Experiments were performed according to the Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Welfare Committee. All efforts were made to reduce the number of animals used and to avoid their suffering. C57BL/6J mice (20–25 g) were housed and acclimated to standard laboratory conditions (12-hour light/dark cycle / lights on at 7:00 AM and off at 7:00 PM) with free access to chow and water. Mice were subjected to one vessel occlusion (i.e. 1 VO) and sacrified as described in (Additional file 1: Figure S1).
For molecular analysis, mice were perfused with saline (0.9% NaCl), brains were removed and immediately frozen in dry ice (Additional file 1: Figure S1; Protocol A). For immunofluorescence, and Thioflavin S analysis, mice were perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, brains were removed and postfixed in 4% PFA (pH 7.4) at 4°C and then immersed in a PFA solution containing 10% sucrose overnight at 4°C. Fixed brains were frozen with dry ice/ethanol mixture, mounted on a microtome (Leica) and cut into 25 μm coronal sections. The collected slices were placed in tissue cryoprotectant solution containing 0.05 M sodium phosphate buffer (pH 7.3), 30% ethylene glycol, and 20% glycerol, and stored at −20°C until analysis (Additional file 1: Figure S1; Protocol B).
Aβ1–42 solution preparation
Monomeric HiLyte Fluor 555-labeled Aβ1–42, and non labelled Aβ1–42 (Anaspec, Fremont, CA, USA) were prepared as previously described . Briefly, lyophilized labelled, or non labelled Aβ1–42, was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (Sigma-Aldrich, St. Louis, MO, USA) to 1 μg/μl, dried under vacuum, and stored at −80°C. Immediately before injection, Aβ1–42 was dissolved in DMSO (Sigma-Aldrich) to 5 μg/μl, and was diluted in 0.9% NaCl sterile solution to obtain final dilutions of either 0.25 ng/μl (functional studies), or 0.1 μg/μl (proof of concept studies), which were always freshly prepared to avoid peptide’s oligomerization, and or degradation.
Induction of mild chronic cerebral hypoperfusion
Four-month-old C57BL/6J mice were subjected to either sham surgery, or to 1 VO by permanently occluding the rCCA, under anesthesia with 2% isoflurane. Briefly, a midline cervical incision was made and the rCCA was exposed and double-ligated with 6–0 silk suture thread (1 VO groups). The sham surgery consisted of a midline cervical incision under isoflurane anesthesia (2%) followed by the exposition of the rCCA (sham group). In both groups, the skin incision was closed with interrupted 6–0 silk sutures and the animals were then placed on a temperature-controlled blanket until they recovered from anesthesia. Twenty-four hours after 1 VO, two groups of mice were re-operated to reperfuse rCCA and reestablish cerebrovascular perfusion or received single intravenous injection, via the tail vein, of high dose of glucose (5 mg/ml, i.e. 5x physiological concentration) (Additional file 1: Figure S1; Protocol C, D).
Human Aβ1-42 injection upon mild chronic cerebral hypoperfusion
For imaging proof of concept studies (Additional file 1: Figure S1; Protocol E) hypoperfused animals received 10 μg of human Aβ1-42 Hilyte Fluor 555 intravenously via the tail vein, and Aβ was visualized by using two-photon intravital imaging in both contralateral, and ipsilateral hemispheres, 3 hours and 48 hours after injection. For functional studies (Additional file 1: Figure S1; Protocol F), hypoperfused animals received 25 ng of human Aβ1-42 Hilyte Fluor 555 intravenously via the tail vein, and Aβ was visualized by using two-photon intravital imaging in both contralateral, and ipsilateral hemispheres, 3 hours and 6 weeks after injection. In order to visualize Aβ deposits 6 weeks after Aβ1-42, Congo Red solution (4 μl; 1.5 μl/min) was injected in the Magna Cisterna 24 hours before imaging, to assure a complete distribution of the dye in the cerebrospinal fluid (CSF), and interstitial fluid (ISF). For molecular analysis studies, hypoperfused animals received 25 ng of non labeled Aβ1-42 intravenously via the tail vein, and were sacrified 24 hours after injection (Additional file 1: Figure S1; Protocol C).
Two-photon intravital microscopy
The two-photon intravital microscopy procedure was performed as described previously . Briefly, mice were deeply anesthetized with 2% isoflurane and mounted in a cranial stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). An incision was made to expose the skull and two small cranial windows were drilled corresponding to the following coordinates: (i) A/P +0.83 mm, M/L +0.5 mm, and A/P +0.83 mm, M/L −0.5 mm relative to the bregma (Additional file 2: Figure S2). All images were acquired on an Olympus FV1000 MPE two-photon microscope dedicated to intravital imaging. The two-photon Mai Tai DeepSee laser (Spectra-Physics, Newport Corp., Santa Clara, CA) was tuned at 950 nm for all the experiments, and the output power was set between 14 and 84 mW. Brain tissues were imaged using an Olympus Ultra 25X MPE water immersion objective (1.05 NA), with filter set bandwidths optimized for CFP (460–500 nm), YFP (520–560 nm), Texas Red/DsRed (575–630 nm) and Qdot 705/800 (669–800 nm) imaging. Detector sensitivity and gain were set to achieve the optimal dynamic range of detection. Using the Olympus Fluoview software (version 3.0a), images with a resolution of 512 × 512 pixels were acquired at different zoom factors (1X to 3X) and at 2.5 frames per second with auto-Hv option enabled, and exported as 24-bit RGB TIF files, while metadata for subsequent automatic setting-detection were exported in a TXT file. Kalman filter was used during scanning in order to reduce background. For each brain hemisphere imaging, a total of 101 frames were acquired from the cortex at a depth ranging from 50 μm (i.e. cortex surface) to 250 μm (inside the cortex), for a total depth of 200 μm. Acquired images were either stacked for 2-dimensional illustrations, or processed using ImageJ image analysis software to produce 3-dimensional illustrations represented as video sections.
Free-floating sections were washed with potassium phosphate-buffered saline (KPBS) (3x, 10 minutes) and then incubated for 20 minutes in a permeabilization/blocking solution containing 4% goat serum, 1% bovine serum albumin (BSA) (Sigma-Aldrich), and 0.4% Triton X-100 (Sigma-Aldrich) in KPBS. Sections were incubated overnight at 4°C with different primary antibodies diluted in the same permeabilization/blocking solution. The following primary antibodies were used: mouse anti-human Aβ monoclonal antibody (1/1500) (6E10, Covance Inc., Princeton, NJ, USA), rat anti-mouse CD31 monoclonal antibody (1/500) (BD Pharmingen, Franklin Lakes, NJ, USA), and mouse anti-Glial Fibrillary Acidic Protein (1/1500) (GFAP) monoclonal antibody (GA5, Chemicon International, Temecula, CA, USA). Afterwards, the sections were rinsed in KPBS (3x, 10 minutes), followed by 2 hours incubation with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody, Alexa Fluor 488-conjugated goat anti-rat secondary antibody, or Cy5--conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Sections were incubated overnight under light protected vacuum to allow an optimal fixation of brain sections on slides. The next day, sections were rinsed in KPBS (3x, 10 minutes), stained with 0.0002% DAPI for 5 minutes, rinsed again in KPBS (3×, 10 minutes), mounted onto SuperFrost slides (Fisher Scientific), and coverslipped with antifade medium composed of 96 mM Tris–HCl, pH 8.0, 24% glycerol, 9.6% polyvinyl alcohol, and 2.5% diazabicyclooctane (Sigma-Aldrich). Epifluorescence images were taken using a Nikon C80i microscope equipped with both a motorized stage (Ludl, Hawthorne, NY, USA) and a Microfire charge couple device color camera (Optronics, Goleta, CA, USA). Confocal laser scanning microscopy was performed with a BX-61 microscope equipped with the Fluoview SV500 imaging software 4.3 (Olympus America Inc., Melville, NY, USA).
Thioflavin S staining
Free-floating sections were stained with 1% Thioflavin S as described previously  with small modifications. Briefly, sections were washed with KPBS (3×, 10 minutes) and then incubated for 8 minutes 1% Thioflavin S solution in distilled water. Sections were washed in 100% ethanol (1×, 1 minute), then in 80% ethanol/water (2×, 1 minute), then rinsed in distilled water (3×, 1 minute), placed in KPBS solution, and finally mounted onto SuperFrost slides, and coverslipped with antifade medium. For immunofluorescent staining combined with 1% Thioflavin S staining, the former was performed as described above, and the time of washing steps with ethanol in the latter were decreased (30 seconds instead of one minute) to not alter antibody-epitope bindings. Stereological analysis was performed as previously described . The contours of the cortex, or hippocampus areas, of both contralateral and ipsilateral were traced as virtual overlay on the steamed images. The number of Thiofalvin S positive brain capillaries was counted in the total structure of five sections per brain (i.e. in all cortex, and hippocampus).
Entrapped IgG in brain capillaries
Free-floating sections were washed with KPBS (3×, 10 minutes) and then incubated for 20 minutes in a permeabilization/blocking solution containing 4% goat serum, 1% BSA (Sigma-Aldrich), and 0.4% Triton X-100 in KPBS. For endogenous IgG detection, sections were incubated for 2 hours with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody. Sections were rinsed in KPBS (3×, 10 minutes), stained with 0.0002% DAPI for 5 minutes, rinsed again in KPBS (3×, 10 minutes), mounted onto SuperFrost slides (Fisher Scientific), and coverslipped with antifade medium. The number of IgG positive brain capillaries was counted in the whole structure of the cortex, and the hippocampus separately. Stereological analysis was performed as described here above.
Brain capillaries isolation
Brain capillaries from contralateral and ipsilateral hemispheres were isolated on dextran gradient as described previously . Contralateral and ipsilateral hemispheres, of each animal, were separated and gently homogenized in a Teflon glass homogenizer in ice-cold microvessel (i.e. capillaries) isolation buffer (MIB; 15 mM Hepes, 147 mM NaCl, 4 mM KCl, 3 mM CaCl2, and 12 mM MgCl2) supplemented with 5% Protease Inhibitor Cocktail (P8340; Sigma) and 1% Phosphatase Inhibitor Cocktail 2 (P5726; Sigma). Homogenates were centrifuged at 1000 rcf for 10 min at 4°C. The resulting pellets were resuspended in 20% dextran (molecular weight, 64,000 to 76,000; D4751, Sigma) in MIB. Suspensions were centrifuged at 4400 rcf for 20 min at 4°C. The resulting crude brain capillaries-rich pellets were resuspended in MIB and filtered through two nylon filters of 120- and 30-μm mesh size (Millipore). The quality of trapped brain capillaries in 30-μm filters was checked. Brain capillaries were stored at −80°C until further use.
For total protein analysis isolated brain capillaries were homogenized and lysed in NP-40 lysis buffer supplemented with 5% Protease Inhibitor Cocktail and 1% Phosphatase Inhibitor Cocktail 2. Lysate samples were sonicated over two cycles lasting 20 s each at 4°C at 40% power. For nuclear protein analysis, nuclear fraction was extracted using commercially available kit (Thermo Scientific, IL, USA). Protein concentrations were measured by means of the Quantipro BCA assay kit (Sigma-Aldrich) according to the manufacturer’s protocol.
Western blot analysis
For total and phosphorylated protein analyses, 2× SDS loading buffer was added to lysate samples containing equal amounts of protein (10 μg). These samples were heated for all protein analysis studies except for those involving ABCB1, for which samples were loaded without heating to avoid aggregation of these highly glycosylated transmembrane proteins. Samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis, with primary antibodies diluted 1:1000 in 5% skim milk (Sigma-Aldrich) and 0.1 M tris-buffered saline–Triton X-100 (TBS-T). The following antibodies were used: Anti-ABCB1 (sc-8313), and -Lamin B (sc-6217) were purchased from Santa Cruz Biotechnology. Anti total GSK3α/β (5676), and total β-catenin (9562) were purchased from Cell Signaling Technology. Anti tyrosine phosphorylated GSK3α/β (ab4797) was obtained from Abcam, anti occludin (71–1500) was purchased from Invitrogen, and anti β-actin (MAB1501) was purchased from Chemicon. Primary antibodies were detected with horseradish peroxidase (HRP)–conjugated secondary immunoglobulin G (IgG) that were diluted 1:5000 in 5% skim milk and TBS-T and revealed by enhanced chemiluminescence plus (ECL) solution (Amersham International). Blots were digitized, densitometrically analyzed with ImageJ image analysis software (NIH), corrected for protein loading by means of Lamin B (nuclear β-catenin), or β-actin (all others) blots, and expressed as relative values comparing ipsilateral (hypoperfused) with contralateral (non hypoperfused).
Results are expressed as mean ± standard error of the mean (SEM). For multiple comparisons statistical analysis was performed using the one-way analysis of variance (ANOVA), followed by Tukey’s post hoc tests. For the comparison between two groups data were analyzed using standard two-tailed unpaired t-test's. A P value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism Version 6 for Windows (GraphPad Software, San Diego, CA, USA).