| Aluminum oxide nanoparticles(Al2O3-NPS)are the most production and widely used nanomaterials.Large numbers of Al2O3-NPS diffusing into atmosphere,are absorbed into the body,which needs to reevaluate toxicity of intranasal instillation Al2O3-NPS,especially toxicity and harm of the explosion of the intranasal instillation Al2O3-NPS during pregnancy.We established models of SD rats and C57BL/6 mice which were intranasal instillation Al2O3-NPS during pregnancy.Pregnant rats and mice were randomly divided into three groups(ddH2O,Al2O3-NPs,AlCl3(0.7mg Al/kg and 1.4mg Al/kg).Pregnant rats were intranasal instillation every day during pregnancy and stopped until offspring were born.Offspring SD rats were sacrificed after being anaesthetized and collected samples respectively on P1 and P21.ICP-MS was used to analysis and detect serum and organs total Aluminum content on P1 and P21.We detect the levels of synaptic associated mRNA and protein(NR2B,PSD-95,SNAP25).After heart perfusion,CA1 area of hippocampus of P21 SD rats were separated prepare TEM samples to observe synaptic structure changes.C57BL/6 mice were conducted learning and memory experiment on P35(step-down test,avoiding darkness test and water maze test)and sacrificed after being anaesthetized and collected samples on P50.Western Blot was used to analyze the levels of proteins(NR2B,PSD-95,SNAP25).P1 total aluminum content in serum shown that Al2O3-NPS group was significantly higher than control group and AlCl3 group.However,no significant differences were found among groups for P21.In liver,lung,spleen,kidney and brain of PI rats,total aluminum content in organs of the Al2O3-NPS group was significantly higher than control group and AlCl3 group.In liver,lung,spleen,kidney,hypothalamus,hippocampus and cerebral cortex of P21 rats,total aluminum content in organs of the Al2O3-NPS group was significantly higher than control group and AlCl3 group.Result of real-time PCR,no significant differences among the groups was found.Significant differences among the groups of the level of proteins(NR2B,PSD-95)of the offspring hippocampus,but no significant differences among the groups of SNAP25,For TEM photos,we found that there were significant differences among the groups on the counts of the synapsis and the thickness of the postsynaptic density.No significant differences on the length of the active area of the synapsis.No significant differences of the mice behaviors were found.Significant differences on the levels of synaptic associated proteins(NR2B,PSD-95)of the C57BL/6 mice hippocampus.No significant differences on the levels of SNAP25.Therefore,we consider that intranasal instillation Al2O3-NPS during pregnancy can change the synaptic microstructure of the CA1 area of hippocampus of the offspring,causing the level of NR2B/PSD-95 proteins changes,but not lead to behavioral changes in the offspring.Part ? Aluminum oxide nanoparticles intranasal instillation during pregnancy causes aluminum accumulation in offspring HippocampusObjectPregnant SD rats intranasal instillation Al2O3-NPS and observe aluminum accumulation in offspring.MethodsTEM and DLS methods were used to Al2O3-NPS characterization.21 pregnant rats keep feeding solely after mating.Pregnant rats were randomly divided into three groups(ddH2O,Al2O3-NPS,AlCl3(0.7mg Al/kg)).Pregnant rats were intranasal instillation every day until offspring born.Offspring SD rats were sacrificed after being anaesthetized and collected samples respectively on P1 and P21 and detect total aluminum content of serum and organs of P1 and P21.ResultsAccording to results of the TEM,Al2O3-NPS spread evenly and average size was 36.7±12.4nm.The dynamic light scattering suggested that Al2O3-NPS aggregated in the ddH2O and hydrodynamic size was 70.1±32.5nm.The zeta potential of Al2O3-NPS was-35.7±4.49mv in the ddH2O.For P1,total aluminum content in the serum of the Al2O3-NPS group was significantly higher than that of the control group and the AlCl3 group(P<0.001,P<0.01);no significant differences among the groups in P21.Liver,lung,spleen,kidney and brain of P1,total aluminum content of Al2O3-NPS group were significantly higher than control group and AlCl3 group(all P<0.001).Liver,lung,spleen,kidney,hypothalamus,hippocampus and cerebral cortex of P21,total aluminum content of organs of Al2O3-NPS group were significantly higher than t control group and AlCl3 group(all P<0.001).Specifically,in liver,lung,spleen and kidney,total aluminum content of Al2O3-NPs group were significantly higher than control group and AlCl3 group(all P<0.001);in hypothalamus,total aluminum content of Al2O3-NPS group and AlCl3 group were significantly higher than control group(P<0.001,P<0.01);in hippocampus,total aluminum content of Al2O3-NPs group and AlCl3 group were significantly higher than control group(P<0.001,P<0.01)and total aluminum content of Al2O3-NPS group were higher than AlCl3 group(P<0.05);in cerebral cortex,total aluminum content of Al2O3-NPS group were significantly higher than control group and AlCl3 group(both P<0.001).ConclusionIntranasal instillation Al2O3-NPs during pregnancy causes aluminum accumulation in serum and organs of offspring,especially in the brain.Part ? Aluminum oxide nanoparticles intranasal instillation during pregnancy causes synaptic structure changes of CA1 of Hippcampus ObjectIn order to study the effect on the synaptic plasticity of offspring,we observed and analyzed synaptic structure changes of the CA1 of offspring hippocampus and the changes of synaptic associated genes and proteins.Methods(1)Total RNA of offspring hippocampus was extracted,and the level of synaptic associated genes(NR2B,PSD-95,SNAP25)were detected by real-time PCR.(2)Protein of offspring hippocampus was extracted,and the level of synaptic associated protein(NR2B,PSD-95,SNAP25)were analyzed by Western Blot.(3)After heart perfusion,CA1 area of hippocampus was separated to make TEM samples,then observed synaptic structure changes.ResultsAccording to results of real-time PCR,there were not significant differences among groups on the level of synaptic associated genes(NR2B,PSD-95,SNAP25)which were in offspring hippocampus.There were significant differences among groups on the level of synaptic associated proteins(NR2B,PSD-95)which were in offspring hippocampus,but no significant differences was found about the level of SNAP25.After grayscale analysis,the level of PSD-95 protein of Al2O3-NPS group and AlCl3 group were significantly higher than control group(both P<0.001),and there was no significant differences between Al2O3-NPs group and AlCl3 group;the level of the NR2B protein of Al2O3-NPs group were significantly higher than control group and AlCl3 group(both P<0.001),and there was no significant differences between control group and AlCl3 group.TEM results shown,counts of the synapsis and thickness of the postsynaptic density were significant differences.But,there were no significant differences among groups on the length of the active area of the synapsis.Specifically,for counts of synapsis,Al2O3-NPs group and AlCl3 group were significantly higher than control group(P<0.05,P<0.001),and AlCl3 group was significantly higher than that Al2O3-NPs group(P<0.05);for the thickness of the postsynaptic density,Al2O3-NPs group and AlCl3 group were significantly lower than control group(P<0.01,P<0.001).ConclusionAl2O3-NPs intranasal instillation during pregnancy causes aluminum accumulation in hippocampus,which cause the changes of the level of synaptic associated protein(NR2B/PSD-95)in offspring hippocampus and synaptic structure changes of CA1 area of hippocampus.All of these can lead to damage of offspring neurodevelopment.Part ? Effects of aluminum oxide nanoparticles intranasal instillation during pregnancy on learning and memory of offspringObjectWe further consider synaptic microstructure changes whether or not lead to behaviors of learning and memory of the offspring changes.Methods30 male and 30 female C57BL/6 mice were used.Pregnant rats feed solely after mated.Pregnant rats were randomly divided into three groups(ddH2O,Al2O3-NPs,AlCl3(1.4mg Al/kg)).Pregnant mice were intranasal instillation every day until offspring were born.Offspring conduct learning and memory experiments on P35(step-down test,avoiding darkness test and water maze test)and sacrificed after being anaesthetized and collected samples on P50.Western Blot was used to analyzed the level of synaptic associated proteins(NR2B,PSD-95,SNAP25).ResultsThere were no significant differences among groups of the wrong numbers of the mice.There were significant differences among groups synaptic associated protein level(NR2B,PSD-95).No significant differences of the level of SNAP25.After grayscale analysis,Al2O3-NPs group NR2B protein level were significantly higher than AlCl3 group and control group(P<0.001,P<0.05),and control group were significantly higher than AlCl3 group(P<0.001);AlCl3 group PSD-95 protein level were significantly higher than Al2O3-NPs group and control group(both P<0.001),no significant differences were found between Al2O3-NPs group and control group.ConclusionAl2O3-NPs intranasal instillation during pregnancy causes synaptic microstructure changes of CA1 area of hippocampus and changes the level of NR2B/PSD-95 protein in hippocampus,not lead to learning and memory behaviors changes of offspring. |
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