Animal Care
All protocols were performed according to the Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Welfare Committee. All experiments were approved by the local committee. All efforts were made to avoid their suffering. All mice were maintained in a pure C57BL/6J background, bred in house, and newborn pups were genotyped with PCR as advised by Jackson Laboratory protocols. All animals were housed up to four per cage in temperature and light-controlled room (12 h-light cycles from 7 am to 7 pm) and were fed (mouse chow) and allowed to drink water ad libitum. All mice were monitored for health status, including weight loss throughout all experimental protocols.
APP model and MDP treatment
APPSwe/PS1 expressing the chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1- dE9) under the control of independent mouse prion promoter elements [B6.CgTg(APPswe,PSEN1dE9)85Dbo/J]. A total of fifty-five 3-months old male APPswe/PS1 transgenic mice and twenty-five age-matched C57BL/6J wild type mice (WT) were utilized. Mice were injected two/three times per week with either MDP diluted in saline (10 or 20 mg/kg) or vehicle (saline 0.9%).
Triple-transgenic model
Mouse strains Cx3cr1gfp[B6.129P-Cx3cr1tm1Litt/J], GFP (under control of the chicken β-actin promoter and cytomegalovirus enhancer) and APPSwe/PS1 (see APP model section) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were injected four times for one week with either MDP diluted in saline (10 mg/kg) or vehicle (saline 0.9%).
Flow cytometry
Blood samples were collected from the submandibular vein and kept in EDTA coated vials on a rotator for < 1 h and fluorescence-activated cell sorting (FACS) analysis was performed as described by (23, 24). FACS and data acquisition were performed using SORP LSR II and FACSDiva softwares (both from BD), respectively. Results were analyzed with the FlowJo software (v10.0.7). For details on flow cytometry protocol, see additional file 5.
Sacrifices
All mice were sacrificed via intracardiac perfusion with 0.9% saline followed by 4% PFA pH 7.4. The brains were then retrieved, post-fixed 10–24 hrs in 4% PFA pH 7.4, and transferred in 4% PFA pH 7.4 + 20% sucrose for a minimum of 15 hours. In another set of experiments, brains were retrieved, and one hemisphere was snap-freeze for protein extraction while the other hemisphere was fixed in 4% PFA pH 7.4 + 20% sucrose. Brains were sliced in coronal sections of 25-µm thickness with a freezing microtome (Leica Microsystems), serially collected in an anti-freeze solution and kept at -20 °C until usage.
Post-mortem analysis
Immunofluorescence
Brain sections were washed four times for 5 min in KPBS and then blocked in KPBS containing 1% BSA, 4% NGS, and 0.4% Triton X-100. The tissues were incubated overnight at 4◦C with the primary Iba-1 antibody (1: 2,000; Wako Chemicals) and monoclonal anti-Aβ (6E10, 1: 3,000; Covance). After washing four times for 5 min in KPBS, tissues were incubated in the appropriate secondary antibody (IgG anti-mouse Alexa 488; Thermofisher and IgG anti-rabbit CY3; Jackson Immunoresearch) for 2 h at room temperature. Following further washes in KPBS and incubation with DAPI, the sections were mounted onto Micro Slides Superfrost Plus glass slides and coverslipped with Fluoromount-G (Electron Microscopy Sciences).
Image Acquisition and Analyses
Image acquisition of fluorescent staining images was performed using a Zeiss LSM800 confocal microscope supported by the Zen software (2.3 system) using the 4 × and 40 × lenses as described previously (25). Number of 6E10, Iba-1 associated with plaques were quantified by unbiased stereological analysis (26) using Stereo Investigator software (version 6.02.1, MicroBrightfield) attached to a Nikon C80i microscope equipped with a motorized stage (Ludl) attached to Microfire CCD color camera (Optronics). Four to six sections were analyzed for each animal.
Soluble Aβ1−42/Aβ1−40 ELISA
Brain levels of soluble Aβ1−42 and Aβ1−40 were quantified by using the Human Amyloid β42 and Human Amyloid β40 Brain ELISA kits (Millipore, Billerica, MA, USA). The experimental procedure was performed according to the manufacturer's instructions (27).
Western blot analysis
Hippocampus and cortex brain proteins were lysates as previously described (27). Proteins were then loaded in 4–15% agarose precast gels (Biorad) and electroblotted onto 0.45 µm Immobilon PVDF membranes. Membranes were immunoblotted with various primary antibodies, as described in Table 1, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and revealed by enhanced chemiluminescence plus (ECL) solution (GE Healthcare Life Sciences). Quantification was done by determining the integrative density of the bands using Thermo Scientific Pierce myImage Analysis Software v2.0. Optical values were normalized over actin.
Table 1
Represents antibodies used for immunoblot analyses and all related information, including the name of the company, molecular weight, species, secondary antibodies, and dilution rates.
Antibody | Company | Molecular Weight | Species | Dilution | Secondary Antibody dilution | Notes |
Actin | Millipore | 42 kDa | Mouse | 1/50 000 | 1/20 000 | |
APP | Millipore | ≈ 100 kDa | Mouse | 1/2000 | 1/5 000 | antigen retrival (Michaud, Hallé et al. 2013) |
Cox-2 | Santa Cruz | ≈ 75 kDa | Goat | 1/1 000 | 1/5 000 | |
Iba-1 | Wako | 18 kDa | Rabbit | 1/1 000 | 1/5 000 | |
ICAM | Santa Cruz | 85–110 kDa | Goat | 1/500 | 1/1 000 | |
LRP1 | CEDARLANE | 85 kDa | Rabbit | 1/1 000 | 1/40 000 | |
MCP1 | Cell Signaling | 13 kDa | Rabbit | 1/1 000 | 1/5 000 | |
PSD95 | Neuromab | 95 kDa | Mouse | 1/2 000 | 1/20 000 | |
Synaptophysin | Thermo Fisher Scientific | 34 kDa | Mouse | 1/10 000 | 1/100 000 | |
Trem2 | R&D Systems | 40 kDa | Rabbit | 1/500 | 1/2 000 | antigen retrival (Michaud, Hallé et al. 2013) |
VCAM | Santa Cruz | 90–100 kDa | Rabbit | 1/1 000 | 1/10 000 | |
NFkB p50 | CEDARLANE | 50 kDa | Rabbit | 1/1000 | 1/10 000 | |
Behavioral tests
Open field
Open field was performed to evaluate anxiety-like behaviors, exploration habits, and also locomotor activity as described by Hui et al. (28). Each mouse was individually recorded and analyzed by the ANY-maze system.
Water T mazeThe Water T maze assay was performed according to Guariglia et al. (29). The pool was filled with 23◦C (± 1◦C) water to a depth of 13 cm, which was 1 cm above the surface of the platform. Mice were trained to swim to a particular arm of the T maze and to remain on a submerged platform for 5 s. Mice had to complete six out of eight trials without error for two consecutive days out of three days to reach the learning criterion. The same criterion was considered for the reversal phase.
Two-Photon Intravital Microscopy Imaging
Prior to the imaging session (5–15 min), blood vessels were labeled by Qdot 705 (Qtracker705, 5% w/v in PBS, Invitrogen, ON, Canada) administered via the tail vein. Animals were anesthetized with the same ketamine/xylazine mixture described above and were placed prone on a small stereotaxic instrument where they were maintained at 37 °C by a temperature controlling device (RWD, Life Science Co., Shenzhen, China). The cranial glass window was covered with few drops of water, and intravital imaging was carried out with an Olympus FV1000 MPE two-photon microscope (Richmond Hill, ON, Canada) equipped with a Mai Tai DeepSee laser (Spectra-Physics, Newport Corp., Santa Clara, CA, USA) tuned at 925 nm. All images were acquired using an Olympus Ultra 25x MPE water immersion objective (1.05 NA), with filter set bandwidths optimized for YFP (520–560 nm), Texas Red/DsRed (575–630 nm) and Qdot 705/800 (662–800 nm) imaging. PMT sensitivity and gain were set in order to obtain a maximal dynamic range of detection. Images were acquired at a zoom factor ranging from 1.0 to 1.5x, with the Olympus Fluoview software (version 3.0a). Kalman filtering was deactivated for time-lapse imaging, and blood vessels were used as landmarks for chronic intravital imaging. All image processing was carried out with ImageJ (US National Institute of Health, Bethesda, MD, USA). The number of GFP-positive crawling cells into blood vessels was manually quantified over time. For details on two-photon intravital microscopy imaging experiments, see the additional file 5 and (22).
Statistics
Data are expressed as the mean ± SEM. Comparison between two groups was conducted using post hoc unpaired t-tests. Comparisons between more than two treatment groups were conducted using either one-way analysis of variance (ANOVA) or two-way repeated-measures ANOVA, followed by Tukey’s post-hoc test. Values were statistically significant if p < 0.05. All analyses were performed using GraphPad Prism Version 6 for Windows (GraphPad Software, San Diego, CA, USA) and SAS 9.4 (SAS Institute Inc., Cary, NC, USA). All panels were assembled using Adobe Photoshop CS5 (version 12.0.4) and Adobe Illustrator CS5 (version 15.0.2).