| With the increasing application of silver nanoparticles(SNPs)in the biomedical field,the toxicity of SNPs on neuron has attracted great attention.Currently,the toxicity of SNPs on neuron is mainly studied by traditional cell biology methods.However,neurons not only have the cytological characteristics of ordinary cells,but also have electrophysiological characteristics,and their functions highly depend on the changes of their electrophysiological characteristics.Therefore,when evaluating the toxicity of SNPs on neuron,we should not only consider the changes of cytological characteristics,but also study the influence of SNPs on electrophysiological characteristics.At the same time,it also shows that it is not comprehensive to use cytological experiments alone to evaluate the toxicity of biomaterials on neurons,and the electrophysiological methods is also an indispensable and important research tool in research.Only by combining them and conducting combined research,can we deeply understand the mechanism of the toxicity of SNPs on neurons.At present,no research on the effect of SNPs on hippocampal neuronal network’s electrical excitability has been reported,There is also no study has employed both electrophysiological and cytological methods to evaluate the toxicity of SNPs on hippocampal neurons.Our group has conducted a series of cytotoxicity studies on SNPs.The effects of external factors and drugs on the electrophysiological excitability of neural networks also have been studied by electrophysiological methods.This paper will combined analysis the effects of SNPs on the cytological and electrophysiological properties of rat hippocampal neurons based on electrophysiological and cytological methods,and discuss the cellular mechanism of the effects of SNPs on neurons.In this paper,voltage threshold measurement method(VTMM),a new method for quantitative measurement of electrical excitability of neural networks and based on MEA(Micro-Electrode Array)technique,was used to study the effect of SNPs on the electrical excitability of hippocampal neuronal networks and rat hippocampal slices.Based on VTMM,the role of NMDA receptor in the effect of SNPs on the electrical excitability of hippocampal neuron network and its mechanism were investigated.Then,two concentrations of SNPs with different cytotoxicity was used and the indexes of ROS content,ATP content,cell apoptosis rate and neurite length in rat hippocampal neurons were studyed.Finally,in order to explain the cellular and molecular mechanism of the changes in electrical excitability,the VTMM test results and the results of cellular/molecular experiments were compared and analyzed.The relationship between the cellular and molecular biological indicators and the voltage threshold in the rat hippocampal neuron network treated with different toxic concentrations of SNPs was studied.The possible cellular molecular mechanism of the effect of SNPs on the electrical excitability of neural networks has been discussed.The study deepens the understanding of the molecular mechanism of toxicity of SNPs on neuron.The main content of this paper is as follows:1)SNPs was prepared by reducing silver nitrate with sodium borohydride.The results of transmission electron microscope and microplate reader showed that the prepared SNPs were uniform in size,without large-scale aggregation,and the particle size was about 20 nm.The primary hippocampal neurons were extracted from embryos staged at E18 from pregnant SD rats.After 2 weeks of culture,the extracted cells were identified.The results showed that most of the cells extracted from the hippocampus were neurons.It was found that SNPs could enter hippocampal neurons through the intracellular uptake experiment of SNPs,which may be the cause of neurotoxicity.MTT method was used to analyze the effect of different concentrations of SNPs on the cell viability of hippocampal neurons.In the following experiments,we chose the non-cytotoxic 5μM and cytotoxic 100 μM SNPs to study its effects on hippocampal neurons.2)The effect of non-cytotoxic 5μM and cytotoxic 100μM SNPs on the electrical excitability of hippocampal neuronal networks and hippocampal brain slices was studied by "VTMM"based MEA.It was found that the effects of 5μM SNPs and 100μM SNPs on hippocampal brain slices were excitatory.The electrical excitability of hippocampal neuronal network at 4-72h induced by 5μM SNPs was excitatory;However,after the 100μM SNPs acted on the hippocampal neuronal network is excited for 4-24 h,its excitability gradually decreases with time,after 48h of action,it began to inhibit the neural network,and with the further extension of time,the inhibition gradually increased.By comparing the voltage threshold(VTh)results of hippocampal neurons and hippocampal brain slices,it was found that the effect of 5μM SNPs and 100μM SNPs on hippocampal neurons and hippocampal brain slices was excitatory after 4h treatment.3)The study explored the role and mechanism of NMDA receptors in the effects of SNPs on the electrical excitability of hippocampal neuronal networks.First,the VTMM was employed to study the role of NMDA receptors in the effects of SNPs on the electrical excitability of hippocampal neuronal networks with the NMDA receptor antagonist MK801.Then,the effects of SNPs on the number of NMDA receptors and the Ca2+content in hippocampal neurons were further investigated,and the relationship between these changes were discussed.The results showed that NMDA receptor played an important role in the influence of SNPs on the electrical excitability of neuronal networks,and the activation of NMDA receptor mediated the toxicity of SNPs on hippocampal neurons.4)In order to further understand the mechanism of the cytotoxicity of SNPs on neurons,this paper studied the effects of non-cytotoxic 5μM SNPs and cytotoxic 100μM SNPs on cell membrane potential difference,ROS,ATP content,apoptosis rate and neurite length.The results showed that except for the light increase of ROS content,the non-cytotoxic 5μM SNPs had no significant influence on the other molecular indicators of hippocampal neurons.The cytotoxic 100μM SNPs led to the increase of ROS content,the decrease of ATP content,cell apoptosis,and the shorten of neuronal neurite length.5)The combined analysis of SNPs on cytological and electrophysiological properties of rat hippocampal neuron,and the mechanism of the toxicity of SNPs on neurons were discussed.It showed that non-cytotoxic 5μM SNPs has an excitatory effect on hippocampal neuronal networks and can trigger the negative feedback regulation mechanism of neurons between the number of NMDA receptors and intracellular Ca2+content.Cytotoxic 100μM SNPs can also excite the electrical activity of neuronal network in a short time(4-24h).The cytotoxicity of 100μM SNPs to hippocampal neurons is increasing with the extension of action time,it leads to the increase of Ca2+ content,ROS content and cell apoptosis,the decrease of ATP content and the shorten of neuronal neurite length.This ultimately leads to a continuous decrease in electrical excitability.This study tried to explain the cellular and molecular mechanism of the change of electrical activity through the change of cellular and molecular indicators.The understanding of the toxic mechanism of SNPs on the central nervous system has been further studied based on the existing research. |
