Introduction

Schistosomiasis is an important parasitic disease caused by trematode worms of the genus Schistosoma. This condition is characterized by high rates of morbidity, cognitive impairment, and incapacitation of affected individuals (Richter 2003). However, Schistosomiasis continues to spread in many parts of the world, especially due to lack of sanitation and irrigation projects (Steinmann et al. 2006). It is estimated that, each year, schistosomiasis afflicts up to 600 million people in 74 tropical and sub-tropical countries, predominantly in the developing world (El Ridi et al. 2014). Among the five different schistosome species, Schistosoma mansoni is the most abundant one in Egypt (Helmy et al. 2009).

Surprisingly, S. mansoni eggs and granulomas were identified in the central nervous system of symptomatic animals (Lambertucci et al. 2014). Central nervous system involvement can occur during acute primary infections (Chitsulo et al. 2000; Amaral et al. 2006). Neurological complications generally occur during chronic hepatointestinal schistosomiasis (Lambertucci 2010). Most pathology in schistosome-infected animals is attributed to the host’s reaction to the eggs which is maximal by the eight week of infection.

In spite of remarkable chemotherapeutic progress and the existence of highly effective molecules such as the acylated quinoline–pyrazine praziquantel (PZQ), there is a spreading of schistosomiasis into new areas. It is generally agreed that chemotherapy, although the mainstay of current schistosomiasis control programs, does have some limitations. In addition, there is increasing concern about parasite resistance development (McManus and Loukas 2008).

Nanotechnology holds promise for medication and nutrition, because materials at the nanometer dimension exhibit novel properties different from those of both isolated atoms and bulk material.

Gold and silver nanoparticles have received special attention because they have found potential application in many fields of chemistry, physics, and biology because they possess an excellent biocompatibility and low toxicity and also because of their unique optical, electrical, and photothermal properties (Perez 2010; Isaac et al. 2013).

Recent interest in using gold nanoparticles (GNPs) in medicine has altered the methods of diagnosis and treatment (Chen et al. 2008; Peng et al. 2012). However, gold has a long history of use in the western world as nervine, a substance that could revitalize people suffering from nervous conditions. In addition, in the sixteenth century, it was recommended for the treatment of epilepsy (Richards et al. 2002).

GNPs have attracted great attention due to their unique electronic, optical, thermal, chemical, and biological properties and their potential catalytic applications in various fields such as biology, medicine, physics, chemistry, material science, and other interdisciplinary fields (Panyala et al. 2009). Although physicochemical properties of nanoparticles are well studied, their biological properties largely remain unexplored (Saritha et al. 2014). So, the aim of this study is to investigate the anti-schistosomal effect of GNPs on the neurotoxicity induced by S. mansoni in mice.

Materials and methods

Animals

Seventy-two male Swiss albino mice were bred under specified pathogen-free conditions and fed a standard diet and water ad libitum. Mice ages were 9 to 11 weeks and were approved by state authorities and followed Egyptian rules for animal protection.

Gold nanoparticles

GNPs have been prepared by chemical reduction method as reported by Turkevich et al. (1951). A solution of HAuCl4 has been used as Au3+ ion precursor, while sodium citrate has been used as both of mild reducing and stabilizing agent. The color of the solution slowly turned into faint pink color, indicating the reduction of the Au3+ ions to Au nanoparticles.

Characterization

Size and morphology of GNPs were done by using transmission electron microscopy (TEM) equipped with high-resolution transmission electron microscope (HR-TEM). Samples for TEM were prepared using the colloidal solution of nanoparticles. The colloidal sample solution was sonicated for 10 min in a bath sonicator before the observation and dipped in carbon-coated copper grid (400 mesh) and dried at room temperature for the morphological analysis. A TEM picture was taken by a JOEL JEM 2000 EX 200 microscope at 200 kV.

Infection

S. mansoni cercariae were obtained from Schistosome Biological Supply Center at Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. Mice were exposed to 100 ± 10 S. mansoni cercariae per mouse by subcutaneous injection method modified by Oliver and Stirewalt (1952).

Experimental design

Animals were divided into six groups of 12 mice per group. Group I served as a control (noninfected control group). Each mouse of this group received 100 μl saline via intrapretonial injection (i.p.) for 10 days. Groups II, III, IV, V, and VI were infected with 100 ± 10 S. mansoni cercariae (Oliver and Stirewalt 1952). On day 46 postinfection (p.i.) with S. mansoni, the animals of groups III, IV, and V were intraperitonially injected with 100 μl of 0.25, 0.5, and 1.0 mg/kg GNPs, respectively, twice per week (on days 46 and day 49 p.i.). Finally, infected animals of group VI were orally administered 100 μl of PZQ (600 mg/kg body weight) on day 46 p.i. at an interval of 24 h for 2 days.

Brain tissue preparation

The animals of all groups were cervically decapitated, on day 56 p.i. with S. mansoni. Brains were rapidly excised from the skulls, blotted, and chilled. Brain tissue was divided into two halves. The first brain half of six mice were used for neurotransmitter estimation, and the second brain half of six mice were investigated for biochemical studies. The brain tissues were weighed, wrapped in plastic films, and quickly stored at −80 °C until used.

For investigating the brain histology, six half brains were fixed in 10 % neutral buffered formalin and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin. For the gene expression analysis, the six half brains were stored in RNAlater (Sigma-Aldrich).

Estimation of neurotransmitter contents

The brain norepinephrine (NE) and dopamine (DA) contents were estimated according to the method of Ciarlone (1978). For NE, flourimeter excitation was adjusted to 300 nm and emission at 480 nm. For DA, the excitation was at 320 nm and emission at 375 nm.

Biochemical studies

Brain tissues were homogenized in ice-cold medium containing 50 mM Tris–HCl and 300 mM sucrose, pH 7.4 (Tsakiris et al. 2004). This homogenate was used for biochemical determinations.

Glutathione

Glutathione (GSH) level in the brain was determined by the method of Ellman (1959). The method based on the reduction of Ellman’s reagent with GSH to produce a yellow compound; the reduced chromogen directly proportional to GSH concentration and its absorbance can be measured at 405 nm.

Nitric oxide

Nitric oxide (NO) level was determined according to the method of Green et al. (1982) where, in an acid medium and in the presence of nitrite, the formed nitrous acid diazotize sulfanilamide is coupled with N-(1-naphthyl)ethylenediamine. The resulting azo dye has a bright reddish-purple color which can be measured at 540 nm.

Malondialdehyde levels

Malondialdehyde (MDA) level was determined according to the method of Ohkawa et al. (1979) by using 1 ml of trichloroacetic acid 10 % and 1 ml of thiobarbituric acid 0.67 % and was then heated in a boiling water bath for 30 min. Thiobarbituric acid-reactive substances were determined by the absorbance at 535 nm.

Quantitative real-time PCR

Total RNA was isolated with TRIzol (Sigma-Aldrich). Quality and integrity of RNA were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A 260nm on the ND-1000 Spectrophotometer (NanoDrop Technologies) (Delic et al. 2010). Template complementary DNA (cDNA) was prepared using High Capacity cDNA Reverse Transcription Kit (Life Technologies, Darmstadt, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Darmstadt, Germany) with TaqMan Gene Expression Master Mix from Life Technologies (Darmstadt, Germany). TaqMan assays (Life Technologies, Darmstadt, Germany) used in the experiment are summarized in Table 1. For quantification, messenger RNA (mRNA) levels were normalized to those of GAPDH. The threshold cycle (C T) value is the cycle number, selected from the logarithmic phase of the PCR curve, in which an increase in fluorescence above background can be detected. ΔC T is determined by subtracting the C T of GAPDH from the C T of the target. The fold change of mRNA expression was determined using the 2−ΔΔCT method (Livak and Schmittgen 2001).

Table 1 Primer assays used for qRT-PCR analysis
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Statistical analysis

The obtained data were presented as means ± standard error. One-way ANOVA was carried out, and the statistical comparisons among the groups were performed with Duncan’s test using a statistical package program (SPSS version 17.0). P ≤ 0.05 was considered as significant for all statistical analysis in this study.

Results

The structural morphology and crystalline character of GNPs were examined via transmission electron microscopy (TEM) equipped with high-resolution transmission electron microscopy (HR-TEM). The obtained images show the corresponding TEM results, which are shown in Fig. 1. The low-magnified image shows that the GNPs are spherical in shape within the range of 10–15 nm in diameter. From the observation, it is depicted that all the GNPs are in definite spherical shape with rough surface and free from agglomeration behavior. Another obtained image represents the high-resolution TEM (HR-TEM) image of GNPs (Fig. 1a), which shows that the lattice difference fringes between two adjacent planes are about 0.235 nm. The obtained lattice difference clearly corresponds to the lattice constant of face-centered cubic (FCC) of GNPs and is analogous with the previously reported information (Balmes et al. 2006; Wang et al. 2009). The crystal lattice fringes of HR-TEM observation (Fig. 1b) again show a confirmation of good crystalline nature of synthesized GNPs, and it is consistent with the low-magnified image of GNPs (Balmes et al. 2006; Wang et al. 2009).

Fig. 1
figure 1

Typical TEM and their corresponding HR-TEM images of synthesized GNPs: a Low-magnification image of spherical GNPs (~10–15 nm) and b HR-TEM image of difference between two lattice fringes, which is ~0.235 nm

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Our microscopic findings revealed marked histopathological impairments, neuronal loss, vacuolated cytoplasm, nuclear hyperchromasia, marked dilated congested blood capillaries accompanied with vessel wall edema, and presence of extravasated red blood cells in brain tissue of schistosome-infected mice as compared to noninfected control group (Fig. 2). On the other hand, the treatment of the schistosome-infected mice with three different doses of GNPs (0.25, 0.5, and 1.0 mg/kg) recorded histological alleviations (Fig. 2).

Fig. 2
figure 2

GNPs induced changes in the histological architecture of mice brain infected with S. mansoni on day 46 p.i. a Noninfected brain with normal architecture, b infected brain tissue with distorted brain parenchyma and pericellular edema, and c–f infected-treated mice with 0.25, 0.5, and 1.0 mg/kg GNPs, respectively. Brain appeared with fewer lesions, and tissue damage appears to be minimized. Sections are stained with hematoxylin and eosin. Bar = 25 μm

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The schistosome-infected mice showed a significant reduction in brain NE content as compared to noninfected control group (Table 2). The treatment of schistosome-infected mice with GNPs at three different doses (0.25, 0.5, and 1.0 mg/kg body weight) induced a significant increase (P ≤ 0.05) in brain NE content as compared to both noninfected control and infected groups. In the same line, the treatment with PZQ cleared a significant increase in the NE content. Also, the S. mansoni infection significantly decreased brain DA content in mice, as compared to control group. A significant decrease (P ≤ 0.05) was noticed in DA content in schistosome-infected mice treated with 0.25 and 0.5 mg/kg GNPs and + PZQ as compared to noninfected control group. On contrary, the administration of the higher dose of GNPs (1.0 mg/kg) resulted in a significant elevation in DA content versus control group. The content of brain DA showed a significant increase in all treated groups as compared to schistosome-infected group (Table 2).

Table 2 Effect of gold nanoparticles on brain neurotransmitters norepinephrine (NE) and dopamine (DA) on S. mansoni-infected mice
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The schistosomiasis induced a significant reduction in brain GSH level as shown in Fig. 3. Likewise, treatment with GNPs (0.25, 0.5, and 1.0 mg/kg) and + PZQ significantly reduced GSH level versus noninfected control group. On the other hand, a significant increase was found in GSH level as a result of GNP injection (0.25 mg/kg) to the infected mice as compared to schistosome-infected mice, while a nonsignificant increase was recorded in brain GSH level due to injection of both doses of GNPs (+0.5 and +1 mg/kg) and + PZQ to the infected mice.

Fig. 3
figure 3

Level of reduced glutathione (GSH) in infected mice brain tissue with S. mansoni and treated with different doses of gold nanoparticles. Values are means ± SEM. Significant against noninfected (-GNPs) group at P ≤ 0.05 (a). Significant against infected (-GNPs) group at P ≤ 0.05 (b)

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A significant elevation was recorded in both levels of brain NO and MDA (Figs. 4 and 5, respectively) of schistosome-infected mice as compared to the noninfected group. Oppositely, treatment with 0.25, 0.50, and 1.0 mg GNPs/kg b. wt and + PZQ to schistosome-infected mice influenced a significant decrease in NO level, while a nonsignificant change was obtained in brain MDA level as a result of PZQ treatment versus infected group.

Fig. 4
figure 4

Changes in brain NO level in the brain of S. mansoni-infected mice and treated with nanogold particles (GNPs) at three different doses. Values are means ± SEM. Significant against noninfected (-GNPs) group at P ≤ 0.05 (a). Significant against infected (-GNPs) group at P ≤ 0.05 (b)

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Fig. 5
figure 5

Gold nanoparticles ameliorated MDA level in brain homogenate of S. mansoni-infected mice. Values are means ± SEM. Significant against noninfected (-GNPs) group at P ≤ 0.05 (a). Significant against infected (-GNPs) group at P ≤ 0.05 (b)

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S. mansoni was able to induce a significant upregulation in Adam23, Glrb, Vdac3, and Cacnb4 mRNA expressions; on contrary, Cabp4 mRNA expression showed a significant downregulation as compared to noninfected group (Fig. 6). Only, Vdac3 and Cabp4 mRNA expressions were significantly increased in the brain of mice infected with S. mansoni, after the administration of GNPs at a dose of 0.25 and 1.0 mg/kg, respectively. The expressions of Adam23, Grlb, and Cacnb4 mRNA were downregulated as a result of GNP treatment at different doses. Meanwhile, Vdac3 mRNA expression cleared a significant reduction as GNP treatment at 0.5 and 1.0 mg/kg.

Fig. 6
figure 6

Gold nanoparticles induced changes in gene expression of mice brain infected with S. mansoni. Expression of Adam23, Glrb, Vdac3, Cacnb4, and Cabp4 in brain tissues was analyzed by quantitative RT-PCR in noninfected mice and S. mansoni-infected mice on day 59 p.i. with and without GNP treatment. Relative expression is given as fold increase in comparison with noninfected control mice. Values are means ± SD. Significant against noninfected (-GNPs) group at P ≤ 0.05 (a). Significant against infected (-GNPs) group at P ≤ 0.05 (b)

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Discussion

Since, schistosomiasis is an endemic disease which, on a global scale, 779 million people are at risk of contracting the disease (Coeli et al. 2013). Therefore, research trials have been carried out to study the new approach to the experimental treatment for schistosomiasis. Somehow, the extensive use of PZQ develops Schistosoma sp. resistant to treatment (Fallon and Doenhoff 1994). Since there are few other options regarding the treatment for schistosomiasis (Doenhoff et al. 2009), in particular for S. mansoni, it is necessary to search for alternative drugs.

In the present study, the schistosomiasis which resulted in a significant reduction in both brain NE and DA contents was recorded in schistosome-infected mice as compared to noninfected mice. Our results are in agreement with Abdel Ghafar et al. (1996) and Bauomy et al. (2013).

The treatment of schistosome-infected mice with different doses of GNPs (0.25, 0.5, and 1.0 mg/kg) caused a significant alleviation in content of NE and DA. Ancient cultures in Egypt, India, and China used gold to treat some diseases (Chen et al. 2008). Moreover, gold complexes with different organic drugs have been tested as ligands against malaria, trypanosomiasis, and leishmaniasis (Navarro et al. 2007). It is worth mentioning that the GNPs have also been used for the diagnosis of Alzheimer’s disease and activated microglia (Nunes et al. 2012).

The oxidative damage in stress could contribute to the degenerative diseases of aging, including brain dysfunction (Liu et al. 1996). The early-onset decline in learning and memory is associated with a very significant increase in two parameters of oxidative stress in the brain, levels of lipid peroxidation and of protein oxidation (Liu et al. 2003).

However, schistosomiasis induced a significant reduction in brain GSH level and a significant elevation in both levels of NO and MDA. These results are in agreement with many studies (de Oliveira et al. 2013; Diab et al. 2013; Bauomy 2014; Bauomy et al. 2014; Dkhil 2014). Also, de Oliveira et al. (2013) cleared that S. mansoni had altered nonenzymatic antioxidant status in the brain. In addition, Bauomy (2014) reported that schistosomiasis induced brain oxidative stress as evidenced by the decrease of GSH level, total antioxidant capacity, and the activity of catalase significantly, while a significant elevation in the levels of nitrite/nitrate and MDA. Furthermore, in different mice organs, S. mansoni infection decreased GSH level, the activities of catalase, and superoxide dismutase significantly and, on contrary, increased NO and MDA levels significantly (Diab et al. 2013; Bauomy et al. 2014; Dkhil 2014).

Moreover, GNP injection at different doses to schistosome-infected mice resulted in a significant increase in GSH level and a significant decrease in NO and MDA levels as compared to schistosome-infected mice. The mechanisms of action of gold drugs are poorly understood (Best and Sadler 1996). Typically, after systemic administration, the nanoparticles are small enough to penetrate very small capillaries throughout the body, and therefore, they could offer the most effective approach to target certain tissues (Braydich-Stolle et al. 2005), such as brain, and can affect the physiology of any cell in an animal body (Brooking et al. 2001).

However, our histological findings emphasized that the GNP treatment to schistosome-infected mice resulted in alleviation of the brain histological impairments. The present records go hand in hand with those of Lasagna-Reeves et al. (2010), where the authors deduced a clear increase on GNP accumulation at increasing doses supporting the possibility to use nanoparticles to target the brain without producing detectable toxicity. This is important for the utilization of GNPs for potential treatment and diagnosis of neurodegenerative disorders. The pharmacokinetic, bioavailability, bioaccumulation, clearance, and toxicity of nanoparticles are likely dependent of the particle composition, size, and surface characteristics.

Since, schistosomiasis increased brain Adam23 gene expression. Sagane et al. (1999) and Mitchell et al. (2001) reported that disruption of the mouse Adam23 induced neurological defects, ataxia, and premature death, indicating that this protein is important for normal brain development. On the other hand, injection of GNPs recorded a significant downregulation in brain Adam23 gene.

Glrb gene encodes the inhibitory human glycine receptor β subunit (Lee et al. 2013). Inhibitory glycinergic synapses are located predominantly in the spinal cord and brainstem (Chalphin and Saha 2010), and disruptions to their function increase the general level of excitability of motor neurons, thus accounting for neonatal hypertonia. Glrb is one of the adult walking behaviors which recorded high expression in the malarial brain (Desruisseaux et al. 2010). These results are in agreement with our records in which schistosomiasis induced a significant overexpression in brain Glrb gene. On contrary, gene amelioration resulted in GNP treatment. In the present result, Vdac3 gene recorded a significant overexpression as a result of schistosomiasis. Cízková et al. (2008) reported that Vdac3 is a mitochondrial gene that is regulated in the malaria brain whose dysfunction is associated with neurological disorders. Our results pointed that GNP and PZQ treatment to schistosome-infected mice induced downregulation in Vdac3 brain gene.

Cacnb4 gene mutations encoding voltage-gated calcium channels (VGCCs) induce diverse neuronal pathologies, such as epilepsy, ataxia, autism, and migraine (Bidaud et al. 2006). It is known that neuroschistosomiasis induced ataxia, headache as a common manifestation (Ferrari 2004).

In the present work, Cacnb4 gene recorded a significant upregulation, while Cabp4 gene showed a significant downregulation as a result of neuroschistosomiasis. It was reported that calcium current through VGCCs controls gene expression (Tadmouri et al. 2012). Barnes and Kelly (2002) deduced that calcium influx through calcium channels triggers neurotransmitter release where increase the release of the neurotransmitters by exocytosis, and this may explain reduced contents of DA and NE in our investigations. VGCCs mediate the influx of calcium ions into the cell upon membrane polarization which control multiple neuronal functions including excitability, synaptic transmission, and activity-dependent gene regulation (Catterall and Few 2008). Cabp4 may be an important regulator of calcium influx and transmitter release in synaptic terminals (Haeseleer et al. 2004). Cabp4 interacts with calmodulin-binding sequences in VGCC which weakly inhibits calcium-dependent inactivation.

Comparing results of treatment of GNPs with those of praziquantel, we can say that treatment with GNPs may be a promising way against neuroschistosomiasis, but further studies are required to know the mechanism of GNP action and the best safe dose for the treatment.

Collectively, GNP treatment for neuroschistosomiasis improved the histopathological changes resulted in the brain of schistosome-infected mice, as well as ameliorated the disturbances in brain neurotransmitters. In addition, GNPs at all investigated doses decreased the neurooxidative stress and regulated the gene expression in the brain of infected mice. Consequently, our results revealed an anti-neuroschistosomal effect in mice infected with S. mansoni.