| Objective:Alzheimer's disease?AD?is the most common type of dementia,which currently affecting more than 45 million people worldwide.Epidemiological studies have shown an obvious sex difference,with higher prevalence of AD in women than men.However,the sex differences in AD pathological features,neuroinflammation and cognitive behavior of AD are still not clear.Also,the molecular mechanisms underlying the sex difference are still unclear.In this study,we used immunohistochemistry,Western blot and Morris water maze test to investigate the sex differences in brain pathological features,cognitive behavior and relevant mechanisms in neuroinflammation and cellular signaling of12-month-old 3xTg-AD mice.Methods:12-month-old male and female APPSwe/PS1M146V/tauP301L?3xTg-AD?mice and wild-type?WT?C57BL/6J mice were randomly divided into the following groups:WT+Male,3xTg-AD+Male,WT+Females,3xTg-AD females+Females.All animals were purchased from the Jackson Laboratory in USA and all animal experimental procedures were approved by the Shanxi Animal Research Ethics Committee.All animals were raised at the Experimental Animal Center of Shanxi Medical University,with comfortable environmental temperature and humidity,and enough food and water.On the basis of the above groups,the mice were first subjected to Morris water maze test according to the experimental requirements,and then the mice were randomly divided into immunohistochemistry experiment group and Western blot experimental group.Half male and female in each subgroup.?1?Immunohistochemistry experiments:animals were anesthetized by intraperitoneal injection with 25%urethane.After performing cardiac perfusion,the brain was taken out,dehydrated,and stored into-80°C refrigerator or paraffin-embedded for preservation.i)Frozen sections:the thickness of the brain slice was 25?m,blocked by 5%goat serum.The primary antibody was incubated overnight at 4°C,followed by incubation with secondary antibody.After DAB coloration,the gradient alcohol dehydration,the xylene transparency,and the brain slice was sealed with neutral gum.Each step was washed 3 times with PBS for 5 minutes each time.The areas of A??6E10?plaques,astrocyte?GFAP?and microglia?Iba-1?in the hippocampus of mice in each group were observed under an ordinary microscope.ii)Paraffin sections:brain slice has a thickness of2?m.After xylene and gradient alcohol dewaxing rehydration,3%H2O2 elimination of peroxidase high pressure antigen repair,and 5%goat serum closure,the brain slices were incubated with primary antibody for 1 hour at 37°C,followed by rabbit kits incubation,DAB coloration,gradient alcohol dehydration,xylene transparency,neutral gum seal.Each step was washed 3 times with PBS for 3 minutes.The number and area of phosphorylated tau protein in hippocampus of each group of mice were observed and compared under ordinary microscope.?2?Western blot experiment:The animals were anesthetized with intraperitoneal injection of 25%urethane.The hippocampal tissues were rapidly taken out from the brain on ice after decapitation of animals,and wrapped in foil and preserved in a-80°C refrigerator.The total protein of hippocampus was extracted and the concentration was measured.After gel preparation,adding sample,electrophoreses,transfer membrane,5%BSA blocking,primary antibody incubation at 4°C overnight,the secondary antibody incubation,exposure with ECL luminescence developing solution,optical density values of target bands?p-PKA,PKA,p-CREB,CREB,p-p38,p38,?-actin,GAPDH?were analyzed by using the Alpha View System.?3?Morris water maze test:Morris water maze test was used to detect the spatial learning and memory ability of animals.After 7 days of environmental adaptation,the mice were subjected to hidden platform test for 5 consecutive days.Then probe trials were carried out on the sixth day,and visual platform test were conducted 24 hours later.The escape latency,swimming speed and swimming time in the target quadrant were recorded.Results:?1?A?immunopositive plaques in hippocampus:A?-immunopositive plaques were deposited in hippocampus in WT mice and 3xTg-AD mice.But A?-area percentage in the hippocampus of 3xTg-AD groups was significantly higher than that of WT groups?P<0.05?.Further,in the 3x Tg-AD groups,the percentage of A?-area in the female3xTg-AD mice was significantly greater?P<0.05?than that in the male 3xTg-AD mice.?2?Neurofibrillary tangles in the hippocampus:neurofibrillary tangles were seen in the hippocampus of WT and 3x Tg-AD mice.But the percentage of p-tau positive cell numbers and the percentage of p-tau-area in the hippocampus of 3x Tg-AD mice?male and female?were significantly higher than that in WT mice?P<0.05 for both?.Moreover,the percentage of p-tau positive cell numbers in the 3x Tg-AD+Female group was significantly larger?P<0.05?than that in 3x Tg-AD+Male group,and the percentage of p-tau-area has also the similar results?P<0.05?.?3?Glial cells in the hippocampal tissue:Although there were activation of glial cells?microglia and astrocyte?in the hippocampus in WT mice and 3xTg-AD mice,the percentage of Iba-1-area in the hippocampus of 3x Tg-AD mice?male and female?was significantly higher than that in WT mice?P<0.05 for both?.Especially,the percentage of Iba-1-area in the 3xTg-AD+Female mice,compared to that in 3xTg-AD+Male mice,was significantly higher?P<0.05?.Similar to microglia reaction,the percentage of GFAP-area in the hippocampus of 3xTg-AD mice?male and female?was significantly higher than that in WT mice?P<0.05 for both?,with a significant increase?P<0.05?in the3xTg-AD+Female group compared to that in the 3xTg-AD+Male group.?4?Expression of p-PKA,p-CREB and p-p38 in the hippocampus:there was an obvious decrease in p-PKA and p-CREB,and an increase in p38-MAPK in 3xTg-AD mice,compared with WT mice.Further,the levels of p-PKA and p-CREB in the3xTg-AD+Female group were significantly?P<0.05?lower than that in the3xTg-AD+Male group.In contrast,the level of p-p38 in the 3x Tg-AD+Female mice was significantly?P<0.05?higher than that in the 3xTg-AD+Male mice.?5?Morris water maze test:In the 1-5 day hidden platform test,with the increase of training day,the escape latency of all mice for finding hidden platform was significantly shortened?P<0.05?.From the third day,the mice of 3xTg-AD group spent significantly longer time in searching for the hidden platform than the WT group mice?P<0.05?,especially in the female 3x Tg-AD group?P<0.05?;in probe trials on the sixth day,the percentage of swimming time in the target quadrant in 3x Tg-AD group was significantly lower than that in the WT mice?P<0.05?,and the female 3x Tg-AD mice were more pronounced?P<0.05?.There was no significant difference in swimming speed between the mice in each group?P>0.05?.In the visual platform test,there was no statistical difference in the time for each group of mice to reach the platform?P>0.05?.Conclusion:?1?Compared with WT mice,12-month-old 3xTg-AD mice had more prominent pathological features in the brains,including not only A?plaques,but also neurofibrillary tangles and neuroinflammation,as well as more severer cognitive impairment.?2?In 3x Tg-AD mice,there were significant sex differences in pathological features,glial inflammation and cognitive behavior,in which female mice showed more severe A?plaques,neurofibrillary tangles,neuronal inflammation and cognitive impairment;?3?The sex difference in pathological characteristics and cognitive behavior of3xTg-AD mice may be related to the difference in activation of PKA-CREB-MAPK signaling pathway.In summary,our experimental results provide important reference for the study of the pathological features and cognitive behavior of AD animals while using different sex,and also provide certain experimental evidence for the application of estrogen replacement therapy. |
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