Introduction

The nanoparticles (NPs, 1 to 100 nm) are ultra-fine materials that have new characters different than that of their bulk forms (Wegner et al. 2006). Therefore, NPs gain new physicochemical and biological properties such as shape, size, distribution, aggregation state, and specific surface areas which give them higher reactivity (Schrand et al. 2010; Azocar et al. 2019).

In recent years, NPs are used in the delivery of drugs, proteins, genes, vaccines, polypeptides, and nucleic acids. Currently, various applications of the drug delivery system via NPs have encountered an enormous position sector like pharmaceutical, medical, and biological (Sahab Uddin and Rashid, 2020; Zahin et al. 2020). However, releasing silver nanoparticles (AgNPs) into the environment from various sources could indirectly affect humans through the food chain (Blaser et al. 2008) and also may cause toxicity to animals (Tang et al. 2008), plants (Lin and Xing 2007), and bacteria (Choi and Hu 2008). AgNPs may enter the body via the respiratory system, gastrointestinal tract, or through the skin and could not easily be eliminated by physiological clearance systems leading to their accumulation in the tissues and organs (Medina et al. 2007). Also, AgNPs may be capable to penetrate the brain by crossing the blood-brain barrier (BBB) causing the impairment of cell functions and inducing cell damage (Tang and Xiong 2009; Tang et al. 2010). Furthermore, AgNPs may interact with the cerebral microvasculature producing neurotoxicity (Trickler et al. 2010; Grosse et al. 2013). In addition to the degeneration of neuritic processes in vitro (Xu et al. 2013), (Cupaioli et al. 2014) demonstrated that NPs can affect Bax protein levels of mitochondria, causing apoptosis or they can accumulate in the lysosomes impairing their degradation capacity, while Huang et al. (2015) revealed that AgNPs could affect the inflammatory gene expression in neural cells. New researches have demonstrated that dyshomeostasis of essential biometals (calcium, magnesium, manganese, copper, zinc, and iron) is impaired and accumulation of toxic metals is usually observed in numerous neurodegenerative diseases including in the case of Alzheimer’s disease. Furthermore, these metals contribute significantly to the metabolism and aggregation of amyloid-beta (Kabir et al. 2021).

Microglia are macrophage-like cells and important mediators of the initial inflammation in the central nervous system that may participate in neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases (Ryu et al. 2002). Normally, over a long period, the nanoparticles can accumulate in the neuronal tissues and microglia, causing necrotic degeneration (Tang et al. 2008), and even aggravate the process of brain pathology (Sharma et al. 2010). Eom et al. 2013 and Struzynski et al. (2014) stated that AgNPs could initiate their toxic effects through the production of different types of free radicals. These free radicals and oxidative stress can cause oxidation of lipids, proteins, DNA injury, and apoptosis (Park et al. 2010; Yin et al. 2013a, b). Recent studies have shown that AgNP exposure implements oxidative stress-mediated neurodegeneration including Alzheimer’s, Parkinson’s, and Huntington’s diseases that are characterized by disorders of the structure and/or function of neurons (Sahab Uddin et al., 2020a). Growing evidence suggested that there is an association between neuronal dysfunction and neuroinflammation in Alzheimer’s disease, coordinated by the chronic activation of astrocytes and microglial cells along with the subsequent excessive generation of the pro-inflammatory molecule (Sahab Uddin et al., 2020b).

Various studies reported that neural damage is associated with dysfunctions of the neurotransmitter system including acetylcholine esterase (AChE) and dopamine (DA) (Stasiuk et al. 2008). Hussain et al. 2006 found that AgNPs could reduce dopamine concentration, causing many risks to the mammalian brain. Also, Wang et al. (2012) showed that decrease in the dopamine levels associated with the increase in reactive oxygen species (ROS) production by AgNPs.

The dielectric properties of the brain tissue include both conductivity and permittivity that provide important information about cell viability. Changes in the cell membrane permeability are associated with the change in the membrane conductance, all leading to cell death. Baruwati et al. (2013) demonstrated that exposure to the green synthesized AgNPs could alter the membrane permeability and stimulate oxidative stress pathways in neurons.

Histologically, Genter et al. (2012) assessed the distribution and toxic potential of AgNPs following intranasal exposure, and it was found that these particles accumulated in the lateral brain ventricles. Several studies have also shown that AgNPs deposit in the brain after exposure via different routes and cause certain types of brain damage (Xu et al., 2015).

As being highly toxic, toxicological effect of AgNP exposure is an important issue concerned by many researchers, and has been tested in various aspects. Of them, Alzheimer’s disease is a chronic neurodegenerative disorder that is marked by cognitive dysfunctions and existence of neuropathological hallmarks such as amyloid plaques and neurofibrillary tangles (Sahab Uddin et al., 2021).

Accordingly, in the present study, we maintained male rats under different citrate-coated AgNP-26.9-nm concentrations throughout 28 days of the experimental period and then measured the brain electrical conductivity and brain dielectric permittivity, neurobiochemical parameters, and antioxidant stress responses of rats to assess the combined neurotoxic effect of AgNPs. Especially, we aimed to test whether chronic neurotoxicity of AgNPs on male rats would be enhanced more under brain electrical conductivity and dielectric permittivity milieu along with histological and ultrastructural alterations. Besides, the present study might provide an additional contribution to neurotoxicological aspects of potential impacts of AgNPs pollution.

Materials and methods

Sampling collection, preparation, and preservation

  1. a.

    Synthesis of citrate-coated silver nanoparticles

Silver nanoparticles were prepared in the Biophysics Department, Medical Research Institute, Alexandria University, Alexandria, Egypt. The particles were processed according to the chemical reduction method of (Pal et al. 2007) based on the reduction of silver nitrate (AgNO3) with sodium citrate.

  1. b.

    Lyophilization of citrate-coated silver nanoparticles

Citrate-coated silver nanoparticles were frozen by vacuum freeze-drying machine work at 230 V, 50 Hz. The obtained solutions were placed in falcon tubes, frozen in liquid nitrogen, and freeze-dried by vacuum freeze drying/lyophilization equipment (LyoCapsule Freeze Dryer™, SP Scientific, New York, USA) at a pressure of 26.5 Pa and 5% saccharine which was used as cytoprotectant.

  1. c.

    Characterization of citrate-coated silver nanoparticles

  1. i.

    UV-VIS spectral analysis

The bio-reduction of pure Ag+ ions in aqueous solution was detected by a sampling of aliquots (0.2 ml) of the colloidal suspension, then diluting the samples with 2 ml deionized water and subsequently measuring UV-VIS spectra of the resulting diluents using UV/VIS Spectrophotometer (Germany). The maximum absorption was scanned at the wavelength of 300 - 800 nm.

  1. ii.

    Transmission electron microscope analysis

Transmission electron microscope (TEM) analysis was employed to visualize the size and shape of AgNPs. A drop of aqueous suspension (50 μl) containing the biosynthesized AgNPs was placed on the carbon-coated copper grids after allowing the water to evaporate, drying in air. TEM micrographs were taken by analyzing the prepared grids on a JEOL TEM instrument.

  1. iii.

    Particle size analysis

The particle size distribution of the AgNPs was determined by laser light scattering on a Beckman Coulter Particle Size Analyzer (N5 submicron particle size analyzer, Japan). The AgNPs were added to the sample dispersion unit containing the stirrer and stirred to reduce the aggregation between them. The mean particle size was measured after experimenting triplicate.

  1. iv.

    Particle agglomeration analysis

Characterization of nanoparticle agglomeration in the deionized distilled water was conducted using a Zetasizer instrument (Nano-ZS, 4 mW He–Ne 633 nm laser).

Animals and experimental approval

Forty-five adult male CD-1 mice (3 months old, weighing 28–32 g) were obtained from the animal house of Medical Research Institute, Alexandria University, Alexandria, Egypt. The males acclimatized for 2 weeks (5 mice/stainless steel cage) at 25 ± 2 °C room temperature, 55 ± 5% humidity, and normal photoperiod day/night. The animals were allowed free access to food and drinking water ad libitum throughout the study.

AgNPs treatment dose

Synthesized prepared citrate-coated AgNPs were orally administered at a concentration of 100 and 1000 mg/kgbw for experimental groups AgNPI and AgNPII, respectively (28 days, 15 mice/each). In the control group, mice have received deionized water. The dose levels were selected based on the LD50 of previous studies (5000 mg/kg) on 28-day oral administration of silver nanoparticle (100 and 1000 mg/kgbw equivalent to 1/50 and 1/5 mg/kgbw, respectively) (Maneewattanapinyo et al. 2011; Adeyemi and Adewumi 2014).

Brain tissue handling techniques

On reaching the empirically determined endpoint, animals were euthanized by intraperitoneally injected ketamine 80 mg/kg and xylazine 10 mg/kg followed by decapitation. The whole brains (controls and treated) were rapidly excised removed and sectioned along sagittal midline into two halves. The right cerebral hemisphere was fixed for microscopical methods. The whole left half was reserved in liquid nitrogen and frozen at − 80 °C for neurochemistry assay.

Total nanosilver content in the brain

The total silver content in the whole brains after 28 days of treatment was quantified measured determined by using an inductively coupled plasma-atomic emission spectrometer (R-R-smz-ICPE-9820 Simultaneous ICP, SHIMADZU EUROPA GmbH, Duisburg F. R. Germany) (Pröfrock and Prange 2012). Data are shown as the average of four independent values.

Energy-dispersive X-ray (EDX) analysis

Identification of the elemental composition of the brain tissues was generated by energy-dispersive X-ray analysis (EDX). It consists of spectra showing peaks corresponding to the elements making up the true composition of the brain samples.

Brain tissue homogenization

AgNP-treated male mice and controls (three experimental replicates per group) were sacrificed after 28 days by decapitation and dissected. The brains were rapidly excised removed, washed in cold buffered phosphate (pH 7.4) and homogenized (Rotary homogenizer BK-HG160, Biobase, China). The homogenized brain tissues were centrifuged at 1600 rpm for 20 min at 5 °C, and the supernatant was immediately stored at −40 °C for further neurochemical assessment.

Neurochemistry methods

  1. a.

    Brain acetylcholine esterase assay

Acetylcholine esterase (AChE, Acetylcholine esterase Assay Kit, Colorimetric, Catalog No. STA-603) activity was assayed in triplicate using the technique of the colorimetric procedure of Ellman et al. (1961). The cholinesterase activities were accomplished at absorbance 670 nm.

  1. b.

    Brain dopamine assay

Quantitatively, dopamine in brain supernatants was measured by a competitive ELISA assay following Bio Vision’s Dopamine ELISA kit (Catalog No. K4219-100) protocol. The average of the triplicate readings for samples was at 450 nm, detection range 1.56–100 ng/ml, and sensitivity <0.938 ng/ml (Anton and Sayre 1964).

  1. c.

    Plasma and brain serotonin assay

In the next morning of the last AgNP intake and fasting overnight, venous blood was gathered from the vena cava by using the catheter in 9-ml ACD-A Vacuette® tubes (Cat. No. 455055, Greiner Bio-One International GmbH, Germany). Following instructions of the manufacturer, competitive enzyme immunoassay (commercial ELISA Kit, ab133053) was used for colorimetric detection of serotonin contents in brain supernatants and concentration of serotonin in plasma (triplicate) (Lee et al. 2014).

Biophysical analysis

Brain electrical conductivity (σ) and brain dielectric permittivity (ε)

One centimeter of the brain tissue from control and the experimental-treated mice was used for dielectric measurements. Capacitance and resistance of the brain tissue were measured using LCR meter (L for inductance, C for capacitance, and R for resistance). This LCR meter operates at a frequency range from 1 to 100 kHz with an accuracy of 0.05%. The relative dielectric permittivity ε′ and real conductivity σ are calculated according to the following equations:

$$ \mathrm{C}={\varepsilon}^{\hbox{'}}{\varepsilon}_0\ \mathrm{A}/\mathrm{d} $$
(1)
$$ G=\sigma \kern0.5em A\kern0.5em /\kern0.5em d $$
(2)

where C is the capacitance in Farad, G is the conductance in Siemens, of the capacitor between the two measuring electrodes, A is the surface area of the electrode in a square meter, d is the separation between the two electrodes in meter, ε: the relative permittivity (Farad/meter), ε0: the permittivity of vacuum (8.85 × 10−12F/m), and σ the electrical conductivity (Siemens/m).

Estimation of brain total antioxidant capacity

By deproteination procedure of 0.1 ml of supernatant by 2 ml of methyl alcohol, vortexed, and centrifuged (30 s, 10,000 r/min/10 min), 1.5 ml methanol and 0.5 ml of DPPH solution were mixed thoroughly with supernatant (colorimetric Kit ab65329). Absorbance at 570 nm was recorded as nanomoles of blank referenced ascorbic acid (Koracevic et al. 2001).

Microscopical methods

  1. a.

    Light microscopy

The right cerebral hemispheres of the brain tissues were cut into 5-mm specimens and fixed in a 10% neutral buffered formalin, dehydrated, cleared by xylem, and embedded in paraffin wax (Bancroft and Gamble 2008). Thereafter, 5-μm coronal sections were stained with hematoxylin and eosin.

  1. b.

    Transmission electron microscopy

One-square millimeter specimens of the right cerebral hemispheres of both control and the experimental mice were immediately transferred to a fixative mixture of 4% paraformaldehyde, 1% glutaraldehyde, and phosphate buffer (0.1 M, pH 7.4) at 4 °C for 24 h. The specimens then were post-fixated using 1% buffered Osmium tetroxide (OsO4) at 4 °C for 2 h. Thereafter, the samples were washed several times with phosphate buffer for 30 min, dehydrated through a combination of ascending grades of ethanol with propylene oxide, and finally blocked in a mixture of 1:1 of Epoxy-Araldite resin mixture. Sixty-nanometer ultrathin sections were obtained with a glass knife on LKB ultramicrotome, mounted on 200 mesh naked copper grids, and subsequently double stained with uranyl acetate and lead citrate for 20–30 min, examined at Joel TEM (Robards and Wilson 1993).

Statistical analysis

Statistical analyses were performed using PASW Statistics 18. Normality test was performed using Kolmogorov-Smirnov test, and independent T-test was used to compare the means of each of the two different studied groups when data were normally distributed, while the Mann-Whitney test was performed to compare the medians of each of the two different studied groups when data were not normally distributed.

Results

Characterization of silver nanoparticles

The results of particle size analyzer for citrate-coated AgNPs showed that more than 90% of the prepared particles are in the average size of 26.9 nm, and about 10% of the particles are oversized with baseline error of 1.35% (Fig. 1A). Examination by the transmission electron microscope revealed that most of the citrate-coated AgNPs are spherical, having smooth and homogeneous surfaces. Analysis of the characteristic state of dispersion of AgNPs using dynamic light scattering showed a low level of agglomeration or adhesion and a high level of uniformity. Further, the polydispersity of most of AgNPs particles is 0.74 (i.e., less than 1), indicating the homogeneous distribution with >90% uniformity in size of the particles (Fig. 1B). UV-visible spectrum analysis showed that the maximum absorption wavelength for the particles is at 422 nm (Fig. 1C).

Fig. 1
figure 1

Characterization data for silver nanoparticles. (A) 90% of the AgNPs show mean particle size of 26.9 nm. The polydispersity of most of AgNPs is 0.74. (B) TEM shows most of the AgNPs are spherical in shape with smooth and homogeneous surfaces and showed a low level of agglomeration with high level of uniformity. (C) UV-visible spectrum analysis shows the maximum absorption wavelength for the particles is at λ max = 422 nm

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Total silver ion content in brain tissue

The results of total brain ion contents of silver revealed a significant increase in a dose-dependent manner being 0.611 ± 0.017 μg/g and 0.851 ± 0.036 μg/g for 100 mg/kg and 1000 mg/kg (AgNPI and AgNPII groups), respectively, compared with the control group (0.206 ± 0.011 μg/g).

Elemental composition of brain

The energy-dispersive X-ray analysis of the brain tissues of both control and the treated mice revealed the presence of many different elements including silver (Ag). These elements are phosphorus (P), sulfur (S), potassium (K), calcium (Ca), sodium (Na), copper (Cu), aluminum (Al), and zinc (Zn). However, considerable high levels and high spectral peaks of silver were recorded in the brain tissues of mice administered with 1000 mg/kgbw of AgNPs, compared with those treated with 100 mg/kgbw, indicating silver accumulation in the brain. No observed silver was detected in the brain of the control mice (Fig. 2).

Fig. 2
figure 2

Energy-dispersive X-ray (EDX) analysis of the brain tissues of control, low dose (AgNP I, 100 mg/kgbw), and high dose (AgNP II, 1000 mg/kgbw) AgNP-treated mice for 28 days

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Total antioxidant capacity

From the analyzed data of Fig. 3, it clearly showed that Ag-26.9 nm significantly decreases the antioxidant capacity of the brain in a dose-dependent manner. The decreased antioxidant capacity was more statistically significant in the dose of 1000 mg/kgbw than the dose of 100 mg/kgbw of silver nanoparticles versus controls.

Fig. 3
figure 3

Effect of 26.9-nm AgNPs on total antioxidant capacity in mice brain tissues of control and treated for 28 days. Data represent means ± SD (n = 15 mice, triplicate). *Significant at P ≤ 0.05 versus control. Control; AgNP I: 100 mg/kg silver nanoparticles; AgNP II: 1000 mg/kg silver nanoparticles

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Dopamine levels

The oral administration of silver nanoparticles significantly decreases the levels of dopamine by 58.1% when the mice of the 1000-mg/kgbw group were compared with the control mice. While in mice of the 100-mg/kgbw group, the impacts of the silver nano on dopamine levels significantly minimize by 26.4% compared with the controls (Fig. 4A).

Fig. 4
figure 4

Effects of 28-day oral administration of 26.9-nm silver nanoparticles (Ag-26.9). (A) The contents of brain monoamines (DA and 5-HT) and AChE activity in cerebral cortex of controls and treated mice. (B) Levels of the plasma 5-HT. Data are expressed as the mean ± SD (n = 15, triplicate). *P ≤ 0.05 versus the control group using Tukey’s post hoc test. DA, dopamine; 5-HT, serotonin; AChE, acetylcholine esterase. Control; AgNP I: 100 mg/kg silver nanoparticles; AgNP II: 1000 mg/kg silver nanoparticles

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Acetylcholine esterase activity

A significant decrease was observed in the mean value of the enzyme activity of acetylcholine esterase in the mice of the AgNPI and AgNPII groups compared to the controls. Notably, the level of acetylcholine enzyme activity was dose-dependent. Moreover, the highest decrease was observed in the 1000-mg/kgbw group (Fig. 4A).

Plasma and brain serotonin levels

In both brain (Fig. 4A) and plasma (Fig. 4B), the levels of serotonin (5-HT) showed a significant decrease in 100 mg/kgbw and 1000 mg/kgbw of silver groups (AgNPI and AgNPII) compared to controls. This decrease being significantly higher by 1000 mg/kgbw (AgNPII) dose than that by 100 mg/kgbw (AgNPI) (Fig. 4A, B).

Brain conductivity (σ) and brain permittivity (ε)

The biophysical findings revealed that the brain conductivity was gradually decreased (dose-dependent) with increasing the frequency of the applied electric field in the brain tissues of mice administered with the two concentrations (100 and 1000 mg/kgbw) of Ag-26.9 nm. This decrease was parallel with the decrease in the relative permittivity that shown to be decreased suddenly and then started to be saturated (Fig. 5A, B).

Fig. 5
figure 5

Biophysical analysis. (A) Variation of the relative permittivity ε′ (F/m) with frequency F (Hz). (B) Variation of the real conductivity σ′ (S/m) with frequency (Hz). Control; AgNP I: 100 mg/kg silver nanoparticles; AgNP II: 1000 mg/kg silver nanoparticles

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Brain histopathology

  1. a.

    Light microscopic observations

The cerebral cortex of mice treated with 100 mg/kgbw of AgNP-26.9 nm showed neuron pyknosis and observable increase of the neuroglia cells (gliosis) located within the moderately vacuolated neuropil (neuropils spongiosis) (Fig. 6B, C ) comparable to control brain tissue (Fig. 6A). However, sections of the cerebral cortex of mice treated with 1000 mg/kgbw Ag-26.9 nm showing the focal area of hemorrhage and the neurons appeared shrunken, containing pyknotic nuclei. Besides, many swelling and hypertrophied neuroglia cells could be found in the injured area of the neuropil spongiosis (Fig. 6D, E).

  1. b.

    Electron microscopic observations

Fig. 6
figure 6

Photomicrographs of the cerebral cortex sections in control and experimental groups. (A) Control group showing normal configurations of the neuronal tissue. (B) and (C) 100 mg/kg group. (D) and (E) 1000 mg/kg group. Notice: the pyramidal-shape neurons with large nuclei (N, short arrow); pyknotic nuclei of neurons (long arrows); area of gliosis (arrowheads); focal area of hemorrhage (F); vacuolation in the neuropil (spongiosis) (NP); vacuolation (V). H&E

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Using the electron microscope, the neurons in the cerebral cortex of the control mice have large centrally located indented nuclei and containing prominent nucleoli (Fig. 7a). The cytoplasm contains numerous free ribosomes and there was a well-developed Golgi saccule adjacent to the nucleus (Fig. 7a). The myelinated axons of nerve cells were distributed in the normal area of neuropil (Fig. 7b). The neuropil contains axon terminals with their large vesicles, mitochondria, and synapses (Fig. 7c).

Fig. 7
figure 7

Electron micrographs of section of cerebral cortex of control mice. (a) Nucleus (N) of neuron; (b) the myelinated axons of nerve fibers (arrows), neuropil (NP), mitochondria (M), nucleus of a neuroglia (Nn); (c) area of neuropile containing processes of nerve fibers, the axoplasm contains rounded mitochondria (M) proper distribution of synaptic vesicles (Vs). × 3000

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The electron micrographs of the cerebral cortex of mice administered with 100 mg/kgbw AgNPs revealed moderate ultrastructural alterations including neuronal shrinkage, an indentation in the nuclear envelope, in addition to the enlargement of the nucleolar size (Fig. 8a). The myelin sheath showed an observable decrease and multiple splitting (separation) of their myelin lamellae with wide spaces in between. The area of neuropil was finely vacuolated because of swelling of the neuronal processes and synapses (Fig. 8b). The synaptic vesicles have well-defined structures, and the vesicles were arranged in the cluster as in the control (Fig. 8c). The electron micrographs of neurons of the cerebral cortex of mice treated with 1000 mg/kgbw AgNPs showed marked irregularities in their nuclear envelope. Small dark nanosilver deposits were observed in the nucleoplasm of neuronal cells, in the blood capillaries, and in many lysosomes (Fig. 9a). Moreover, a myelin-like body (myelin figure) contained remnants of synaptic vesicles or disturbed mitochondria were observed, and it represents the initial stage of macro-autophagosomes (Fig. 9b). In the area of neuropil, severe degenerative changes were seen in the myelinated axons. This was in the form of defective, disruption, irregular myelination, splitting, and loss of lamellar structure around the nerve fibers. The axoplasm contains swollen irregular mitochondria with destroyed cristae. Moreover, the results revealed local swelling in synapses, and aggregation of the free synaptic vesicles, which were loosely located in the neuropil and were not surrounded by the presynaptic membrane (Fig. 9c).

Fig. 8
figure 8

Electron micrographs of sections of cerebral cortex of mice treated with 100 mg/kgbw AgNPs. (a) Indentation in nucleus of neuron (arrow); neuropil area (NP). (b) Nucleolus (Nu); mitochondria (M); (c) myelin showed an observable decrease and multiple splitting (separation) of their myelin lamellae with wide spaces in between (arrows). × 3000

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Fig. 9
figure 9

Electron micrographs of sections of cerebral cortex of mice treated with 1000 mg/kgbw AgNPs, showing: (a) marked irregularities in the nuclear envelope (arrowhead) of the neuronal nucleus (N); the blood capillary (Cap) contains small dark nanosilver deposits; (b) chromatolysis (arrow); dilatation of rER; (c) nanosilver deposits in lysosomes (S); hypertrophied mitochondria (M); (d) the axoplasm contains swollen mitochondria (M); (e) loss of lamellar structure of the myelinated axons (arrow); deposition of the nanosilver granules (S). × 3000

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Discussion

The present assays infer that oral gavage with 26.9 nm citrate-coated AgNPs was absorbed, distributed, and then eventually traversed the blood-brain barrier in vivo inducing neurotoxicity. Exposure mode (ingestion, intranasal, intraperitoneal) and particle size together with characteristics of surface charge activity, hydrophilicity, and lipophilicity are substantial factors contributing to the toxicity of nanomaterials (Liu et al. 2012; Wang et al. 2015; Wen et al. 2016; Islam et al. 2017; Iqbal et al. 2018). Besides, the physicochemical characteristic is a bioactive agent that can reinforce the ability of nanomaterials to enter the cell. In the present study, the particle size of 26.9 nm indicates that nanoparticles are small and toxic because of their preferable mobility inside the BBB (Osborne et al. 2013) wherein directly disturb BBB and could be easily transported by the trans-synaptic neurons. This is consistent with the previous studies that revealed 25-nm silver nanoparticles cause significant neurotoxicity both in vivo and in vitro (Rahman et al. 2008, 2009).

Regarding the accumulation of AgNPs in the present findings, the possible mechanism can be proposed as the sulfur molecules are rich in thiol groups and intent to bind with Ag substrates. The formation of new bonds leads to sulfur molecules tightly adsorbing on the surfaces of AgNPs particles. In this case, the chemical enhancements derived from AgNPs make up for the efficiency of the surface effect of Ag metals (Guo et al. 2019). Further, the disposition of AgNPs could be ascribed to the damage of cell integrity and increase in-stream opening the gates (Antsiferova et al. 2019). According to Wen et al. (2016), accumulation of AgNPs depends on its surface oxidation to release silver nano ions. Zhang et al. (2011) suggested traversing the nanoparticles across the BBB through dissolving membranes of the endothelial cells. Unlike the present data, the high accumulation of brain silver may be surmised by the difference in size average (56 nm, 60 nm) and the variation in the value of doses (Kim et al. 2008, 2010). Therefore, size-dependent increases of silver accumulation in the brain indicate that the orally absorbed silver nanoparticles have the bioavailability to penetrate and distribute in the blood-brain circulation and the possible deposition that may be due to particle size as opposed to ionized silver. On the other hand, the high total brain contents of silver in the present study might be interpreted by the powerful affinity of nanoparticles to the thiol group of free brain sulfur and sulfur-linked proteins forming thiol-silver complex (Liu et al., 2010) which also may contribute to the explanation of the neurotoxic effects of nanosilver (Elle et al. 2013). This great affinity of silver to sulfur could also indicate the high merging of silver ions with sulfur-rich biological constituents (Loeschner et al. 2011).

A decrease of neurotransmitter concentrations was detected in AgNP-treated rats suggesting that the AgNP has a differential tight binding to neurotransmitters and more bracing architecture in which can be rapidly restrained in and out through the synapse trafficking (Varela et al. 2016). Besides, organic cation transporters (OCT) are facilitative transporters that are mediating the translocation of small organic cations across the basolateral membranes (Wagner et al. 2016). In the present findings, the significantly decreased choline, serotonin, and dopamine concentrations may be attributable to downregulation of protein and mRNA expression levels of the OCT, indicating that OCT is the target for silver nano. The present findings conflict Hadrup et al. (2012) who found an increase in the concentration of the dopamine in rat brain suggesting a differential effect of silver on dopamine relying on the length of exposure. According to findings in Parkinson’s disease, the decreased brain dopamine concentrations following of AgNP administration could be explained by an increase in apoptosis of dopaminergic neurons (Savitt et al. 2006; Ehringer and Hornykiewicz 2012). Further, in vitro data suggest that AgNPs change the regulation of mRNA levels of dopamine-related expressed genes during neural cell differentiation (Wang et al., 2009a, b; Powers et al. 2010, 2011). Probably, this indicates distinct variations in the effects of silver nanoparticles on the regulation of neurotransmitter-related genes associated with normal brain activity. Crossing, distribution, and deposition of AgNPs in the presynaptic endings could provide another possible evidence of deficiency of neurotransmitters (Fedorovich et al. 2010; Tang et al. 2010). Also, the deficit neurotransmitters may suggest axonal defects (dysplasia) due to mutation in gene expression of Growth Associated Protein 43 (GAP43) that critical motif of the axon and synaptic terminals (Rosskothen-Kuhl and Robert-Benjamin 2014; Liu et al., 2020a, b).

In the present study, the brain acetylcholinesterase (AChE) activity significantly declined in both low and high doses indicating that AgNPs cause nervous toxicity (Bugata et al. 2019). However, the precise mechanism identifying that the AgNPs inhibit cholinesterase activities is still not understood in relating to interactions between nanoparticles and enzyme. It has been suggested that adsorption of the AChE on AgNPs surface and the subsequent embracing of enzyme configuration is the primary mechanism influenced by the distribution of surface charges on an enzyme (Wang et al., 2009a, b; Šinko et al. 2014). There is evidence that impairment of brain serotonin (5-HT) may be prompted by the sustained extreme attrition of tryptophan (Neumeister et al. 2002). ElMazoudy et al. (2015, 2020) reported that severe tryptophan depletion could lead to degradation of neural 5-HT levels suggesting the tight binding of tryptophan to the surface of nanoparticles (Hernández et al. 2018). As silver nanoparticles possess superior biophysical properties with regard to electrical conductivity and relative permittivity (Khan et al. 2011), so, the decrease of neurotransmitters that are parallel with the decrease in electrical conductivity and dielectric permittivity of the brain refers to a deficit in brain circuitry.

The decreased electrical conductivity and relative permittivity in the present results may be attributed to the disturbances in dipoles of nanosilver relating to the field direction of the brain (Zhang et al. 2010). The electron-trapping and molecular ionization of nanofluid mechanisms may be also implicated in the reduction of electrical conductivity and relative permittivity (cerebrospinal fluid) (Prasad et al. 2006; Fal et al. 2016). Additional studies showed that reduced conductivity and relative permittivity may be attributable to the morphological defects in myelin sheaths and mutation in the gene expression of myelin glycoproteins suggesting that the nerve fibers (myelinated) may be a target of AgNPs (Dabrowska-Bouta et al. 2019). Furthermore, Schmid and Überbacher (2005) and Peyman (2011) concluded that impaired myelination observed in the present findings may be the cause of the decline in the values of permittivity and conductivity.

Neurotoxicity induced via high oxidative stress in the present study is assigned to the stimulation of downregulation of CAMKII (CAMK2A/CAMK2B) protein expression, which results in synapse deformation, apoptosis, and perturbation of signal transduction and the demise of axons and neural dendrites (Chen et al. 2011; Mockett et al. 2011; Hamdani et al. 2013). These pathways suggest that nanosilver might be closely implicated and extremely mediate molecular signalling of the synaptic development and morphogenesis through the calcium/calmodulin-regulated kinase mechanisms. Besides, the oxidative stress damages dopaminergic neurons via excessive releasing of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and their inflammatory mediators (nitric oxide synthase and cyclooxygenase-2) and that explains the decrease in dopamine activity in the present findings (Malú and Matthew 2010; Joshi and Singh 2018). Thereby, the damage of dopaminergic nerve cells and abnormal signal transduction activating further neuroglia are essential tools for identifying mechanisms of neurodegenerative diseases and potential treatments. Meanwhile, the damage of dopaminergic regulation of neurons may attribute to influence and permanent oxidative stress that probably provoke inflammation-related transcription factors (Tarique et al. 2016). The caspase3 enzyme is another oxidative stress-mediator factor essential for the apoptotic death in diverse neurodegenerative diseases through regulation of endogenous mechanisms implicated in the caspase3-dependent apoptosis transcription factors referring to a critical role of AgNPs in the normal regulation of neuronal apoptosis in vivo (Nijhawan et al. 2000; Bazan-Peregrino et al. 2007; Yin et al., 2013a, b).

In the present findings, the deficiency of the neurotransmitters could reflect the inefficiency of nerve fibers as transmission pathways which may due to damage to the neuronal cytoskeleton, shortening of nerve fibers, or reduction in the number of nerve nodes (axon diameter) induced by damage of neurofilament protein-H (NF-H), tubulin-α, or tubulin-β, causing obstruction of the axonal transport and nerve conduction velocity (Ayaz et al. 2012; Kengaku 2018; Liu et al., 2020a, b). Therefore, the suppressed dopamine, acetylcholine, and serotonin in present findings could be a local injury induced by AgNPs on nerve fibers (Wang et al. 2003). In this context, inhibition of neurotransmitters in vivo is dependent on the brain electric conductivity and dielectric permittivity. So, the present results provide many conclusions related to perceive the intended mechanism of how the brain can regulate the physicochemical conditions to control signal transductions in real time. On the other hand, Michel et al. (2017) reported that the alterations of brain biophysical properties reflect brain toxicity and indicate a disturbance of brain water contents causing tissue necrosis, tissue abscess, and tissue cysts. Further, decreased brain conductivity may be attributed to median impedances of gray and white matter cerebral tissues wherein gray matter shows lower median impedances in neurodegenerative diseases (Koessler et al. 2017).

Conclusions

From the present findings, we can surmise that profilings of disruption in brain electric conductivity and dielectric permittivity, oxidative stress, deficiency in brain neurotransmitters, and robust damage of myelinated nerve fibers firmly proved the neurotoxic hazards of AgNPs. The results also revealed the existence of robust toxicity of AgNPs at 100- and 1000-mg/kg dose levels and particle size 29.6 nm indicating the probable capability of crossing-over the BBB. The deposition of silver in brain organ was higher in the AgNP-treated groups than in the control rats indicating the ionic bioavailability of silver rather than nanoparticles. Finally, present findings suggest the deficit of neurotransmitters may be via axonal and dendrites defects and/or disruption in neurotransmitter-transporter gene expression as well as brain circuitry that raise a serious explanation of brain toxicity like aging, Parkinson’s disease, and Alzheimer’s disease. Therefore, the electric conductivity and dielectric permittivity profiling may be helpful in layout brain toxicity studies as indications on the dynamics and kinetics in brain tissues and could provide decisive information and comprehension by determining signal transduction levels in potential neurodegeneration for exposure aspects.