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Ecotoxicology and Environmental Safety

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Spirulina platensis attenuates the associated neurobehavioral andinflammatory 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, Egyptb Pharmacology Department, Faculty of Veterinary Medicine, Zagazig University, Egyptc Veterinary Public Health Department, Faculty of Veterinary Medicine, Zagazig University, EgyptdHistology and Cytology Department, Faculty of Veterinary Medicine, Zagazig University, Egypte Laboratory of Anatomy, Department of Biomedical Sciences. Graduate school of Veterinary, Hokkaido University, Sapporo, Japan

A R T I C L E I N F O

Keywords:Caspase-3Comet assayHSP70LeadOpen field testSpirulina platensis

A B S T R A C T

Heavy metals are well known as environmental pollutants with hazardous impacts on human and animal healthbecause of their wide industrial usage. In the present study, the role of Spirulina platensis in reversing the oxi-dative stress-mediated brain injury elicited by lead acetate exposure was evaluated. In order to accomplish thisaim, rats were orally administered with 300mg/kg bw Spirulina for 15 d, before and simultaneously with anintraperitoneal injection of 50mg/kg bw lead acetate [6 injections through the two weeks]. As a result, the co-administration 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 wasobserved that Spirulina diminished the lead content that accumulated in both the blood and the brain tissue ofthe exposed rats, and reduced the elevated levels of oxidative damage indices, and brain proinflammatorymarkers. Also, because of the Spirulina administration, the levels of the depleted biomarkers of antioxidantstatus 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 ofHSP70 and Caspase–3 in both cerebrum and cerebellum tissues. Collectively, our findings demonstrate thatSpirulina 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 organ-isms (Vigani et al., 2015). Spirulina is a photosynthetic cyanobacteriumthat is used commercially as a dietary supplement and food additive.Currently, the annual production of Spirulina exceeds 3000 t on a dryweight basis and is acknowledged by several companies in variousnations. The extensive production of Spirulina is owing to its originalchemical composition [proteins, polyunsaturated fatty acids, and vita-mins]. Besides, it is a source of bioactive components, for example,phycocyanin, β-carotene, and allophycocyanin, which possess anti-in-flammatory and antioxidant properties (Wang et al., 2007). Also, it isable to manage numerous diseased conditions, including diabetes andobesity (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 itsideal safety profile in human and animal studies (Karkos et al., 2011).Spirulina contains non-toxic cyanobacteria, but the methods of culti-vation without proper quality controls permit contamination by othertoxin producer species prompting the presence of harmful cyanotoxinswhich have general health concerns. Roy-Lachapelle et al. (2017) de-tected the presence of cyanotoxins in various commercially availableproducts containing Spirulina at levels exceeding the tolerable dailyintake levels, showed the significance of better screening by the ap-propriate authorities of algal-based food supplements like spirulina toguarantee the safety of these products.

The studies conducted in the past using Spirulina platensis as asupplement have proved that it has an ability to encounter the multi-organ toxicity caused by various medications and chemicals (Simseket al., 2009; Banji et al., 2013). Spirulina also reversed the modifiedimmune response and hepatic damage prompted by the herbicideatrazine in the common carp (Khalil et al., 2017, 2018). It has been

https://doi.org/10.1016/j.ecoenv.2018.03.068Received 6 February 2018; Received in revised form 22 March 2018; Accepted 24 March 2018

⁎ Corresponding author at: Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, 44511 Zagazig, Egypt.E-mail address: resamah@zu.edu.eg (S.R. Khalil).

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hypothesized that the modulating potency of Spirulina, which includesthe central nervous system, could be considered a part of the ther-apeutic regimes for neurodegenerative diseases, such as, Alzheimer'sand Parkinsonism (Pabon et al., 2012), nigrostriatal dopamine systeminjury (Strömberg et al., 2005), cerebrovascular accident models(Thaakur and Sravanthi, 2010), and for neurotoxicity by means of freeradical generation to protect the dopaminergic neurotransmission(Tobón-Velasco et al., 2013).

Lead is an environmental contaminant causing occupational healthhazards that can be perceived in the environment and biological sys-tems as it is widely used in industry (El-Nekeety et al., 2009). Bothenvironmental and occupational exposures cause significant and ser-ious medical issues in the industries where lead and lead-based com-ponents are used for the production of ammunition, bearing metals,bronze and brass, cable covering, sheet lead, and solder. Besides, leadcan be used in ceramics, weights or ballast, containers or tubes, oxides,and gasoline additives (Schwartz and Levin, 1991). Although the use oflead as an additive in household paint has ceased, the lead-containingpaint is nonetheless found in buildings built before the 1960s(Bradberry, 2016). Lead can enter the body by inhaling, by ingestingthe contaminated soil and water, and by consuming food. After ab-sorption, lead accumulates in several tissues, such as kidney, liver,brain, and bones, and impacts various biological activities at the mo-lecular, cellular, and intracellular levels, which may result in morpho-logical perturbations that cannot be reversed even after the levels oflead have diminished in the body (Flora et al., 2006).

Studies on both animals and humans have demonstrated lead-in-duced deleterious effects in hepatic tissue (Khalil et al., 2018), cardi-ovascular, 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 nervoussystem, including hindered cognition and memory, as well as debili-tated peripheral nerve functions (White et al., 2007). Despite the var-ious studies dealing with lead toxicity, the exact mechanisms throughwhich lead causes the neurotoxic effects are not yet fully understood.Oxidative stress, membrane alterations, cell signaling deregulation, andneurotransmission impairment are viewed as key perspectives involvedin lead neurotoxicity (Sanders et al., 2009). Moreover, certain in vitroand in vivo studies demonstrated that lead could give rise to neural cellapoptosis (Baranowska-Bosiacka et al., 2013; Sharifi et al., 2010).

Considering the involvement of oxidative stress in lead-inducedneurotoxicity (Chander et al., 2014), it is reasonable that the admin-istration of an antioxidant may be an imperative therapeutic approachto deal with lead toxicity. Stemmed from this notion, the present studywas undertaken to explore the putative protective potency of Spirulinaagainst the lead-induced neurotoxicity by addressing the behavioralresponses, probing into oxidative stress, tissue inflammatory reactions,and cellular death, and seeking a possible link with histopathologicalchanges in the brain tissue.

2. Material and methods

2.1. Test compounds and chemicals

Lead acetate (99.6% purity) was purchased from El-NasrPharmaceutical Chemical Co. ( Egypt). Spirulina platensis, as a blue-green powder, was obtained from EL-Hellow for Biological Productssupplier (Egypt). The Spirulina powder was subjected to heavy metalsanalysis like lead, mercury, cadmium, and arsenic by using flameatomic absorption spectrophotometer, these metals are the most likelyto contaminate Spirulina. Heavy metal contents in the tested Spirulinasample were all within the permissible levels (0.29, 0.048, 0.09 and0.037 ppm, respectively). Therefore, the tested Spirulina sample was

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

2.2. Animals and experimental design

Forty male Sprague–Dawley rats (150–200 g) obtained from thelaboratory animals farm of Faculty of Veterinary Medicine (ZagazigUniversity, Egypt), were used in the present study. The animals weremaintained in clean stainless steel cages under controlled environ-mental conditions. The animals had free access to water and feedthroughout the experimental period, and they were accommodated tothe laboratory conditions for two weeks before being used in the ex-periments. The experiments were performed following the guidelines ofthe National Institutes of Health (NIH) for the Care and Use ofLaboratory Animals in scientific investigations and approved by theEthics 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 in-volved rats that were intraperitoneally (IP) injected with distilled wateras a vehicle. Spirulina-administered animals received Spirulina orally ata dose of 300mg/kg bw for 30 days via gastric tube (Simsek et al.,2009). Lead acetate-exposed animals were injected (IP) with leadacetate at a dose of 50mg/kg bw (three times a week) for two weeks(Ahmed et al., 2013). The animals in the Spirulina/Lead acetate co-treated group were orally administered with Spirulina for 15 days be-fore and 15 days simultaneously with an IP injection of lead acetate atthe same course as previously described. On the day of sacrifice, thefinal body weight of each rat was recorded by weighing each animal inall the groups for estimating the body weight changes.

2.3. Behavioral response evaluation

In order to evaluate the behavioral changes provoked by leadacetate exposure and/or Spirulina administration, the rats were trans-ferred to the testing room in their home cages and kept there for about30min prior to testing to accommodate them to the testing-room con-ditions. All behavioral tests were done in the same testing room on thelast day of the experimental period. The experimenter was blind to/unaware of the identity of the animal groups. Behavioral analyses, in-cluding the open field test, swimming performance, grip strength, andinclined plane, were performed with the different experimental groupsas described ahead.

2.3.1. Open field testThe rats were set individually in the center of the open field arena

and behavioral parameters were recorded for 5min, beginning 2minafter setting the animal in the test cage. The open field apparatus wasthen thoroughly cleaned with 5% ethanol before placing the next rat, toeliminate the possible cueing effects of the odors left by previous ani-mals. The behavioral indices observed in this test were ambulation orlocomotion frequency (the number of floor sections entered with twofeet), rearing frequency (the number of times the animal stood on itshind legs), stereotype counts (the number of grooming movements),and immobility (freezing) duration (total time in second withoutspontaneous movements). The open field contained a central square orarea that was considered an unprotected area for rats; thus, the numberof entries into this area reflected the level of anxiety-like behavior(Contó et al., 2005). The observations were recorded between 10:00and 12:00 a.m.

2.3.2. Swimming performance testSwimming performance test was carried out by putting each animal

at the middle of the glass aquarium for observation for 5–10 s, wherethe swimming performance was scored according to the position of noseand head on the surface of water as follows: 0 F, head and nose below

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the water surface; 1, nose below the water surface; 2, nose and top ofthe 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 thebottom of the ears (Schapiro et al., 1970).

2.3.3. Inclined planeRats 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). Theinclined plane performance was measured with a standard protractor tothe nearest 5 degrees. The trial ceased when the rat started to slipbackward. Hence, there was no particular trial duration. The angle atwhich the rat began to slip downward was recorded. The results of twotrials were averaged for each rat. Both the trials were separated by aone-hour interval.

2.3.4. Grip strength testThe rat's forepaw strength was monitored by having it to hold a

wood dowel (5 mm diameter) that was held horizontally and raised sothat the animal supported its body weight, as described by Andersenet al. (1991). Time taken to release the grip was recorded in seconds. Allrats attempted to grip the dowel during this grip strength testing. Theresults of two trials separated by a one-hour interval were averaged foreach rat.

2.4. Blood and brain tissue collection

At the time of sacrifice, whole blood samples were collected fromthe medial canthus (orbital vessels) of the experimental rats in heparin-containing tubes for evaluating the levels of lead. Brain tissue speci-mens were obtained, dissected, rinsed with sterile physiological saline(0.9% NaCl), weighed, and then divided into four sets. The first one washomogenized and the homogenates were centrifuged at 664×g at 4 °Cfor 15min to obtain the supernatants, which were then used for de-termining the antioxidant, oxidative stress and inflammatory indices.The second one was used to determine the levels of lead. The third onewas used to prepare the cells for DNA-damage investigation (cometassay), by setting it in 1mL of cold Hank's balanced salt solution con-taining 20mM ethylenediaminetetraacetic acid (EDTA) and 10% di-methyl sulfoxide (DMSO), and then mincing it into fine pieces to ac-quire cell suspensions. The final set was fixed in 10% neutral bufferedformalin for histopathological and immunohistochemical analysis.

2.5. Assessment of lead levels in blood and brain tissue

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

2.6. Evaluation of antioxidant biomarkers in brain tissue

The superoxide dismutase (SOD and catalase CAT) activities and thelevels of reduced glutathione (GSH) were estimated according tomethods described previously (Misra and Fridovich, 1972; Sinha, 1972;Beutler, 1963).

2.7. Evaluation of oxidative stress biomarkers in brain tissue

Lipid peroxides (MDA) were measured as described by Ohkawaet al. (1979). Protein carbonyl was assessed using a colorimetric assaykit (Cayman's Chemical Company, Ann Arbor, USA) according to Levine

et al. (1994). The comet assay was conducted according to the methoddescribed by Singh et al. (1988). Fifty randomly selected cells per slidewere 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 visualscoring method and the computer image analysis was performed usingComet Assay Software Project.

2.8. Evaluation of inflammatory markers in brain tissue

Rat ELISA kits were used for the detection of inflammatory markersin the brain tissue–nitric oxide, NO; tumor necrosis factor-α, TNF-α;interleukin–10, IL-10. The kits were obtained from MyBioSource (SanDiego, California, USA) (Catalog No.: MBS010567, MBS704859, andMBS269138 for NO, TNF-α, and IL-10, respectively). The quantitativedetection of such indices was done following manufacturer's instruc-tions.

2.9. Histopathological study

2.9.1. Light microscopyBrain specimens (cerebrum and cerebellum) from the control and

treated groups were processed for histopathological investigation usinga light microscope (Olympus BX51 microscope, Olympus Inc., Japan)according to Bancroft and Gamble (2008). This process involved sec-tioning the tissues, staining with hematoxylin/eosin (H&E) dye, andviewing under the light microscope.

2.9.2. Immunohistochemical analysisBrain tissue (cerebrum and cerebellum) sections were processed for

immunohistochemical analysis and incubated overnight at 4 °C with arabbit polyclonal anti-caspase (Caspase–3) primary antibody (1:100) oranti-heat shock protein 70 (HSP70) primary antibody (1:100) in phos-phate-buffered saline (PBS). After three thorough washes with PBS,they were incubated with a goat anti-rabbit IgG biotin-conjugatedsecondary antibody (1:2000) for 20min at 32 °C. After further in-cubation with horseradish peroxidase-labeled streptavidin, antibodybinding was visualized using 3,3′-diaminobenzidine, and the sectionswere counterstained with hematoxylin.

2.10. Data analysis

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

3. Results

3.1. General observations, body weight changes, and brain/body weightratios of rats in response to lead acetate exposure and Spirulinaadministration

During the experiments, neither deaths nor any clinical symptomswere recorded in the groups. However, anorexia and low activity wereobserved, mainly in the lead-exposed group. Lead-exposed rats showeda significant decline in the body weight changes relative to the controlgain. Spirulina administration induced an improvement in rats bylowering the decrease in the body-weight gain in Spirulina/Lead co-treated group. On the other hand, there was no significant difference inthe relative brain weight (brain somatic index) among all the experi-mental groups (Table 1).

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3.2. Behavioral responses in response to lead acetate exposure and Spirulinaadministration

3.2.1. Open field testThere was a significant increase in the locomotors activity re-

presented by a significant increment in ambulation, which was asso-ciated with a lowering of freezing time in rats after exposure to leadacetate, compared with control rats. Spirulina co-administration withlead exhibited a significant modulation in freezing time; however, it didnot affect ambulation behavior. Rearing and grooming behaviors didnot show any significant changes as a result of lead exposure.Meanwhile, these rats exhibited more entries and time spent in thecentral arena than the control rats. Spirulina supplementation sig-nificantly maintained the rats' behavior in the central arena in the co-treated group, compared to the lead-exposed rats (Fig. 1).

3.2.2. Grip strength, inclined plane, and swimming performance testsThe data for swimming performance, inclined plane, and grip time

in the rats exposed to lead displayed significant deficits in comparisonto the corresponding data recorded in the control animals, indicatingthat the rats treated with lead experienced sensorimotor deficits. InSpirulina/Lead co-treated animals, Spirulina was unable to modulatethese deficits, which were observed to be non-significantly different incomparison with the lead-exposed animals (Fig. 2).

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

Lead acetate exposure significantly increased the levels of lead inblood and the brain tissue, with greater lead accumulation occurring inthe brain tissue when compared with the levels recorded in the controlgroup. The supplementation of Spirulina with lead significantly de-creased its levels in blood and the brain tissue, relative to the lead-exposed group. In comparison with the control level, it exhibited non-significant differences in the blood lead levels; whereas, in the braintissue, it exhibited significantly higher lead levels than the control(Table 1).

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

Lead exposure significantly decreased the SOD, CAT activity, andGSH level in the brain tissue, compared to control group. On the otherhand, co-administration of Spirulina with lead enhanced the SOD andCAT activity and increased the GSH level. Such improvement was sig-nificant in Spirulina/Lead co-treated group in comparison with thevalues recorded in the lead-exposed group, although it did not attainthe control value (Table 2).

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

Parameters Experimental groups

Control Spirulina Lead acetate Spirulina/Lead acetate

Body weight change (gm) 22.66 ± 1.76a 22.00 ± 1.80a −10.16 ± 2.61c 15.00 ± 1.46b

Relative brain weight (%) 0.87 ± 0.022 0.94 ± 0.015 0.91 ± 0.015 0.95 ± 0.028Blood lead level (ppm) 0.11 ± 0.005b 0.05 ± 0.01b 0.54 ± 0.02a 0.09 ± 0.012b

Brain lead level (ppm) 0.23 ± 0.021c 0.20 ± 0.035c 0.89 ± 0.043a 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).

Fig. 1. Effect of pre and simultaneous Spirulina supplementation (300mg/kg bw for 30 days, orally) on open field behavior (ambulation, freezing time, rearingfrequency, grooming frequency, central arena entrance frequency and time spent) in rat exposed to Lead acetate (50mg/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).

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3.5. MDA, PC, and DNA damage levels in the brain tissue of rats in responseto lead acetate exposure and Spirulina administration

MDA (a lipid peroxidation product) levels were significantly ele-vated in the brain tissue of lead-exposed rats, compared to the controlgroup. Co-supplementation of Spirulina with lead significantly de-creased the MDA levels compared to the lead-exposed group. Themodulation recorded in the Spirulina/Lead co-treated group depicted atrend toward normal value. However, it did not match the values ofcontrol. Brain protein carbonyl (a protein oxidative damage product)level was significantly increased in the lead-exposed group, comparedto the control group. Nevertheless, the Spirulina/Lead group exhibiteda significant reduction in the PC level, compared to the level recordedin the lead-exposed group; this level was, however, non-significantlydifferent from the value recorded in the control rats, indicating thatSpirulina co-treatment significantly prevented carbonyl formation inthis group (Table 2).

The findings of DNA damage revealed a significant elevation in thetail length, tail DNA %, and tail moment in the brain tissues of the lead-exposed animals, relative to controls. Spirulina supplementation alonedisplayed a non-significant decline in the level of potential DNA da-mage. The modulatory potency of Spirulina was confirmed by a sig-nificant 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 tolead acetate exposure and Spirulina administration

An exposure to lead resulted in a significant increase in NO andTNF-α levels in the brain tissue of lead-exposed group. Although theseindices exhibited a significant decrease in the Spirulina/Lead co-treatedgroup compared to the lead-exposed group, the level of NO wasnonetheless significantly different from the recorded in the control, andthe TNF-α level was non significantly different than of the controlgroup.

Spirulina supplementation was observed to significantly elevate thelevels of interleukin–10 in the brain tissue when compared with controlgroup. Lead exposure induced a significant decrease in the IL-10 levelscompared to the control values. This decrease was modulated, as itexhibited a non-significant difference compared to the control values,in the Lead/Spirulina co-administered group (Table 2).

3.7. Histopathological and immunohistochemical findings

Histopathological observations of the H&E-stained paraffin-em-bedded brain sections revealed normal meninges and meningeal bloodvessels in both control and Spirulina-administered group. In the lead-exposed group, congestion and hemorrhage were observed in the me-ningeal blood vessels. In the Spirulina/Lead co-treated group,

Fig. 2. Effect of pre and simultaneous Spirulina supplementation (300mg/kgbw for 30 days, orally) on grip strength time, inclined plane test and swimmingperformance test in rat exposed to Lead acetate (50mg/kg bw, every other dayfor two weeks, IP). Columns carrying different superscripts (a, b,c) are sig-nificantly different (P < 0.05) (mean ± SE, n= 6).

Table 2Effect of pre and simultaneous Spirulina supplementation (300mg/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 (50mg/kg bw, every other day for two weeks, IP).

Parameters Experimental groups

Control Spirulina Lead acetate Spirulina/Lead acetate

SOD activity (U/gm) 395.23 ± 14.27a 427.0 ± 15.71a 235.42 ± 9.30c 299.17 ± 8.58b

CAT activity (U/gm) 0.80 ± 0.03a 0.77 ± 0.02a 0.46 ± 0.03c 0.62 ± 0.02b

GSH level (mmol/gm) 2.51 ± 0.14a 2.66 ± 0.15a 1.41 ± 0.05c 1.80 ± 0.05b

MDA (nmol/gm) 5.38 ± 0.25a 5.20 ± 0.32a 10.85 ± 0.44c 6.94 ± 0.32b

PC (nmol/gm) 0.134 ± 0.02b 0.112 ± 0.02b 0.30 ± 0.03a 0.19 ± 0.01b

NO (µmol/gm) 8.64 ± 0.42c 8.44 ± 0.43c 18.70 ± 0.39a 13.21 ± 0.11b

TNF-α (Pg/gm) 0.19 ± 0.01bc 0.13 ± 0.02c 1.34 ± 0.10a 0.34 ± 0.02b

IL-10 (Pg/gm) 0.17 ± 0.01 b 0.29 ± 0.01a 0.086 ± 0.01c 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).

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meningeal edema and congestion of blood vessels were observed in theregion between cerebellum and cerebrum. Moreover, the choroidplexus exhibited normal structure in both control and Spirulina-ad-ministered group. In the lead-exposed group, the congestion of thechoroidal blood capillaries and proliferation of the choroidal epitheliallining were prominent. In the Spirulina/Lead co-treated group, only thecongestion of choroidal blood capillaries was observed. The cerebellumin both control and Spirulina-administered group displayed normallyarranged cerebellar cortical layers, including the outer molecular, theinner granular, and the middle layer of pear-shaped Purkinje cells thataligned regularly in-between the former layers. In the lead-exposedgroup, the middle Purkinje-cells layers displayed a fewer number ofintact Purkinje cells that exhibited disorganized alignment, along withthe presence of a few necrotic Purkinje cells and perineural edema. Inthe Spirulina/Lead co-treated group, the Purkinje cells exhibited se-verely disorganized alignment (Fig. 4).

Immunohistochemical analysis for the detection of heat shock pro-tein (HSP) 70 in cerebrum and cerebellum revealed numerous bloodvessels with the positive reaction to HSP70 in the Spirulina/Lead co-treated and lead-exposed groups, compared with the control andSpirulina-administered groups. Additionally, the immunohistochemical

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

4. Discussion

The naturally occurring sources of antioxidants have been used invarious in vitro and in vivo studies, and have demonstrated promisingoutcomes 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 mod-ulating the neurotoxic alterations induced by subacute exposure to leadacetate. The main outcome of this study was that an IP injection of leadacetate resulted in a significant oxidative stress-mediated damage andbrain injury, which was inferred from the altered behavioral, bio-chemical, histopathological, and immunohistochemical indices underinvestigation. Besides, the supplementation of Spirulina provided aneuroprotective 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

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 andSpirulina-administered groups, cells with a tail (DNA fragments) in Lead-exposed and Spirulina/Lead co-treated groups. B. Effect of pre and simultaneous Spirulinasupplementation (300mg/kg bw for 30 days, orally) on DNA damage indices (Comet assay) in the brain of rat exposed to Lead acetate (50mg/kg bw, every other dayfor two weeks, IP). Columns carrying different superscripts (a, b,c) are significantly different (P < 0.05) (mean ± SE, n= 6).

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exhibited a significant loss of body weight. This might be a conceivabledirect effect of lead on the gastrointestinal tract, resulting in the ma-labsorption of supplements, or of a diminished food intake as lead in-fluences food satiety signals, causing premature termination of foodintake during a meal (Minnema and Hammond, 1994). Nonetheless,certain other studies have demonstrated that exposure to lead has noimpact on body weight (Salehi et al., 2015; Wang et al., 2015). Spir-ulina significantly minimized the recorded weight loss, which mighthave been a result of providing the body with essential nutrients pre-sent in Spirulina, such as high-quality proteins, vitamin, and aminoacids (Sharma et al., 2007), and these nutrients might have a beneficialrole in restoring the body weight and health.

Rats exposed to lead in the present study displayed enhanced lo-comotors activity, evidenced by higher numbers of ambulation, lowerfreezing duration, increased number of entries and time spent in thecenter; however, this behavior was not indicative of lowered anxiety aswe also observed an increase in locomotion. As mentioned in the pre-vious reports, only an increase in the central locomotion or an increasein the time spent in the central arena without any modification in totallocomotion can be translated as an anxiolytic effect (Prut and Belzung,2003). Likely, this trend suggests dopaminergic impairment (Nasutiet al., 2007).

This concept is anchored to the fact that dopamine is a neuro-transmitter 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 ofneurological processes and prompt debilitating behavioral disorders(Jones and Miller, 2008). Assuredly, lead has been demonstrated toalter a number of neurotransmitter systems, including the dopamine,norepinephrine, epinephrine, serotonin, and c-aminobutyric acid

systems. Lead-induced consequences on the dopaminergic system in-corporate changes in the synthesis, turnover, and reuptake of dopamineand 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 fromthe presynaptic nerve endings as it mimics the action of Ca++. Therelease of the stored neurotransmitter is achieved by Ca++-dependentphosphorylation of the cytoskeleton proteins such as synapsin I (Baherand Greengard, 1987), consequently lowering the levels of neuro-transmitter discharged from the secretory vesicles. Additionally,Struzyńska and Rafałowska (1994) demonstrated that lead can assaultthe synaptic neurotransmission in two ways: either by depressing theCa–KCl-evoked release of GABA, dopamine, and histidine or by a se-lective stimulation of spontaneous release of GABA and dopamine,however, not histidine.

Furthermore, the lead exposure in the present study induced a sig-nificant sensorimotor impairment that was confirmed by the inclinedplane performance, and the forepaw grip of the exposed animals. Theseneurobehavioral deficits may reflect dysfunction in multiple anatomicalareas in the CNS, peripheral nervous system, or skeletal muscles(Maurissen et al., 2003). In the present study, a marked decline in thegrip strength score was observed, which delineates motor neurotoxicity.On the other hand, the cerebellum is an essential structural target re-lated to lead-mediated neurotoxicity (Adhami et al., 1996); therefore,in this regard, the cerebellar damage has been pointed out as a pivotalevent related to lead-induced motor deficits (Bortolozzi et al., 1999).Such behavioral alterations are in strong concurrence with our histo-pathological findings demonstrating the loss of neuronal cells andPurkinje cells, neuronal degeneration, and cerebral edema; all of which

Fig. 4. Photomicrograph of brain tissue of: Control (A) and Spirulina-administered (B) groups showing normal histological structured normal meninges and me-ningeal 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 pear-shaped purkinjie cells that aligned regularly in-between the former layers. Lead-exposed group (C) showing less number of intact middle purkinje cell layers thatshowing 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).

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have been reported previously to have occurred because of the impactof lead on rat brain (Moneim et al., 2011). On the contrary, Ashafaqet al. (2016) demonstrated that lead-treated rats did not exhibit anyneuromuscular impairment when tested for the grip test.

Lead-induced aberrant behavior was partially reversed withSpirulina, which prevented alterations in the neural organization. Ourfindings indicated that Spirulina exerted its protective action plausiblyby decreasing the concentration of lead in the body, as evidenced by thedecline in the elevated levels of lead in the exposed rats, as well as byinhibiting the toxicity of lead. Along these lines, Banji et al. (2013) hadsuggested that Spirulina obstructed the cytoarchitectural changes in thebrain tissues induced by fluoride exposure, and enhanced the avail-ability of thyroid hormones, which in turn might be responsible for theimproved behavior. These results are in accordance with the previousstudy demonstrating behavioral disturbances upon lead exposure andtheir subsequent reversal through antioxidant treatments (Sansar et al.,2011).

In the present study, elevated lead levels were observed in the wholeblood and the brain tissue of lead acetate-exposed rats, which dimin-ished upon Spirulina supplementation, indicating that Spirulina couldreduce lead levels in both blood and the brain tissue in rats. Kaushalet al. (1996) recorded that nearly 15% of the administered lead doseremained in the body of the rat following the IP injection. Furthermore,the decrease in the lead levels caused by Spirulina administration wasmostly related to the chelating capacity of Spirulina for heavy metals,

such as cadmium, iron, and fluoride (Hutadilok-Towatana et al., 2008;Bermejo-Bescós et al., 2008). Indeed, Spirulina has a rapid lead ad-sorption rate and high lead adsorption capacity, and it enhances theelimination 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 ofmitochondrial functions through the expansion of the intracellular le-vels of calcium, and declining the activities of the electron transportchain components, changing the mitochondrial energy metabolism andresulting in a free radical generation (Gleichmann and Mattson, 2011).It is well known that the disruption of the prooxidant/antioxidantbalance is the core mechanism through which lead damages varioustissues (Chander et al., 2014).

In our findings, the damage was clearly demonstrated by the im-provement in the formation of lipid peroxidation and protein oxidationproducts, which was accompanied by depletion in the antioxidant en-zymes’ activities, and the GSH levels in the brain tissue of rats uponexposure to lead. Several enzymes in the antioxidant defense systemsmay secure the imbalance between the prooxidants and antioxidantsinduced by lead exposure, and most of them become inactive due to thedirect binding of lead with their sulfhydryl groups (Rendón-Ramírezet al., 2014), altering their function and suppressing their activities(SOD, CAT, and glutathione) (Ercal et al., 2001). Furthermore,

Fig. 5. Photomicrograph of HSP70 Immunohistochemistry in the cerebrum (Bar = 200 µm) and cerebellum (Bar = 40 µm) of: Control (A) and Spirulina –ad-ministered (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 Immunohistochemistryin 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 moderateimmunostaining to Caspase-3(Dashed arrow) (Bar = 100 µm).

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Spirulina administration reversed the alterations induced by leadacetate in the antioxidant enzymes, and these findings were similar tothose in the previous study (Ponce-Canchihuamán et al., 2010). Sucheffect was achieved by improving the brain tissue antioxidant capacitymechanisms because of better antioxidant supply, thus reducing theoxidative damage represented by the reduction of MDA and PC for-mation.

Moreover, lead promotes DNA damage in the brain tissue of theexposed rats. Lead has been accounted to be aneugenic, clastogenic,and mutagenic; it induces chromosomal aberrations, DNA damage, andmicronuclei and nuclear alterations (Bonacker et al., 2005). In thepresent study, administration of Spirulina significantly lessened theDNA damage induced by lead exposure. Bhat and Madyastha (2000)revealed that the polysaccharides in Spirulina improved both the repairactivity of the damaged DNA excision and the unscheduled DNAsynthesis. Similarly, phycocyanin and phycocyanobilin in Spirulinapossess a potent anti-cyclooxygenase–2 and antioxidant activity toscavenge peroxynitrite, and therefore, can lessen the OONO–-inducedoxidative damage to DNA (Abou-El Fotoh et al., 2013). Besides, theprotection against genotoxicity may be explained by the fact that si-multaneous treatment with Spirulina would allow interception of thefree radicals generated by lead before they can reach DNA and inducegenotoxicity. This is confirmed by the data obtained in the presentstudy which demonstrated that Spirulina pretreatment reduced thelead-induced lipid peroxidation and prevented the reduction in anti-oxidant indices.

Spirulina ameliorated the effects of lead on the brain tissue by fa-cilitating the displacement of lead, resulting in reduced lead accumu-lation in the body, through its potential antioxidant efficacy via itsradical-scavenging ability, which was evident from a 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and β-carotene linoleic acid assay(Banji et al., 2013). The ability of Spirulina to neutralize the oxidativestress 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, xanthophyllphytopigments, magnesium, selenium, and manganese, which also ex-hibit 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) inthe rat brain suggest that lead enhances the inflammatory process. Ourresults were in line with those obtained by Liu et al. (2012), who ob-served that lead exposure significantly increased microglia activation,and up-regulated the release of cytokines, including TNF-α, IL-1β, andiNOS, in these cells. Spirulina supplementation evoked anti-in-flammatory effects through the reversal of NO and TNF-α generatedand the suppressed IL-10 levels because of lead exposure in rats. Suchpotency may be attributed to c-phycocyanin, which may impact thefunction of mast cells which are a critical source for the synthesis andrelease of prostaglandin D2, ROS, leukotrienes, and cytokines, such asTNF-α, which trigger the release of interleukin (IL)–1β, IL–6, as well asblock 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, suppressesinducible nitric oxide synthase expression, and lessens liver microsomallipid peroxidation (Romay et al., 2003). Also, the constituents presentin Spirulina, provide high nutritive value and a potent anti-in-flammatory activity other than the antioxidant potency (Alvarengaet al., 2011). As anti-inflammatory activities, phycocyanin inhibits theproinflammatory cytokines, such as TNFα, and also the cycloox-ygenase–2 (COX–2) expression (Romay et al., 1998).

The biochemical data together with histopathological observations(an altered normal-tissue architecture of brain) obviously showed thatlead acetate injured the brain; however, the structure of the brain wasrestored with the co-treatment with Spirulina, proposing the efficiencyof Spirulina to rescue brain from lead-induced damage. These histo-pathological outcomes suggest that lead induces cell death, which was

confirmed by an elevation in the MDA levels in the lead-exposed ani-mals, demonstrating membrane damage. Besides, lead readily crossesthe blood-brain barrier (BBB) and directs structural alteration of theBBB components, which further results in morphological and functionalchanges in the brain architecture (Prasanthi et al., 2005). Morpholo-gical perturbations observed in the brain tissue were supported byprevious neuropathological data, which revealed pathological changesfollowing the lead exposure (Chander et al., 2014; Bazrgar et al., 2015).Besides, Spirulina's restoration potency in the brain tissue was reliableeven in terms of biochemical outcomes. This implies that Spirulina ef-fectively prevented the lead-induced neurotoxic damage in rat.

Oxidative stress initiates apoptosis in various cell types. In order toinvestigate the relationship between oxidative stress and the expressionprofiles of the apoptotic-mediated pathway, a pro-apoptotic gene pro-duct, Caspase–3, was selected for immunohistochemical analysis. Theapoptotic process is regulated by the expression of several proteins,including the members of the family of caspases. Our biochemicalfindings suggest a crosstalk between lead-induced apoptosis and acti-vation of oxidative stress-responsive cell signaling. In fact, DNA damageand DNA strand breaks, have been known to occur during the process ofapoptosis, 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 andpotentiate 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 ob-served in the Spirulina/Lead co-treated group. These findings suggestthat Spirulina could serve as a biologically effective therapeutic agentthat can increase cell survival in the rat brains through Caspase–3down-expression and cell death suppression.

In response to cellular stress, the expression of HSPs elevates dra-matically. It is notable as a pervasive adaptation mechanism in organ-isms that enables them to survive and adapt to different environmentalstressors. The immunohistochemical data obtained for HSP70, whoseexpression was increased in the brain tissue in response to the stressgenerated by lead exposure as depicted by positive staining for HSP70in the cerebrum and cerebellum, reflected the activation of this in-tracellular buffer system, which responds to the oxidative stress whenthe antioxidant enzyme exhaustion occurs (Kalmar and Greensmith,2009).

In conclusion, Spirulina has displayed a protective role against leadacetate-mediated brain damage, probably by scavenging the free radi-cals and chelating lead, thereby reducing the lead burden in rats. Thus,it is worthwhile using Spirulina as a food supplement in the geo-graphical areas with high lead exposure in order to minimize the risk ofneurological alterations. Nevertheless, further investigation is requiredfor elucidating the molecular mechanism through which Spirulina ex-hibits this protective effect against the subacute toxicity of lead.

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