Study Of Toxicity And Mechanisms Of CdTe Quantum Dots On Rat Hippocampus

Presently,the novel nanotechnology has changed the field of medicine significantly.As we know,nanotechnology,showing a huge applicable potential,could be used to solve a number of medical problems through providing specific diagnosis and treatments.Quantum dots(QDs),as a common nanomaterial,provide long stable and multiple detection signals due to their excellent optical property.However,it is important to assess the bio-safety of QDs before they are allowed to be used in the bio-medical studies,expeically in the clinical medicine.So far,there have been some studies relevant to the biological effects of QDs,but the number of studies on the toxic effects of them on the central nervous system(CNS)is limited.Since the critical applications of QDs on the neuroscience basic study or the diagnosis and therapy of nervous system disorder,it is urgent and necessary to do a systematic investigation of the toxic effects of CdTe QDs on the CNS.According to the above consideration with our group's previous achievements on the hepato-,renal-and respiratory-toxicity of MPA-modified CdTe QDs,we decide to investigate the toxic effects of CdTe QDs on the rat hippocampus and the underlying mechanisms of QDs causing these effects on the genetic,biomolecular,cellular,model animal and whole mammal animal levels.The thesis would attempt to answer the following parts:1.The electrochemical method is used to synthesis the water-soluble MPA-modified CdTe QDs with high quality.In order to assess the physical and chemical properties of QDs,some instruments and methods,including transmission electron microscopy(TEM),ultraviolet-visible light spectrophotometer,fluorescence spectrophotometer,X-ray diffractometer and Malvern size analyzer,etc.,are used.2.The toxic effects of CdTe QDs on model animals called Caenorhabdits elegans(C.elegans)are studied in the first time.Some indicators are used to evaluate the effcts of CdTe QDs on the behaviors relevant to nervous system,including location behavior,pharyngeal pumping,defecation,learning and memory.Based on the results,the possible ways in which CdTe QDs involved to cause these effects are investigated as well.3.The toxic effects of CdTe QDs on primary cultured rat hippocampal neurons are studied after then.The tested indicators include the cell viability,apoptosis,intracellular ROS generation and intracellular calcium balance and dose-effect relationship.Meanwhile,considering the unique physical and chemical properties,the toxicity difference of the two size CdTe QDs is discussed.Based on the results,The complex mechanisms of CdTe QDs causing the high level of intracellular calcium are specially investigated from the angle of neuro-electrophysiology.4.The toxic effects of CdTe QDs on rat hippocampus are studied thoroughly.We observe the changes of rats' learning and memory ability after CdTe QDs exposure,and try to find the reason from hematological and pathological researches.The whole RNA sequencing(RNA-seq)is used to figure out the biomolecular toxic mechanisms of CdTe QDs.Based on the results,three signaling pathways in which CdTe QDs might involved to cause toxic effects were studied deeply.They are PI3K-Akt and MAPK signaling pathways that are relevant to the foundation of learning and memory,and NF-?B singaling pathway that is relevant to inflammatory response.The main finding of the work described as follows:1.The water-soluable MPA-modified CdTe QDs used in this study are prepared by the electrochemical method.The CdTe QDs with average sizes of 2.2 nm and 3.5 nm are based on the water bath temperature reaction for 2 h and 20 h,respectively.They represente excellent optical property,showing green and red fluorescence.The fluorescent intensity would still be strong for at least four weeks The results of TEM show that QDs are spherical,the particle dispersion is uniform,and the stability is good.The surface charges of the two QDs are both negative.Taken these results together,the MPA-modified CdTe QDs meet all requirements of all experiments in this study.2.The model animal study show that CdTe QDs exposure induce behavioral defects,including alterations to location,pharyngeal pumping and defecation intervals,as well as impaired chemotaxis-based learning and thermotaxis-based memory.The toxic effects of CdTe QDs treated for 72 h are significantly stronger than those for 24 h.Further investigations suggest that CdTe QDs exposure can induce these toxic effects through affecting the transporters and receptors of glutamate,serotonin and dopamine in C.elegans at the genetic level.Otherwise,we also observe the excessive ROS generation in QD-treated nematodes,which suggests the important role of oxidative stress in the neurotoxic effects of CdTe QDs.However,owning to the complexcity of neurotransmissions and the diversity of genes,it is still neccesary to evaluate the neurotoxic effects of CdTe QDs and the underlying mechanism in traditional mammal animals through classical and innovative toxicological researches.3.In the cultured cells researches,we observe that CdTe QDs can reduce the cell viability of primary cultured rat hippocampal neurons,and induce apoptosis in a dose-dependent manner.The reason of CdTe QDs causing these effects is CdTe QDs improve the ROS production in neurons,resulting in oxidative stress damage and intracellular calcium imbalance.After then,the antioxidative NAC is used to confirm the excessive intracellular ROS generation is one of action mechanisms to cause these toxic effects.Considering the complexity of intracellular calcium level increase,apart from the oxidative stress,patch clamp technique,a classical neuro-electrophysiological method,is applied to figure out more ways of CdTe QDs causing intracellular calcium imbalance.The results show that CdTe QDs exposure increase the number of activated HVA calcium channel number in neural membranes,and inhibit their inactivation,therefore promoting calcium ions to flow from outside to inside of cells.Meantime,CdTe QDs exposure can increase the current amplitude of sodium channel,accelerate the activation and deactivation of sodium channel,and slow its recovery after deactivation,resulting in calcium ions inflow through calcium and sodium exchanges in the cell membrane.Finally,the several calcium imbalance inevitably resultes in neuron impairments.However,the findings from in vitro study are still not perfect,it is indispendsable to assess the toxic effects of CdTe QDs in the CNS of living rodents.4.In the in vivo study,intrahippocampal injection of CdTe QDs is used to stimulate a successfully clinical application of QDs for diagnosis and therapy of neurological disorders in the hippocampus.As the tested CdTe QDs are not conjugated with any biomolecules,the toxic effects and potential mechanisms in the CNS would be clear and explicit.Additionally,it assures the integrity of this thesis since using the same QDs in all experiments.The findings show that CdTe QDs exposure induce the impairments of learning efficiency and special memory,which might be associated with the pathological changes and disruption of ultrastructure of hippocampal neurons and synapses.Apart from explaining the toxic effects of CdTe QDs in the pathological level,we obtaine the possible target genes of CdTe QDs through novel technique---RNA-seq analysis.According to the information from RNA-seq,Toll-like receptor-signaling pathway,which includes three pathways,is focued on right here.The results show that CdTe QDs exposure inhibit the phosphorylation of proteins AKT and ERK1/2 in the PI3-Akt and MAPK signaling pathways,which directly or indirectly downregulate the expression level of nuclear protein c-FOS to damage synapses plasticity and inhibit the LTP genenration,resulting in rat learning and memory.Otherwise,CdTe QDs activate the MyD88/TLR2/NF-?B pathway and NLRP3 inflammasome in microglial cells that are immune cells in the CNS,which increase pro-inflammatory cytokine IL-1? secretion,resulting in inflammatory reaction.Since the study showing the neurotoxicity of QDs is limited,the purpose of this thesis is to investigate the effects of CdTe QDs on the rat hippocampus and the underlying toxic mechanisms.Herein,the findings show the toxic effects of CdTe QDs on the cultured hippocampal neurons,microglial cells,nervous system of model animals C.elegans and hippocampus of living rats.Some reported ways,such as ROS generation and neurotransmission disorder,in which CdTe QDs involve to cause these effects are confirmed.Some toxic molecular mechanisms,especially Toll-like receptor-signaling pathway,is deeply investigated.This thesis can provide not only a series of indicators as toxicological endpoints to evaluate the neurotoxicity of QDs,but also references for further neurotoxicological studies and toxicological studies involving more than one organ or biological system.