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.