The Neuroprotective Effects And Mechanism Of Adiponectin Against The Learning And Memory Impairment In Triple Transgenic Alzheimer’s Disease Model Mice

Objective:To observe whether adiponectin(APN)could effectively ameliorate the new object recognition,working memory and spatial memory impairment,alleviate the anxiety,reverse the hippocampal synaptic plasticity suppression,and influence the expression of NF-κB,IL-1β,TNF-α and IL-10 in 9-month-old triple transgenic Alzheimer’s disease(3xTg-AD)model mice.In addition,the neuroprotective effects of adiponectin against AD andits relationship to neuroinflammation were also investigated.Methods:The 9-month-old 3xTg-AD mice and C57BL/6J mice were used in the experiments.All mice randomly divided into four groups(n=8 for each group): WT+Saline,WT+APN,3xTg+Saline and 3xTg+APN group.Each mouse was intracerebroventricular injected adiponectin or saline for 10 days prior to behavioral tests,and maintaining the injection until the electrophysiological recording finished.Four different experiments were included in the behavioral tests(1)Open field test:Each mouse was placed in the center of open field to freely explore 5 min.The total distance of each mouse moved and the percentage of time spent in center or peripheral region were recorded.(2)Novel object recognition test: Second day after open field test,each mouse was firstly subject to a 10 min acquisition trial,with two identical objects in the open field.Five hours after free exploration finished,one original object was changed into another one with the same material,but different color and shape.Then the mouse explored another 10 min.The time each mouse explored familiar or novel object was recorded,and the recognition index and discrimination index were calculated.Recognition index = time exploring novel object or familiar object /(time exploring both the novel and familiar objects)×100%.Discrimination Index =(time exploring the novel object-time exploring familiar object)/(time exploring both the novel object and familiar objects)×100%.(3)Y maze test: On the third day,the mice were placed into the intersection of thethree arm of the Y maze and let them freely explore 8 min.The total number of the arm entries and the order of arm-entry were recorded,with the percentage of right spontaneous alternation was calculated.(4)Morris water maze: Firstly,the 5 days of hidden platform test was performed.Each mouse should swim 4 times a day for consecutive 5 days,with the swimming track,swimming speed and escape latency(time to find hidden platform)were recorded.At the sixth day,the probe trial test was carried out,with the swimming track and swimming time in target quadrant were recorded.Then,the time to find the platform and swimming speed in visible platform test were recorded.After the behavioral experiments completed,the mice were anesthetized and fixed on the stereotaxic apparatus for in vivo electrophysiological recording.The field excitatory postsynaptic potential(fEPSPs)was recorded 30 minutes,with the paired stimulus used to record paired-pulse facilitation(PPF),then the long term potentiation(LTP)was induced by high frequency stimulation(HFS)and fEPSPs was recorded for another 60 minutes.The effects of AD related gene mutation and adiponectin pretreatment on synaptic plasticity and presynaptic neurotransmitter release were observed.Finally,the mice were decapitated and the expression of NF-κB,IL-1β,TNF-α and IL-10 in the hippocampus of mice were tested by using Western blot.Results:(1)Adiponectin could significantly ameliorate the impairments of recognition memory,working memory and spatial memory,and alleviate the anxiety in 9-month-old3xTg-AD mice.In the open field test,compared to WT+Saline group,the percentage of time spent in center of 3xTg+Saline group was significantly lower(P<0.001),and the time spent in peripheral region obviously increased(P<0.001),indicating that 3xTg mice were anxious.However,in 3xTg+APN group,the time spent in the center increased and the time spent in the peripheral region decreased(P<0.01),indicating that the administration of APN effectively alleviate the anxiety observed in 3xTg mice.In the novel object recognition test,the novel object recognition index in WT+Saline and 3xTg+APN group was significantly higher than the old object recognition index(P<0.05),but there was not significant difference between the novel and old object recognition index in 3xTg+Saline group.In addition,the discrimination index of 3xTg+Saline group were significantly lower than WT+Saline and 3xTg+APN group(P<0.01).These results indicated that adiponectin could effectively prevent the recognition memory impairment in 3xTg mice.In Y maze test,the percentage of right spontaneous alternation in 3xTg+Saline group was lower than WT+Saline group and 3xTg+APN group(P<0.01),but the total arm entries of mice in four groups was similar(P>0.05),which indicating that adiponectin could prevent working memory impairment in 3xTg mice.In the Morris water maze test,the escape latency of the mice in each group to find hidden platform was decreased with the extension of training days.On the day 2-5,the escape latency of mice in 3xTg+Saline group was significantly longer than WT+Saline group(P<0.05).On the day 3-5,the escape latency of mice in3xTg+APN group was significantly shorter than 3xTg+Saline group(P<0.05).In the probe trials,compared to WT+Saline group,the swimming time in the target quadrant of 3xTg mice injected with saline was significantly decreased(P<0.001),and adiponectin pretreatment increased the percentage of swimming time in the target quadrant of 3xTg mice.In addition,there was no significant difference in swimming speed and the time to visible target among all group(P >0.05).These results indicated that adiponectin could protect against the spatial learning and memory impairment in 3xTg mice.(2)Adiponectin reversed hippocampal synaptic plasticity depression in 9-month-old3xTg-AD mice.There was no significant difference in fEPSPs slope before HFS and PPF ratio among four groups(P>0.05).After HFS applied,LTP always successfully induced in four groups of mice.However,the fEPSPs slope in 3xTg+Saline group began to decrease20 min after HFS.At 30 and 60 min after HFS,the fEPSPs slope in 3xTg+Saline group were all significantly decreased than WT+Saline group and 3xTg+APN group(P<0.05).The results above indicated that AD related gene mutation and adiponectin pretreatment did not affect basal synaptic transmission and presynaptic neurotransmitter release,but adiponectin pretreatment effectively reversed the hippocampal synaptic plasticity depression in 3xTg mice.(3)Adiponectin alleviated the neuroinflammation in the hippocampus of 3xTg-AD mice.Compared to WT+Saline group,the expression of proinflammatory cytokines,such as NF-κB,IL-1β and TNF-α,in the hippocampus of 3xTg mice significantly increased(P<0.01),whereas the expression of anti-inflammatory factor IL-10 significantly decreased(P<0.001).After adiponectin pretreatment,the expression of NF-κB,IL-1β and TNF-α in the hippocampus of 3xTg-AD mice decreased(P<0.05),while the expression of IL-10 increased(P<0.01).Conclusion:The recognition memory,short-term working memory and long-term spatial memory in 9-month-old 3xTg-AD mice were significantly impaired,accompanying with the anxiety.In addition,the LTP depression and neuroinflammation also observed in the hippocampus of 3xTg-AD mice.However,adiponectin pretreatment effectively ameliorated the multiple learning and memory impairment,prevented the anxiety,reversed the LTP suppression,and alleviated hippocampal neuroinflammation in 3xTg-AD mice.The neuroprotective effects of adiponectin exerted in AD might be related to the downregulate the expression of proinflammatory cytokines and upregulate the secretion of anti-inflammatory factors.