The Effects And Mechanism Of Nanosized Titanium Dioxide On Synaptic Plasticity In Wistar Rats

Nanotechnology involves the creation and manipulation of materialsat the nanoscale level to create unique products that exploit novelproperties. Nanomaterials range in size from1to100nm. The newphysical and chemical properties of novel engineered nanoparticle makethem extremely attractive for using in applications such as medicalscience, drug applications, agricultural and defense industries. Nanosizedtitanium dioxide （nano-TiO₂）, one of the most widely used nanoparticles,has now been produced in a large industrial scale and used widely inpaints, paper and plastics, as well as in food additives and colorants.Despite wide ranges of applications, there is a serious lack ofinformation on the impact of nano-TiO₂on human health and theenvironment. The extensive uses in medical research and industrialapplications highlight that there are many routes for the nano-TiO₂to potentially enter into human bodies, such as through inhalation（respiratory tract）, ingestion （gastrointestinal tract）, dermal penetration（skin） and injection （blood circulation）. With the ultrafine size andunusual properties, nano-TiO₂can enter human body and cross biologicalbarriers such as blood brain barrier （BBB） relatively unimpeded. Thereby,the studies to evaluate the effects of nano-TiO₂on the central nervoussystem （CNS） are allimportant.In this study, we investigated the effects of nano-TiO₂on the spatialcognition capability of Wistar rats by intraperitoneal injection.Meanwhile, the cytotoxicology of nano-TiO₂and the effects of nano-TiO₂on the ion channels of hipocampal CA1neurons were detected inexperiments. Part1the effects of nano-TiO₂on the spatial cognition capability ofWistar ratsObjective: To investigate the effects of nano-TiO₂on synaptic plasticityand spatial cognition of Wistar rats, and discuss the underlingmechanism.Material and methods: Rats were randomly divided into three groups:normal control group,5mg/kg nano-TiO₂group and50mg/kg nano-TiO₂group. Rats were treated intraperitoneally with different concentrations ofnano-TiO₂suspension once a day for3weeks. After that, the Morriswater maze （MWM） test was employed to evaluate the spatial cognitivefunction. The long term potentiation （LTP） from Schaffer collaterals toCA1region in the hippocampus was recorded. The Ti content in thehippocampus was tested by the inductively coupled plasma-massspectrometry （ICP-MS）. In addition, the activity of superoxide dismutase（SOD）， glutathione peroxidase（GSH-Px） and the content of malondialdehyde （MDA） in hippocampus were detected to approach thepossible mechanism of neuron damage caused by nano-TiO₂.Results:1. The ICP-MS test showed that the Ti content in the hippocampus wasincreased in a concentration-dependent manner when rats wereexposed to nano-TiO₂, which indicated that nano-TiO₂could crossBBB and accumulate in the hippocampus region.2. The MWM test showed that the escape latencies in place navigationphase was significantly increased in50mg/kg nano-TiO₂groupcompared to those of5mg/kg nano-TiO₂group and the control group（P<0.01）. Moreover, in spatial probe phase, the time percentage intarget quadrant was significantly decreased in50mg/kg nano-TiO₂group （P<0.05）; there was no significance between5mg/kgnano-TiO₂group and control group. And the number of platformlocation crossings was also decreased in both the50mg/kg nano-TiO₂ group and the5mg/kg nano-TiO₂group compared to that of controlgroup.3. The LTP test indicated that the slope of excitatory postsynapticpotential （EPSP） was lower in50mg/kg nano-TiO₂group than that of5mg/kg nano-TiO₂group and control group （P<0.05）.4. In50mg/kg nano-TiO₂group and5mg/kg nano-TiO₂group, theactivity levels of superoxide dismutase （SOD） and glutathioneperoxidase （GSH-Px） were significantly decreased while the contentof malondialdehyde （MDA） was significantly increased comparedwith those of control group.Conclusion:Nano-TiO₂can cross the BBB and accumulate in the hippocampus.Meanwhile, nano-TiO₂could decrease LTP from Schaffer collaterals toCA1region in the hippocampus and impair the spatial cognitivecapability of rats, which may partly due to oxidative stress damage of neurons caused by nano-TiO₂. Part2cytotoxicolgy of nano-TiO₂on PC12cellsObjective: To investigate the cytotoxicology of nano-TiO₂on PC12cells.Methods: PC12cells were cultured with different concentrations ofnano-TiO₂for6,12,24and48h. The inverted phase contrast microscopewas used to observe the morphology of nano-TiO₂treated PC12cells,and the cellular viability was tested by3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Furthermore, the flowcytometry was used to detect the apoptosis of PC12cells and the level ofintracellular reactive oxygen species （ROS） after cells were treated bynano-TiO₂. The ROS scavenger N-（2-mercaptopropionyl）-glycine （N-MPG） was used to approach the relationship between the cellapoptosis and intracellular ROS accumulation.Results:1. When PC12cells were incubated with different concentrations ofnano-TiO₂for24h, there was a significant change in the cellmorphology, and the cell number was reduced in aconcentration-dependent manner.2. Results of MTT assay showed that the cellular viability wassignificantly decreased in concentration and time dependent mannerafter cells were treated with nano-TiO₂.3. Results of flow cytometry test: The treatment of nano-TiO₂induced theapoptosis of PC12cells and increased the level of intracellular ROS.4. The ROS scavenger N-MPG improved the cellular viability andreduced the damage caused by nano-TiO₂. Conclusion:Nano-TiO₂could cause the accumulation of intracellular ROS andinduce the cell apoptosis successively. Part3the effect of nano-TiO₂on voltage-gated ion channels in rathippocampal CA1neuronsObjective: To approach the effect of nano-TiO₂on voltage-gated ionchannels in rat hippocampal CA1neuronsMethods: The effects of nano-TiO₂on voltage-gated sodium currents （INa）and voltage-gated potassium currents were studied in rat hippocampalCA1neurons using the voltage clamp technique in the whole-cellconfiguration. Meanwhile, the effects of nano-TiO₂on the actionpotential （AP） were detected by the current clamp technique in thewhole-cell configuration.Results:1. The amplitude of INawas significantly inhibited by nano-TiO₂in aconcentration-dependent manner, which produced a positive shift inthe inactivation curve of INaand delayed the recovery of INafrominactivation. The activation curve was not affected. 2. The amplitude of transient outward potassium current （IA） and delayedrectifier potassium current （IK） was significantly inhibited bynano-TiO₂. Meanwhile, the steady-state inactivation curve of IAwasshifted to the left by nano-TiO₂, and the recovery of IAfrominactivation was delayed. There were no significant shift in theactivation curve of IAand IKafter nano-TiO₂treatment.3. In the present of nano-TiO₂, the peak amplitude and overshoot of theevoked single action potential were decreased, but the half-width wasincreased. Furthermore, the firing rate of repetitive firing wasdecreased.Conclusion:Nano-TiO₂could alter the excitability of neurons by impairingfunctions of voltage-gated sodium channels （VGSCs） and Kv, andthereby induce damages of neurons.