Chronic neuropathic pain caused by nerve injury provokes peripheral and central sensitization [1]. That is, patients with neuropathic pain involve emotional disorders such as anxiety and depression [2]. As well, depression is an outspread chronic psychiatric disorder that interferes with social life and work performance [3]. According to World Health Organization (WHO), depression is estimated to become the second leading cause of death and disability in humans by 2020 [4]. The most presently available antidepressant treatments are designed to target the serotonergic and/or the noradrenergic system in brain [5]. However, many of these antidepressants treatments have a slow onset of action (over 2 weeks) and are effective in only 50% of patients, with serious adverse reactions [6]. This may reflect the heterogeneity of the mechanisms underlying depression, drawing attention to novel therapeutic targets that are not addressed by current antidepressants [5]. Despite the dominance of neurotransmitter research in biological psychiatry, psychoneuroimmunology has developed over recent decades, investigating the role of immune dysfunction and inflammation in major psychiatric disorders [7]. Conjointly, accumulating evidence suggests that cytokines, critical to the neuroimmune response in neuropathic pain, play a central role in the etiology of depressive symptoms [8]. Furthermore, rodent models of depression are also associated with elevated levels of inflammation in the periphery and brain [9]. Thereby, modulation of peripheral inflammation and associated immune signaling pathways may provide an effective way to treat depression [5]. Immunomodulatory drugs may have efficacy for depressive symptoms that are comorbidly associated with inflammatory disorders [10]. Over time, so many immunomodulators were introduced like adjuvants, cytokines, glucocorticocoids, minerals, herbs, vaccines and others [11]. The most extensively investigated immunoadjuvant is Bacillus Calmette-Guerin (BCG) [12]. The first observation for the use of BCG in enhancement of immune response was evidenced by use of mycobacteria in preparation of Freund’s adjuvant in the late 1950s [13]. Furthermore, improvement in the efficacy of specific immunotherapy (SIT) has been attempted by the addition of adjuvants, including BCG [14].

On the other hand, medicinal plants, with lower economic costs and fewer side effects, have proved their curative effect in treating many clinical symptoms [15], including mental illness [16,17]. Indeed, many plants are traditionally used by native people for the treatment of neurological health problems [3]. Additionally, the special importance of total plant extracts is that they are easily available products, without purification to apply them in possible prevention of diseases [18]. The red algae Sphaerococcus coronopifolius extract was reported to possess antioxidant, anti-inflammatory, antitumoral and anxiolytic-like activities [19-21]. The present study was therefore designed to evaluate the potential effects of aqueous extract of S. coronopifolius in combination with BCG on depression-like behavior in female Wistar rats.

In the present study, behavioral, biochemical and histological approaches were applied to investigate the protective effects of S. coronopifolius crude extract in combination with BCG on ligature-induced depression in female Wistar rats. The choice of females in this study is focused on the fact that stress is more likely to affect females than males [30,31]. Previous studies have shown sex differences in pain perception and in the prevalence of chronic pain [32,33]. In addition, females have a higher prevalence of psychological complications associated with chronic pain [34]. Furthermore, persistent neuropathic pain is associated with emotional disorders, such as anxiety and depression [1,2]. Previous reports using rodents have shown that pain lasting more than 4 weeks induces emotional dysfunction, such as anxiety or depression-like behavior [35,36]. In the present work, depression-like behavior was observed in female Wistar rats 3 weeks after sciatic nerve injury. Our results are in accordance with those of Nishinaka et al. [37] in which female mice exposed to MSSI (social isolation after weaning) exhibited depression-like behavior within 3 weeks after partial sciatic nerve injury (PSL).

Nowadays, natural products, especially polyphenols, have received growing interest due to their potential benefits in treating psychiatric disorders [5]. In this work, we opted for the use of the crude extract despite the fact that conventional drug development claimed the use of a single active principles isolated from plants [38]. Nevertheless, there are instances when attempts to isolate the bioactive metabolites from plant extracts ultimately prove fruitless, since overall bioprospecting effects often rely on synergistic and multivalent interactions between plant components [39]. Therefore, aqueous extract of S. coronopifolius exerted positive effect on depression-like behaviors, which may be related to its richness in antioxidant polyphenols [19]. Different polyphenolic compounds have been observed to have antidepresssant-like effects in rodents and human [40]. The substantial antioxidant and anti-inflammatory activities of polyphenols and their ability to modulate synaptic plasticity may contribute to their mechanisms of action [41,42].

Recently, it has been established that BCG vaccination exerted neuroprotection in several CNS diseases models [43,44]. Further, its role as an immune adjuvant in the therapy of mood disorders may be more promising. Nevertheless, there are many studies reported that BCG induced a depression-like phenotype in rodents [45,46]. In fact, it has been demonstrated that the BCG-induced depressive-like behavior was dose-dependent and the illness (adverse health effect) was observed only in mice receiving no less than 107 CFU [47]. Accordingly, the role of BCG as an adjuvant has been examined in our study at low dose in a combination with algae, which showed promising results in the treatment of depression. The dose of 105 CFU was selected because of its effectiveness in immune activation, without adverse health effects [25]. Moreover, it was reported that neonatal BCG vaccination at a dose of 105 CFU elevated neurogenesis, synaptic plasticity, cognitive function, and the levels of neurotrophic factors in mice housed in a standard environment [48].

The forced swimming test (FST) is a behavioral despair test useful for probing the pathological mechanism of depression and for the evaluation of antidepressant drugs [49]. Antidepressants achieved their effects by reducing immobility time and increase or prolong active escape behaviors [39]. It has been shown that the antidepressant-like actions in FST, which were mediated by the stimulation of 5-HT neurotransmission, induced the increase of active swimming period, whereas the augmentation of NE neurotransmission was related to the enhanced climbing activity [50]. In our study, treatment with algae extract produced a decrease in immobility time accompanied by an increase in climbing time, which suggest the stimulation of the neurotransmission mediated by NE. Noradrenaline plays an important role in the pathophysiology of depression and is used as an effective treatment target [39]. However, the combined treatment with algae extract and BCG showed an increase in swimming time, which indicate the activation of 5-HT receptors. As well, several antidepressants produce their effect by modulation of brain 5-HT levels [39]. It is important to point out that there is no significant difference in immobility time between the group treated by algae extract alone and that treated by the combination treatments, which suggest that immunomodulatory responses that might have been induced by BCG require more than two weeks.

The open field test (OFT) is used in combination with the FST to discard non-specific actions of antidepressant treatments [51]. It was reported that drugs inducing increase in motor activity may give a ‘false’ positive effect [52]. This test evaluates the reaction of an animal towards a new and spacious environment. It creates a conflict between the tendency of rodents to find a place to hide, and their aversion to open spaces [53]. The results from open field test suggested that the combination of algae extract and BCG, at the doses that produced an anti-immobility response, did not show significant locomotor stimulation or inhibition. Therefore, the reported antidepressant-like effect of these treatments was not due to its psychostimulant action [54]. It has been reported also that low-molecular proanthocyanidins from grapes, applied to mice, exerted antidepressant-like effects in behavioral despair tests but did not affect locomotor activity [55]. Our results have showed that the combination of algae extract with BCG increased rearing in comparison with non-treated rats, which supports the antidepressant-like activity of this this combination. It has been found that normal rats show increased rearing and grooming in a novel open field, which is driven by the instinct interests of rats to explore a novel environment [56].

Latterly, oxidative stress was linked with pathophysiology of depression. Oxidative stress damage involves the oxidation of proteins, nucleic acids, membrane lipids, etc. by free radicals, leading to cell degeneration and even death, which is one of the important mechanisms of depression [57]. The brain has a large potential oxidative capacity but a limited ability to counteract oxidative stress. Cells in the central nervous system are more sensitive to toxic effects of ROS, than cells in other organs of the body [58]. The present study revealed an important decrease in GST and CAT activities in brain tissue of ligated female rats. It should be noted also that after 2 weeks of treatment with algae extract in combination with BCG, GST and CAT activities remain significantly lower than that in the control group. In line with our results, Sarandol et al. [59] showed that six weeks of antidepressant treatment with either reboxetine, sertraline, or venlafaxine did not change oxidative-antioxidative system parameters. Furthermore, it was suggested that long-term treatment of at least 12 weeks is needed for oxidative and anti-oxidative system parameters to be changed [60].

Currently, increased appetite and weight gain have been identified as a common symptom of depression as well as other disorders involving emotional distress [61]. In our study, the female rats with depression showed an increased body mass. Indeed, studies on animals under stress conditions have pointed out that food intake could be either stimulated or inhibited [62,63]. In contrast, it was observed a decrease in body weight in LIG+ BCG group 3 days after BCG inoculation accompanied with a decrease in motor activity. These core symptoms constitute the sickness behavior, or behavioral changes that accompany immune response [64]. However, these sickness behaviors return to normal by a few days after inoculation of BCG [22,47]. In our case, the female rats started to regain weight 2 weeks after inoculation. Interestingly, the group treated with a combination of algae extract and BCG showed a balance of body weight, which suggest the beneficial effects of algae extract on sickness behaviors- induced by inoculation of BCG.

The histological analysis of the spleen is particularly important because it gives insight to the presence of the spleen structural components that make the cell potential on which depends its defensive function [65]. Chronic stress has been described to induce structural alterations in splenic lymphoid tissue in rats [66]. Granulomatous inflammation, which is a distinctive pattern of chronic inflammatory reaction characterized by focal accumulation of activated macrophages, can generally develop an epitheloid appearance [67]. Epitheloid cells can fuse to form giant cells with diameter of 40-50 µm. These giant cells have a large mass of cytoplasm with 20 or more nuclei arranged haphazardly or in a horseshoe pattern peripherally-the Langhans type [67]. Along with, chronic stress is known to promote a disruption of the HPA axis, usually indicated by increased levels of corticosterone in the serum of rodents [68]. Thus, hypertrophy of the adrenal gland was observed [69]. In the present investigation, increased adrenal activity as shown by increased adrenal weight in ligatured rats indicate the activation of the HPA axis. Nonetheless, normal weight of adrenal gland in rats treated by S. coronopifolius extract alone or combined with BCG indicated that these treatments prevented the depression-induced activation of adrenocortical activity. Supplementary, the repeat restraint and exogenous corticosterone have low adrenal gland weight [66].

In summary, the present study examined the mechanisms of the potential antidepressant-like actions for S. coronopifolius extract in combination with low dose of BCG. This study elicited algae extract as the principal drug for the treatment, whereas BCG served as an adjuvant. Our results provides the first evidence indicating that aqueous extract of S. coronopifolius exhibited antidepressive-like effect while BCG inoculation provided no significant benefit, suggesting that immunomodulatory responses that might have been induced by BCG require more than two weeks. Obviously, the combination treatment of algae extract with BCG can attenuated depression-like behaviors in stressed female rats through its anti-oxidant and anti-inflammatory properties following at least three weeks. Further investigations should be geared towards using herbal immunomodulators in order to evaluate a potential synergistic effect with algae extract in the treatment of depression.