| Background"Nanotechnology" is one of the key technologies in twenty-first Century, it plays a revolutionary change in the development of modern society. "Nano materials" is defined as a presence in the non binding state, or aggregation, or with one or more coalescence material, its exterior dimensions in the range of 1-100 nm.With the rapid development of nano technology, nanoparticles (NPs) based on its good biological compatibility and lower cost become one of the most widely used materials, and widely used in industry, agriculture, cosmetics and clothing and to apply in the healthcare and life sciences.The size of nanomaterials is similar to DNA, proteins, viruses and biological molecules, the biological effects of some nanoparticles including the mechanism of interactions between organisms and the environment, the mechanism is still not well understood. People have a lot of studies in vitro or in vivo, to explore the interaction of nanomaterials and biological macro molecules, cells, organs and tissues, only found that biological toxicity effect of the nanomaterials may be caused by the mechanism of oxidative stress and inflammatory mechanisms. At the same time, the physical and chemical characteristics of nanomaterials also has a significant impact, including size, shape, surface charge, chemical composition, surface modification, metal impurities, agglomeration and dispersion, degradation, and the protein crown formation.There is no doubt that these characteristics have increased the complexity of toxicology. Recent in vitro and in vivo data show that nanoparticles can enter through the respiratory tract inhalation, absorption in the digestive tract, skin to skin contact, drug injection, planting body releases or other means into the human body, and then through the lymphatic circulation and blood circulation or nerve reach the second target organ, the possible biological toxicity. There is evidence that inhaled or intranasal instillation of nanoparticles can directly enter the olfactory lobes of the brain via the olfactory epithelium and neurons, to enter the central nervous system, induce significant inflammatory reaction, this pathway including olfactory organ or trigeminal nerve system, starting in brain regions, passing the blood brain barrier, finally distribute to the nasal olfactory epithelium or respiratory epithelium. Zhang found that the intranasal instillation of TiO2 nanoparticles, particles can enter the cerebral cortex and hippocampus through olfactory pathway. In addition, Andrea found that the sub-acute and sub-chronic inhalation of ZnO nano particles, particles will be highly soluble in the lung tissue, and Zn can enter blood circulation through translocation.Visceral sensory pathway of the nervous system is divided into general visceral sensory pathway and special visceral sensory pathway, including olfactory and gustatory pathway. Nanoparticles can be by nasal instillation into the olfactory cells, to enter the olfactory pathway, caused by the neurotoxic effects of animals. Considered gustatory nerve and olfactory nerve have such similarity, maybe nanoparticles can also go through the gustatory nerve by lingual instillation.Therefore, this study intends to use adult male Wistar rats with lingual instillation. Materials were selected as ZnO NPs and TiO2 NPs suspension, administration for 30 days, to explore whether nanoparticles can go into the gustatory nerve pathway by lingual instillation, and evaluate the central neurotoxicity in animals.Objection1. To explore whether the nanoparticles can enter the body through the animal lingual instillation.2. To explore the content of nanoparticles by lingual instillation in animal in vivo deposition.3. To assess the oxidative stress related to central neurotoxicity of nanoparticles.4. To explore the influence of nanoparticles to central neurotoxicity through morphological observation and immunohistochemical analysis.5. To explore the influence of nanoparticles on the cognitive function of spatial memory and learning in rats by Morris water maze.The thesis is composed of the following two chapters:Chapter 1 The nanoparticles enter into Wistar rats by gustatory nerve pathwayMaterials and Methods1. Characterization of nano materials1) To observe the surface morphology by transmission electron microscope (TEM) Using TEM to observe the morphology and structure of ZnO NPs and TiO2 NPs, and calculate the average particle size2) Analysis of chemical constituents of nano materials To analyse the chemical composition of ZnO NPs and TiO2 NPs by the Energy Dispersive Spectromete (EDS).3) Analysis of X-Ray Diffraction (XRD) Analysis of ZnO NPs and TiO2 NPs by XRD at room temperature, using Cu-K alpha Ni filter.4) Analysis of the Zeta potential and the agglomeration of nanomaterials The ZnO and TiO2 NPs nanoparticles dispersed in sterile water, vortexed for lmin, and configured for suspension. Measurement of nanoparticle suspension for Zeta potential and hydrodynamic size.2. The construction of animal model and detection of elements in nerve tissue and brain tissue1) Preparation of ZnO NPs and TiO2 NPs suspension ZnO NPs and TiO2 NPs powder were weighing 1.5 g respectively, pour into 100 mL serum bottle marked "ZnO NPs" and "TiO2 NPs" with 30 mL sterile water. Used turbine vigorously mixing, to formed the concentration as 50 mg/mL. Both serum bottle put into the hot water, when the water temperature reached 85 to 100℃, with the balance respectively weighing 0.3 g hydroxypropyl methyl cellulose (HPMC) according to the proportion of 1% were added separately to the ZnO NPs and TiO2 NPs serum bottle.2) Ultrasonic mixing Configuration of mixed suspension into the ultrasound machine, frequency was adjusted to 95%, the temperature was adjusted to 40℃, ultrasound for 90 min, repeated ultrasound. After each administration before configuration good mixed suspension heated again, turbine vigorously mixing, the bottom of the bottle without precipitation, into the ultrasound machine super sound for about 30 minutes, until the drug evenly dispersed without deposition or obvious agglomeration.3) Animals selection Weight purchased from the animal center of Southern Medical University. A total of 30 adult male Wistar rats, the nanometer zinc oxide group and nanometer titanium dioxide group and control group with 10 rats in each group, rat body healthy, normal activity, hair color and normal densities.4) Anesthetic preparation Took 1.0 g of sodium pentobarbital into the serum bottle containing 100 mL of physiological saline, made the concentration of anesthetic as 1%, and mixed until no visible particles.5) Lingual instillation Intraperitoneal injection of 1%pentobarbital sodium with dosage of 0.48 mL/100 g. Then took the rats in lateral position on the operating table, pulling out the tongue from the side of the mouth. Experimental group were divided into two groups, respectively instillation with ZnO NPs and TiO2 NPs with dose 5 mg/100 g. Control group instillation with equivalent sterile water. Administration time was 1,3,5,7......27,29 days, sacrificed in the thirtieth days, and tissues were extracted to detect.6) Detection of elements in nerve tissue and brain tissue Extracted the chorda tympani and glossopharyngeal nerve, cerebellun, brainstem, cerebral cortex, hippocampus, detected Zn content in ZnO NPs group, Ti content in TiO2 NPs group, Zn and Ti content in control group by inductively coupled plasma mass spectrometry (ICP-MS).7) Statistical analysis SPSS 20.0 software was used for statistical analysis. All data are expressed as mean±standard deviation. Comparisons between groups were tested by one-way analysis of variance (ANOVA), least significant difference (LSD) post hoc tests when the variance was regular, otherwise Dunnett’s T3 test was used. All differences were considered significant when P< 0.05.3. Detection of brain tissue and nerve tissue by transmission electron microscope Hippocampus, cerebral cortex and nerve tissue in ZnO NPs group, TiO2 NPs group and the control group of rats were extracted, fixed into the fixative, then observed tissue in the presence of nanoparticles and tissue damage.Results1. ZnO NPs and TiO2 NPs were in high purity, no impurities, the particle sizes were about 50 nm, and prepared suspension dispersed uniformly, no obvious aggregation or precipitation.2. Lingual instillation with ZnO NPs suspension, TiO2 NPs suspension, sterile water, administration time were 1,3,5,7......27 and 29 days. Rats were sacrificed in the thirtieth days, extracted chorda tympani, glossopharyngeal nerve, cerebral cortex, hippocampus, cerebellum, brainstem, heart, liver, spleen, lung and kidney.3. Using ICP-MS detection of ZnO NPs experimental group Zn, TiO2 NPs in the experimental group Ti, were compared with the control group of Ti and Zn element found experimental group Ti and Zn element were significantly higher than that of control group, and the difference was statistically significant.4. Detection of nerve tissue and brain tissue by transmission electron microscopy, nanoparticles were deposited in ZnO NPs and TiO2 NPs group. The control group had no obvious deposition of nanoparticles.5. TEM observed that both brain and nerve tissue in ZnO NPs and TiO2 NPs group were injuried, such as mitochondria swelling, crista fragmentation, endoplasmic reticulum swelling, glial cell nuclear pyknosis, nuclear fast crack, neurons sag, cytoplasmic free plate layer loose solution, formation of vacuoles, myelin axonal atrophy, vacuolization, visible autophagosome and lysosome formation. Control group did not appear any injury in the brain and nerve tissue.Chapter 2 To investigate the neurotoxicity of nanoparticles through the gustatory neural pathways in the brainMaterials and Methods1. Oxidative stress damage in brain tissue1) The frozen samples were extracted, and the ZnO NPs, about brain 0.2 g of TiO2 NPs in the control group, physiological saline, as far as possible ensure samples in 4℃ environment, using tissue homogenate prepared homogenate (1:9 V/V), about 3-5 times, each time interval 30-60 s, a total of about 10 min, centrifuged at the speed of 2500 R/min. The supernatant was collected.2) The Nan jing Jian cheng kit were used to detect the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) glutathione (GSH) glutathione peroxidase (GSH-Px) and GSH/GSSG. According to the instructions to add the appropriate reagents. For a plate in the 96 hole plate, using fluorescence eliasa measurement of fluorescence intensity, excitation wavelength and emission wavelength.3) Expression of oxidative stress-related genes Collected the brain tissue, uptook total RNA, reversed transcription to cDNA, and tested oxidative stress-related genes such as Dhcr7, Cyp51, Gsr, Noxl, Sod2, Nqo1, Fmo2 by qRT-PCR.β-actin acted as internal reference.4) Statistical analysis SPSS 20.0 software was used for statistical analysis. All data are expressed as mean ±standard deviation. Comparisons between groups were tested by one-way analysis of variance (ANOVA), least significant difference (LSD) post hoc tests when the variance was regular, otherwise Dunnett’s T3 test was used.. All differences were considered significant when P< 0.05.2. Morphological observation and immunohistochemical analysis1) The removal of the brain tissue was fixed, paraffin embedded, by slicing, slice thickness of about 4 μm.2) The sections were stained with hematoxylin and eosin (HE), then observed under the optical microscope.3) Immunohistochemical analysis with the slices stained by Ki-67 to the presence of proliferation cells,8-OHDG to the DNA damage cells and GFAP to astrocytes, all of which were randomly selected in cerebral cortex and hippocampus region were photographed.3. Analysis of neurotoxicity of nanoparticles in animals behavior1) Water maze was a diameter of about 2 meters of steel pipe concrete, a depth of about 80 cm, the water temperature constant at 26℃, adding opaque black ink filled with water after. The pool was divided into four quadrants:Northeast, Southeast (NE) (SE) (SW), southwest and Northwest (NW). Built in a circular platform (10 cm diameter), a central water level control quadrant,2 cm above the platform position in the round.2) Recorded the searching time and motion platform for the rats in the water maze on 1-8 days.3) Using the software SPSS 20.0 the obtained data were statistically analyzed. The determination results of the repeated measures analysis of variance (ANOVA) and multivariate variance analysis was used to compare the differences between different time points and different groups of pairwise, homogeneity of variance test, a=0.05 level test.4) Expression of learning and memory-related genes Collected the brain tissue, uptook total RNA, reversed transcription to cDNA, and tested oxidative stress-related genes such as Sgkl, Drd2, Trappc4, Gnaq by qRT-PCR. β-actin acted as internal reference.Results1. Oxidative stress damage in brain tissue1) ZnO NPs group and TiO2 group NPs SOD activity was significantly lower than the control group. ZnO NPs group and TiO2 NPs group MDA were significantly higher than the control group. ZnO NPs group and TiO2 NPs group GSH, GSH-Px, GSH/GSSG were significantly lower than the control group.2) Compared to control group, most expression of oxidative stress-related genes showed up regulations or down regulations in ZnO NPs group and TiO2 NPs group.2. Morphological observation and immunohistochemical analysis1) Under the microscope observation, HE staining of ZnO NPs and TiO2 NPs group of brain cortex and hippocampus had tissue dissolution, indicating that pathological changes in ZnO NPs and TiO2 NPs group.2) Under the microscope observation showed that Ki-67,8-OHDG and GFAP staining after ZnO NPs group and TiO2 NPs group in cerebral cortex and hippocampus, the number of proliferation cells was lower than that of control group, the number of DNA damage cells was higer than that of control group, and the number of astrocytes was higher than that of control group, indicating that the experimental group had brain injury.3. Analysis of neurotoxicity of nanoparticles in animals behavior1) In 1,2,3,5,8 days, time to find the platform ZnO rats in NPs group were significantly longer than the control group. On the sixth day, the rats in ZnO NPs group went through the platform and the residence time was shorter than the control group with significant difference.2) In 2,3,4,5,8 days, time to find the platform TiO2 rats in NPs group were significantly longer than the control group, with statistical difference. On the sixth day, the rats in TiO2 NPs group go through the platform and the residence time was shorter than the control group with significant difference.3) Compared with control group, most expression of oxidative stress related genes have significant difference in ZnO NPs group and TiO2 NPs group.Conclusion1. Nanoparticles can deposite in the brain after lingual instillation in Wistar rats for 30 days.2. The oxidative stress kits find that the SOD activity decreases, MDA increases, GSH, GSH-Px and GSH/GSSH decrease, indicating oxidative stress in the brain tissue damage is caused by ZnO NPs and TiO2 NPs nanoparticles.3. Morphological observation shows that tissue disolution in cerebral cortex and hippocampus in ZnO NPs and TiO2 NPs group.4. Immunohistochemical analysis shows that in cerebral cortex and hippocampus prolification cells decrease and DNA damage increase in ZnO NPs and TiO2 NPs group.5. Behavioral analysis shows that rats cognitive and learning function in ZnO NPs and TiO2 NPs group are worse than the control group,which indicates that nanoparticles cause cognitive and learning function disorders. |
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