Effect Of Neuronal Damage And Inflammatory Cytokines Caused By Aβ_1-42 Oligomers With Different Molecular Weight In AD Rats

Objective:1.To investigate the toxic effect of beta-amyloid_1-42 oligomers(Aβ_1-42)with different molecular weight on the hippocampal neurons in Alzheimer’s disease（AD）rats;2.To study the influential effect of Aβ_1-42 with different molecular weight on inflammatory cytokines which were secreted from the hippocampal tissues of different AD rats.Methods:1.The preparation of Aβoligomers and monomers:Aβ₁-₄₂ peptide was dissolved in cold HFIP,while volatiled in the fume hood tobe,the forming film was dissolved in anhydrous DMSO and diluted into PBS aged at 37℃for 7d.Aβ_1-42 oligomers distribution was dvided into two groups according to different molecular weight,including high molecular weight（molecular weight>30KD）and low molecular weight（molecular weight 10-30KD）by 30KD and 10KD overspeed centrifugal tube.Aβ1-₄₂ peptide was dissolved in cold PBS,and the resulting solution was monomer group.2.To make AD Animal model:Eight-weeks-old female SD rats（n=120）were randomly assigned into four groups:high molecular weight group,low molecular weight group,monomer group and control group.According to"stereotactic brain atlas",stereotaxic instrument was used tolocat the point of the CA2 on both sides of hippocampe in rats,and three different groups of Aβ_1-42oligomers and PBS were injected into CA2 of hippocampe in rats （concentration was 2μg/μl）to establish different AD models.3.Morris water maze test:Two months later,the changes of learning ability and memory of experimental rats were observed through orientation navigation test.4.To observe the pathological changes in the hppocampal tissue:10 rats were randomly selected from each group to collect brain tissue via heart perfusion and made into tissue slice.The morphological changes of the hppocampal neurons in the rats was observed through HE staining,and the expression of CD68（CD68 was used to mark activated glial cells）was detected by Immunohistochemistry.5.The expression of inflammatory cytokines in hippocampal tissue:The remaining rats were sacrificed directly and the hippocampal tissue was made into tissue homogenate,then the expression of inflammatory cytokines including IL-18,IL-1βand TNF-αwere tested by ELISA and Western-Blot.Results:1.Results from Morris water maze experiment:Escaping latency of modeling before/after:high molecular weight group（16.4±3.8s/76.4±4.2s）,low weight group（14.0±4.2s/56.3±3.7s）,monomer group（15.5±5.7s/25.5±6.2s）,control group（14.8±5.6s/ 15.9±5.4s）;Before model established,escaping latency of all groups had no statistical difference（all P alues>0.05）;Compared with those before modeling,the escaping latency of each model group after modeling had statistical difference（P<0.05）;After modeling,compared with the each other,high molecular weight group>low weight group>monomer group>control group,P<0.05,the escaping latency of those model groups had statistical difference（P<0.05）;2.Results of HE staining:Compared with the control group,the three modeling groups showed different degree of neuronal loss and glial cell hyperplasia in rat hippocampus,high molecular weight group was the most obvious one,low molecular weight group was more obvious than the monomer group,and the monomer group was the lowest one.3.The expresion of CD68 in hppocampal tissue:CD68 presented different expression level in AD model groups with the highest expression in high molecular weight group and the lowest expression in monomer group;4.The expression of inflammatory cytokines:（1）The results of Western-Blot:The expression trends of IL-18 and IL-1βwere alike,but gray level ratio of high molecular weight group was the highest,and the monomer group was the lowest one;（2）The results of ELISA staining:The expression trend of IL-18、IL-1βand TNF-αwere alike,concentration of the model group were higher than the control group,concentration of high molecular weight group was the highest,monomer group was the lowest one.Conclusions:1.Aβ_1-42 oligomers have toxic effect on hippocampal neurons with obvious damage to the learning ability and memory of the rats;2.Aβ_1-42 oligomers can activate the CD68 labeled cells and promote the expression of inflammatory cytokines,such as IL-18,IL-1β and TNF-α;3.The effect of neurotoxicity and learning cognitive impairment is related to the molecular weight of Aβ_1-42 oligomers,the higher molecular weight of Aβ_1-42 oligomers,the stronger neurotoxicity;4.The experimental results show that the pathogenesis of AD is associated with Aβ_1-42 oligomers,especially to high molecular weight oligomers,which mechanism is remained unclear,and further studies are necessary in this field.