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Research of Graphene Oxide on Nervous System Toxicity in Sprague Dawley Rats in Vivo and in Vitro

Author: ZhaoXiangQin
Tutor: WangShaLi
School: Chongqing Medical University
Course: Physiology
Keywords: graphene oxide neural stem cells neurons toxicity invitro and vivo experiments
CLC: R114
Type: Master's thesis
Year: 2013
Downloads: 54
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Abstract

Objective：Graphene oxide is the derivative oxidized from graphene,ten to several hundreds nanometers or even microns in size, having goodstability in water and higher fluorescence quenching efficiency and strongadsorption capacity, with a large number of hydroxyl group, epoxy group insurface and oxygen-containing reactive group, such as carboxyl group,carbonyl group in marginal. The unique physical and chemical propertiesmake them possible to be modified and functionalization for achieving avariety of functions, such as targeted drug loading and sustained release etc.Therefore, GO shows the inestimable prospect in tumor targeted therapy,cell imaging, biosensors and biological detection, favored by thebiomedical field.Although nanotechnology brings great benefits to humans, it may alsopotentially bring dangerous to humans and ecosystems at the same time.Nanoscale particles are easily enter in vivo, just as small molecules shuttlefreely between all parts of the body, then rapidly distribute in variousorgans and tissues of the body except the brain, and interact with the tissue, cells, organelles and biological macromolecules (such as proteins), whichwill result in dysfunction of the tissue or cells and affect the health of theorganism. Studies have found that nanomaterials can penetrate theblood-brain barrier. Kreuter found that polysorbate-80wrapped doxorubicinnanoparticles administrated by intravenous injection can penetrate BBBthrough brain capillary endothelial cells’ endocytosis. Oberd rster alsoconfirmed that nanomaterials can also enter the central nervous systemthrough the olfactory nerve pathway or transported directly to the brain bysensory nerve endings. Once nanoparticles enter into the nervous system,they can activate microglia to produce ROS-induced oxidative stress andinflammation, which will induce neurotoxicity-mediated neural tissuedamage. CNS is the most important organ in the body, which integrates theinformations of body’s tissues and organs and regulates the bodyphysiological activity and brain senior activities. Therefore, it is veryimportant to evaluate the biosecurity of nanomaterials on the nervous systemfor clinical implications, which determines the application prospects ofnanomaterials to a large extent.Little has been reported whether GO entered into the body can penetratethe blood-brain or blood brain barrier and affect the nervous system.Therefore, this study comprehensively evaluate the effect of GO sized100～500nm on the nervous system in vitro and in vivo, which may provideobjective and scientific experimental basis for GO applications in many fields.PART ONE NEUROTOXICITY OF GRAPHENE OXIDETO SPRAGUE DAWLEY RATS IN VIVOMethods:1. Identify the characterization of GO1.1Using high resolution transmission electron microscopy to observe thesurface characteristics of GO;1.2Using Raman spectroscopy to detect surface groups of GO;1.3Using UV scanning to observe GO’s absorption peak.2Animal experiments32healthy and clean SD rats (4-5weeks of age, weight95±10g) wererandomly divided into4groups, including control group (saline) and3GOexperimental groups (2.5,5and10mg/kg/d weight, respectively), eachgroup consisted of8rats was treated with intravenous injection once a dayfor5days continuously,2days off to test the experiments indicators for4weeks continuous injection. Animals were sacrificed after the water mazetest. During the experiment the following indicators were observed andrecorded:2.1General situations: animal activities and mental condition, daily foodconsumption and body weight. 2.2The following experimental indicators were tested in theintravenous GO before and7d,14d,21d,28d, respectively.Open-Field Test: To observed neuropsychiatric activities of experimentalanimals;Functional observational battery (FOB): to comprehensively evaluatethe autonomic behavior, activity and excitability of central nervoussystem, neuromuscular response and sensory-motor function ofexperimental animals.2.3On29thof the experiment, Morris water maze to detect learning andmemory as well as space exploration of laboratory animals.2.4The rat tissues (brain, cerebellum, sciatic nerve and spinal cord lumbarenlargement) were dissected after water maze test and observed thehistopathological changes by optical and transmission electron microscopyafter fixing, embedding, sectioning and staining.Result:1. All rats were generally in good condition, no death and disease, there wereno significant difference in daily food consumption and body weight of eachdose group (p＞0.05).2. Open-Field Test: There were no significant differences in horizontal andvertical movement of the experimental animals（(p＞0.05).3. FOB: There were no significant differences in activity and neuromuscularmeasures, sensorimotor and excitability measures, and autonomic measures of each dose group (p＞0.05).4. Morris water maze: Compared with the control group, there were nosignificant difference in escape latency and swimming distance in GOexperimental groups（p＞0.05）; there was no significant difference inswimming time in original platform quadrant, the percentage of theswimming distance in the original platform quadrant to the total distance andthe number of platform location crossings in all groups (p＞0.05).5. Histopathological observations: No GO sheets were observed in brain,cerebellum, sciatic nerve and spinal cord lumbar enlargement and nosignificant histopathological changes were found in these tissues underoptical microscopy or TEM.Conclusion：No GO sheets existed in the central and peripheral nervous system,suggesting that GO can not pass the blood-brain and blood brain barrier,therefore, for the experimental animals:1. There were no apparent effect on growth and development,neuropsychiatric conditions, motor and sensory.2. There were no apparent effects on learning and memory, and spaceexploration abilities.3. There were no significant histopathological changes in the central andperipheral nervous system. PART TWO THE EFFECT OF GO ON NEURAL STEMCELLS AND HIPPOCAMPAL NEURONSMethods:1. Identify the characterization of GO: just as the above.2. The primary cultured NSCs and hippocampal neurons cultured for6~7days were seeded in6-well or96-well plates, respectively, which randomlydivided into six groups: control (without GO) and GO groups (treated withGO of10,25,50,100and200μg/mL, respectively) for24h or120h. Thefollowing experimental indicators were observed:1. During culture period, the morphological characteristics of the cells wereobserved under inverted phase contrast microscope.2. After cultured for24h or120h:Ultrastructure changes of cells were examined by TEM;Cell viability were analyzed by MTT;Cell mortality was evaluated by Trypan blue exclusion;Apoptosis and intracellular ROS were measured by flow cytometer;Membrane integrity was evaluated by LDH assay.Result:1. Cell morphology and ultrastructure: Compared with the controlgroup, there were no significant difference between the NSCs and neurongroups. 2. Cell viability, Cell mortality, Membrane integrity, Cell apoptosis andAssay of intracellular ROS: There were no statistically significantdifferences between the groups, under the dosing conditions at both timepoints.Conclusion:GO exposure did not induce visible toxicity in neurons or NSCs in both24and120h treatments. The underlying mechanisms may be partly due toGO’s inability to enter NSCs and neurons and/or its inability to induce anoxidative stress response, suggesting that GO had good biocompatibilitywith CNS.Combining the vitro and vivo experiments, we believe that the GO mayhave good biocompatibility on the nervous system and is a safe nanomaterialfor nervous system.

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