Silver nanoparticles (AgNPs) are emerging as one of the fastest growing nanotechnology product categories. However, the impact of silver nanoparticles (AgNPs) on the central nervous system is a topic with mounting interest and concern and the facts remain elusive. This work focused on the in vitro and in vivo neurotoxicity of silver nanoparticles. The aim of our work is to assess the biological safety of silver nanoparticles, provide basic data for their applications and establish feasible methods to investigate the neurotoxicological effects of nanomaterials.
      First, the neurotoxicity of commercial AgNPs to rat cerebellum granule cells (CGCs) and the corresponding molecular mechanism are closely investigated. It was demonstrated that AgNPs induced significant cellular toxicity to CGCs in a dose-dependent manner without damaging the cell membrane. Flow cytometry analysis with the Annexin V-FITC/propidium iodide (PI) staining indicated that the apoptotic proportion of CGCs upon treatment with AgNPs was greatly increased compared to the negative control. Moreover, the activity of caspase-3 was largely elevated in AgNP-treated cells compared to the negative control based on the ELISA assay. AgNPs were demonstrated to induce oxidative stress, reflected by the massive generation of reactive oxygen species (ROS), the depletion of antioxidant glutathione (GSH), and the increase of intracellular calcium. Western blot assay suggested the activation of caspase-3, indicating caspase-dependent apoptosis was induced in response to AgNPs treatment. Taken together, it is demonstrated that AgNPs substantially impaired the survival of primary neuronal cells through apoptosis coupled to oxidative stress, depending on the caspase activation-mediated signaling pathway.
      Based on in vitro studies, we futher evaluated the in vivo toxicity of silver nanoparticles. The neonatal SD rats, as the animal model, were administered with AgNPs by intranasal instillation. We demonstrated that 3-month AgNPs exposure caused cerebellar ataxia like symptom in SD rats which was evidenced by dysfunction of motor coordination and impairment of locomotor activity. Observation of cerebellum section revealed that AgNPs could provoke destruction of cerebellum granular layer with concomitant proliferation or activation of glial cells. AgNPs treatment decreased the calcium channel protein (CACNA1A, or Cav2.1-α1A-subunit) levels in cerebellum without changing the potassium channel protein (KCNA1) levels. In vitro experiments showed that AgNPs treatment significantly reduced the expression of CACNA1A in CGCs.
      As an antioxidant, vitamin E (VE) was demonstrated to antagonize the neurotoxicity of AgNPs both in vitro and in vivo through the depletion of oxidative damage. Taken together, AgNP-induced rat motor dysfunction is associated with decreased expression of CACNA1A and introduction of vitamin E (VE) was able to efficiently attenuate the abnormal neuronal penotype through antagonizing oxidative stress caused by the nanoparticles.