Influences Of Nanoparticle Zinc Oxide In The Electrophysiological Properties Of Hippocampal Neurons And The Mechanism Of The Biological Effects On PC12 Cells

Nanomaterials have been defined as material with one dimension of less than 100 nm. Due to their small size and the special structure, they have many new physical and chemical properties, such as small size effect, the large specific surface, high reactivity, quantum size effects. With the industrialization of nanotechnology, a variety of nanomaterials have a broad range of applications across industry, agriculture, manufacturing, military, medical and many other fields because of their excellent properties and novel functions. Nanomaterials are also widely used in daily life products, such as cosmetics, food, fabrics, coatings, antibacterial materials, etc., and public exposure to nanoparticles are increasing daily. Therefore, the biological effects and safety issues of nanomaterials have also been raised, especially the potential negative impacts of nanoparticles on human health, living environment and social security. Toxicological studies have shown that nanoparticles could enter into the human body through several distinct routes including inhalation, ingestion, and dermal penetration. Subsequently they could elicit toxicological effects at different levels of biological systems. Recent studies have shown that the central nervous system (CNS) is an important target organ for nanoparticles. Therefore, the effects of nanoparticles on the CNS have also gained more attention. Nanoparticles can cross most strong biological barriers such as blood-brain barrier and via the olfactory neuronal pathway enter into the CNS and accumulate in different brain regions. So the hippocampus is also the main target for nanoparticles.Breaking achievements and great attention have been gained in the important applications of Nano-ZnO because of its unique optical, electrical and magnetic properties. Meanwhile, Nano-ZnO have excellent applications in cosmetics, textiles, paints, ceramics, catalysis, antibacterial, medical diagnostics. Some researchers have demonstrated that nanostructures of ZnO were toxic to the bacteria, the aquatic biota or eco-relevant species, the mammalian cells and mammals. However, little is known about the possible impacts of manufactured nano-ZnO on the CNS. In the present study, differentiated PC12 cells induced by nerve growth factor (NGF) were used to investigate the cytotoxicity of the nano-ZnO. The viability of the cells was observed by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the generation of ROS for the cells was evaluated by a fluorometry assay. The apoptosis of cells were detected and analyzed by flow cytometry.The neurons in the CNS express various types of ion channels, and the cell electrical activity caused by the opening of ion channels are the basis of physiological function. Ion channels involved in transmitter release, hormone secretion, signal transduction, metabolic regulation and cell growth as well as learning and memory and other important physiological processes, and they are also targets for many toxins and drugs. Therefore, whole-cell patch clamp technique was used to study the effects of nano-ZnO on ion channels of hippocampal pyramidal neurons, including voltage-gated sodium channels, potassium channels, calcium channels and neuronal excitability, and investigate the potential mechanism of nano-ZnO on the CNS. The main results are as follows:1. MTT cell viability assay:Nerve growth factor (NGF) induced differentiation of PC12 cells were incubated with different concentrations of the nano-ZnO (10-6 g/ml,10-5 g/ml,10-4 g/ml) for the periods 6,12,24 hours, and cell viability was decreased in a dose-dependent and time-dependent manner. Nano-ZnO (10-4 g/ml) caused a significant decrease in cell viability (P<0.05).2. Changes in intracellular ROS levels:PC12 cells were incubated with different concentrations of the nano-ZnO (10-6 g/ml,10-5 g/ml,10-4 g/ml) for 6h, and then treated by GENMED working solution and GENMED preserving fluid. Using the inverted fluorescence microscope, setting the excitation wavelength at 490nm and 530nm, the fluorescence intensity of nano-ZnO treatment group gradually increased with the increasing concentration of nano-ZnO compared to that of the control group. The result indicated that intracellular ROS levels were increased.3. The proportion of apoptotic cells by flow cytometry analysis:PC12 cells were incubated with different concentrations of the nano-ZnO (10-6 g/ml,10-5 g/ml,10-4 g/ml) for 24 hours, and the apoptosis rate of PC12 cells was increased from 8.75% in control group to 34.24% (P<0.05) after the cell exposure to the nano-ZnO (10-4 g/ml) for 24h, However, a pre-treatment to the cells using MPG (3 mM) and then treatment to PC12 cells 24h with nano-ZnO (10-4 g/ml) reduced the cellular apoptosis to 15.78% (P<0.05). The result indicated that pre-treated the ROS scavenger can inhibit the nano-ZnO induced apoptosis in PC 12 cells.4. Protective effects of ROS scavengers:PC12 cells were pre-treated with MPG (3 mM) for 30 min and then were incubated with nano-ZnO (10-4 g/ml) for 24h.The viability of PC 12 cells was significantly increased compared to that of the nano-ZnO treatment group (P<0.05).5. Effects of nano-ZnO on voltage-gated sodium channels in hippocampal pyramidal neurons:In the present of final concentration of 10-4g/ml nano-ZnO, the peak amplitudes of INa were increased considerably from-50 mV to+20 mV(P<0.05), and the current-voltage curve of sodium current (INa) was decreased. Meanwhile, the values of Vh for inactivation of Ina before and after addition of nanoparticle ZnO are-52.54±0.43 mV and-54.67±0.39mV (P<0.01); The time constants (r) before and after addition of nano-ZnO were 5.40±0.19 ms and 3.95±0.15 ms (P<0.01), respectively. However, the steady-state activation curve of INa was not shifted by the nano-ZnO.6. Effects of nano-ZnO on voltage-gated potassium channels in hippocampal pyramidal neurons:The amplitudes of transient outward potassium current (IA) were increased by the nano-ZnO solution (10-4g/ml), while the current-voltage curve of delayed rectifier potassium current (Ik) was significantly increased from+20 mV to +90 mV (P<0.05). However, it is apparent that the nano-ZnO solution didn't shift the steady-state activation curve of IA and IK, and neither had significant effects on the inactivation and the recovery from inactivation of IA.7. Effects of nano-ZnO on high-voltage activated (HVA) calcium currents in hippocampal pyramidal neurons:HVA calcium currents had a tendency of enhancement (run up) in the first 5 minutes of the recording in both the experimental and control groups, and were followed by a decline (run down) with time. At 10 minutes of the recording,10-4g/ml nano-ZnO first altered the current-voltage curve and the peak amplitudes of HVA calcium currents (P<0.05), and at the end of 30 minutes the peak current amplitudes were increased significantly from-15 mV to+15 mV(P<0.05) compared to that of the control group. But there were no statistically significance the steady-state activation curve and the steady-state inactivation curve of HVA calcium currents compared with that of the control group.8. Effects of nano-ZnO on excitable activity in hippocampal pyramidal neurons excitability:the results of current clamp showed that peak amplitude and overshoot of the evoked single action potential were increased in the presence of the 10"4g/ml nano-ZnO solution (P<0.05) and half-width was diminished (P<0.05). Simultaneously, a prolonged depolarizing current injection enhanced repetitive firing evoked firing rate(P<0.05) and firing rate of spontaneous firing was also increased (P<0.05).The main conclusions are as follows:1. Nano-ZnO can cause a significant decrease in cell viability in a dose-dependent and time-dependent manner and promote apoptosis in PC 12 cells; while the increase of intracellular ROS are one of potential mechanisms of cellular apoptosis induced by nano-ZnO.2. Nano-ZnO could involve in the neuronal apoptosis caused by the loss of cytoplasmic K+ due to increased K+ efflux through delayed rectifier potassium channels.3. Nano-ZnO could cause the elevation of cytosolic calcium levels by up-regulation HVA calcium channels, which would increase the generation of intracellular ROS, and consequently, promote neuronal apoptosis.4. Nano-ZnO could enhance the neuronal excitability by increasing the peak amplitudes of INa and promoting the inactivation and the recovery from inactivation of INa, which may involve in depolarization-induced neuronal injury by activation of voltage-gated Na+ channels. Meanwhile, nano-ZnO would potentially perturb cellular calcium homeostasis by up-regulation HVA calcium channels, and contribute to the neurodegenerative process.5. Nano-ZnO could enhance the neuronal excitability by increasing the peak amplitudes of INa, IK and HVA calcium currents, and therefore increase the peak amplitude and overshoot of the evoked single action potential and diminish half-width. Simultaneously, firing rate of repetitive firing and spontaneous firing were aslso increased. The decrease of peak amplitude and overshoot of the subsequent action potentials of repetitive firing may due to the dysfunction or deficiency of Na+-K+-ATPase.