AQP4-/-/APP/PS1 mice were established as previously described . 2- or 3-month-old AQP4-/-/APP/PS1 mice, APP/PS1 mice, AQP4-/- mice and WT mice were used in this experiment. Animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Nanjing Medical University. All of the animals were handled according to approved by the Institutional Animal Care and Use Committee (NMU).
Anesthetized mice were fixed to a stereotaxic frame. For intracisternal tracer injection , the membrane of the cisterna magna was exposed and carefully pierced by a pulled glass capillary with a 10 μm tip diameter attached to a syringe. Then, 5 μl of 0.5% Texas Red-dextran (TR-d3, 3 kD, ThermoFisher, #D3328) dissolved in artificial cerebrospinal fluid (CSF) was injected, at a rate of 1 μl/min by a micropump (TJ-2A/L07-2A, Suzhou Wen Hao Chip Technology Co. Ltd., Jiangsu, China). For intrastriatal injection , the skin was opened, and the skull exposed. A burr hole was made with a hand-held drill on the coordinates anterior/posterior: +0.5 dorsal/ventral: +3.25 medial/lateral: +2.0 mm from bregma. The pulled glass capillary was implanted into the brain parenchyma. A one-microliter volume of 0.5% TR-d3 was then perfused, at a rate of 0.1 μl/min via the micropump. The micropipette remained in place for 5 min, then withdrew very slowly to avoid any possible backflow. Thirty minutes after intracisternal, or 1 h after intrastriatal injection, and with a second supplementary anesthesia, mice were transcardially perfused with fixative containing 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The brains and deep cervical lymph nodes (dcLNs) were removed and post-fixed for 24 h.
Cerebral injection of clodronate liposomes or adeno-associated virus (AAV) encoding apoE siRNAs
Clodronate liposomes (Liposoma, Amsterdam, The Netherlands; #P22JO419) were injected into the frontal cortex to deplete local microglia, as previously reported . Three-month-old APP/PS1 mice and AQP4-/-/APP/PS1 mice were intracerebrally administrated 1.5 μl clodronate liposomes, or PBS liposomes, for 5 min (coordinates relative to Bregma: +1.34 mm anterior/posterior, ±1.00 mm medial/lateral, and +1.00 mm dorsal/ventral). The microsyringe was withdrawn 2 min after injection. In order to down-regulate expression of apoE in the prefrontal cortex, a single dose (1010 viral particles in 1.5 μl) of AAV encoding siRNAs targeting for apoE was administrated into the frontal cortex of two-month-old APP/PS1 mice or AQP4-/-/APP/PS1 mice with the same stereotaxic parameters mentioned above. A subgroup of APP/PS1 mice and AQP4-/-/APP/PS1 received intracerebral injection of AAV encoding a non-sense sequence was used as controls. Mice were sacrificed for the Aβ-related pathological analyses, 5 days after clodronate liposome injection or 1 month after AVV injection, respectively.
Section and tissue preparation
Following anesthesia, mice were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde by perfusion pump (Cole-parmer, USA). Brain tissues and dcLNs were fixed in 4% PFA overnight and dehydrated with 20% sucrose solution dissolved in 0.01 M PBS for 3 days. Sections were cut at 30 μm on a cryostat (Leica, Wetzlar, Germany) and mounted onto gelatin-coated slides. For CSF tracer experiments, PFA post-fixed forebrain tissues were coronally sliced on a vibratome (Leica) at 100 μm and mounted onto gelatin-coated slides in sequence. For biochemical analyses, anesthetized mice were euthanized by decapitation. The cerebral cortex was promptly dissected. Tissues were flash frozen in liquid nitrogen and stored at −80 ℃ awaiting analysis.
Brain slices were blocked for 1 h at room temperature with 5 % BSA, incubated with primary antibodies including : rabbit anti-NeuN (1:500; Abcam, #ab177487), mouse anti-glial fibrillary acidic protein (GFAP) (1:800; Millipore, #AB3594), rabbit anti-GFAP (1:400; Abcam, #ab7260), rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba-1) (1:1000; Wako, #019-19741), rabbit anti-Aβ1-40 (1:250; CST, 12990), mouse anti-total Aβ (Aβ1-16) (1:1000; Covance, # 803001), rabbit anti-AQP4 (1:400; Millipore, #AB3594), rabbit anti-apoE (1:200; Abcam, #ab20874) or mouse anti-apoE (1:200; Abcam, #ab1906) overnight at 4 ℃. After extensive rinsing, sections were incubated for 2 h at room temperature with AF488-conjugated donkey anti-mouse IgG (1:1000, ThermoFisher, #A21202), AF488-conjugated donkey anti-rabbit IgG (1:1000, ThermoFisher, #A21206), AF555-conjugated donkey anti-rabbit IgG (1:1000, ThermoFisher, #A31572), AF555-conjugated donkey anti-mouse IgG (1:1000, ThermoFisher, #A31570), or AF647-conjugated donkey anti-mouse IgG (1:1000, ThermoFisher, #A31571). The sections were washed for 3×5 min in PBS containing 1.5 μM 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, #D21490), then cover slipped with buffered PBS/glycerol.
Cerebral cortex extracts were loaded onto 10-16 % Tris/tricine SDS gels, and transferred to nitrocellulose membranes before overnight incubation with one of the following primary antibodies: mouse anti-β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) (1:1000; Millipore, #MAB5308), anti-PS1 (1:1000; Sigma-Aldrich, #PRS4203), anti-neprilysin (NEP) (1:1000; Millipore, #AB4348), anti-insulin-degrading enzyme (IDE) (1:1000; Abcam, #ab32216), anti-low-density lipoprotein receptor-related protein (LRP1) (1:1000; Abcam, #ab92544) and anti-apoE (1:1000; Abcam, #ab20874) or GAPDH (1:1000; Bioworld; #AP0063). Horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories) were used, and bands were visualized using ECL plus detection system. GADPH was utilized as an internal control for protein loading and transfer efficiency.
Quantitative analyses of images
Fluorescence micrographs were captured by a digital microscope (DM4000B, Leica) with constant exposure time, offset, and gain for each immunofluorescent staining marker, and analyzed by the Image-Pro Plus 6.0 Analysis System (Media Cybernetics Inc., San Francisco, CA, USA). The frontal cerebral cortex in each section was manually delineated. The area of positive signal of GFAP, Ibal, Aβ1-40, total-Aβ, thioflavin-S, or TR-d3 was measured using the interest grayscale threshold analysis with constant settings for minimum and maximum intensities for each staining marker . The percentage area of positive signal was calculated by dividing the area of positive signal to the total area in the region of interest. The percentage area of TR-d3 in the dcLNs was also detected with the same method. For analysis of total-Aβ (or apoE) accumulation within neurons, astrocytes and microglia, the percentage of area covered by double positive signal for total-Aβ (or apoE) and NeuN, GFAP or Iba1 were calculated, respectively. For analysis of AQP4 polarization, the mean AQP4-immunoreactive intensity at the regions immediately abutting vessels or pia maters and correspondingly adjacent parenchymal domains was measured. AQP4 polarization was obtained by comparing expression ratios of AQP4 at perivascular or pia surface versus parenchymal domains [9, 19].
All data were expressed as means ± SEM. Using SPSS software, version 16.0 (SPSS Inc., USA), and analyzed by Student’s t-test or ANOVA followed by Newman-Keuls post-hoc multiple comparison test as indicated in the figure legends.