant defense system parameters in the spinal cord of CCI rats.

Animals and housing
The statistical population of the present study consists of all rats in the Laboratory Animal Breeding Center of Iran University of Medical Sciences, of which thirty two 8-week-old male albino Wistar rats with an average weight of 200-250 g were initially purchased and stored in the animal room with free access to water and food at 23 + 2 °C, under conditions of 12 h of light and 12 h of darkness (starting light at 6 a.m. and off at 6 p.m.). As stated in the variation of the female sex hormones from cycle to cycle and their impact on pain sensation in this study, male rats were used.
The animals were randomly allocated to the four experimental groups: control group, neuropathy group with CCI model, CCI + nanoselenium, CCI + PBS as vehicle (n = 8 per group, 32 rats totally). Caregivers were blended into the grouping. All behavioral tests were blindly achieved between 10:00 a.m. and 02:00 p.m. for each experimental group. The experiments adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP) and were approved by the Ethical Committee of the University (IR.IUMS.REC 1396.3147).

Animal surgery
Bennett and Xie designed the CCI model to generate neuropathic pain symptoms [12]. In brief, the rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) at a dose of 8 to 1 before the surgery. Then, hairs parallel to the iliac spine were shaved and an incision shaped in the left thigh. After the sciatic nerve appeared, four loose knots were applied to the nerve with 0.4-mm chromic tread at a distance of 1 mm from each other. The knots did not obstruct the circulation through the epidural vasculature. The muscles and skin were sutured by the silk thread separately. When the rats recovered, they transferred to their cages. All animals underwent behavioral evaluation before starting the experiment and were considered as a pretest.

Drugs and pharmacological treatments
The selenium nanoparticles used in this research were purchased from Nanomaterials Pioneers (NANOSANY, Mashhad, Iran) and have an average size of 30-nm water-soluble colloids with 99% purity, which is confirmed by TEM-SEM photo and X-ray analysis done. The amount of 0.5 mg/kg of nanoselenium particles was dissolved in 0.5 ml PBS, and the day after surgery was injected intraperitoneally to rats for 14 consecutive days. In this manner, the vehicle group received 0.5 ml PBS intraperitoneally. The doses of the drugs have been selected based on previous studies [9,11].

Experimental design
The day of CCI surgery was considered the zero day of the study. Behavioral pain assessment carried out before the starting study and 7, 14, 21, and 28 days after surgery for the ipsilateral hind paws in all experimental groups. The pain behavior assessed for contralateral hind paw using these tests, but the results were not significant between groups, and we did not show them in "Results" sections. The treatment with nanoselenium or vehicle started on the day after surgery for 14 consecutive days. Lastly, the rats were scarified, and the spinal cords were removed and dissected on an ice-chilled glass plate.

Assessment of mechanical allodynia
Mechanical allodynia was tested by Von frey filaments (Stoelting, Wood Dale, IL, USA). The filaments 4.56, 4.74, 4.93, 5.07, 5.18, 5.46, and 5.88 were straight-up tried to the midplantar skin in the up and down method with sufficient force to cause slight bending against the paw. Withdrawal threshold was measured by consequently increasing and decreasing the stimulus power, and the mean withdrawal threshold was calculated by the Dixon nonparametric test [13].

Assessment of thermal hyperalgesia
The thermal withdrawal threshold was evaluated by using the radiant heat test (Hargreaves apparatus, 7370, Ugo Basile, Comerio, Italy) [14]. At first, rats were put in a Plexiglas box and habituated for 20 min. Then a heat beam is radiant to the plantar surface of the hind paw. The period between heat radiation and the reaction of rats like a paw lifting is considered the withdrawal latencies. The endpoint is detected automatically thereby removing the observer bias. To avoid tissue injury, the length of the heat radiation was fixed at 25 s. The trial was done three times for each rat at least 1 min interval, and the average values were calculated for statistical analysis.

Biochemical evaluation
Based on previous studies, MDA is considered a marker of lipid peroxidation, while catalase and SOD are the indicators of antioxidant defense systems (enzymatic and nonenzymatic) [15,16].

Tissue preparation
At the end of the examination, the animals were deeply anesthetized with ketamine (100 mg/kg, IP) and xylazine (10 mg/ kg, IP). After that, about 0.5 cm length of the L4-L5 spinal cord levels was dissected and quickly stored at − 80 °C. Finally, the sections were defrosted for testing and prepared for each test according to the test instructions.

Assessment of spinal level of MDA
For this part, the MDA level was determined by the MDA assay kit (Teb Pazhouhan Razi (TPR), Tehran, Iran) and that national quality was certificated. For this purpose, the homogenated spinal cord tissues were centrifuged at 1600 × g for 10 min at 4 °C. After that, the supernatant was used for analysis. In the next part, BHT was added to supernatants, and 100 µM of the samples was carried to tubes containing sodium dodecyl sulfate. The solution of TBA (0.5%, w/v), sodium hydroxide and acetic acid were then added to each reaction mixture. Then, while the tubes were caped, these were placed on boiling water. After 1 h, immediately the tubes were placed on an ice bath for 10 min to stop reactions. Finally, the tubes were centrifuged at 10,000 × g for 10 min at 4 °C. MDA concentration was measured by a plate reader at 532 nm. The MDA content of samples was determined by the MDA standard curve and showed as nmol/mg of protein.

Assessment of spinal level of SOD
The activity of SOD was measured by a commercial kit (Teb Pazhouhan Razi (TPR), Tehran, Iran) and that national quality was certificated, according to the method of the manufacturer. The reagents and the supernatants of spinal cord tissues were prepared and added to each well. After that, a working solution was added to each well, and other solutions were added, respectively, according to the kit method. After the shaking and incubation at 37 °C for 20 min, the absorbance was assessed at 450 nm by a plate reader. The activity of SOD was showed as units of enzyme per mg of protein.

Assessment of spinal catalase activity
The activity of catalase was measured using a catalase activity kit (Teb Pazhouhan Razi (TPR) Tehran, Iran) and that national quality was certificated. Briefly, after preparing the supernatant, we allowed all the solutions in the kit to reach room temperature. Then, according to the method of the manufacturer in the kit, the desired solutions and standards were prepared and added to the plate wells, respectively. After that, the standard dilutions were poured into the wells and the samples were added respectively; the plate surface was covered and kept at room temperature for 5 min. Finally, the absorbance of the samples was read by the plate reader at 540 nm. By drawing a standard curve, the amount of catalase activity in each sample was calculated.

Data and statistical analysis
Differences between the groups to analyze pain-related behavior were calculated using repeated measurement two-way ANOVA with a post hoc test (Bonferroni's test). The mean comparisons for biochemical data were calculated by one-way ANOVA with post hoc test (Tukey's test) with SPSS 21 in experimental groups. Data presented as mean ± SEM. P < 0.05 was considered as significant.

Effect of CCI and chronic nanoselenium treatment on pain-related behavior
CCI induced mechanical allodynia and thermal hyperalgesia in rats, for at least 28 days, as compared with the control group (Fig. 1).
The result determined the chronic nanoselenium treatment which improved pain-related behavior in CCI rats. After 14 days of treatment, nanoselenium treatment increased the mechanical allodynia (P < 0.001, F = 29.82) and thermal hyperalgesia (P < 0.001, F = 37.86) thresholds in CCI + nanoselenium rats compared to CCI + vehicle group.
There was no significant difference in the mechanical and thermal thresholds between the vehicle-treated and CCI groups.

Effect of CCI and chronic nanoselenium treatment on lipid peroxidation and antioxidant defense system parameters in spinal cord
The biochemical result showed chronic administration of nanoselenium could alter lipid peroxidation and antioxidant Fig. 1 The effect of chronic nanoselenium treatment on mechanical allodynia (A) and thermal hyperalgesia (B) in CCI rats. Nanoselenium or vehicle treatment started 1 day after surgery and continued for 14 days. **P < 0.01 and ***P < 0.001 indicated the differences between CCI + nanoselenium vs CCI + vehicle groups. Data analyzed by two-way ANOVA with a post hoc (Bonferroni's test) and expressed as mean ± SEM of 8 animals per group defense system parameters in addition to relieving allodynia and hyperalgesia in the chronic constriction injury in rats. CCI surgery was associated with increases in MDA level, a reduction in SOD, and catalase levels as in the spinal cord (Fig. 2). While ANOVA and post hoc analyses revealed that, after 14 days of nanoselenium treatment, MDA level significantly decreased (P < 0.01, F = 33.16) and catalase and SOD levels increased (P < 0.05, F = 10.17, P < 0.001, F = 13.43, respectively) in the spinal cord of CCI rats. The mean level of MDA in the spinal cord of CCI rats treated by the vehicle was 1 ± 0.06 nmol/mg and in the nanoselenium-treated rats was 1.69 ± 0.06 nmol/mg. Also, the mean levels of the catalase and SOD in the spinal cord of CCI rats treated by nanoselenium were 7.33 ± 0.08 u/mg of protein, 5.38 ± 0.21 u/mg of protein, and the vehicle group was 6.29 ± 0.21 u/ mg of protein and 2.67 ± 0.08 u/mg of protein, respectively.
There was no significant difference in the spinal levels of MDA, SOD, and catalase activity between the vehicletreated and CCI groups.

Discussion
The current data suggested chronic nanoselenium administration is associated with alleviation of neuropathic painrelated behavior and altered the spinal level of MDA, catalase, and SOD in the sciatic nerve constriction model of rats.
Neuropathic pain provides by a lesion or disease of the somatosensory system. When neuropathic pain lasts for a prolonged period, it causes biochemical, anatomical, and functional changes, not only in the injured area but also in the spinal cord and supraspinal structures such as the anterior cingulate cortex, medial prefrontal cortex, thalamus, amygdala, and hippocampus. Maladaptive neural connectivity and neuroplastic changes result in nociceptive pathways and pain perception alterations. Therefore, in addition to pain-related symptoms, patients also suffer from neuropathic pain comorbidities such as mood and cognitive disorders [1,17,18] Based on previous studies, targeting neuronal plasticity changes in somatosensory pathways is a major direction for finding pain-relieving medication [17,19]. In this assay, we focus on spinal cord alterations as a main pain transmission pathway; other investigators also observed changes in oxidative-stress parameters in the spinal cord of CCI rats [20][21][22].
In the future, we decide to investigate the effect of nanoselenium administration on related behavior and chemical  A, B, and C, respectively). Nanoselenium or vehicle treatment started 1 day after surgery and continued for 14 days. Biochemical evaluation performed 28 days after CCI surgery.*P < 0.05, **P < 0.01 indicate the differences between CCI + nanoselenium vs CCI + vehicle groups. Data analyzed by oneway ANOVA with a post hoc test (Tukey's test) and expressed as mean ± SEM of 3 animals per group ▸ structure of the hippocampus in CCI rats. Allodynia and hyperalgesia are the common signs in patients suffering from neuropathic pain. We performed the CCI model to induce typical chronic neuropathic pain symptoms like clinical symptoms in neuropathic patients [12]. Our result showed mechanical and thermal threshold decreased 2 weeks after surgery and raised gradually up to week 4. Previous data identified chronic constriction injury of the sciatic nerve resulting in obvious mechanical allodynia and thermal hyperalgesia that peaked 2 weeks after surgery and gradually decreased up to week 7. Consequently, in the CCI model, pain-related behaviors could be partly reversible [23][24][25].
In the present study, we investigated the possible analgesic effect of nanoselenium on neuropathic pain-related behavior in rats. These results suggested that chronic nanoselenium treatment could raise the withdrawal threshold and latencies in behavioral pain-related tests and reliving neuropathic pain symptoms in sciatic nerve injury. Previous study reported that organoselenium compound synergistically could increase the analgesic effect of opioids in neuropathic pain [26]. Another assays similarly indicated the antinociptive effect of organoselenium compound in peripheral pain [5]. Since nanoparticles of selenium were determined to be less toxic, more stable, and bioavailable than selenium [10], we studied the antinociceptive effect of nanoselenium on peripheral neuropathic pain. Our data showed the analgesic effect of long-term administration of nanoselenium remained up to 2 weeks after drug discontinuation so that rats in the second and third weeks after CCI still had a higher mechanical and thermal threshold compared to vehicle-treated animals.
Common pathologies for neuropathic pain are oxidative stress, reduction of endogenous antioxidant enzyme activity, mitochondrial dysfunction, and neuroinflammation. Proteotoxic stresses trigger the cellular stress response. However evidence suggests that the direction of the cellular stress response may provide strategies to protect cells from damage. Nutritional antioxidants and phytochemicals can activate vintages, such as heme oxygenase, Hsp70, thioredoxin reductase, and sirtuins. Activation of the vintage system may cause the reduction of pro-oxidant conditions and tolerance of cellular stress [27][28][29][30]. While decreased antioxidant defenses and the ability to repair oxidative damage help increase oxidant production and may lead to neuropathic pain caused by oxidative stress [31][32][33]. The current data displayed CCI led to an increase in the spinal level of MDA and a reduction in the levels of catalase as well as SOD. Epineural ischemia occurs due to compression of the sciatic nerve, which in turn induces oxidative stress that is not limited to the affected peripheral region and resulted in changes in the excitability of spinal cord cells and develops central hypersensitivity. So, CCI caused hyperalgesia and allodynia and is associated with alteration in the spinal levels of oxidative stress factors and lipid peroxidation [34].
We observed that treatment with nanoselenium for 14 days, in addition to improving the behavioral symptoms of neuropathic pain, was associated with a significant decrease in MDA levels and an increase in catalase and SOD levels in the spinal cord of rats. Selenium is commonly present in inorganic or organic form and has a crucial role in human health through the various selenoprotein functions. Selenoproteins exist in enzymes like glutathione peroxidase and modulate the immune system, antioxidants, and metabolism. Selenium increases glutathione peroxidase activity, which causes lipid peroxidation in CNS [35]. Documents have reported the potential role of selenoproteins against ROS and the association between cellular redox state and the activation of cyclo-oxygenases (COX) and lipoxygenases (LOX). Selenoproteins involve in nitrosative stress responses: GPxs and TrxRs metabolize nitrosothiols and peroxynitrite. Previous studies have identified selenium as a hormetic chemical with a biphasic dose-response, toxic at high doses but beneficial at low doses. Optimal levels of selenoproteins may be clinically helpful for the inflammatory disorders associated with high peroxidase activity [29,36,37].
Traditional supplements of selenium almost have low absorption and raised toxicity. However, nano-sized elemental selenium particles have marked bioactivity and biosafety properties and used as a food additive or a therapeutic agent without significant side effects in medicine [4,38,39]. Nanoselenium is known to have superior antioxidant effects than selenium and has more effective results in inducing lipid peroxidation and causes less oxidative stress. It was showed that nanoselenium acted as an antioxidant and decreased reactive oxygen species scavenging enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase thus made its neuroprotective effects in CNS [4,17]. Other studies suggested that selenium therapy could affect oxidative stress by controlling Ca 2+ influx via the TRPM2 and TRPV1 channels in the neuropathic rats [6,40].
Because antioxidants could detoxify a variety of reactive oxygen species in some neurological diseases, it seems that chronic administration of nanoselenium with the same mechanism may alleviate related pain behaviors in CCI rats [41]. In other words, the antinociceptive effect of nanoselenium may be connected to alteration in lipid peroxidation and oxidative stress factors in sciatic nerve injury. Consistent with our findings, it is reported that free radical scavengers, like ascorbic acid, vitamin E, and selenium compounds alone, or in combination with other medications, behave as neuroprotective agents and might prevent the development of peripheral neuropathies and pain [5,9,11,17,26].
In conclusion, our findings suggest that chronic administration of nanoselenium could influence oxidative parameters in the spinal cord and alleviate pain-related behavior in CCI rats.