| Objective Anti-beta-amyloid (AP) immunotherapy is effective in removing brain Aβ, but has shown to be associated with detrimental effects. To avoid severe adverse effects such as meningoencephalitis brought by amyloid beta vaccine with adjuvant,and take advantage of amyloid beta antibody’s therapeutic effect on Alzheimer’s disease sufficiently, our group has developed a new Alzheimer vaccine with mutanted amyloid beta1-42peptide stimulating dendritic cells (DC). Amyloid beta with Freund’s adjuvant was inoculated at same time to act as positive control. Our previous work has confirmed that DC vaccine can induce adequate anti-amyloid beta antibody in PDAPP Tg mice safely and efficiently. DC vaccine can improve impaired learning and memory ability of Alzheimer animal model, do not cause microvasculitis, microhemorrhage and meningoencepalitis in animal model. The exact mechanism by which immunotherapy reduces Aβ deposition remains unknown. In this report, we studied the mechanism of the vaccine.Methods:PDAPPV717I transgenic mice were used in these studies, mice with punch number using random number table were divided into PFDM group (mice with mutanted Aβ1-42peptide stimulating dendritic cells treatment,12) Adjuvant group (mice with Freund’s adjuvant Aβ1-42treatment as positive control,12), DC group (mice with dendritic cells alone treatment as negative control of PFDM group,12), PBS group(mice with PBS treatment as negative control of Adjuvant group,12) Dendritic cells (DC) were obtained from C57/B6mouse femur and tibia. All the Tg mice were injected intraperitoneally. First vaccinations were given every two weeks for a total of three times After the treatment was done, the samples of brains were collected, fixed, paraffin-embedded and then4μm paraffin sections were done. The nuclear hormone liver X receptor (LXR), a membrane-bound protein tyrosine phosphatase (CD45), the ATP-binding cassette family of active transporters (ABCA1), The receptor for advanced glycation endproducts (RAGE), The b-site APP-cleaving enzyme (BACE1) were checked with immunstaining as well as Aβ. In the five tissue sections analyzed per animal, between five and ten of the densest areas under the microscopic field were submitted for quantitative analysis, namely the CA1, CA2, CA3, DG, Rad of the hippocampus and the cortex on coronal plane sections. Pixel intensities (gray level) ranged from0(densest-stained pattern; i.e., black) to255(lightest-stained pattern; i.e., white). An individual blinded to the experimental condition of the study performed all measurements.Results:(1) Aβ burden:PFDM group (0.18±0.035) and positive control group (0.29±0.078) was significantly reduced between the two vaccines no significant meaning. Aβ expression of DC group(4.44±0.57) and PBS group(4.74±0.66) hardly changed.(2) LXR burden:LXR in PFDM group (0.046±0.0068) and DC group(0.040±0.0083) were significantly higher than the adjuvant group (0.010±0.0035) and PBS group (0.012±0.0032).(3)ABCA1burden:ABCA1in PFDM group expressed the highest (9.28±1.04), the DC group (4.39±0.60) was significantly higher than the adjuvant group (1.52±0.56) and PBS group (1.42±0.54).(4) CD45burden:Expression in PFDM group was0.90±0.12, significantly higher than the adjuvant group (0.75±0.14), the DC group (0.20±0.055) and PBS group (0.20±0.043). Adjuvant group and DC group and PBS group is a significant difference, P value was less than0.001.(5) RAGE burden:Adjuvant group was the lowest (0.59±0.22), and were significantly different when compared with the other three groups. PFDM group (1.75±0.39) was significantly lower than the DC group (2.02±0.47) and PBS group (4.72±0.47).(6) BACE burden:PFDM group (1.09±0.32) was higher than the DC group (0.88±0.11) and PBS group (0.89±0.084); Adjuvant group was (1.08±0.29) higher than the PBS group (P<0.05).Conclusion:The results indicate that:(1) The reduction of Aβ via the DC vaccine occurs through multiple factors to achieve a new immune balance.(2) The beneficial results of our DC vaccine, which did not produce side effects, may be due to the LXR/ABCA1path.(3) DCs alone play a role in balancing the process of clearing the A(3generated while preventing unnecessary side effects. Objective Amyloid plaques are one of the pathological hallmarks of Alzheimer’s disease (AD). The visualization of amyloid plaques in the brain is important to monitor AD progression and to evaluate the efficacy of therapeutic interventions. The aim of this research is to test nontoxic contrast agents with great specificity and sensitivity, capable of rendering reliable MR images upon Aβ in brains.Methods:Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (μMRI) in AD transgenic mice, where we used intra-carotid and femoral mannitol to enhance blood brain barrier (BBB) permeability. In the present study, we used ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Aβ1-42peptide and polyethylene glycol (PEG) to detect amyloid deposition for in vivo7-T uMRI by femoral intravenous injection. We chose12-18month-old mice for in vivo and ex vivo uMRI and matched-age wild type mice for comparison with the same dose of USPIO-PEG-Aβ1-42. As the negative control, the Tg mice with USPIO or USPIO-Aβ1-42injection were used. USPIO-PEG-Aβ1-42was dissolved in300μl in0.9%sodium at a dose of0.2mmol Fe/kg body weight immediately before infusion. The nanoparticals were infused via the right femoral vein at a rate of60μl/min. Upon4hours, an in vivo7-TμMRI was employed for2hours and20minutes. The brains of the animals were subsequently extracted, and then an overnight PLP fixation and preservation with DMSO in preparation of ex vivo brain scanning. A3D gradient multi-echo sequence was used for imaging with a100μm isotropic resolution. The amyloid plaques detected by T2*-weighted MRI were confirmed with matched histological sections. Furthermore, two different quantitative analyses were used. For the in vivo images, T2*were used for measurement quantitative. The ex vivo scans were examined with voxel-based morphometry (VBM) using statistical parametric mapping (SPM) for comparison. The brain was cut by40um and stained (Aβ and Perl’s staining).Furthermore, two transgenic mice were injected with the same dose of USPIO-PEG-Aβ1-42into the internal carotid artery to evaluate whether the mode of injection is a deciding factor for the deposition of the contrast agent.Results The region of interest-based quantitative measurement of T2*values showed contrast injected Tg mice had significantly reduced T2*values compared to wild-type mice and control Tg mice with USPIO or USPIO-Aβ1-42injection. The result showed a remarkable difference between the flat region and the hippocampus region. The regional differences seen in VBM comparing USPIO-PEG-Aβ1-42injected Tg mice and wild type mice correlated with the amyloid plaque distribution histologically. Contrasting with no differences between the two groups of mice with USPIO-PEG-Aβ1-42and USPIO-Aβ1-42injection in regions of the brain with amyloid deposition.With double staining on pathology images, a good binding between USPIO-PEG-Aβ1-42and brain A(3exhibits. Following adequate treatment with USPIO-PEG-Aβ1-42on the experimental group, both in vivo and ex vivo senile plaques in rat brains produced high quality images.Conclusion:Our results demonstrated that both approaches were able to identify the differences between AD transgenic mice and wild type mice, after injected with USPIO-PEG-Aβ1-42. USPIO-PEG-Aβ1-42itself can get across the Blood-Brain Barrier to detect the Aβ1-42in the brain of Tg mice and responded dark spot no matter the images were in vivo or ex vivo. The feasibility of using less invasive intravenous femoral injections for amyloid plaque detection in AD transgenic mice facilitates using this method for longitudinal studies in the pathogenesis of AD. |
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