Effect Of Aβ_25-35 To Rat’s Neuron And Microglia And Mechanism

Alzheimer’s disease which is also called senile dementia, is adegenerative disease of the central nervous system, mainly showing cognitivedysfunction and memory impairment. Though the pathogenesis of AD hasbeen unclear, the idea that amyloid beta-protein is the initiate factor has beenaccepted. Currently,the study of mechanisms of Alzheimer’s disease inducedby amyloid beta-protein focus on inflammation, apoptosis and oxidative stressof neurons. Since1970experts began to realize that microglia cells areimmune cells of the central nervous system, play an important role in thebody’s innate immunity and the inflammatory response of a variety ofneurodegenerative disorders. In recent years,related studies have shown thatmicroglia play a double-edged sword role in neuropathology changes ofAlzheimer’s disease. On the one hand, amyloid beta-protein can be eliminatedby activated microglia through phagocytosis, reducing the toxicity of amyloidbeta-protein to neurons. On the other hand, excessive activated microglia cangenerate a number of inflammatory mediators,such as IL-1β、TNF-α and so on.Formation of chronic inflammatory response induced by the inflammatorymediators, in turn, accelerates the development and degradation of course ofAlzheimer’s disease. The overall animal experiment focuses on analysis of therelations among amyloid beta-protein deposits, neuronal damage and thefunction of microglia in Alzheimer’s disease. The aim of experiment in vivois to analyze the role of microglia to neuronal damage, Aβ eliminationthrough dynamic observation of Aβ deposition,neuron damage,activatedmicroglia. Recent research has shown that amyloid beta-protein may inducedneurotoxicity by adding significant neuronal apoptosis in rat hippocampus.The toxicity of amyloid beta-protein to neurons in vivo and in vitro dependson the aggregation and concentrationa.In order to find new target of treatmentof Alzheimer’s disease with an experimental basis,using immunocytochemical and Western Blot method, comparative analysis on effect and mechanism ofamyloid beta-protein on neural apoptosis by in vitro cell culture.Part1Effect of amyloid beta-protein(25-35) to neuron and microglia inhippocampus of ratsObjective:To study the change of neuron and microglia of hippocampus in differenttime after injection amyloid beta-protein(25-35)in hippocampus of rats.Analysis on effect and mechanism of amyloid beta-protein on neural injuryand the role of microglia.Methods:1Experiment group and management:40male Wistar rats were randomlydivided into five groups(control, Aβ treated2thday group, Aβ treated7thdaygroup, Aβ treated15thday group, Aβ treated21thday group). On the eighthday saline (5μl,10μg)or Aβ(5μl,10μg) was injected to bilateral hippocampus inthe control group or the Aβ treated group.2Model preparation: Aggregated state Aβ(5μl,10μg) was injected inhippocampus CA1region of rat(AP-3.5mm，ML±2mm，DV2.7mm) usingbrain stereotaxis technique in10min.Finaly, needle retained5min.3Morris water maze test: Keeping water above the the surface ofplatform1.5cm in the tank. Maintaining the water temperature at (21±1)degrees centigrade. Adding ink to the tank up to ensure that the rats would notsee the platform. Keeping reference constant around the maze during theexperiment, automatic camera synchronized recording trajectories of rats.One week after injection Aβ morris water maze was performed for6d(twiceone day) to record the latency finding the hidden platform, on the6thday, theplatform was moved to record the times crossing the platform area, timestaying in the platform quadrant, ratio of path in platform quadrant to thewhole path during2min.4Specimen management:Rats of each group were infused withparaformaldehyde, brain tissue was performed external fixation for24h, paraffin imbedding. The thickness of coronal slicesis5μm,including thehippocampus part,for thionine-Nissl body stain and immunohistochemistry.5Statistical analysis：SPSS17.0software was used to make statisticalanalysis.Results:1Effect of Aβ on rat’s morris water maze test: The latency of controlgroup in3th,4th,5thwere13.57，8.68，7.09s respectively, the latency of Aβtreated15thday and21thday group were significantly longer than those ofcontrol(P<0.05). On the6thday, the average times of control group crossingthe platform was11.13.While the average times of Aβ treated15thday and21thday group crossing the platform were4.25,4.38. Compared with control group,Aβ treated15thday and21thday group were significantly decreased.2Effect of Aβ on neuron in hippocampus CA1region of rats: Cellarranges uncluttered, cell morphology was normal, nucleus did not stain.cytoplasm shaded deep in control. The injection of Aβ in rats from2to7days,15days21days, the damage degree of neuron increased gradually, while glialcells increased. The damage degree of neuron and the number of glial cells inthe Aβ treated21thday group was less than the Aβ treated15thday group.3Aβ and CD11b expression in hippocampus CA1region: The result ofimmunohistochemistry showed the deposition of Aβ reached the highest valuefrom2thday to7thday in the Aβ treated group,while the deposition of Aβ wason the decline from15thday to21thday. The sites distribution of microglia anddeposition of Aβ were located in the similar regions. Compared with thecontrol group,the morphology and quantity of activated microglia in the fourdifferent time Aβ treated group were significant differences(P<0.05). Quantityof activated microglia was no significant differences between Aβ treated2thday and7thday group (P>0.05),then the quantity of activated microglia was onthe decline from7thday to21thday.Conclusion:It could be concluded that the injection of Aβ into hippocampus CA1region could cause severe neural damage in CA1region, decrease learning and memory ability; Injection of Aβ can activate microglia in hippocampus andinduce the morphological change.MG could phagocytose Aβ.There is a directproportion correlation between the sedimentation of Aβ and activities ofmicroglia.Part2Effect of amyloid beta-protein(25-35)to apoptotic proteins expressionon PC12cellsObjective:To explore the effect of different concentration (0.1、1、2、5、10、20μmol/L) amyloid beta-protein(25-35)to apoptotic proteins expression onPC12cells.Methods:PC12cells were cultured with different concentration aggregated Aβ25-35for48h, MTT assay was used to detect cell activity. The hoechst33258method was used to detect the PC12cell apoptosis by fluorescencemicroscope. Immunocytochemical staining and western blotting were used todetect the expression of cell apoptotic protein,including Bax,Bcl-2,Caspase-3.Results:MTT assay showed that the impact on cell growth inhibitory activity ofAβ25-35was a concentration-dependent relationships.In vitro, the growthinhibition activity to PC12cell was strengthened with the increase of theconcentration of Aβ25-35. Hoechst33258staining showed that PC12cellapoptosis was a concentration-dependent relationships with Aβ25-35. Theresults of immunocytochemical staining and western blotting showed that theexpression of Bcl-2protein was on decline with the increasing concentrationof Aβ25-35.,while the expression of Bax and Caspase-3protein were on the rise.Conclusion:Aβ25-35could induce PC12cell apoptosis, the mechanism might bedecrease of Bcl-2protein expression and increase of Bax and Caspase-3protein expression.