Effects Of G-CSF Combined With VEGF On Synaptic Plasticity Of APP Transgenic Mice Model

Backgounds:Alzheimer disease has no effective treatment now. Granulocyte colony stimulating factor (G-CSF) is one of main cytokines, which can modulate marrow hematopoiesis, can mobilize marrow stem cells, promote proliferation, and migration to the brain. The stem cells may improve intellectual function of AD mice model. Vascular endothelial growth factor (VEGF) combined with VEGF-receptor can induce endothelial cells proliferating, migrating and promoting the development of vascular net. VEGF can also directly influence neurons, promote synaptic extension. It has the abilities to protect and regerate neuron. The results of human neural stem cells culture in vitro and mature mice brain study in vivo showed that G-CSF can induce receptor-mediated neural precursor cells proliferating, and differentiating. G-CSF can activate STAT3and STAT5, increase expressions of VEGF and it's receptor VEGFR-2/flk-1. VEGFR-2, which is a tyrosine kinase inhibitor, can block neural regeneration induced by G-CSF. We suppose that G-CSF combined VEGF may have synergetic effect to promote proliferation, migration and differentiation of marrow stem cells, and modulate synaptic plasticity. These reports have not been found so far. Our study was aimed to observe APP-transgenic mice memory function by Morris water maze testing, then investigated the expressions of stem cells marker CD133and NCAM by immunohistochemistry, And detected neural regeneration marker GAP-43and synaptic marker synaptophysin(SYN) by double fluorescent labeling.Discuss the effects of G-CSF combined VEGF on memory impairment, stem cells' proliferation,neural regeneration and synaptic plasticity. Materials and methodsAnimal grouping and administration:54APP transgenic mice were randomly divided into3groups:the G-CSF combined with VEGF group, the G-CSF group and the control group. Injections of VEGF(8μg/Kg.d) for3days were peritoneally given to mice in the G-CSF combined with VEGF group, then injections of G-CSF (50μg/Kg.d) for7days were subcutaneously given; injections of PBS for3days were peritoneally given to mice in the G-CSF group, then injections of G-CSF (50μg/Kg.d) for7days were subcutaneously given; injection of PBS for3days were peritoneally given to mice in the control group, then injection of normal saline for7days were subcutaneously given.Morris water maze testingThe Morris water maze task was performed to obtain the spatial memory impairment of PDGF-hAPPV7171transgenic mice, according to the method of Morris. The escape latency was analyzed the morning before the injection and on the7th,14th, and28th days after the last day of the injection. One hour after the last trial, the mice performed probe tests during which the platform was removed from the pool. The mice were allowed to swim for60s to test whether they remembered the original position of the platform. The time spent traveling in the target quadrant (where the platform was previously located) were recorded and the number of crossings over a point where the platform had been was counted.Brain specimen collecting:injection of10%chloral hydrate (0.38g/kg) were peritoneously given to mice in all groups in7day,14day,28day after administration, then perfusion with PBS and4%paraformaldehyde were made until all limbs were rigor. The brains were removed and fixed in4%paraformaldehyde for20hours, then moved into30%and20%sucrose solution for24hours. The fixed brains were cut into15μm thick slices across coronal hippocampus section. The slices were examined by immunohistochemistry.CD133immunohistochemistry:According to the unstruction manual of CD133antibody, next is the detail procedure:the frozen slices were put in the normal temperature for30min, then washed with PBS5min X3. Incubate5-10min in the 3%H2O2solution to get rid of endogenous peroxidase activity, then washed with PBS5min. Put into1%H2O2to block endogenous peroxides in normal temperature for10-20min, then washed with PBS3min X3. Incubate in the normal serum for20min at normal temperature. Dripped CD133antibody and stay overnight at4, then washed with PBS3min. Dripped CD133second antibody at room temperature for30min, then washed with PBS3min. Colorate with0.04%DAB and0.03%H2O2for5-10min, then washed with water and dyed with haematoxylin, mounting at last.BrdU/GAP-43double fluorescent labeling:frozen slices were warmed in normal temperature, than washed by0.3%Tritonx-100for30min, PBS3x5min.2MHC137℃60min, washed by PBS3x5min. The slices were covered with5%BSA confining liquid for20min at normal temperature. Discarded more liquid on the slices and didn't wash the slices again. Dripped1:100diluted BrdU first antibody, and stay overnight at4℃, washed with PBS (PH7.2-7.6)2min3min x3. dripped1:100diluted GAP-43first antibody, and stay overnight at4℃. washed with PBS (PH7.2-7.6)2min3min x3. Dripped the mixed liquor coat-anti-mouse TRITC couple second antibody and coat-anti-rabbit FITC couple second antibody at37℃for20min, then washed with PBS(PH7.2-7.6)2min3min x3, mounting at last. BrdU and GAP-43positive cells were observed with fluorescence microscope, then count the number of BrdU/GAP-43positive cells and BrdU positive cells. Calculate the percentage of BrdU/GAP-43positive cells and BrdU positive cells.BrdU/SYN immunofluorescence double tagging labeling:frozen slices were rewarmed in normal temperature, than washed by0.3%Tritonx-100for30min, PBS3minX32MHCl37centigrade for60min, washed by PBS3min X5. The slices were covered with5%BSA confining liquid for20min at normal temperature. Discarded more liquid on the slices and didn't wash the slices again. Drpped1:100diluted BrdU first antibody, and stay overnight at4, washed with PBS (PH7.2-7.6)2min. dripped1:100diluted SYN first antibody, and stay overnight at4. washed with PBS (PH7.2-7.6)2min. Dripped the mixed liquor coat-anti-mouse TRITC couple second antibody and coat-anti-rabbit FITC couple second antibody at37for20min, then washed with PBS(PH7.2-7.6)2minX3, mounting at last. BrdU and SYN positive cells were observed with fluorescence microscope, then count the number of BrdU/SYN positive cells and BrdU positive cells. Calculate the percentage of BrdU/SYN positive cells and BrdU positive cells.NCAM immunohistochemistry:According to the instruction manual of NCAM antibody, next is the detail procedure:the frozen slices were put in the normal temperature for30min, then washed with PBS5min3min x3. Incubate5-10min in the3%H2O2solution to get rid of endogenous peroxidase activity, then washed with PBS5min3min x3. Put into1%H2O2to block endogenous peroxides in normal temperature for10-20min, then washed with PBS3min3min x3. Incubate in the normal serum for20min at normal temperature. Dripped NCAM antibody and stay overnight at4℃, then washed with PBS3min x3. Dripped NCAM second antibody at room temperature for30min, then washed with PBS3min3min x3. Colorate with0.04%DAB and0.03%H2O2for5-10min, then washed with water and dyed with haematoxylin, mounting at last.statistical treatment:CD133immunohistochemistry:Count the number of CD133positive cells in5random non-overlapped visual fields of the CD133immunohistochemistry hippocampus slices, then calculate mean gray scales with Leica QwinV3image analysis system. BrdU/GAP-43double fluorescent labeling: Count the number of BrdU/GAP-43double labeling positive cells and BrdU positive cells in5random non-overlapped visual fields of the double labeling hippocampus slices, then calculate the percentage and mean. BrdU/SYN immunofluorescence double tagging labeling:Count the number of BrdU/SYN double tagging positive cells and BrdU positive cells in5random non-overlapped visual fields of the double tagging hippocampus slices, then calculate the percentage and mean. SPSS10.0was adopted to conduct statistical ananlysis. The results were expressed with x±s. Double t test was adopted to show the differences between two groups. P<0.05is regarded as statistic significance. NCAM immunohistochemistry: Count the number of NCAM positive cells in5random non-overlapped visual fields of the NCAM immunohistochemistry hippocampus slices, then calculate mean gray scales with Leica QwinV3image analysis system.Results:Part1:G-CSF combined with VEGF ameliorates the memory impairment of APP transgenic mice1. Before G-CSF/VEGF treatment, the escape latencies of the G-CSF-treated group and G-CSF/VEGF-treated group were similar to the control group (G-CSF/VEGF-treatment group:61.22±25.09s, G-CSF-treatment group62.26±22.07s, control group:60.98±17.11s,P>0.05). After the intraperitoneal administration of G-CSF/VEGF or PBS, we found that G-CSF/VEGF significantly shortened the escape latencies in PDGF-hAPPV7171transgenic mice compared toG-CSF and control group on the7th,14th, and28th day after G-SCF/VEGF treatment.(On the7th day, G-CSF/VEG F-treatment group:23.73±12.50s,G-CSF-treatment group:27.19±4.07s, control group:46.07±7.21s, P<0.05; on the14th day, G-CSF/VEGF-treatment group:20.22±9.28s, G-CSF-treatment group:26.48±5.29s, control group:42.99±11.70s, P<0.01; on the28th day, G-CSF/VEGF-treatment group:20.81±10.46s, G-CSF-treatment group:24.97±3.61s,control group:45.54±9.55s, P<0.01).2. Three main patterns of search behaviors were used by mice to find the platform:1) spatial search strategies,2) systematic but non-spatial search strategies, or3) strategies involving repetitive looping paths. After the administration of G-CSF/VEGF or PBS, we observed that search strategy use differed markedly between G-CSF/VEGF-treated, G-CSF-treated and control mice. The mice of control group used a mixture of strategies, including spatial, systematic but non-spatial, and a relatively high proportion (23%on the7th day,39%on the14th day,25%on the28th day) of repetitive looping paths strategies. The fraction of spatial strategies use stayed relatively constant at29%to33%. In contrast, G-CSF-treated mice used a high proportion (45%on the7th day,55%on the14th day,63%on the28th day) of spatial strategies along with some systematic but non-spatial strategies (38%on the7th and 30%on the14th days,20%on the28th day) and very few repetitive looping paths strategies (17%on the7th day,15%on the14th and17%on the28th days). G-CSF/VEGF-treated mice used a higher proportion (68%on the7th day,76%on the14th and80%on the28th days) of spatial strategies along with some systematic but non-spatial strategies (23%on the7th and13%on the14th days,10%on the28th day) and very few repetitive looping paths strategies (9%on the7th day,11%on the14th and10%on the28th days). A chi-square test showed that G-CSF/VEGF-treated mice preferentially choose spatial strategies while repetitive looping paths strategies are used more often by control mice than G-CSF-treated and control transgenic mice, P<0.05.3. After removal of the platform, we found that the time spent traveling in the target quadrant of G-CSF/VEGF-treated mice was longer than that of G-CSF-treated mice and untreated mice. Compared to the untreated PDGF-hAPPV717I transgenic mice, the mean value of the percentage of the total time in the target quadrant was significantly increased on the14th and28th days after VEGF treatment (on the14th day, G-CSF/VEGF-treatment group:59.24±18.69, G-CSF-treatment group:56.24±17.19%,control group:35.12±14.13%, P<0.05; on the28th day, G-CSF/VEGF-treatment group:54±14.37%, G-CSF-treatment group:48.42±16.41%, control group:35.67±9.79%,P<0.05). Our results demonstrated that the number of crossings over a point where the platform had been was significantly increased in the VEGF-treated group compared to the untreated PDGF-hAPPV717I transgenic mice on the14th and28th days (on the14th day, G-CSF/VEGF-treatment group:6.5±3.81, G-CSF-treatment group:4.5±1.64control group:2.67±2.16, P<0.05; on the28th day, G-CSF/VEGF-treatment group:6.23±1.89, G-CSF-treatment group:5.23±1.61, control group:3.24±1.63, P<0.05). However, no difference was observed on the7th day.Part2Effects of G-CSF combined with VEGF on proliferation of stem cells and neural regeneration of transgenic mice modelCD133immunohistochemistry:CD133was expressed mainly in the surface of stem cells and showed buffy. The number of CD133positive cells varied according to different time points. The CD133expressions were at low level in day7,day14and day28in the control group. The expressions of CD133in G-CSF combined with VEGF group increased compared with G-CSF group and control group (P<0.05). BrdU/GAP-43immunofluorescence double tagging labeling:The slices were observed with fluorescence microscope. TRITC labeled BrdU positive cells were showed red, and GAP-43positive cells showed green. BrdU/GAP-43double positive cells showed yellow, which can be seen in day7after administration. Double labellde positive cells increase gradually, and reach the pick (17.36±3.29)%in day28after administration. The percentage of BrdU/SYN positive cells in G-CSF combined with VEGF group and G-CSF group were increased significantly compared with the control group and G-CSF group (P<0.05).Part3Effects of G-CSF combined with VEGF on synaptic plasticity of APP transgenic mice modelBrdU/SYN immunofluorescence double tagging labeling:The slices were observed with fluorescence microscope. TRITC labeled BrdU positive cells were showed red, and SYN positive cells showed green. BrdU/SYN double positive cells showed yellow, which can be seen in day7after administration. Double labellde positive cells increase gradually, and reach the pick (14.47±1.38)%in day28after administration. The percentage of BrdU/SYN positive cells in G-CSF combined with VEGF group and G-CSF group were increased significantly in day14and day28compared with the control group (P<0.05).NCAM immunohistochemistry:NCAM was expressed mainly in the surface of stem cells and showed buffy. The number of NCAM positive cells varied according to different time points. The NCAM expressions were at low level in day14and day28in the control group. The expressions of NCAM in G-CSF combined with VEGF group increased compared with G-CSF group and control group (P<0.05).Conclusion1. G-CSF combined with VEGF administration could ameliorate the learning and memory impairment in PDGF-hAPPV7171transgenic mice in vivo. 2. G-CSF combined with VEGF can improve the expression of stem cell marker CD133and neual regeneration marker GAP-43, promote the proliferation and neural regeneration in hypocampus area of the APP-transgenic mice.3. G-CSF combined with VEGF may increase synaptic mareker NCAM and SYN, so can promote synaptic function and improve synaptic plasticity.