Spirulina platensis attenuates the associated neurobehavioral and inflammatory response impairments in rats exposed to lead acetate

Spirulina platensis attenuates the associated neurobehavioral and inflammatory response impairments in rats exposed to lead acetate

Ecotoxicology and Environmental Safety 157 (2018) 255–265 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal h...

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Ecotoxicology and Environmental Safety 157 (2018) 255–265

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Spirulina platensis attenuates the associated neurobehavioral and inflammatory response impairments in rats exposed to lead acetate


Samah R. Khalila, , Hesham A. Khalifab, Sabry M. Abdel-Motalb, Hesham H. Mohammedc, Yaser H.A. Elewad,e, Hend Atta Mahmoudb a

Forensic Medicine and Toxicology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt c Veterinary Public Health Department, Faculty of Veterinary Medicine, Zagazig University, Egypt d Histology and Cytology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt e Laboratory of Anatomy, Department of Biomedical Sciences. Graduate school of Veterinary, Hokkaido University, Sapporo, Japan b



Keywords: Caspase-3 Comet assay HSP70 Lead Open field test Spirulina platensis

Heavy metals are well known as environmental pollutants with hazardous impacts on human and animal health because of their wide industrial usage. In the present study, the role of Spirulina platensis in reversing the oxidative stress-mediated brain injury elicited by lead acetate exposure was evaluated. In order to accomplish this aim, rats were orally administered with 300 mg/kg bw Spirulina for 15 d, before and simultaneously with an intraperitoneal injection of 50 mg/kg bw lead acetate [6 injections through the two weeks]. As a result, the coadministration of Spirulina with lead acetate reversed the most impaired open field behavioral indices; however, this did not happen for swimming performance, inclined plane, and grip strength tests. In addition, it was observed that Spirulina diminished the lead content that accumulated in both the blood and the brain tissue of the exposed rats, and reduced the elevated levels of oxidative damage indices, and brain proinflammatory markers. Also, because of the Spirulina administration, the levels of the depleted biomarkers of antioxidant status and interleukin–10 in the lead-exposed rats were improved. Moreover, Spirulina protected the brain tissue (cerebrum and cerebellum) against the changes elicited by lead exposure, and also decreased the reactivity of HSP70 and Caspase–3 in both cerebrum and cerebellum tissues. Collectively, our findings demonstrate that Spirulina has a potential use as a food supplement in the regions highly polluted with heavy metals.

1. Introduction Microalgae have been known for centuries as food and animal feed; they grow in freshwater and marine ecosystems as filamentous organisms (Vigani et al., 2015). Spirulina is a photosynthetic cyanobacterium that is used commercially as a dietary supplement and food additive. Currently, the annual production of Spirulina exceeds 3000 t on a dry weight basis and is acknowledged by several companies in various nations. The extensive production of Spirulina is owing to its original chemical composition [proteins, polyunsaturated fatty acids, and vitamins]. Besides, it is a source of bioactive components, for example, phycocyanin, β-carotene, and allophycocyanin, which possess anti-inflammatory and antioxidant properties (Wang et al., 2007). Also, it is able to manage numerous diseased conditions, including diabetes and obesity (Anitha andChandralekha, 2010), arthritis (Kumar et al., 2009), cardiovascular diseases (Deng and Chow, 2010), and allergies (Vo et al., 2012). Moreover, Spirulina is generally accepted to be safe for ⁎

consumption because of its long history of usage as a food source and its ideal safety profile in human and animal studies (Karkos et al., 2011). Spirulina contains non-toxic cyanobacteria, but the methods of cultivation without proper quality controls permit contamination by other toxin producer species prompting the presence of harmful cyanotoxins which have general health concerns. Roy-Lachapelle et al. (2017) detected the presence of cyanotoxins in various commercially available products containing Spirulina at levels exceeding the tolerable daily intake levels, showed the significance of better screening by the appropriate authorities of algal-based food supplements like spirulina to guarantee the safety of these products. The studies conducted in the past using Spirulina platensis as a supplement have proved that it has an ability to encounter the multiorgan toxicity caused by various medications and chemicals (Simsek et al., 2009; Banji et al., 2013). Spirulina also reversed the modified immune response and hepatic damage prompted by the herbicide atrazine in the common carp (Khalil et al., 2017, 2018). It has been

Corresponding author at: Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, 44511 Zagazig, Egypt. E-mail address: [email protected] (S.R. Khalil).

https://doi.org/10.1016/j.ecoenv.2018.03.068 Received 6 February 2018; Received in revised form 22 March 2018; Accepted 24 March 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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considered to be safe. For experimental use, a working solution of the test compounds was prepared by diluting them with distilled water.

hypothesized that the modulating potency of Spirulina, which includes the central nervous system, could be considered a part of the therapeutic regimes for neurodegenerative diseases, such as, Alzheimer's and Parkinsonism (Pabon et al., 2012), nigrostriatal dopamine system injury (Strömberg et al., 2005), cerebrovascular accident models (Thaakur and Sravanthi, 2010), and for neurotoxicity by means of free radical generation to protect the dopaminergic neurotransmission (Tobón-Velasco et al., 2013). Lead is an environmental contaminant causing occupational health hazards that can be perceived in the environment and biological systems as it is widely used in industry (El-Nekeety et al., 2009). Both environmental and occupational exposures cause significant and serious medical issues in the industries where lead and lead-based components are used for the production of ammunition, bearing metals, bronze and brass, cable covering, sheet lead, and solder. Besides, lead can be used in ceramics, weights or ballast, containers or tubes, oxides, and gasoline additives (Schwartz and Levin, 1991). Although the use of lead as an additive in household paint has ceased, the lead-containing paint is nonetheless found in buildings built before the 1960s (Bradberry, 2016). Lead can enter the body by inhaling, by ingesting the contaminated soil and water, and by consuming food. After absorption, lead accumulates in several tissues, such as kidney, liver, brain, and bones, and impacts various biological activities at the molecular, cellular, and intracellular levels, which may result in morphological perturbations that cannot be reversed even after the levels of lead have diminished in the body (Flora et al., 2006). Studies on both animals and humans have demonstrated lead-induced deleterious effects in hepatic tissue (Khalil et al., 2018), cardiovascular, immune, and reproductive systems (Patra and Swarup, 2004; Shah and Altindag, 2005; Teijón et al., 2006), and nerve dysfunction (Ademuyiwa et al., 2007). Among the organ systems influenced by lead, the nervous system is particularly sensitive to lead exposure, which, therefore, has received much research consideration for a long time. Exposure to low doses of lead causes injurious impacts on the nervous system, including hindered cognition and memory, as well as debilitated peripheral nerve functions (White et al., 2007). Despite the various studies dealing with lead toxicity, the exact mechanisms through which lead causes the neurotoxic effects are not yet fully understood. Oxidative stress, membrane alterations, cell signaling deregulation, and neurotransmission impairment are viewed as key perspectives involved in lead neurotoxicity (Sanders et al., 2009). Moreover, certain in vitro and in vivo studies demonstrated that lead could give rise to neural cell apoptosis (Baranowska-Bosiacka et al., 2013; Sharifi et al., 2010). Considering the involvement of oxidative stress in lead-induced neurotoxicity (Chander et al., 2014), it is reasonable that the administration of an antioxidant may be an imperative therapeutic approach to deal with lead toxicity. Stemmed from this notion, the present study was undertaken to explore the putative protective potency of Spirulina against the lead-induced neurotoxicity by addressing the behavioral responses, probing into oxidative stress, tissue inflammatory reactions, and cellular death, and seeking a possible link with histopathological changes in the brain tissue.

2.2. Animals and experimental design Forty male Sprague–Dawley rats (150–200 g) obtained from the laboratory animals farm of Faculty of Veterinary Medicine (Zagazig University, Egypt), were used in the present study. The animals were maintained in clean stainless steel cages under controlled environmental conditions. The animals had free access to water and feed throughout the experimental period, and they were accommodated to the laboratory conditions for two weeks before being used in the experiments. The experiments were performed following the guidelines of the National Institutes of Health (NIH) for the Care and Use of Laboratory Animals in scientific investigations and approved by the Ethics of Animal Use in Research Committee (Zagazig University, Egypt). The animals were randomly assigned into four experimental groups; therefore, each group consisted of ten animals. The control group involved rats that were intraperitoneally (IP) injected with distilled water as a vehicle. Spirulina-administered animals received Spirulina orally at a dose of 300 mg/kg bw for 30 days via gastric tube (Simsek et al., 2009). Lead acetate-exposed animals were injected (IP) with lead acetate at a dose of 50 mg/kg bw (three times a week) for two weeks (Ahmed et al., 2013). The animals in the Spirulina/Lead acetate cotreated group were orally administered with Spirulina for 15 days before and 15 days simultaneously with an IP injection of lead acetate at the same course as previously described. On the day of sacrifice, the final body weight of each rat was recorded by weighing each animal in all the groups for estimating the body weight changes. 2.3. Behavioral response evaluation In order to evaluate the behavioral changes provoked by lead acetate exposure and/or Spirulina administration, the rats were transferred to the testing room in their home cages and kept there for about 30 min prior to testing to accommodate them to the testing-room conditions. All behavioral tests were done in the same testing room on the last day of the experimental period. The experimenter was blind to/ unaware of the identity of the animal groups. Behavioral analyses, including the open field test, swimming performance, grip strength, and inclined plane, were performed with the different experimental groups as described ahead. 2.3.1. Open field test The rats were set individually in the center of the open field arena and behavioral parameters were recorded for 5 min, beginning 2 min after setting the animal in the test cage. The open field apparatus was then thoroughly cleaned with 5% ethanol before placing the next rat, to eliminate the possible cueing effects of the odors left by previous animals. The behavioral indices observed in this test were ambulation or locomotion frequency (the number of floor sections entered with two feet), rearing frequency (the number of times the animal stood on its hind legs), stereotype counts (the number of grooming movements), and immobility (freezing) duration (total time in second without spontaneous movements). The open field contained a central square or area that was considered an unprotected area for rats; thus, the number of entries into this area reflected the level of anxiety-like behavior (Contó et al., 2005). The observations were recorded between 10:00 and 12:00 a.m.

2. Material and methods 2.1. Test compounds and chemicals Lead acetate (99.6% purity) was purchased from El-Nasr Pharmaceutical Chemical Co. ( Egypt). Spirulina platensis, as a bluegreen powder, was obtained from EL-Hellow for Biological Products supplier (Egypt). The Spirulina powder was subjected to heavy metals analysis like lead, mercury, cadmium, and arsenic by using flame atomic absorption spectrophotometer, these metals are the most likely to contaminate Spirulina. Heavy metal contents in the tested Spirulina sample were all within the permissible levels (0.29, 0.048, 0.09 and 0.037 ppm, respectively). Therefore, the tested Spirulina sample was

2.3.2. Swimming performance test Swimming performance test was carried out by putting each animal at the middle of the glass aquarium for observation for 5–10 s, where the swimming performance was scored according to the position of nose and head on the surface of water as follows: 0 F, head and nose below 256

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et al. (1994). The comet assay was conducted according to the method described by Singh et al. (1988). Fifty randomly selected cells per slide were investigated. Imaging was done using a fluorescence microscope (Zeiss Axiovert L410 Inc., Germany) fitted with a CCD camera (Olympus Inc., Japan). The comets were examined using a visual scoring method and the computer image analysis was performed using Comet Assay Software Project.

the water surface; 1, nose below the water surface; 2, nose and top of the head at or above the water surface and ears below the surface; 3, similar to score two except that the water line was at the mid-ear level; and 4, similar to score three except that the water line was at the bottom of the ears (Schapiro et al., 1970). 2.3.3. Inclined plane Rats were put on a flat plane in a horizontal position with the head facing the side of the board to be raised (Yonemori et al., 1998). The inclined plane performance was measured with a standard protractor to the nearest 5 degrees. The trial ceased when the rat started to slip backward. Hence, there was no particular trial duration. The angle at which the rat began to slip downward was recorded. The results of two trials were averaged for each rat. Both the trials were separated by a one-hour interval.

2.8. Evaluation of inflammatory markers in brain tissue Rat ELISA kits were used for the detection of inflammatory markers in the brain tissue–nitric oxide, NO; tumor necrosis factor-α, TNF-α; interleukin–10, IL-10. The kits were obtained from MyBioSource (San Diego, California, USA) (Catalog No.: MBS010567, MBS704859, and MBS269138 for NO, TNF-α, and IL-10, respectively). The quantitative detection of such indices was done following manufacturer's instructions.

2.3.4. Grip strength test The rat's forepaw strength was monitored by having it to hold a wood dowel (5 mm diameter) that was held horizontally and raised so that the animal supported its body weight, as described by Andersen et al. (1991). Time taken to release the grip was recorded in seconds. All rats attempted to grip the dowel during this grip strength testing. The results of two trials separated by a one-hour interval were averaged for each rat.

2.9. Histopathological study 2.9.1. Light microscopy Brain specimens (cerebrum and cerebellum) from the control and treated groups were processed for histopathological investigation using a light microscope (Olympus BX51 microscope, Olympus Inc., Japan) according to Bancroft and Gamble (2008). This process involved sectioning the tissues, staining with hematoxylin/eosin (H&E) dye, and viewing under the light microscope.

2.4. Blood and brain tissue collection At the time of sacrifice, whole blood samples were collected from the medial canthus (orbital vessels) of the experimental rats in heparincontaining tubes for evaluating the levels of lead. Brain tissue specimens were obtained, dissected, rinsed with sterile physiological saline (0.9% NaCl), weighed, and then divided into four sets. The first one was homogenized and the homogenates were centrifuged at 664×g at 4 °C for 15 min to obtain the supernatants, which were then used for determining the antioxidant, oxidative stress and inflammatory indices. The second one was used to determine the levels of lead. The third one was used to prepare the cells for DNA-damage investigation (comet assay), by setting it in 1 mL of cold Hank's balanced salt solution containing 20 mM ethylenediaminetetraacetic acid (EDTA) and 10% dimethyl sulfoxide (DMSO), and then mincing it into fine pieces to acquire cell suspensions. The final set was fixed in 10% neutral buffered formalin for histopathological and immunohistochemical analysis.

2.9.2. Immunohistochemical analysis Brain tissue (cerebrum and cerebellum) sections were processed for immunohistochemical analysis and incubated overnight at 4 °C with a rabbit polyclonal anti-caspase (Caspase–3) primary antibody (1:100) or anti-heat shock protein 70 (HSP70) primary antibody (1:100) in phosphate-buffered saline (PBS). After three thorough washes with PBS, they were incubated with a goat anti-rabbit IgG biotin-conjugated secondary antibody (1:2000) for 20 min at 32 °C. After further incubation with horseradish peroxidase-labeled streptavidin, antibody binding was visualized using 3,3′-diaminobenzidine, and the sections were counterstained with hematoxylin. 2.10. Data analysis

2.5. Assessment of lead levels in blood and brain tissue

Data were expressed as mean ± SE, one way-ANOVA followed by Tukey's multiple comparisons post hoc test using the SPSS 16.0 computer program was performed to compare mean values of Lead-exposed groups and Spirulina-administered groups versus control one. A value of p < 0.05 was considered as statistically significant.

In order to perform the lead levels assay, whole blood (200 µL) and brain tissue samples (100 mg) were digested with 4 mL of nitric/perchloric acid mixture at room temperature for 24 h. Then, the samples were heated at 80 °C for 2 h in a water bath, cooled to room temperature, filtered, and diluted with deionized water before analysis (Julshman, 1983). The amount of lead was quantified using Buck Scientific 210VGP flame atomic absorption spectrophotometer; the suitable wavelength for the lead was 220.35 nm.

3. Results 3.1. General observations, body weight changes, and brain/body weight ratios of rats in response to lead acetate exposure and Spirulina administration

2.6. Evaluation of antioxidant biomarkers in brain tissue The superoxide dismutase (SOD and catalase CAT) activities and the levels of reduced glutathione (GSH) were estimated according to methods described previously (Misra and Fridovich, 1972; Sinha, 1972; Beutler, 1963).

During the experiments, neither deaths nor any clinical symptoms were recorded in the groups. However, anorexia and low activity were observed, mainly in the lead-exposed group. Lead-exposed rats showed a significant decline in the body weight changes relative to the control gain. Spirulina administration induced an improvement in rats by lowering the decrease in the body-weight gain in Spirulina/Lead cotreated group. On the other hand, there was no significant difference in the relative brain weight (brain somatic index) among all the experimental groups (Table 1).

2.7. Evaluation of oxidative stress biomarkers in brain tissue Lipid peroxides (MDA) were measured as described by Ohkawa et al. (1979). Protein carbonyl was assessed using a colorimetric assay kit (Cayman's Chemical Company, Ann Arbor, USA) according to Levine 257

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Table 1 Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on body weight changes, relative brain tissue weight and Lead level in rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Parameters

Body weight change (gm) Relative brain weight (%) Blood lead level (ppm) Brain lead level (ppm)

Experimental groups Control


Lead acetate

Spirulina/Lead acetate

22.66 ± 1.76a 0.87 ± 0.022 0.11 ± 0.005b 0.23 ± 0.021c

22.00 ± 1.80a 0.94 ± 0.015 0.05 ± 0.01b 0.20 ± 0.035c

−10.16 ± 2.61c 0.91 ± 0.015 0.54 ± 0.02a 0.89 ± 0.043a

15.00 ± 1.46b 0.95 ± 0.028 0.09 ± 0.012b 0.54 ± 0.032b

Means within the same row (in each parameter) carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n = 6).

3.2. Behavioral responses in response to lead acetate exposure and Spirulina administration

3.3. Lead levels in blood and the brain tissue of rats in response to lead acetate exposure and Spirulina administration

3.2.1. Open field test There was a significant increase in the locomotors activity represented by a significant increment in ambulation, which was associated with a lowering of freezing time in rats after exposure to lead acetate, compared with control rats. Spirulina co-administration with lead exhibited a significant modulation in freezing time; however, it did not affect ambulation behavior. Rearing and grooming behaviors did not show any significant changes as a result of lead exposure. Meanwhile, these rats exhibited more entries and time spent in the central arena than the control rats. Spirulina supplementation significantly maintained the rats' behavior in the central arena in the cotreated group, compared to the lead-exposed rats (Fig. 1).

Lead acetate exposure significantly increased the levels of lead in blood and the brain tissue, with greater lead accumulation occurring in the brain tissue when compared with the levels recorded in the control group. The supplementation of Spirulina with lead significantly decreased its levels in blood and the brain tissue, relative to the leadexposed group. In comparison with the control level, it exhibited nonsignificant differences in the blood lead levels; whereas, in the brain tissue, it exhibited significantly higher lead levels than the control (Table 1).

3.2.2. Grip strength, inclined plane, and swimming performance tests The data for swimming performance, inclined plane, and grip time in the rats exposed to lead displayed significant deficits in comparison to the corresponding data recorded in the control animals, indicating that the rats treated with lead experienced sensorimotor deficits. In Spirulina/Lead co-treated animals, Spirulina was unable to modulate these deficits, which were observed to be non-significantly different in comparison with the lead-exposed animals (Fig. 2).

Lead exposure significantly decreased the SOD, CAT activity, and GSH level in the brain tissue, compared to control group. On the other hand, co-administration of Spirulina with lead enhanced the SOD and CAT activity and increased the GSH level. Such improvement was significant in Spirulina/Lead co-treated group in comparison with the values recorded in the lead-exposed group, although it did not attain the control value (Table 2).

3.4. SOD, CAT activity, and GSH content in the brain tissue of rats in response to lead acetate exposure and Spirulina administration

Fig. 1. Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on open field behavior (ambulation, freezing time, rearing frequency, grooming frequency, central arena entrance frequency and time spent) in rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n = 6). 258

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3.5. MDA, PC, and DNA damage levels in the brain tissue of rats in response to lead acetate exposure and Spirulina administration MDA (a lipid peroxidation product) levels were significantly elevated in the brain tissue of lead-exposed rats, compared to the control group. Co-supplementation of Spirulina with lead significantly decreased the MDA levels compared to the lead-exposed group. The modulation recorded in the Spirulina/Lead co-treated group depicted a trend toward normal value. However, it did not match the values of control. Brain protein carbonyl (a protein oxidative damage product) level was significantly increased in the lead-exposed group, compared to the control group. Nevertheless, the Spirulina/Lead group exhibited a significant reduction in the PC level, compared to the level recorded in the lead-exposed group; this level was, however, non-significantly different from the value recorded in the control rats, indicating that Spirulina co-treatment significantly prevented carbonyl formation in this group (Table 2). The findings of DNA damage revealed a significant elevation in the tail length, tail DNA %, and tail moment in the brain tissues of the leadexposed animals, relative to controls. Spirulina supplementation alone displayed a non-significant decline in the level of potential DNA damage. The modulatory potency of Spirulina was confirmed by a significant reduction in all DNA damage indices in the co-treated group, relative to those exhibited in the lead-exposed rats (Fig. 3). 3.6. NO, TNF-α, and IL-10 levels in the brain tissue of rats in response to lead acetate exposure and Spirulina administration An exposure to lead resulted in a significant increase in NO and TNF-α levels in the brain tissue of lead-exposed group. Although these indices exhibited a significant decrease in the Spirulina/Lead co-treated group compared to the lead-exposed group, the level of NO was nonetheless significantly different from the recorded in the control, and the TNF-α level was non significantly different than of the control group. Spirulina supplementation was observed to significantly elevate the levels of interleukin–10 in the brain tissue when compared with control group. Lead exposure induced a significant decrease in the IL-10 levels compared to the control values. This decrease was modulated, as it exhibited a non-significant difference compared to the control values, in the Lead/Spirulina co-administered group (Table 2). Fig. 2. Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on grip strength time, inclined plane test and swimming performance test in rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n = 6).

3.7. Histopathological and immunohistochemical findings Histopathological observations of the H&E-stained paraffin-embedded brain sections revealed normal meninges and meningeal blood vessels in both control and Spirulina-administered group. In the leadexposed group, congestion and hemorrhage were observed in the meningeal blood vessels. In the Spirulina/Lead co-treated group,

Table 2 Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on antioxidant biomarkers (SOD, CAT activities and GSH level), oxidative stress biomarkers (MDA, and PC levels) and inflammatory response indices (NO, TNF-α and IL-10 levels) in the brain of rat exposed to Lead acetate (50 mg/ kg bw, every other day for two weeks, IP). Parameters

SOD activity (U/gm) CAT activity (U/gm) GSH level (mmol/gm) MDA (nmol/gm) PC (nmol/gm) NO (µmol/gm) TNF-α (Pg/gm) IL-10 (Pg/gm)

Experimental groups Control


Lead acetate

Spirulina/Lead acetate

395.23 ± 14.27a 0.80 ± 0.03a 2.51 ± 0.14a 5.38 ± 0.25a 0.134 ± 0.02b 8.64 ± 0.42c 0.19 ± 0.01bc 0.17 ± 0.01 b

427.0 ± 15.71a 0.77 ± 0.02a 2.66 ± 0.15a 5.20 ± 0.32a 0.112 ± 0.02b 8.44 ± 0.43c 0.13 ± 0.02c 0.29 ± 0.01a

235.42 ± 9.30c 0.46 ± 0.03c 1.41 ± 0.05c 10.85 ± 0.44c 0.30 ± 0.03a 18.70 ± 0.39a 1.34 ± 0.10a 0.086 ± 0.01c

299.17 ± 8.58b 0.62 ± 0.02b 1.80 ± 0.05b 6.94 ± 0.32b 0.19 ± 0.01b 13.21 ± 0.11b 0.34 ± 0.02b 0.14 ± 0.01b

Means within the same row (in each parameter) carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n = 6). 259

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Fig. 3. A. Fluorescent microscopic images of cells derived from brain tissue of rat from the experimental groups showing, cells with intact DNA in control and Spirulina-administered groups, cells with a tail (DNA fragments) in Lead-exposed and Spirulina/Lead co-treated groups. B. Effect of pre and simultaneous Spirulina supplementation (300 mg/kg bw for 30 days, orally) on DNA damage indices (Comet assay) in the brain of rat exposed to Lead acetate (50 mg/kg bw, every other day for two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n = 6).

observations for Caspase–3 revealed the presence of a few apoptotic cells in the cerebrum and cerebellum of the Spirulina/Lead co-treated and lead-exposed animals. These cells were rarely observed in the control and Spirulina-administered groups (Fig. 5).

meningeal edema and congestion of blood vessels were observed in the region between cerebellum and cerebrum. Moreover, the choroid plexus exhibited normal structure in both control and Spirulina-administered group. In the lead-exposed group, the congestion of the choroidal blood capillaries and proliferation of the choroidal epithelial lining were prominent. In the Spirulina/Lead co-treated group, only the congestion of choroidal blood capillaries was observed. The cerebellum in both control and Spirulina-administered group displayed normally arranged cerebellar cortical layers, including the outer molecular, the inner granular, and the middle layer of pear-shaped Purkinje cells that aligned regularly in-between the former layers. In the lead-exposed group, the middle Purkinje-cells layers displayed a fewer number of intact Purkinje cells that exhibited disorganized alignment, along with the presence of a few necrotic Purkinje cells and perineural edema. In the Spirulina/Lead co-treated group, the Purkinje cells exhibited severely disorganized alignment (Fig. 4). Immunohistochemical analysis for the detection of heat shock protein (HSP) 70 in cerebrum and cerebellum revealed numerous blood vessels with the positive reaction to HSP70 in the Spirulina/Lead cotreated and lead-exposed groups, compared with the control and Spirulina-administered groups. Additionally, the immunohistochemical

4. Discussion The naturally occurring sources of antioxidants have been used in various in vitro and in vivo studies, and have demonstrated promising outcomes with regard to their effects on metal-induced toxicity (Moustafa et al., 2012; Khalil et al., 2013, 2018; Khalil and Hussein, 2015). The present study deals with the efficacy of Spirulina on modulating the neurotoxic alterations induced by subacute exposure to lead acetate. The main outcome of this study was that an IP injection of lead acetate resulted in a significant oxidative stress-mediated damage and brain injury, which was inferred from the altered behavioral, biochemical, histopathological, and immunohistochemical indices under investigation. Besides, the supplementation of Spirulina provided a neuroprotective efficacy through the modulation of these impairments, when given prior to and simultaneously with lead acetate. In the present study, animals that were exposed to lead acetate 260

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Fig. 4. Photomicrograph of brain tissue of: Control (A) and Spirulina-administered (B) groups showing normal histological structured normal meninges and meningeal blood vessels (Bar =100 µm). Lead-exposed group (C) showing congestion and hemorrhage in the meningeal blood vessels (Arrow) (Bar = 40 µm). Spirulina/Lead co- treated group (D) showing meningeal edema, and congestion of the blood vessels in the region between cerebellum and cerebrum (Arrow) (Bar = 200 µm). Photomicrograph of choroid plexus of: Control and Spirulina-administered (A&B) groups showing normal histological structured normal choroid plexus (Bar =200, 40 µm). Lead-exposed group (C) showing congestion of the choroidal blood capillaries and proliferation of the choroidal lining epithelial (Arrows) (Bar = 40 µm). Spirulina/Lead co-treated group (D) showing congestion of the choroidal blood capillaries only (Star) (Bar = 40 µm). Photomicrograph of cerebellum: Control (A) and Spirulina-administered (B) groups showing normally arranged cerebellar cortical layers including outer molecular, inner granular and middle pearshaped purkinjie cells that aligned regularly in-between the former layers. Lead-exposed group (C) showing less number of intact middle purkinje cell layers that showing disorganized alignment (Arrowheads) with the presence of some necrotic purkinje cells (Arrow) and perineural edema. Spirulina/Lead co- treated group (D) showing a disorganized alignment of the purkinje cells (Arrowheads) (Bar = 50 µm) (Stain: H &E).

systems. Lead-induced consequences on the dopaminergic system incorporate changes in the synthesis, turnover, and reuptake of dopamine and its metabolites, as well as in the number of dopamine receptors (ATSDR, 1999). Along these lines, Bressler and Goldstein (1991) demonstrated that, at the neuronal level, lead alters the release of neurotransmitters from the presynaptic nerve endings as it mimics the action of Ca++. The release of the stored neurotransmitter is achieved by Ca++-dependent phosphorylation of the cytoskeleton proteins such as synapsin I (Baher and Greengard, 1987), consequently lowering the levels of neurotransmitter discharged from the secretory vesicles. Additionally, Struzyńska and Rafałowska (1994) demonstrated that lead can assault the synaptic neurotransmission in two ways: either by depressing the Ca–KCl-evoked release of GABA, dopamine, and histidine or by a selective stimulation of spontaneous release of GABA and dopamine, however, not histidine. Furthermore, the lead exposure in the present study induced a significant sensorimotor impairment that was confirmed by the inclined plane performance, and the forepaw grip of the exposed animals. These neurobehavioral deficits may reflect dysfunction in multiple anatomical areas in the CNS, peripheral nervous system, or skeletal muscles (Maurissen et al., 2003). In the present study, a marked decline in the grip strength score was observed, which delineates motor neurotoxicity. On the other hand, the cerebellum is an essential structural target related to lead-mediated neurotoxicity (Adhami et al., 1996); therefore, in this regard, the cerebellar damage has been pointed out as a pivotal event related to lead-induced motor deficits (Bortolozzi et al., 1999). Such behavioral alterations are in strong concurrence with our histopathological findings demonstrating the loss of neuronal cells and Purkinje cells, neuronal degeneration, and cerebral edema; all of which

exhibited a significant loss of body weight. This might be a conceivable direct effect of lead on the gastrointestinal tract, resulting in the malabsorption of supplements, or of a diminished food intake as lead influences food satiety signals, causing premature termination of food intake during a meal (Minnema and Hammond, 1994). Nonetheless, certain other studies have demonstrated that exposure to lead has no impact on body weight (Salehi et al., 2015; Wang et al., 2015). Spirulina significantly minimized the recorded weight loss, which might have been a result of providing the body with essential nutrients present in Spirulina, such as high-quality proteins, vitamin, and amino acids (Sharma et al., 2007), and these nutrients might have a beneficial role in restoring the body weight and health. Rats exposed to lead in the present study displayed enhanced locomotors activity, evidenced by higher numbers of ambulation, lower freezing duration, increased number of entries and time spent in the center; however, this behavior was not indicative of lowered anxiety as we also observed an increase in locomotion. As mentioned in the previous reports, only an increase in the central locomotion or an increase in the time spent in the central arena without any modification in total locomotion can be translated as an anxiolytic effect (Prut and Belzung, 2003). Likely, this trend suggests dopaminergic impairment (Nasuti et al., 2007). This concept is anchored to the fact that dopamine is a neurotransmitter involved in a variety of CNS processes, including cognition, motor activity, reward, mood, attention, and learning. Consequently, any changes in its transmission may unfavorably influence a variety of neurological processes and prompt debilitating behavioral disorders (Jones and Miller, 2008). Assuredly, lead has been demonstrated to alter a number of neurotransmitter systems, including the dopamine, norepinephrine, epinephrine, serotonin, and c-aminobutyric acid 261

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Fig. 5. Photomicrograph of HSP70 Immunohistochemistry in the cerebrum (Bar = 200 µm) and cerebellum (Bar = 40 µm) of: Control (A) and Spirulina –administered (B) groups, showing weak immunostaining of few cells. Lead–exposed group (C) showing an intense positive reaction to HSp70 in numerous blood vessels (Arrows). Spirulina/Lead co-treated group (D) showing moderate immunostaining to HSp70 (Dashed arrow). Photomicrograph of Caspase-3 Immunohistochemistry in cereberum and cerebellum (Bar = 40 µm) of: Control (A) and Spirulina– administered (B) groups, showing weak immunostaining of few apoptotic cells. Lead– exposed group (C) showing an intense presence of apoptotic cells positive to the Caspase-3 reaction (Arrows). Spirulina/Lead co-treated group (D) showing moderate immunostaining to Caspase-3(Dashed arrow) (Bar = 100 µm).

such as cadmium, iron, and fluoride (Hutadilok-Towatana et al., 2008; Bermejo-Bescós et al., 2008). Indeed, Spirulina has a rapid lead adsorption rate and high lead adsorption capacity, and it enhances the elimination of heavy metals from the body (Banji et al., 2013). Lead toxicity is believed to be mainly mediated by oxidative stress, through the collection and auto-oxidation of 5-aminolevulinic acid, with the production of a superoxide anion and hydrogen peroxide (Zhao et al., 2007). Lead toxicity likewise includes the alteration of mitochondrial functions through the expansion of the intracellular levels of calcium, and declining the activities of the electron transport chain components, changing the mitochondrial energy metabolism and resulting in a free radical generation (Gleichmann and Mattson, 2011). It is well known that the disruption of the prooxidant/antioxidant balance is the core mechanism through which lead damages various tissues (Chander et al., 2014). In our findings, the damage was clearly demonstrated by the improvement in the formation of lipid peroxidation and protein oxidation products, which was accompanied by depletion in the antioxidant enzymes’ activities, and the GSH levels in the brain tissue of rats upon exposure to lead. Several enzymes in the antioxidant defense systems may secure the imbalance between the prooxidants and antioxidants induced by lead exposure, and most of them become inactive due to the direct binding of lead with their sulfhydryl groups (Rendón-Ramírez et al., 2014), altering their function and suppressing their activities (SOD, CAT, and glutathione) (Ercal et al., 2001). Furthermore,

have been reported previously to have occurred because of the impact of lead on rat brain (Moneim et al., 2011). On the contrary, Ashafaq et al. (2016) demonstrated that lead-treated rats did not exhibit any neuromuscular impairment when tested for the grip test. Lead-induced aberrant behavior was partially reversed with Spirulina, which prevented alterations in the neural organization. Our findings indicated that Spirulina exerted its protective action plausibly by decreasing the concentration of lead in the body, as evidenced by the decline in the elevated levels of lead in the exposed rats, as well as by inhibiting the toxicity of lead. Along these lines, Banji et al. (2013) had suggested that Spirulina obstructed the cytoarchitectural changes in the brain tissues induced by fluoride exposure, and enhanced the availability of thyroid hormones, which in turn might be responsible for the improved behavior. These results are in accordance with the previous study demonstrating behavioral disturbances upon lead exposure and their subsequent reversal through antioxidant treatments (Sansar et al., 2011). In the present study, elevated lead levels were observed in the whole blood and the brain tissue of lead acetate-exposed rats, which diminished upon Spirulina supplementation, indicating that Spirulina could reduce lead levels in both blood and the brain tissue in rats. Kaushal et al. (1996) recorded that nearly 15% of the administered lead dose remained in the body of the rat following the IP injection. Furthermore, the decrease in the lead levels caused by Spirulina administration was mostly related to the chelating capacity of Spirulina for heavy metals, 262

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confirmed by an elevation in the MDA levels in the lead-exposed animals, demonstrating membrane damage. Besides, lead readily crosses the blood-brain barrier (BBB) and directs structural alteration of the BBB components, which further results in morphological and functional changes in the brain architecture (Prasanthi et al., 2005). Morphological perturbations observed in the brain tissue were supported by previous neuropathological data, which revealed pathological changes following the lead exposure (Chander et al., 2014; Bazrgar et al., 2015). Besides, Spirulina's restoration potency in the brain tissue was reliable even in terms of biochemical outcomes. This implies that Spirulina effectively prevented the lead-induced neurotoxic damage in rat. Oxidative stress initiates apoptosis in various cell types. In order to investigate the relationship between oxidative stress and the expression profiles of the apoptotic-mediated pathway, a pro-apoptotic gene product, Caspase–3, was selected for immunohistochemical analysis. The apoptotic process is regulated by the expression of several proteins, including the members of the family of caspases. Our biochemical findings suggest a crosstalk between lead-induced apoptosis and activation of oxidative stress-responsive cell signaling. In fact, DNA damage and DNA strand breaks, have been known to occur during the process of apoptosis, which was clearly detected in the current study. Moreover, the increase in the MDA levels is an indication of lipid peroxidation, which could cause structural damage to mitochondrial membranes and potentiate their dysfunction, activating the release of cytochrome c, prompting the initiation of a variety of caspases (Nicholls and Budd, 2000). Furthermore, a declined Caspase–3 expression level was observed in the Spirulina/Lead co-treated group. These findings suggest that Spirulina could serve as a biologically effective therapeutic agent that can increase cell survival in the rat brains through Caspase–3 down-expression and cell death suppression. In response to cellular stress, the expression of HSPs elevates dramatically. It is notable as a pervasive adaptation mechanism in organisms that enables them to survive and adapt to different environmental stressors. The immunohistochemical data obtained for HSP70, whose expression was increased in the brain tissue in response to the stress generated by lead exposure as depicted by positive staining for HSP70 in the cerebrum and cerebellum, reflected the activation of this intracellular buffer system, which responds to the oxidative stress when the antioxidant enzyme exhaustion occurs (Kalmar and Greensmith, 2009). In conclusion, Spirulina has displayed a protective role against lead acetate-mediated brain damage, probably by scavenging the free radicals and chelating lead, thereby reducing the lead burden in rats. Thus, it is worthwhile using Spirulina as a food supplement in the geographical areas with high lead exposure in order to minimize the risk of neurological alterations. Nevertheless, further investigation is required for elucidating the molecular mechanism through which Spirulina exhibits this protective effect against the subacute toxicity of lead.

Spirulina administration reversed the alterations induced by lead acetate in the antioxidant enzymes, and these findings were similar to those in the previous study (Ponce-Canchihuamán et al., 2010). Such effect was achieved by improving the brain tissue antioxidant capacity mechanisms because of better antioxidant supply, thus reducing the oxidative damage represented by the reduction of MDA and PC formation. Moreover, lead promotes DNA damage in the brain tissue of the exposed rats. Lead has been accounted to be aneugenic, clastogenic, and mutagenic; it induces chromosomal aberrations, DNA damage, and micronuclei and nuclear alterations (Bonacker et al., 2005). In the present study, administration of Spirulina significantly lessened the DNA damage induced by lead exposure. Bhat and Madyastha (2000) revealed that the polysaccharides in Spirulina improved both the repair activity of the damaged DNA excision and the unscheduled DNA synthesis. Similarly, phycocyanin and phycocyanobilin in Spirulina possess a potent anti-cyclooxygenase–2 and antioxidant activity to scavenge peroxynitrite, and therefore, can lessen the OONO–-induced oxidative damage to DNA (Abou-El Fotoh et al., 2013). Besides, the protection against genotoxicity may be explained by the fact that simultaneous treatment with Spirulina would allow interception of the free radicals generated by lead before they can reach DNA and induce genotoxicity. This is confirmed by the data obtained in the present study which demonstrated that Spirulina pretreatment reduced the lead-induced lipid peroxidation and prevented the reduction in antioxidant indices. Spirulina ameliorated the effects of lead on the brain tissue by facilitating the displacement of lead, resulting in reduced lead accumulation in the body, through its potential antioxidant efficacy via its radical-scavenging ability, which was evident from a 2,2-diphenyl-1picrylhydrazyl (DPPH) scavenging and β-carotene linoleic acid assay (Banji et al., 2013). The ability of Spirulina to neutralize the oxidative stress could be attributed to the blend of antioxidants present in it, including phycocyanins, β-carotene, vitamin E and C, and chlorophyll. Additionally, Spirulina contains riboflavin, α-lipoic acid, xanthophyll phytopigments, magnesium, selenium, and manganese, which also exhibit antioxidant potency (Bermejo-Bescós et al., 2008). The promotion of the proinflammatory mediators (NO and TNF-α) and the reduction in the levels of the anti-inflammatory one (IL-10) in the rat brain suggest that lead enhances the inflammatory process. Our results were in line with those obtained by Liu et al. (2012), who observed that lead exposure significantly increased microglia activation, and up-regulated the release of cytokines, including TNF-α, IL-1β, and iNOS, in these cells. Spirulina supplementation evoked anti-inflammatory effects through the reversal of NO and TNF-α generated and the suppressed IL-10 levels because of lead exposure in rats. Such potency may be attributed to c-phycocyanin, which may impact the function of mast cells which are a critical source for the synthesis and release of prostaglandin D2, ROS, leukotrienes, and cytokines, such as TNF-α, which trigger the release of interleukin (IL)–1β, IL–6, as well as block the phosphorylation of p38 mitogen-activated protein kinases (MAPK), which in turn regulate the synthesis of cytokines (Shih et al., 2009). Furthermore, Spirulina diminishes nitrite generation, suppresses inducible nitric oxide synthase expression, and lessens liver microsomal lipid peroxidation (Romay et al., 2003). Also, the constituents present in Spirulina, provide high nutritive value and a potent anti-inflammatory activity other than the antioxidant potency (Alvarenga et al., 2011). As anti-inflammatory activities, phycocyanin inhibits the proinflammatory cytokines, such as TNFα, and also the cyclooxygenase–2 (COX–2) expression (Romay et al., 1998). The biochemical data together with histopathological observations (an altered normal-tissue architecture of brain) obviously showed that lead acetate injured the brain; however, the structure of the brain was restored with the co-treatment with Spirulina, proposing the efficiency of Spirulina to rescue brain from lead-induced damage. These histopathological outcomes suggest that lead induces cell death, which was

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