Animals
A group of homozygous 3×Tg-AD mice were obtained by purchasing breeding pairs from Jackson Laboratory (USA). C57/B6 mice were from Beijing Vital River Laboratory Animal Technology Co., Ltd., China as wild- type (WT) mice. The mice were grown in a sterile environment in the Laboratory Animal Centre of China Medical University, with light/dark cycle of 12 hours. In addition, experiments were performed according to the guidelines of the Animal Care and Use Committee of the China Medical University.
Peptide synthesis and preparation
The gene was identified based on the cDNA sequence of a gene 3-10 in GenBank Fragment synthesis :5 '-TT-Ecori-Cozak-ATG (initial Bcodon)
(Aβ3-10)10-TAG(stop codon)-NotI-XhoI-GG-3′, and 10 × Aβ3-10, was
cloned of mammalian expression vector by EcoRI/ (pcDNA3.1)
XhoI restricts websites.The recombinant plasmid was verified by NotI/
EcoRI digestion and gel electrophoresis.The correct plasmid sequence is
confirmed by nucleotide sequence analysis (Shanghai GeneCore Bio)technology.Recombinant plasmid was amplified in DH5 receptive cells of E. coli and purified using the E.z.n.TM Fastfilter plasmid - free Maxi kit (OMEGA, USA).
Immunization of Mice by In Vivo Electroporation
The 3×Tg-AD mice were randomly divided into two groups and immunized with p(Aβ3–10)10-MT vaccine (n = 10) and PBS (n = 10) respectively. The PBS group was used for positive group while C57/B6 mice was used for negative group. Mice with p(Aβ3–10)10-MT were intramuscular injected into the left leg and immunized with 10 times every 3 weeks. After anaesthetized, a pair of 26 electrode needles was inserted into the muscle 5 mm,covering the DNA injection sites, electrical pulses were transmitted using an electric pulse generator (ECM830, BTX Harvard Apparatus company, USA), Output of 6,75V pulses at a rate of one pulse per 200 ms[14].Orbital venous blood samples were taken for biochemical examination before the first immunization and 10 days after each immunization.
Immunoreactivity of antisera and response to Aβ polymers
Humoral immune response was detected by enzyme-linked immunoabsorbent assay(ELISA)[15]. In brief, we use 96-microwell plates coated with GST-Aβ proteins to detect the Aβ peptide in serum samples diluted with PBS at 1:1,000. Microtiter wells were treated with blocking buffer (5.0 % goat serum, 1 % BSA, and 0.05 % Tween-20 in PBS)and left at room temperature for 2 hours. Standard curve was produced by continuous dilutions of the standard 6E10 antibody (monoclonal anti-Aβ antibody, Covance).For western blot analysis, oligomeric Aβ was prepared as described by Dahlgren et al[16]. The peptide was dissolved in 1 mM hexafluoroisopropanol (Sigma) and removed under vacuum in a Speed Vac(Savant, Holbrook, NY). The remaining peptide was in 5 mM in dimethyl sulfoxide (Sigma) concentration. Free ham F-12 medium(Mediatech, Herndon, VA)was added with phenol red at 100 lM and maintained at 4 °C for 24 h. The sample dilution buffer(Carlsbad, CA) and 16.5% Tris–Tricine SDS–PAGE separation for NuPage sample. Western blotting was performed using induced anti-sera and an enhanced chemiluminescence system (Amersham, Arlington Heights, IL) as described previously[17].
Morris Water Maze Test
As described previously, Morris water maze is conducted in a 150cm round pool which was fulled with white milk, 40cm in diameter, quiet in the centre with room temperature 22℃[18]. The water maze test was performed within 2 weeks of immunization.The experiment was carried out in a circular tank 1.25 m in diameter and 0.4 m high with a digital acquisition camera for monitoring animal behaviour and a computer program for data analysis (ZH0065, Zhenhua Biological Equipment, China).The mice were trained on the visible platform for two days, then on the hidden platform for two days, and were withdrawn from the platform a day later for the probe test.Each mouse was placed in the water from the same position in each quadrant and reached the platform 60 seconds later.Finally, the escape delay, the number of times in the platform position has been crossed, and the percentage of time spent in the target quadrant are analyzed.
Immunohistochemistry of Aβ ,Tau,Neuron
Immunohistochemical analysis and quantitative staining sections were described previously[19].Aβ plaques were detected using a monoclonal anti-Aβ antibody 6E10. HT7 recognizing epitopes 159–163 (1,40, Thermo Scientific, Waltham, MA, USA) detected total tau, AT8 and AT180 recognize phosphorylated tau levels. AT8 identified Ser202/Thr205 site while AT180 identified phosphorylated Thr231 site seperately. Quantification of HT7- ,AT8-positive and AT180-positive neurons were quantified and the number of pixels (A.U.) represents total tau (HT7) or hyperphosphorylated tau (AT8 and AT180) load.Both cortex and hippocampus were performed of Aβ plaques and tau protein. In order to better identify neurodegenerative sensitive neurons, neuron-specific nuclear antigen NeuN antibody and pPKR antibody were used as markers.To be representative and accurate, images were analyzed using ImageJ(developed by W.S. Rasband, National Institutes of Health, Bethesda MD, USA, 1.47g) to obtain protein loads (percentage of staining rea).pPKR was performed on thirty cortical gray matter fields magnification ×20 from each brain region to determine the number of NeuN-positive neurons in each field.
Prussian blue for cerebral hemorrhage
To stain the microhemorrhage, series of coronal sections of the brains of laboratory and control mice were mounted on gelatin coated slides and stained with Prussian blue working fluid as described earlier [20].Briefly, parts of the brain were incubated with the same amount of mixture distilled H2O potassium ferrocyanide and 20% hydrochloric acid for 30 minutes.The subsequent sections are washed with water, redyed with a quick 10 minute red solution, rewashed with water, dehydrated and covered with glass slides using Depex mounting media (BDH Laboratory Supplies, England).
Immunofluorescence to detect relationship between microglial and Aβ
For double immunofluorescence staining, the frozen sections were placed in fetal bovine serum(1:20) and incubated at room temperature for 1 h.Sections were incubated overnight at room temperature with monoclonal anti-Aβ antibody 6E10 (dilution, 1:1000) and rabbit anti Iba-1(1:500). The mixture of goat anti-mouse sections were incubated for 2h at room temperature in a mixture of fluorescein isothiocyanate-conjugated goat anti-mouse IgG and Texas Red-conjugated goat anti-rabbit IgG after rinsed with PBS with fluorescein isothiocyanate for 2h. Then the sections were rinsed and covered with PBS/glycerol buffer. The fluorescence micrographs were captured and analyzed by confocal laser microscope.
Western Blot
We used western blot determine the level of Aβ oligomers, tau protein, calpain and five synaptic proteins in the cerebral homogenate from immunized mice.Protein samples were isolated on 12% SDS-PAGE gel and transferred to a PVDF membrane. In 20 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 0.05% Tween 20 (TBS-T), 10% skim milk closed membrane was washed with TBS-T.Then antibody 6E10 (1:1500),Tau5 (mouse tau antibody 1:200, Thermo scientific, Rockford, IL), AT8 (mouse anti-PHF tau antibody 1:200, Thermo scientific, Rockford, IL),AT180 (mouse anti-PHF tau antibody 1:500 Thermo scientific, Rockford, IL), anti-dynamin 1 (C16, 1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-PSD-95 (1:500, Invitrogen, CA), anti-synaptophysin (L128, 1:500, Bioworld Technology, Inc., MN, USA), anti-synapsin (1:500, Bioworld Technology, Inc.) or anti-β-actin (Sigma, MO) for 1 h at 37°C. Then, the membrane was incubated with HRP-conjugated IgG (GBI, WA) and protein was detected with enhanced chemiluminescence reagents (ECL, Pierce, IL). Density was performed using the Image J Software.We considered the value obtained in the untreated control group to be 100%.The values of each group are expressed as mean ±SE (n = 10) .
Statistical analysis
All statistical parameters were expressed as mean ±SE. We used SPSS19 to analyze immunological, neuropathological and behavioral results.Single factor analysis was used to test significant variance (ANOVA) for statistics.P < 0.05 was considered statistically significant different.