Silver nanoparticles (AgNPs) cause degeneration of cytoskeleton and disrupt synaptic machinery of cultured cortical neurons
© Xu et al.; licensee BioMed Central Ltd. 2013
Received: 16 April 2013
Accepted: 15 June 2013
Published: 19 June 2013
Silver nanoparticles (AgNPs), owing to their effective antimicrobial properties, are being widely used in a broad range of applications. These include, but are not limited to, antibacterial materials, the textile industry, cosmetics, coatings of various household appliances and medical devices. Despite their extensive use, little is known about AgNP safety and toxicity vis-à-vis human and animal health. Recent studies have drawn attention towards potential neurotoxic effects of AgNPs, however, the primary cellular and molecular targets of AgNP action/s remain to be defined.
Here we examine the effects of ultra fine scales (20 nm) of AgNPs at various concentrations (1, 5, 10 and 50 μg/ml) on primary rat cortical cell cultures. We found that AgNPs (at 1-50 μg/ml) not only inhibited neurite outgrowth and reduced cell viability of premature neurons and glial cells, but also induced degeneration of neuronal processes of mature neurons. Our immunocytochemistry and confocal microscopy studies further demonstrated that AgNPs induced the loss of cytoskeleton components such as the β-tubulin and filamentous actin (F-actin). AgNPs also dramatically reduced the number of synaptic clusters of the presynaptic vesicle protein synaptophysin, and the postsynaptic receptor density protein PSD-95. Finally, AgNP exposure also resulted in mitochondria dysfunction in rat cortical cells.
Taken together, our data show that AgNPs induce toxicity in neurons, which involves degradation of cytoskeleton components, perturbations of pre- and postsynaptic proteins, and mitochondrial dysfunction leading to cell death. Our study clearly demonstrates the potential detrimental effects of AgNPs on neuronal development and physiological functions and warns against its prolific usage.
Nanoparticles are ultra-fine materials (range of 1-100 nm in length or diameter) that have gained enormous popularity in modern technology, medical health care, and commercial products [1–3]. Silver nanoparticles (AgNPs) are one of the most commonly used metal-nanoparticles, which possess potent antibacterial and antifungal characteristics. AgNPs have been used extensively as an antimicrobial agent in cosmetics, textiles and the food industry, as well as a disinfectant for medical devices and for coating home applicance . AgNPs upon entering the human body can be systemically distributed throughout, and may affect organs like the lung, liver, spleen, kidney and the central nervous system (CNS) [5–7]. Although various organs can rid themselves of AgNPs, these particles tend to reside for a considerable time, and exhibit a longer half-life within the brain than in other organs . AgNPs could gain access to the CNS through the upper respiratory tract via the olfactory bulb  or through the blood–brain barrier (BBB) [5, 8, 10] and accumulate in various brain regions [4, 11]. AgNPs are also known to cause inflammation and disruption of the BBB . Although the translocation of AgNPs into the brain through the BBB is fairly low under normal condition, its accumulation is augmented under pathological conditions such as meningitis, stroke, or systemic inflammation [8, 13]. Therefore, there exist potential health risks within the brain when exposed to, or upon consumption of AgNP-containing substances.
In the past several years, researchers have begun to explore the potential neurotoxicity of AgNPs using animal models and primary neuronal cell cultures. For instance, studies have reported that animals treated with AgNPs exhibited cognitive impairment, motor deficits and cellular alterations in the brain . In AgNP-treated zebrafish embryos, AgNPs have been found to mainly distribute in the brain, heart, and the blood. Accordingly, AgNPs resulted in cardiorespiratory arrhythmicity, slow blood flow, and impaired body movement and development [14, 15]. In mixed primary neuronal cell cultures of mouse frontal cortex, AgNPs have been found to induce acute intracellular calcium rise followed by a strong oxidative stress response and cytotoxicity in both neurons and glial cells . Glial cells were found in this study to be more vulnerable to AgNP toxicity than neurons. Other studies have revealed that AgNPs could alter excitatory glutamatergic synaptic transmission and receptor functions . It could also change cellular excitability by affecting voltage-gated sodium  and potassium channels  in primary CA1 neurons from mice.
Despite the potential effects of AgNP neurotoxicity cited above, it is still not known whether these nanoparticles could differentially affect brain tissues in the early developmental stage versus later growth phases. It is also unclear whether AgNPs affect fundamental structural and functional components such as the cytoskeleton, mitochondria and synaptic machinery. In the present study, we first examined the effect of AgNPs on neurite outgrowth and cell viability during both early (< 6 days in culture) and more mature (> 10 days) developing stages. We found that AgNPs (20 nm) reduced cell viability in both the early and later stage of cultures in a concentration-dependent manner. Specifically, AgNPs not only inhibited the sprouting of neuronal branches and elongation of neurites, but also caused fragmentation and degeneration of mature neurons. Our data further demonstrated that AgNP neurotoxicity involves the perturbation of structural and/or functional integrity of cytoskeletal components, mitochondria and synaptic proteins.
AgNPs inhibit neuronal extension, neuritic overlap, and compromise cell viability of cultured rat cortical cells
Our data thus demonstrate that AgNPs not only inhibit neuritic initiation, extension and overlap, but also cause degeneration of well-established neuronal structures leading to cell death.
AgNPs induce alterations in cytoskeletal components in rat cortical neurons
We next examined whether AgNPs affect glial cytoskeletal components. To do this, cells were first cultured for 4 days and were then exposed to AgNPs at different concentrations. On day 7, control and AgNP-treated cultures were immunostained with antibodies of β-tubulin (green) and GFAP (red), a fluorescent marker that specifically labels astrocytes. Our representative fluorescent images in Figure 5C show that under the control conditions, healthy neuronal soma (indicated by a solid arrow) and neurites (indicated by an open arrow) were surrounded by glial cells (indicated by asterisks). The glial cells in the control were intensely labeled with both the β-tubulin antibody (green) and the GFAP (red) antibody. However cells that were treated with AgNPs at 10 μg/ml compromised neuronal soma morphology (indicated by a solid arrow) as well as the neurites (indicated by an open arrow). Notably, the fluorescent intensity of red GFAP and green β-tubulin in glial cells (indicated by asterisks) was remarkably affected by AgNPs (Figure 5C). In summary, our data demonstrate that the major components of neuronal and glial cytoskeletal proteins, β-tubulin and F-actin, were affected by AgNPs.
AgNPs induce alterations in pre- and postsynaptic proteins in rat cortical neurons
Assessment of mitochondrial integrity after AgNP treatment
The present study provides the first morphological and cellular evidence that exposure to AgNPs results in a reduction in synaptic proteins, cytoskeletal integrity, mitochondria functionality and cell viability in a dose-dependent manner. Specifically, AgNPs not only inhibited neurite extension and overlap during the early stage of neuronal development, but also caused degeneration of neuritic processes or aberrant aggregations of cell bodies in well-established neurons and their networks. AgNPs-induced neurotoxicity involved altering cytoskeletal proteins (e.g. β-tubulin and F-actin), dissolution of synaptic proteins (e.g. synaptophysin and PSD-95), and compromising of mitochondria function. Our data show that the AgNP – induced reduction of cellular viability occurs in all types of cells in the primary culture. Recent awareness has raised concerns regarding the impact of environmental factors (e.g. heavy metals, pesticide etc) on human and animal health involving a wide range of diseases including cancer, liver, lung, and kidney diseases as well as brain disorders. Our study together with several published accounts thus cautions against the chronic and extensive use of AgNPs in products that may come in direct contacts with all living organisms [7, 13–15].
With the noteworthy maturation of nanotechnology during the last decade, nanoparticle products will continue to be used increasingly in our everyday commercial products, industrial processes and medical applications. Such extensive and unregulated exposure to ultrafine-size substances released either into the atmosphere and/or water system, food and therapeutic products holds potential hazardous risks not only to humans but also to all organisms [27–30]. Recent studies have revealed that AgNPs, one of the most commonly used metal nanoparticles, cause severe developmental deficits not only in aquatic animals but also aquatic plants [31, 32]. Owing to their antibacterial properties, AgNPs have been predominately used for the development of medicines, drug delivery systems and medical device coatings [4, 7]. AgNPs can be translocated to the blood stream and distributed throughout vital organs such as the liver, kidney, lung and brain [4, 5, 11]. AgNPs can cross the BBB and cause BBB inflammation and increase in permeability indicating the potential risk for toxicity to the brain [5, 10, 12]. While the literature is silent on the precise concentrations of AgNPs found in the brain as it crosses the BBB, several studies have reported that the rate of nanoparticle translocation into the brain can be significantly increased under certain pathological conditions, such as infection, meningitis, systemic inflammation etc [8, 13]. Our choice of AgNP concentration and size was based on previously established works. AgNPs (20 nm) were used due to their known high cytotoxic properties with respect to permeating and damaging cerebral microvascular structures, as compared with larger particles (40 nm and 80 nm) . Similarly, smaller nanoparticles (20 nm) have also been shown to induce higher levels of cellular oxidative damage . Previous experiments conducted with 20-40 nm AgNPs used concentrations ranging from 1 to 100 μg/ml to examine the potential hazardous effects of AgNPs with primary neuronal cells [16–18, 33]. In these studies, AgNPs have been found to inhibit neuronal sodium and potassium currents at 10 μg/ml, disturb neuronal calcium homeostasis at 5 μg/ml, and reduce dopamine concentration at 50 μg/ml. Based on these studies, we pursued to identify the effects of chronic exposure to AgNPs (20 nm) at the low to medium doses of 1, 5, 10 and 50 μg/ml on the primary rat cortical cell viability, cytoskeletal frameworks, key synaptic proteins, and mitochondrial function.
Because all brain functions rely critically upon the normal development of neuronal structures and network circuitry, any perturbation of these processes will render the nervous system dysfunctional. In the course of neuronal development, cell viability and neurite outgrowth are two fundamental and essential factors enabling neurons to reach their potentials targets and establish functional communications. These steps rely upon the integrity of cytoskeletal structures. Specifically, cytoskeletal components play imperative roles in neuronal architecture formation and maintenance such as neurite outgrowth, axon guidance, information transmission, and functional synaptic circuitry establishment [34–36]. The reduced number of stained branches and neurite processes by β-tubulin and F-actin antibodies in cells cultured in the presence of AgNPs indicate that AgNPs may inhibit neurite initiation and sprouting by disturbing the assembly/disassembly of these cytoskeletal proteins. The intact cytoskeleton also plays an indispensible role in the localization and trafficking of synaptic machinery (neurotransmitters/receptor proteins) and intracellular organelles including the mitochondria. The degeneration of microtubule and neurofilament components by AgNPs in a well-established neuronal network may directly or indirectly contribute to AgNP-induced loss of synaptic proteins (eg. PSD-95 and synaptophysin, Figure 6) and injury of mitochondria (Figure 7). One would need to be aware that AgNPs might first cause impairment of synaptic proteins and/or mitochondria which may in turn, deprive the fundamental cytoskeletal structures of their vitality source and hence the ensuring collapse. Future studies using time-lapse imaging in combination with fluorescent tagging techniques will be required to investigate these possibilities further. Nevertheless, our data does indicate that by curtailing the growth patterns of neurons, AgNP may not only prevent normal brain development but also preclude neuronal plasticity, learning and memory that relies upon new growth of process and synapses.
We found that exposure to AgNPs exerted detrimental effects on all types of cells including glial cells present in the primary, rat cortical cultures. It is well appreciated that glial cells not only play crucial supportive roles in the development and maintenance of neuronal structures, but that they also actively communicate with neurons to enable synapse formation, synaptic transmission, plasticity and synaptic homeostasis [37, 38]. The pronounced loss of glial cells or their ability to form layers in AgNP-treated cultures (Figure 3) indicates that the glial cell morphology can be severely compromised by AgNPs. This finding is further supported by our immunostaining of glial cell cytoskeletal β-tubulin and GFAP (Figure 5C) in which glial microtubules and intermediate filament proteins were compromised in AgNP-treated culture, while intense green labeling of glial β-tubulin and GFAP was evident in control cultures. This data suggests that components of the glial cytoskeleton are the major target for AgNP-induced toxicity in glial cells. Our findings are consistent with a previous study demonstrating that the glial cell astrocytes were more sensitive to AgNP insult than neurons . Glial cells also play guiding and adhesion roles during neuronal network formation [39–41], hence any absence of preferred neuronal adhesion to glia may contribute to AgNP-induced aggregation of cell bodies and neurofibrillary processes that were observed in our study (Figure 3G and 3H). In addition, a lack of proper innervations and communications between neurons and neuroglia may also markedly deprive them of their glial trophic support. Moreover, in the absence of glial neuro-protection, the neurotoxic effects of AgNPs may have also been exacerbated. These in turn may also impact synaptic transmission, deteriorate synaptic components, and eventually lead to cell death. Synaptic damage has been implicated in a variety of brain disorders, including traumatic nerve injury, stroke, and many neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases [42–44]. Alterations to synaptic structures rank among the earliest notable features in the commencement of the cognitive decline characterized and represented in the co-morbidity of Alzheimer’s diseases . In fact, studies have shown that normal animals treated with AgNPs exhibited reduced cognitive/motor functions and altered cellular structures in the brain . This study together with our data showing the AgNP toxicity to nervous tissues at the cellular, molecular and system levels during both developing and mature stages advise against the potential chronic exposure to AgNPs not only in young people whose brains undergo rapid development, but also in adults whose cognitive functions require continued growing of new neuronal networks.
Taken together, this study demonstrates the potentially devastating effects of AgNPs on cell survival, synaptic protein localization, cytoskeletal proteins, and mitochondrial integrity. Based on the fact that AgNPs have emerged as an important class of nano materials for a wide range of industrial and medical applications, our study highlights not only an urgent need to assess their potential health risks at the whole organism level but also to develop strict policies for their usage.
Rat cortical cultures
All animal procedures were approved by the University of Calgary Animal Care Committee. Conditions met with the standards established by the Canadian Council on Animal Care. The primary culture of rat cortical cells was made using Sprague–Dawley rat pups at postnatal day zero. Dissociated cortical neurons were plated onto cover slips coated with poly-D-lysine (30 μg/ml, Sigma P6407) and Laminin (2 μg/ml, Sigma L2020). Cortical neurons were cultured in neurobasal medium (Invitrogen, no. 21103-049) supplemented with 2% B27 (Invitrogen, no. 17504-044), L-Glutamine (200 mM) (Invitrogen, no. 25030-081), 4% FBS (Invitrogen, no. 12483-020), and penicillin-streptomycin (Invitrogen, no. 15140-122). Approximately 80% of the culture media was replaced every 3-4 days. Cultures were maintained at 37°C in an incubator circulated with air and 5% carbon dioxide.
To study the effect of AgNPs on neuronal process initiation, neurite outgrowth and overlap, freshly dissociated cortical neurons were cultured either in the absence (control) or presence of different concentrations of AgNPs (1, 5, 10 and 50 μg/ml) for 3 days. Neurite outgrowth and cell viability were evaluated on day 3. To examine the effect of AgNPs on newly established neuronal processes and overlaps, cells were first cultured for 4 days to allow for the establishment of neurite outgrowth and network. Neurons were then exposed to AgNPs at different concentrations for 2 days, and the effects of AgNPs were examined. To study the effect of AgNPs on well-established mature network, 10 day-old cells cultured on cover slips were exposed to different concentrations of AgNPs for two days and the effects were evaluated on day 12.
Live/dead cell viability assay for cortical cells
To determine and quantify the impact of AgNPs on cell viability, cortical cultures that were maintained under control or drug-treated conditions were subsequently loaded with the LIVE⁄DEAD® Viability⁄Cytotoxicity Kit (Molecular probes, L-3224) for 15 mins at room temperature (21-22°C). This two-color assay was developed based on the fact that intracellular esterase activity and an intact plasma membrane are unique characteristics of live cells. The LIVE⁄DEAD® Viability⁄Cytotoxicity Kit discriminates live from dead cells by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Preparations were visualized using confocal microscopy (LSM 510 Meta, Zeiss, Germany) under a 20× objective at 488 nm excitation (green) and 548 nm (red) wavelength and images were collected using a band pass filter (560-615 nm). The number of cells labeled with both colors was subsequently counted using imageJ software.
Immunochemistry and confocal microscopy
To stain the cytoskeletal proteins of β-tubulin and F-actin, cultured cells were fixed for 1 h with pre-warmed 4% paraformaldehyde and subsequently washed four times with 1× PBS and permeabilized for 1 h with incubation media (IM) (0.5% Triton in 1× PBS with 10% goat serum). Preparations were then incubated overnight at 4°C with a monoclonal anti-β-tubulin antibody produced in mouse (1:500) (Sigma, T0198). The next day, cells were rinsed twice with 1× PBS and incubated with Alexa Fluor® 488 goat anti-mouse IgG secondary antibody (1:100) (Invitrogen, A-11001) for 1 h at room temperature (21-22°C) under dark conditions. Cultures were subsequently rinsed two times with 1× PBS and incubated for 30 minutes with rhodamine phalloidin (1:20) (Invitrogen, R415) at room temperature. Following two washes with 1 × PBS and one quick rinse with double distilled H2O, cover slips were mounted using MOWIOL mounting media with 4′6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). Samples were viewed using confocal microscopy (LSM 510 Meta, Zeiss, Germany) under a 20× or 63× oil objective at 488 nm (green, β-tubulin) and 548 nm (red, F-actin) excitation wavelengths. Images were collected using a band pass filter (560-615 nm). To stain glial cytoskeletal proteins, the mouse monoclonal anti-β-tubulin antibody (1:200) (Sigma, T0198) and rabbit monoclonal anti-GFAP antibody (1:200) (Biomedical Technologies Inc., BT-575) were stained following procedures as described above. The secondary antibodies were Alexa Fluor® 488 goat anti-mouse IgG antibody (1:100) (Invitrogen, A-11001) and Alexa Fluor® 546 goat anti-rabbit IgG antibody (1:100) (Invitrogen, A-11010). Image acquisition parameters (e.g. laser intensity, gain settings, pinhole sizes, exposure time etc) for control and drug-treated neurons were kept the same. Negative control experiments were performed at the same time to test the specificity of ß-tubulin, F-actin, and GFAP antibodies. No immunofluoresecence was detected when primary antibodies were excluded from the staining procedures (see Additional file 1A and 1B). To verify whether neurons exhibited autofluorescence, unstained cells were excited with all lasers (633, 488, 546 nm) of the confocal microscope and no autofluorescence was observed (data not shown).
To stain the synaptophysin and PSD-95, cultured cells were fixed with pre-warmed 4% paraformaldehyde and 15% picric acid at room temperature (21-22°C) for 20 mins. Cells were subsequently washed four times with 1× PBS and permeabilized for 1 h with blocking incubation media (0.1% Triton in 1× PBS with 5% goat/donkey serum and 2% BSA). Preparations were then incubated overnight at 4°C with the monoclonal anti-PSD-95 antibody produced in mouse (1:2000) (NeuroMab, 75-028) and an anti-synaptophysin monoclonal antibody produced in rabbit (1:500) (Abcam, Ab52636). The next day cells were rinsed twice with 1× PBS. Cells were then incubated with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (1:100) (Invitrogen, A-11001) and Alexa Fluor® 546 goat anti-mouse IgG secondary antibody (1:100) (Invitrogen, A11030) for 1 h at room temperature (21-22°C) under dark conditions. Following two washes with 1× PBS and one quick rinse with double distilled H2O, cover slips were mounted using MOWIOL mounting media with 4′6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). Samples were viewed using confocal microscopy (LSM 510 Meta, Zeiss, Germany) under a 63× oil objective at 488 nm excitation (green, synaptophysin) and 548 nm (red, PSD95) wavelength. Images were collected using a band pass filter (560-615 nm). To assess the level of synaptophysin and PSD-95 staining, image acquisition parameters (laser intensity, gain settings, pinhole sizes, exposure times etc) for control and drug-treated neurons were kept the same. As a specificity control for the immunostaining, no immunofluorescence was observed when primary antibodies of synaptophysin and PSD-95 were excluded from the above staining procedures (see Additional file 1C and 1D).
To assess the impact of AgNPs on the integrity and function of mitochondria, cortical cultures were first maintained in control medium for 4 days and cells were then exposed to AgNPs for another 2 days. On day 6, control or drug-containing medium was subsequently washed off twice with warm Hanks’ Balanced Salt Solution (HBSS) containing sodium bicarbonate, calcium, and magnesium that also included HEPES (10 mM), L-glutamine (2 mM) and succinate (100 μM) to support healthy mitochondrial function in live cells. Cells were then incubated in a dye-loading solution containing calcein AM (1 μM) and MitoTracker Red CMXRos (200 nM) for 15 mins at 37°C. Fluorescence images of mitochondrial integrity (red) and cell viability (green) were collected using a Nikon Eclipse C1si Spectral Confocal microscope with motorized stage (Nikon Instruments Inc., Melville, NY, United States) at the excitation wavelength of 561 nm (red, with a 590/50 emission filter) and 488 nm (green, with a 515/30 emission filter) through a 60× water objective. Again, image acquisition parameters for control and drug-treated neurons were kept the same.
Silver nanoparticles (AgNPs) were purchased from nanoComposix (Sandiego, CA, USA). All chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).
Data was analyzed statistically using one-way analysis of variance (ANOVA) as appropriate. Post hoc analysis was conducted using Tukey’s test. Values were considered statistically significant at the level of P < 0.05. The data is presented as mean ± S. E.M. Each experiment was replicated a minimum of four times; the actual number of replicates for each experiment is listed in the corresponding figure legend or in the text.
This work was supported by Natural Sciences and Engineering Research Council (NSERC) grants to both FX and NIS. We are grateful for Lina Chen in Dr. Gerald W. Zamponi’s laboratory for the excellent technical support with rat cortical cell culturing.
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