Neuropathic pain is one of the major chronic health problems, with allodynia and hyperalgesia being the most common signals of pain 1. Failure in response to standard treatments is a serious challenge in treating these conditions 23. There are different etiologies for neuropathic pain but one of the most common causes is diabetic neuropathy; in fact, 50-60% of diabetic patients have polyneuropathy 4. Several complicated and similar mechanisms have been associated with peripheral and central neuropathic pain. These mechanisms include increased release of excitatory neurotransmitters, modulation of descending inhibitory pathways, inflammatory reactions and oxidative stress 567, all of which may change with time thereby increasing complications 8. With regard to these complications, all of the approaches presented above have not shown sufficient evidence of effective neuropathic pain treatment 39. Furthermore, it should be noted that in addition to the varied side effects, standard pharmacologic medications also have serious interactions with each other, which must be considered. Therefore, alternative methods of suppressing and treating neuropathic pain are necessary.

Plants have many valuable constituents that enable them to improve cellular function in healthy and diseased states. Plants and medicinal herbs have been used for a long time ago throughout the world for promoting health and as therapeutic agents in various disorders, such as cancer 10, neuropathy 1112, and Parkinson’s disease 13. Among plants, Spirulina platensis is a marine microalgae which belongs to the class of cyanobacteria 14, known for containing rich resources of carotenoids, trace elements, and vitamins 15. Spirulina has many positive biological features, including anti-inflammatory and antioxidant 1617, antimicrobial 18, antitumoral 19 and immunomodulatory properties. It was reported that Spirulina alleviates proinflammatory cytokine TNF-alpha in the brain tissues of aged rats 17. The antioxidant property of Spirulina is ascribed to its C-phycocyanin component 2021, which is a potent antioxidant.

There are several animal studies demonstrating the protective effects of Spirulina against environmental chemotoxicity and pharmacologic-induced oxidative stress 22. For example, it has been reported that Spirulina reduces neuronal injury due to oxidative stress and ischemia 2324. It has also been reported that Spirulina protects different organs against toxicity of heavy metals and pharmacologic agents 252627. Indeed, Spirulina completely blocked MPTP (1-methyl-4-phenyl-1, 2, 3, 6- tetrahydropyridine)-induced oxidative stress, partially reduced MPTP-induced Parkinson’s disease symptoms in mice, and improved nigrostriatal dopamine pathway injury (caused by 6-hydroxydopamine (6-OHDA)) in rats 2328. In light of these effects, it seems that the protective effects of Spirulina are, at least, due to its antioxidant properties, although other mechanisms may also play a role. Given the aforementioned impact of the role of oxidative stress and inflammation in peripheral neuropathic pain 6, and the antioxidative properties of Spirulina microalgae, the aim of this study was to evaluate whether orally administered Spirulina could suppress neuropathic pain induced by chronic constriction injury (CCI) in rats. To our understanding, no other study has examined the effects of Spirulina therapy on CCI-induced neuropathic pain thus far.

In this study, adult male Wistar rats weighing 200-220 grams were used. Animals were obtained from the animal house of Semnan University of Medical Sciences. The rats were kept in an environment where humidity, temperature, and light-dark cycle were controlled. Food and water were sufficiently available to the animals. All experimental procedures were performed, as approved by the local ethical committees on animal research of the Semnan University of Medical Sciences, under permit number 1394.217.

Spirulina platensis microalgae were provided from Qeshm Microalgae Biorefinery Company (Qeshm Island, Iran). Spirulina was in a powder form. Spirulina was dissolved in physiologic saline at concentrations of 200, 400, and 800 mg/kg and was administered intragastrically to the rats in a volume of 2 ml by a gavage tube. The dosage of Spirulina platensis evaluated in our study were: 0, 200, 400, and 800 mg/kg.

Induction of neuropathy was performed through the method described by Bennett and Xie 29. Animals were anesthetized using intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture. Using a surgical blade, a 2-cm incision was made on the left thigh and the sciatic nerve was exposed and then released from the surrounding tissues. Using catgut chromic sutures 4-0, 4 loose ligations were made around the common sciatic nerve at 1-mm intervals. At the end, the incisures sites (muscle and skin) were separately sutured with silk 4-0. In the sham group, after exposing the sciatic nerve only, muscle and skin were sutured.

In this study, 74 male rats were used in two experiments. In the first experiment, 37 rats were randomly assigned to 6 groups (Intact, Sham, Neuropathy, Neuropathy with Spirulina (200 mg/kg), Neuropathy with Spirulina (400 mg/kg), and Neuropathy with Spirulina (800 mg/kg)) for behavioral and oxidative stress tests at 14 days post surgery. In the second experiment, another 37 rats were assigned to the same groups and tested at 21 days post surgery. Saline or Spirulina gavages were initiated on the post surgery day and continued on to 14 and 21 days after surgery in the first and second experiments, respectively. Spirulina was dissolved in physiologic saline and was intragastrically administered in a volume of 2 ml by a gavage tube. Four rats were paralyzed and thus excluded from the study. The study design is demonstrated in Table 1.

Pain response was evaluated using the following tests:

Paw withdrawal response was detected using the von Frey’s hairs (Stoelting, Wood Dale, IL, and USA) which were described by Ren et al. 30. Von Frey’s hairs are calibrated filaments that, depending on their thickness, apply a certain force (in grams) to the surface to which they are pressed. The filaments were applied with ascending style on the dorsal surface of the injured hind paws. The experiment began with a low force and if there was no response, a greater force was applied. If the animal withdrew its foot three times per five stimuli delivered, that force was considered as a response. A stimulus equivalent to 60 g was considered as the cut-off force.

Figure 1

The animals were placed in the plantar test device and after habituation, infrared radiation was applied to the plantar surface of the injured paw with an intensity of sixty. Infrared radiation was displayed every five minutes in three rounds. Withdrawal latency to each thermal stimulus was recorded and the average of three rounds was considered as response. Sixty seconds was set as cut-off point of the test 29.

Figure 2

Oxidative stress was evaluated using malondialdehyde (MDA) and ferric reducing ability of plasma (FRAP) assay of the rats’ blood.

Blood samples were prepared from the rats’ hearts while they were anesthetized. Using a centrifuge with 2000 rpm for 10 min, the serum was removed from the blood and kept at −80°C until use in the MDA and FRAP assays.

The MDA was assayed in rat serum. Thiobarbituric acid method was used to measure lipid peroxidation 31. Peroxidation of unsaturated fatty acids increases through oxidative stress, and various aldehydes (including MDA) are produced. MDA (as a marker of oxidative stress) reacts with thiobarbituric acid in acidic regions at high temperatures. The maximum absorption was evaluated at 535 nm using a spectrophotometer.

For detecting FRAP, measurement of total antioxidant capacity (TAC) in the rat serum was used. The ability of the serum to reduce Fe ³⁺ ions to Fe ²⁺ is the basis of this method and the output is a blue complex solution which shows the maximum light absorption at 593 nm wavelength 32.

One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were used for analyzing the data. All data were expressed as mean ± SEM of the detected variables; P<0.05 was considered significant.

Our results revealed that Spirulina can suppress neuropathic pain induced by CCI. The results were divided in two parts- behavioral and biochemical.