| PartⅠVEGF ameliorates the memory impairment in APP transgenic miceOBJECTIVE:Alzheimer’s disease (AD), the primary common cause of dementia in the geriatric population, is characterized by memory loss and cognitive decline. Currently, there are not radical treatments. Vascular endothelial growth factor (VEGF) is an established angiogenic trophic factor. After binding with the VEGF receptor, VEGF induces endothelial cells to proliferate, migrate, survive, and assemble into an interconnected vessel network. The administration of VEGF reduced infarct size, improved neurological performance, stimulated angiogenesis in the striatal ischemic penumbra, and enhanced the maturation of stroke-induced cortical neurogenesis and dendritic formation of newborn neurons in a rat model. However, little is known about the restorative effect of VEGF for AD in vivo.MATERIALS AND METHODS1. Animals and Drug treatmentThirty-six PDGF-hAPPV717I transgenic mice were divided randomly into two groups, a VEGF-treated and control group. The VEGF-treated group was intraperitoneally (i.p.) injected with 8μg/kg·d of VEGF (R&D Systems) for three consecutive days as described previously, the control group was injected i.p. with an equal volume of PBS. The eighteen PDGF-hAPPV717I transgenic mice of each group were divided randomly into three subgroups for analysis on the 7th,14th, or 28th days after the last day of VEGF injection.Morris water maze testingThe Morris water maze task was performed to obtain the spatial memory impairment of PDGF-hAPPV717Itransgenic mice, according to the method of Morris. The escape latency was analyzed the morning before the injection and on the 7th,14th, and 28th 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 for 60 s 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.RESULTS1. Before VEGF treatment, the escape latencies of the VEGF-treated group were similar to the control group (VEGF-treatment group:62.26±22.07s, control group:60.98±17.11s, P>0.05). After the intraperitoneal administration of VEGF or PBS, we found that VEGF significantly shortened the escape latencies in PDGF-hAPPV7171 transgenic mice compared to controls on the 7th,14th, and 28th day after VEGF treatment. (On the 7th day, VEGF-treatment group:30.91±12.30s, control group:46.07±7.21 s, P＜0.05; on the 14th day, VEGF-treatment group:24.95±10.40s, control group: 43.03±11.70 s, P＜0.01; on the 28th day, VEGF-treatment group:25.68±8.38 s, control group:45.54±9.55 s, 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, or 3) strategies involving repetitive looping paths. After the administration of VEGF or PBS, we observed that search strategy use differed markedly between VEGF-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 (21% on the 7th day,29% on the 14th day,21% on the 28th day) of repetitive looping paths strategies. The fraction of spatial strategies use stayed relatively constant at 29% to 33%. In contrast, VEGF-treated mice used a high proportion (58% on the 7th day,63% on the 14th day,67% on the 28th day) of spatial strategies along with some systematic but non-spatial strategies (33% on the 7th and 14th days,29% on the 28th day) and very few repetitive looping paths strategies (8% on the 7th day,4% on the 14th and 28th days). A chi-square test showed that VEGF-treated mice preferentially choose spatial strategies while repetitive looping paths strategies are used more often by control mice than VEGF-treated PDGF-hAPPV717I transgenic mice.3. After removal of the platform, we found that the time spent traveling in the target quadrant of VEGF-treated mice was longer than that of untreated mice. Compared to the untreated PDGF-hAPPV7171 transgenic mice, the mean value of the percentage of the total time in the target quadrant was significantly increased on the 14th and 28th days after VEGF treatment (on the 14th day, VEGF-treatment group:53.89±21.72%, control group: 31.95±15.93%, P＜0.05; on the 28th day, VEGF-treatment group:49.72±18.15%, control group:32.78±4.79 s, 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-hAPPV7171 transgenic mice on the 14th and 28th days (on the 14th day, VEGF-treatment group:6.5±1.64, control group:2.67±2.16, P＜0.05; on the 28th day, VEGF-treatment group:5.83±1.72, control group:2.83±1.33, P＜0.05). However, no difference was observed on the 7th day.CONCLUSION:These data indicate that VEGF administration could ameliorate the learning and memory impairment in PDGF-hAPPV717I transgenic mice in vivo. Part II VEGF induced angiogenesis in brain of APP transgenic miceOBJECTIVE:Accumulating evidence implies that vascular changes may play an essential role in the pathogenesis of AD. Cerebral blood flow (CBF) is usually significantly reduced in AD patients. Correspondingly, the cortical capillary densities are decreased and the microvessels are narrowed and torn off, especially microvessels close to the senile plaques. The deposition of Aβ(beta-Amyloid) evokes vasoconstriction, reduces the blood flow, and even induces endothelial cell damage. Meanwhile, neurovascular unit impairment and vessel constriction may reduce Aβclearance across the blood-brain barrier (BBB), which leads to Aβaccumulation in the blood vessels and in the brain parenchyma, eventually inducing tissue injury, neuronal death and cognitive decline. A number of studies suggest that a lower level of VEGF might confer a susceptibility to neurodegeneration and vascular dysfunction in AD. In this part, we observed whether an intraperitoneal injection of VEGF relieved the deficiency of VEGF in the brain of AD model and ameliorated the cognitive impairment of AD mice.MATERIALS AND METHODS1. Animals and Drug treatmentThirty-six PDGF-hAPPV717I transgenic mice were divided randomly into two groups, a VEGF-treated and control group. The VEGF-treated group was intraperitoneally (i.p.) injected with 8μg/kg·d of VEGF for three consecutive days as described previously, the control group was injected i.p. with an equal volume of PBS. At the same time, all mice received daily i.p. injections of 50 mg/kg BrdU for 10 consecutive days. The eighteen PDGF-hAPPV7171 transgenic mice of each group were divided randomly into three subgroups for analysis on the 7th,14th, or 28th days after the last day of injection.2. Flow CytometryBlood samples (0.5 ml) were collected from the retro-orbital venous plexus into tubes with ethylenediaminetetraacetic acid (EDTA) anticoagulant on the 7th,14th, and 28th days after the last administration of VEGF. Samples were analyzed on a BD FACSCalibur flow cytometer with FACStationCell software (Becton Dickinson). CD34+ cells were recorded as a percentage of the total gated lymphocyte population.3. Tissue processingThree mice from each group were anesthetized with 10% chloral hydrate (0.38 g/kg, i.p.) on the 7th,14th, and 28th days after the last administration of VEGF and perfused transcardially with PBS and ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH=7.2). The brains were removed and post-fixed in the same fixative for 20 hours at 4℃and transferred to 30% and 20% sucrose/PBS for 24 hours. Fifteen-micrometer sections were prepared on a cryostat. The sections through the hippocampus were collected.4. Immunohistochemistry analysisDouble immunofluorescence staining of CD34/VEGFR-2, vWF/VEGFR-2 and vWF/BrdU was performed.To detect the expression of vWF in the mouse brain, immunohistochernical staining was performed. Immunofluorescence staining of CD34 was used for detect CD34-positive cells in brain of mice.RESULTS1. VEGF stimulated angiogenesis in the brains of PDGF-hAPPV71TI transgenic miceWe found a significant increase in blood vessels in the hippocampus and cortex, after treatment with VEGF compared to the control group on the 7th and 14th days. The numbers of vWF-positive vascular in hippocampus of control group were 2.67±0.71 on 7th day and 2.44±0.88 on 14th day. The numbers of vWF-positive vascular in cortex of control group mice were 6.33±1.41 on 7th day and 6.11±1.66 on 14th day. However, The number of vWF-positive vascular in hippocampus of VEGF-treatment group mice were 6.00±0.87 on 7th day and 10.11±1.54 on 14th day. The numbers of vWF-positive vascular in cortex of control group mice were14.55±1.66 on 7th day and 16.22±1.99 on 14th day. Moreover, the number of vWF-positive vascular in the hippocampus and cortex increased reached a peak on the 14th day after treatment with VEGF. Subsequently, the number of blood vessels decreased gradually. On the 28th day after treatment with VEGF, the number of blood vessels in the hippocampus of VEGF-treated PDGF-hAPPV7171 transgenic mice was not different from control.We found that some vWF-positive vessels co-localized with BrdU in the hippocampus and cortex of VEGF-treated PDGF-hAPPV717I transgenic mice, which indicated the neovasculature formation after VEGF-treatment. We checked the expression of VEGFR-2 on vWF-positive cells in the brain by double-staining with vWF and VEGFR-2 on the 14th day. Our results demonstrated almost all of the vWF-positive cells in the hippocampus expressed VEGFR-2 in the hippocampus of VEGF-treated mice.To test if EPCs were recruited to the brain after the administration of VEGF, double-immunostaining with CD34+/VEGFR-2+ was performed. We observed double-labeled cells with CD34 and VEGFR-2 in the hippocampus and cortex of PDGF-hAPPV7171 transgenic mouse brain treated with VEGF.2. VEGF stimulated CD34+ cell proliferation in the peripheral bloodAfter treatment with VEGF, the mean percentage of CD34+ cells in the peripheral blood of mice was elevated. Seven days after treatment with VEGF, the percentage of CD34-positive cells in the peripheral blood was 1.04±0.89%, which was 15-fold higher than the controls. This high percentage of CD34-positive cells was maintained until day 14. On day 28, the percentage of CD34-positive cells (0.25±0.1%) decreased compared to day 14. No difference in the percentage of CD34-positive cells in the peripheral blood was observed between VEGF-treatment group and control group on day 28.CONCLUSION:These data indicated that VEGF could mobilize EPCs into the peripheral blood and home them into the brains and enhance angiogenesis in the hippocampus and cortex of VEGF-treated PDGF-hAPPV7171 transgenic mice. PartⅢThe effect of VEGF on ChAT expression and Aβdeposition in brain of APP transgenic miceOBJECTIVE:To identify the molecular basis for the rescue of cognitive function by VEGF, we analyzed the number of ChAT-positive cells and ChAT protein expression in the NBM of PDGF-hAPPV717I transgenic mice with or without VEGF treatment. To investigate whether the amelioration of memory impairment in PDGF-hAPPV717I transgenic mice was related to the accumulation of Aβ1-42, we performed immunohistochemistry to check the deposition of Aβ1-42 in the hippocampus and cortex and vascular of PDGF-hAPPV717I transgenic mice.MATERIALS AND METHODS1. Animals and Drug treatmentAs same as partⅡ.2. Tissue processingThree mice from each group were anesthetized with 10% chloral hydrate (0.38 g/kg, i.p.) on the 7th,14th, and 28th days after the last administration of VEGF and perfused transcardially with PBS and ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH=7.2). The brains were removed and post-fixed in the same fixative for 20 hours at 4℃and transferred to 20% and 30% sucrose/PBS for 24 hours. Fifteen-micrometer sections were prepared on a cryostat. The sections through the hippocampus and nucleus basalis of Meynert (NBM) were collected. Three mice were anesthetized with 10% chloral hydrate (0.38 g/kg, i.p.), and the brains were carefully removed quickly. The tissues were stored at-80℃for Western blot analysis.3. Immunohistochemistry analysisTo detect the expression of Aβ1-42 and ChAT in the mouse brain, immunohistochemical staining was performed using standardized methods.4. Western blot analysisWestern blot analysis was carried out according to standard methods.RESULTS 1. VEGF enhanced the level of ChAT in the brains of PDGF-hAPPV717I transgenic miceWe found the number of ChAT-positive cells in the NBM of VEGF-treated PDGF-hAPPV717I transgenic mice increased compared to the PBS-treated controls (P<0.05). The results of the Western blot also showed that the expression of ChAT in the NBM increased and was significantly higher than the controls on the 7th,14th and 28th days after treatment with VEGF (P<0.05).2. VEGF decreased the Aβdeposition and Aβlevel of in the brains of PDGF-hAPPV717I transgenic miceWe performed immunohistochemistry to check the level of Aβ1-42 in the brains of PDGF-hAPPV7171 transgenic mice. On the 7th,14th and 28th days after VEGF treatment, immunohistochemistry showed that the immunoreactivity of Aβ1-42 was significantly decreased in the hippocampus, cortex and vascular of VEGF-treatment PDGF-hAPPV7171 transgenic mice compared to the controls (P＜0.05).CONCLUSION:VEGF enhanced the level of ChAT in NBM and decreased the deposition of Aβ1-42 in the hippocampus, cortex and vascular of VEGF-treatment PDGF-hAPPV717I transgenic mice. |
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