| PART ONE THE EFFECTS OF ANTI-LINGO-1 ANTIBODY ON BEHAVIOR AND HIPPOCAMPAL SYNAPSE CHANGES IN CHRONIC STRESS RATS WITH COGNITIVE FUNCTION IMPAIRMENTObjective To investigate the effects of anti-Lingo-1 antibody on the behaviors and synapse of the rats with cognitive function impairment after chronic stress and find a new method to treat cognitive function impairment after chronic stress.Methods Male Sprague Dawley(SD)rats aged 4-6 weeks were randomly divided into the control group and the model group after one week of adaptive feeding.The model group was randomly divided into the Chronic stress group and the Anti-Lingo-1 group.The Anti-Lingo-1 group was given 8 mg/kg Anti-Lingo-1 antibody once a week for 3 weeks.The weight of rats in each group was measured at the same time every week.After 3 weeks of Anti-Lingo-1 antibody treatment,the Morris water maze test and open field test were used to evaluate the behavior levels of the Control group,the Chronic Stress group and the Anti-Lingo-1 group.After the behavioral test,5 rats in each group were randomly selected.After anesthesia,4% paraformaldehyde was injected into the heart for fixation and brain tissue separation.One hemisphere of rats was randomly selected and dehydrated with gradient concentration sucrose water before being embeded with OCT embedding agent.After that,60 μm thick serial sections were cut along the coronal plane by the frozen section machine.Finally,6 groups of continuous equidistant sections were obtained.According to the stereological principle,the Sp~+ spines were stained with immunohistochemistry.Then,the PSD95 protein was detected with fluorescence semi-quantitative method.Results 1.Weight: After baseline adjustment,the body mass of the Chronic stress group was significantly lower than that of the control group at the fourth week(P = 0.000).After 3 weeks of anti-Lingo-1 antibody treatment,the body weight of the Control group,the Chronic Stress group and the Anti-Lingo-1 group were significantly different(P = 0.000),but there was no significant difference between Chronic Stress group and Anti-Lingo-1 group(P = 0.130).2.Morris water maze: After 3 weeks of Anti-Lingo-1 antibody treatment,there was no significant difference among the three groups in the navigation test(P = 0.966).On the last day of the Morris water maze test,there were significant differences in the number of times crossing platforms in the Control group,the Chronic Stress group and the Anti-Lingo-1 group(P = 0.000).Compared with the Control group,the number of times crossing platforms in the Chronic stress group was lower(P = 0.004).The number of times crossing platforms in Anti-Lingo-1 group was significantly increased when compared to the Chronic Stress group(P =0.000).3.Open field test: There was no significant difference among the Control group,the Chronic Stress group and the Anti-Lingo-1 group in the percentage of central distance(P > 0.05).4.Spinophilin~+(Sp~+)spines: The total number of Sp~+ spines in the Chronic stress group was significantly decreased compared with the Control group in the DG,the CA1 and the CA3(P = 0.000,P = 0.012,P =0.001).The total number of Sp~+ spines in the Anti-Lingo-1 group was significantly increased compared with the Chronic Stress group(P =0.023,P = 0.016,P = 0.022).Compared with the Control group,the density of Sp~+ spines in the Chronic Stress group decreased significantly in the DG,the CA1 and CA3(P = 0.001,P = 0.034,P = 0.000).Compared with the Chronic Stress group,the density of Sp~+ spines in the Anti-Lingo-1 group was significantly increased in the DG,the CA1 and CA3(P = 0.015,P =0.044,P = 0.006).5.PSD95 protein: The expression of PSD95 protein in the DG,CA1 and CA3 of hippocampus in the Chronic Stress group was significantly lower than that in the Control group(P = 0.001,P = 0.000,P = 0.000).The expression of PSD95 protein in the DG,CA1 and CA3 of hippocampus in the Anti-Lingo-1 group was significantly higher than that in the Chronic Stress group(P = 0.001,P = 0.001,P = 0.000).Conclusions Chronic stress led to the decline of the ability of spatial memory and the number of synapses in hippocampus.3 week Anti-Lingo-1antibody treatment could significantly improve the cognitive function through protecting synapse loss.PART TWO THE EFFECTS OF ANTI-LINGO-1 ANTIBODY ON BEHAVIOR AND HIPPOCAMPAL SYNAPSE CHANGES IN TRANSGENIC MOUSE MODEL OF EARLY ALZHEIMER’S DISEASE.Objective To investigate the effects of Anti-Lingo-1 on behavioral changes in early AD mice and the mechanism for the effects in order to further study the mechanism of Anti-Lingo-1 delaying the decline of cognitive function in early AD.Methods Ten month old transgenic APP/PS1 male mice and ten month old wild type mice(WT+NS)were used.The 10 month old APP/PS1 mice were randomly divided into the APP/PS1 + LA group and the APP/PS1 + NS group.Mice in the APP/PS1 + LA group were intraperitoneally injected with Anti-Lingo-1 antibody(10 mg/Kg/d)for 8weeks.Meanwhile,the WT+NS group and the APP/PS1+NS group were injected with the same dose of normal saline once a week for 8 weeks.After 8 weeks of Anti-Lingo-1 antibody or saline injection,the Morris water maze was used to test the spatial learning and memory ability of mice.The Y maze was used to test the working memory function of mice.Then,five mice were randomly selected from each of the three groups for perfusion.The randomly selected side of the brain was sectioned along the coronal plane with the frozen section machine.According to the stereological principle,the samples were collected and the Sp~+ spines were stained with immunohistochemistry.Then the number and activated status of microglia in hippocampus were detected with fluorescence semi quantitative method.The average number of synapses phagocytized by microglia in the hippocampus of the three groups was counted.Results 1.Morris water maze: There was no significant difference in time spent in the visible platform testing among the WT+NS group,the APP/PS1+NS group and the APP/PS1+NS group(P > 0.05).Compared with the WT + NS group,the escape latency time of APP/PS1 + NS group was significantly increased(P = 0.007),the frequency of platform location crosses was significantly decreased(P = 0.004),time spent in the target quadrant was significantly decreased(P = 0.001),the percentage of swimming paths in the target quadrant was significantly decreased(P =0.001),the swimming speed in the space exploration experiment was significantly decreased(P = 0.000),and the total path distance in the space exploration experiment was significantly decreased(P = 0.000).2.Y maze: Compared with the WT + NS group,the alternation rate in the APP/PS1 + NS group was significantly lower(P = 0.001).Compared with the APP/PS1 + NS group,the alternation rate in the APP/PS1 + LA group was significantly higher(P = 0.006).3.Stereological counting of spinophilin~+(Sp~+)spines: Compared with the WT + NS group,the total number of Sp~+ spines in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was significantly decreased(P = 0.000,P = 0.000,P = 0.002).Compared with the APP/PS1+ NS group,the number of Sp~+ spines in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was significantly increased(P =0.007,P = 0.008,P = 0.020)。4.Immunofluorescence staining results of Iba1~+ microglia: Compared with the WT + NS group,the number of Iba1~+ microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was significantly decreased(P = 0.015,P = 0.027,P = 0.008).The number of Iba1~+microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was significantly higher than that in the APP/PS1 + NS group(P =0.015,P = 0.018,P = 0.033).5.Immunofluorescence staining of Iba1~+CD68 labeled activated microglia: The number of activated microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was higher than that in the WT +NS group(P = 0.000,P = 0.000,P = 0.00).After treatment with antibody,the number of activated microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was lower than that in the APP/PS1 + NS group(P = 0.000,P = 0.005,P = 0.000).6.Immunofluorescence staining results of PSD95 protein: The expression of PSD95 protein in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was lower than that in the WT + NS group(P =0.000,P = 0.000,P = 0.000).After treatment with antibody,the expression of PSD95 protein in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was higher than that in the APP/PS1 + NS group(P =0.009,P = 0.034,P = 0.004).7.Immunofluorescence staining of SYP protein: The expression of SYP protein in DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was lower than that in the WT + NS group(P = 0.000,P = 0.000,P= 0.000).After treatment with antibody,the expression of SYP protein in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was higher than that in the APP/PS1 + NS group(P = 0.000,P = 0.002,P =0.009).8.Three dimensional reconstruction by confocal laser: The average number of synapses phagocytized by microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + NS group was significantly higher than that in the WT + NS group(P = 0.000,P = 0.000,P = 0.000).Compared with the APP/PS1 + NS group,the average number of synapses phagocytized by microglia in the DG,CA1 and CA3 of hippocampus in the APP/PS1 + LA group was significantly decreased(P = 0.005,P = 0.008,P = 0.012).Conclusions 8 weeks of Anti-Lingo-1 treatment could delay the decline of learning and working memory ability in early AD mice.Anti-Lingo-1 treatment could not only significantly reduce the loss of synapses,but also reduce the number of microglia and inhibit the activation of microglia.The results suggested that the effects of Anti-Lingo-1antibody in hippocampus of early AD mice may be related to the phagocytosis of microglia. |
PDF Full Text Request