Study The Effect Of Slit2 Over Expression On Alzheimer’s Disease Mice Model And Its Related Mechanism

[Objective]Early results suggest us that over expression of Slit2 can cause memory damage, neuronal loss and metabolites abnomal, etc. Similar to Tg2576 mouse model of AD. Hence,in this study, transgenic mice over expressing Slit2 protein and Tg2576 AD mouse model transferred by human origin APP695 gene were hybridized. Established AD mouse model of Slit2 protein over expression. By testing the behavior of the mice, studying the tissue pathology, molecular biology and other aspects to explore the effect of Slit2 protein on Alzheimer’s disease.[Method]1. Construction and behavioral studies of AD model mice over expression of Slit2.Use the6-8-week-old transgenic mice for breeding, after PCR gene identification,screened Slit2-Tg mice, Tg2576 mice, Tg2576;Slit2 mice and wild-type(WT) mice.Which were used for subsequent experiments.Behavioral tests of 3, 6, 9-month-old mice, including step through test, morris water maze test, open field test and elevated plus maze test.2. Study on pathological features of AD model mice over expressing of Slit2. After the behavioral test, each group of mice were anesthetized to collect experimental material.HE staining to observe morphological changes in mice brain;Immunohistochemistry and Western Blot assay to detect the expression of the beta amyloid protein in mice brain;ELISA of brain tissue and serum to detect levels of Aβ1-40 and Aβ1-42. Meanwhile, brain acetylcholinesterase expression and activation of microglial cells was observed by Immunohistochemistry and Immunofluorescence.3. Research on the mechanisms of Slit2 over expression in AD. Detection of vascular function by HE staining, Evans Blue experiments and in vivo imaging experiments.Then,using immunohistochemistry to detect A β transporter RAGE and P-g P expression.[Result]1. PCR results showed that Tg2576;Slit2 mice are clear bands at 1000 bp and 428 bp,Slit2-Tg mice only in clear bands at 1000 bp, Tg2576 mice have a clear strip only at 428 bp band, WT mice without the gene, so no strip show.2. Behavioral results showed that, step through test showed Tg2576; Slit2 mice wrong more often, have shorter latency times compared to Tg2576 mice. In morris water maze test, the Tg2576;Slit2 mice performance the escape latency of long trend,the platform quadrant percentage, percentage of time were reduced compared to Tg2576 mice, and there are significant differences. Further,used the behavioral experiments to detect their anxiety, the open field maze test results showed the percent reduction in the central region in Tg2576;Slit2 mice compared to Tg2576 mice,it can be seen at 9 months.By the elevated plus maze test, results can be seen that,in 9 months, compared with the Tg2576 mouse, the percentage of Tg2576; Slit2 mice in the open arms reduced and there are significant differences.3.HE staining results can be seen that,WT mice brain neurons complete, a larger number of cells, arranged in row; compared with WT mice, the remaining groups were neurons with vacuolar degeneration, the cells loose arrangement where in Tg2576; Slit2 mouse is more serious.4. Immunohistochemistry and Western Blot analysis of mice brain beta amyloid showed,in Tg2576; Slit2 mice brain, beta amyloid expression was significantly increased. Meanwhile,Western blot results quantitatively explain Tg2675;Slit2 mice brain beta amyloid content were significantly higher than the Tg2675 mice.Four group of the 12-month-old mice brain tissue and serum to detected Aβ1-40 and Aβ1-42 content showed,after Slit2 over expression,Tg2576;Slit2 mice brain of Aβ1-40, Aβ1-42 was significantly higher than Tg2576 mice,whereas in serum levels Aβ1-40, Aβ1-42 have no significant difference. In the final result,Aβ1-40 ratio in brain tissue and serum was significantly increased, but Aβ1-42 in the brain the ratio of serum is not significantly increased.5. The Immunohistochemical method to detect the cortex and hippocampus acetylcholinesterase expression of 12-month-old mice.we found that,the content of ACh E in the brain of Tg2576;Slit2 mice was significantly higher than Tg2576 mice.6. Immunofluorescence and Immunohistochemistry combined to detect the activation of microglia in brain of 12-month-old mice.We found that,in the cortex region, microglial activation was obvious. Compared with WT mice, the Tg2576 mouse, Tg2576; Slit2 mouse cortical cell body swelling appeared, indicating that microglia activation obvious.7. HE staining showed Slit2-Tg mice choroid plexus structural damage occurs.Evans Blue and in vivo imaging test illustrate increased BBB penetration in Slit2-Tg mice.Immunohistochemical method to detect the brain Aβ transporter of RAGE and P-g P expression in each group mice of 12 months had found that RAGE expression has been enhanced and the expression of p-gp obvious reduced in Tg2576;Slit2 mice.[Conclusion]1. The experiment successfully constructed Slit2 overexpression of AD model mice(Tg2576;Slit2).2. Tg2576;Slit2 mice have more obvious obstacles for learning and memory than the Tg2576 mice, and performance anxiety.3. Tg2576;Slit2 mice was more vulnerable to deposit Aβ protein in brain, while acetylcholinesterase increased and expression of inflammatory cell activation.4. The mechanism may that Slit2 over expression leads to the damage of the blood brain barrier, allowing the expression of Aβ transporters on the blood-brain barrier appears abnormal.5. Tg2576; Slit2 mice,which was an animal model to study the cerebral vascular injury in Alzheimer’s disease.