6-month-old and 9-month-old APP1/PS1 double transgenic AD model mice (9 each) [provided by Jiangsu Changzhou Cavens Experimental Co., Ltd. (license number: SCXK (Su) 2016-0010)] and 9 wild-type (WT) mice (C57BL/6 J) were used [provided by Beijing Sibefu Biotechnology Co., Ltd. (license number: SCXK (Jing) 2019-0010)]. The animals were kept in an SPF animal room at a constant temperature (23 °C ± 2 °C), and the conditions included the alternation of white light and dark (8:00 a.m. to 8:00 p.m. white light; 8:00 p.m. to 8:00 a.m. dark) and free access to food and water. The mice used in this experiment were all males, which met the SPF clean-level standard for laboratory animals issued by the Ministry of Health. Male Sprague–Dawley (SD) rats (30–550 g, approximately 3 weeks old) were provided by the Animal Experiment Center of Tongji Medical College, Huazhong University of Science and Technology. During the experiment, raising and obtaining animal materials complied with the relevant rules and regulations of the Animal Management Committee of Huazhong University of Science and Technology and the International Association for Animal Research (IASP) on the management and protection of experimental animals.
Antibodies and reagents
The following antibodies were used in this study: β-amyloid (20.1) (Santa Cruz Biotechnology); PDGFRβ (Abcam and CST); NG2 (Abcam); LRP1 (Abcam); CD36 (ABclonal); CD31 (CST); GLUT1 (Abcam); CD36 (BD); β-actin (ABclonal); HSP60 (Santa Cruz Biotechnology); Tim23 (ABclonal); LC3B (Sigma–Aldrich); cleaved-caspase 3 (CST); caspase 3 (ABclonal); BAX (ABclonal); BCL2 (ABclonal); GPx4 (Proteintech); xCT (Proteintech); NOX1 (Proteintech); GAPDH (Proteintech); ferritin (Beyotime); and a Mitophagy Antibody Sampler Kit (CST). Aβ1-40 (ChinaPeptides Co., Ltd.), BODZPY 581/591 (Invitrogen), Mito-FerroGreen (Dojindo), LysoTracker Red (Invitrogen), HiLyte Fluor™ 555-Aβ1-40 (Anaspec), FITC-Aβ1-40 (Bachem), and FITC-dextran (Sigma–Aldrich) were also used.
Mouse brain PET/CT imaging
Abnormal changes in brain 18F fluorodeoxyglucose (18F-FDG) uptake are often used as a sensitive indicator of the BBB . The procedure used was as follows. (1) One day before the experiment, the mice had not fasted from food and water. (2) Gas anesthesia (2% isoflurane) and tail vein injection of 200 ± 10 µCi 18F-FDG were used. (3) Then, scanning was immediately started, and PET was performed first, followed by CT. (4) PET was performed in dynamic mode with head scan for 10 min; CT was performed in normal mode. (5) The reconstruction conditions were as follows: PET, two iterations, 12 subsets, slightly filtered with time division of 1 min*10; CT, FDK, image size of 256, FOV of 1.0. (6) After scanning, the mice were placed in a lead-shielded room, restored to a regular diet, and fed normally after 10 18F half-lives. The mean standardized uptake value (SUV) was calculated using the following formula: mean pixel value with the decay-corrected region-of-interest activity (μCi/kg)/(injected dose [μCi]/weight [kg]).
We used 4% sodium pentobarbital [50 mg/kg, intraperitoneal (i.p.)] to anesthetize the mice and then fix them on the operating table. After thorough disinfection with 75% alcohol, tissue scissors were used to cut the skin along the midline of the mouse sternum from bottom to top. The sternum was cut to fully expose the heart. Care was taken to prevent damage the blood vessels. After the diaphragm was carefully broken, toothless long forceps were used. After carefully clamping and fixing the heart, we quickly inserted the perfusion needle from the apex of the heart into the left ventricle, paying attention to the angle and depth of the needle to avoid penetrating the interventricular septum. Then, we cut a small opening in the right atrial appendage with ophthalmological scissors, opened the perfusion valve, infused precooled saline from the left ventricle, and allowed flow out from the right atrial appendage through the large circulation; the outflow was colorless. The lungs, liver, and mucous membranes were whitish. The mice were perfused and fixed with 4% paraformaldehyde at 4 °C for approximately 10 min. Finally, the intact brain tissue was carefully removed and soaked in 4% paraformaldehyde for histological analysis.
Immunofluorescence of brain slices
Brain slices prepared from the indicated groups of mice were subjected to double immunofluorescence staining. The primary antibodies used in our studies were mouse anti-β-amyloid (20.1) (1:25, Santa Cruz Biotechnology), rabbit anti-PDGFRβ (1:100, Abcam), and rabbit anti-CD36 (1:100 ABclonal). All images were obtained, and colocalization was visualized, using a Nikon confocal microscope.
Cell isolation, generation, culture, and activation
In this experiment, the bEnd.3 cell line was purchased from ATCC and was passaged once every 3–4 days according to the company’s requirements. Brain pericytes were extracted according to a previous extraction method [31, 32] and improved. Each time, 5 male SD rats at approximately 3 weeks of age were used. The SD rats were from Huazhong provided by the Animal Experiment Center, Tongji Medical College, University of Science and Technology. After anesthetizing the rat (as described previously), we immediately removed the brain. Then, the whole brain was placed in prechilled 75% alcohol for 3–5 min for disinfection, the cerebral cortex was removed, 500 µl each of 0.1% type II collagenase and DNase I (1000 U/ml) was added and the brain tissue was digested a constant temperature water bath at 37 °C for approximately 1 h. Finally, the capillaries were separated with Percoll and seeded in a 6-well plate containing a unique medium for pericytes. After 72 h, the medium was changed for the first time, and new medium was added every 2 days. PDGFR-β and CD31 antibodies were used for immunostaining.
Primary cells were incubated with 500 nM human HyLyte Fluor™ 555-labeled Aβ1-40 (Anaspec, Fremont, CA) for 2 h in serum-free media as previously described . Following treatment, the cells were rinsed twice with 1X PBS without calcium and magnesium. We observed the fluorescence associated with Aβ under a fluorescence microscope and superimposed the cells under white light to observe the uptake of Aβ. The cells on the slides were fixed and processed for imaging according to the abovementioned immunocytochemistry protocol. After incubation with the primary anti-CD36 antibody (1:200), the cells were stained with DAPI to observe the colocalization of CD36 and Aβ1-40.
Construction of in vitro BBB model
After separate culture of the BBB model, bEnd.3 cells (1.0 × 105 cells/cm2) were placed on the front side of the transwell and then cultured. For the contact cocultivation model, we referred to a previous method [34, 35]. Transwell cell membranes (24 wells, 0.4 μm) were seeded with pericytes (1.0 × 104 cells/cm2) on the reverse side. After 6 h, the pericytes were firmly attached, bEnd.3 cells (1.0 × 105 cells/cm2) were seeded on the front side of the transwell cell membrane and cultured. Pericytes and bEnd.3 cells were in contact through the small holes between the membranes. Generally, the fluid was changed every 2 days, and the BBB model was established for use in approximately 6 days.
Transendothelial electrical resistance (TEER) determination
The TEER was measured with an EVOM2 resistance meter (WPI Company) . After calibration as needed, we placed the long end of the electrode in the culture medium outside the cell insert and the short end in the upper culture medium of the cell (taking care not to contact the cell with the wall of the culture plate). The dynamic TEER value was used as the criterion for forming tight cell junctions: Ω measurement = Ω actual measurement- Ω blank. The area was used to calibrate TEER: Ω/cm2 = Ω measurement/S, where S is the area of the bottom film of the board .
BBB permeability test
Permeability tests with dextran or Aβ labeled with fluorescein isothiocyanate (FITC) [32, 38, 39] were performed using FITC-dextran (molecular weight 40 kDa) and FITC-Aβ1-40 to evaluate the permeability of the in vitro BBB to larger molecules. After the BBB model was established, 100 nM or 1 µM Aβ1-40 was added to the lower chamber of the transwell container in the experimental group, and then assay buffer containing 5 µM FITC-dextran or FITC-Aβ1-40 (1 µM) was added. We further cultivated the cells in the lower chamber of the BBB system. At 0, 30, 60, 90, 120, and 180 min after the addition of fluorescein, 50 μl of the medium was collected from the upper chamber and supplemented with an equal volume of medium. A fluorescence microplate reader was used to detect the fluorescence intensity of the sample (excitation wavelength 485 nm; emission wavelength 520 nm). According to the standard curve of FITC-dextran/Aβ, the relative fluorescence unit was converted to the value of ng/ml, and the background fluorescence and serial dilution were corrected during the experiment.
Aβ1-40 was mixed to 1 mM with hexafluoroisopropanol. The sample was vortexed to dissolve the mixture, stored in a fume hood to air dry overnight, resuspended in DMSO, mixed with 1 mM liquor, and aggregated at 37 °C. This sample was stored at − 80 °C and diluted with culture medium to the required concentration for use.
Gene silencing with lentivirus-mediated siRNA
LV-CD36-RNAi was obtained from GeneChem. According to the manufacturer’s protocols, the lentivirus and negative control sequences were transduced into pericytes. The virus was added according to the appropriate multiplicity of infection (MOI). Puromycin (2 µg/ml) was added after 72 h to select cells. After 1 week of screening, the cells were passaged at 1:2 in T25 bottles, and the medium was changed every 2 days. qRT–PCR and immunoblotting experiments were used to detect the silencing efficiency of siRNA, which was then used for the follow-up experiments.
Analysis of reactive oxygen species (ROS) production
After cell treatment, a lipid ROS fluorescent probe (BODIPY 581/591, 50 µM) was added for 1 h or an intracellular Fe2+ probe (Mito-FerroGreen, 5 µM) working solution was added, and the cells were incubated at 37 °C. After culture for 30 min, the cells were washed 3 times with serum-free medium. Finally, the cells were evaluated using a multifunctional fluorescence microplate reader.
ROS fluorescent probe
Dihydroethidium (DHE), a superoxide anion fluorescent probe, is bound to DNA by ROS oxide in living cells, and the fluorescence is red. The Reactive Oxygen Species Assay Kit (also called ROS Assay Kit) contains the fluorescent probe DCFH-DA. ROS can oxidize this probe into green-emitting DCF in cells. The probe loading steps were as follows: DHE and DCFH-DA were diluted with serum-free medium to 5 µM and 10 µM, respectively. The cell supernatant was aspirated, the cells were washed twice with PBS, and DHE or DCFH-DA was added to cover the cells. Then, the cells were placed in a 37 °C cell incubator and incubated for 60 min. The cells were washed twice with PBS before observation, and images were obtained with a fluorescence microscope to remove the probes in the supernatant.
Detection of mitochondrial ROS (MitoSOX) and mitochondrial membrane potential (Δψ)
The Δψ and MitoSOX assays were performed according to the manufacturer’s specifications. The cells were treated as described above. After stimulation with Aβ1-40, the cells were separated using trypsin and centrifuged at 3000 rpm for 5 min. Then, the cells were resuspended in pericyte culture medium and stained as follows. Mitochondrial ROS were incubated with 5 µM MitoSOX at 37 °C for 15 min, and mitochondrial membrane potential was determined with 100 nM TMRM at 37 °C for 30 min. After washing, the cells were subsequently measured using an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software.
Annexin V-FITC/PI double-label staining to detect cell apoptosis
The culture medium was discarded, and the cells were digested with 0.25% trypsin without EDTA at room temperature. After the cells were round and floating under the microscope, the digestion was immediately stopped with complete culture medium, and the cell suspension was transferred to a flow tube.
The sample was centrifuged at 1000 rpm, and the precipitate was left after 5 min.
The cells were washed twice with precooled PBS and resuspended at 1 × 106 cells/ml in 1 × buffer.
One hundred microliters of the resuspended cell solution was placed in a flow tube.
Then, 5 µl of FITC Annexin V and 5 µl of PI were added.
The sample was vortexed gently and incubated for 5 min at room temperature in the dark.
Then, 400 µl of 1 × buffer was added to each tube for testing within 1 h. The cells were subsequently measured using an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software.
The Annexin V-FITC/PI double labeling results were analyzed using FlowJo software, and the cells were categorized in the following four quadrants: lower left, live cells; lower right, early apoptosis; upper right, late apoptosis; and upper left, cell fragments. Data collection and analysis were performed.
Measurement of adenosine-5ʹ-triphosphate (ATP) levels
ATP levels were determined using an ATP Bioluminescence Assay Kit following the manufacturer’s instructions. Briefly, the supernatant was discarded, lysis solution was added, and the samples were shaken to fully lyse the cells (0.2 ml was added to each well of the 6-well plate). After lysis, the cells were transferred to a 1.5 ml EP tube and placed in a 4 °C centrifuge at 12,000 rpm for 10 min. After washing, the protein concentration was determined by the BCA method. First, 100 µl of ATP detection working solution was added to each well of a black 96-well plate. Then, 20 µl of sample or standard (drawn ATP standard curve) was added and mixed quickly. The samples were immediately tested in multifunctional microplate reader (luminometer) mode. The ATP concentration was converted to µmol/µg according to the protein concentration.
Detection of superoxide dismutase (SOD) in the cell supernatant
The protocol was performed according to the SOD detection kit manual (Nanjing Jiancheng Bioengineering Institute, A001-3). First, after the cells were treated according to the above method, the cell supernatant was transferred to a 1.5 ml EP tube and centrifuged at 2000 rpm for 5 min. After the cells were lysed, the protein concentration was determined by the BCA method, and the samples were used for subsequent experiments. After thorough mixing, the mixture was incubated in a 37 °C incubator for 20 min and measured at 450 nm with a microplate reader. Finally, the OD value was converted to U/ml.
Glutathione peroxidase (GSH-PX) detection
With a GSH-PX test kit (Nanjing Jiancheng Bioengineering Institute, A005), the enzyme activity can be calculated according to the consumption of reduced glutathione in the enzymatic reaction. Cell protein was extracted according to the above method, and BCA assays were used to measure the protein concentration of GSH-PX. First, the optimal sampling concentration was determined, and an inhibition rate of 45% to 50% was taken as the optimal sampling concentration. The inhibition rate formula was as follows: inhibition rate = (nonenzyme tube-enzyme tube) OD value/nonenzyme tube-enzyme tube OD value × 100%, and the result was between 15 and 55%. Then, the OD value of each tube was measured at a wavelength of 412 nm according to the instructions, and the GSH-PX activity was calculated.
Iron content detection
We used an iron colorimetry kit (Elabscience Biotechnology Co., Ltd.); ferrous iron and pyridine combine to form a pink complex, and the amount of iron ions is proportional to the color. According to the above method, the cell protein was extracted, and BCA was used to measure the protein concentration for quantification of iron ion levels. The protocol was as follows. (1) The blank tube contained 500 µl of deionized water; the standard tube had 500 µl of standard application solution at a concentration of 2 mg/l, and the measurement tube had 500 µl of the sample to be tested. The samples were separately added to 5 ml EP tubes. (2) Then, 1.5 ml of iron developer was added to each tube (1), and the tubes were vortexed, mixed well, and boiled in boiling water at 100 °C for 5 min. (3) The samples were cooled with running water and centrifuged for 10 min at 2300×g. (4) Then, 250 µl of each supernatant was added to a 96-well plate, and the absorbance was measured at a wavelength of 520 nm. The iron content was calculated according to the following formula: iron content (mg/gprot) = (sample-blank) OD value/(standard-blank) OD value × standard concentration (2 mg/l) ÷ test sample protein concentration (gprot/l).
CCK-8 assay of cell proliferation
This assay was conducted following the instructions of the Cell Counting Kit-8 (Dojindo, CK04). The steps were as follows. (1) First, 100 µl of cell suspension was prepared in a 96-well plate, and the culture plate was placed in an incubator for 24 h (at 37 °C, 5% CO2). (2) After Aβ1-40 treatment, the sample was added to the culture plate. (3) The culture plate was incubated in an incubator for 0, 6, 12, 18, and 24 h. (4) Then, 10 µl of CCK-8 solution was added to each well. (5) The culture plate was incubated in the incubator for 2 h. (6) The absorbance was measured at 450 nm with a microplate reader.
Immunofluorescence and confocal imaging
After the various treatments, primary cultured mouse cells seeded on coverslips were fixed with 4% ice-cold paraformaldehyde at room temperature for 15 min followed by permeabilization with 0.1% Triton X-100/PBS. After incubation in blocking solution (2% BSA/PBS) at room temperature for 1 h, the cells were incubated with primary antibody at 4 °C overnight. This step was followed by three washes in PBS. The cells were then incubated with goat anti-rabbit IgG-Alexa 594 (Abcam), goat anti-rabbit IgG-Alexa 488 (Abcam), goat anti-mouse IgG-Alexa 594 (Abcam), goat anti-mouse IgG-Alexa 488 (Abcam), or goat anti-mouse IgG-Alexa 594 (Arigo) for 1 h at room temperature. After the cells were washed with PBS 3 times, the coverslips were mounted onto glass slides using Fluorescent Mounting Medium. For visualization of acidic lysosomal compartments, cells were stained with LysoTracker Red (Thermo Fisher Scientific) for 1 h according to the manufacturer’s instructions. After incubation with LysoTracker Red, cells were fixed in 4% PFA/PBS for 15 min at room temperature. The cells were incubated with primary antibody at 4 °C overnight. The cells were then washed in PBS 3 times followed by staining with 2 mg/ml Hoechst 33,342 for 5 min at room temperature. The cells were washed with PBS four times, and coverslips were mounted on glass slides using Fluorescent Mounting Medium. All confocal images were acquired and processed using a confocal microscope (Nikon).
Proteins from animal brains were homogenized, and cells were lysed with RIPA buffer. The samples were loaded on SDS–PAGE (8–12% acrylamide) gels, and the total protein concentration was determined by BCA assays. The same amount of protein (20–40 mg) was loaded and run on SDS–PAGE gels, and the proteins were then transferred to nitrocellulose membranes (0.45 or 0.22 μm), which were blocked with 5% nonfat dry milk in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (PBST) at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibodies directed against target proteins overnight at 4 °C. The final dilutions for primary antibodies were as follows: after three quick washes in PBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Abcam) diluted 1:3,000 in PBST for 1 h. The final detection of immunoreactive bands was developed using an enhanced chemiluminescent Western blot system (ECL) with exposure to a chemiluminescence imaging system (UVP). The immunoblotting signal intensity was measured using ImageJ 64 software.
For scanning electron microscopy, the cells were grown and processed as described above. First, the cells were fixed with 2.5% glutaraldehyde at 4 °C overnight and then washed three times with PBS. After being fixed with 1% osmium acid for 1.5 h at 4 °C, the samples were dehydrated in a graded ethanol series and embedded in acrylic resin at 60 °C for 48 h. Then, 70-nm ultrathin sections were mounted on a nickel grid. The samples were stained with uranyl acetate for 10–20 min and lead citrate for 5–10 min and rinsed thoroughly with distilled water. A 120 kV Hitachi transmission electron microscope was used to capture images of the sample.
Quantitative real-time PCR and sequencing
Total RNA was extracted from cells using TRIzol (Invitrogen) and further washed using RNase-free water. RNA was reverse-transcribed with a PrimeScript RT Reagent kit (TaKaRa) for cDNA synthesis, and the cDNA was then amplified by real-time PCR with a SYBR Premix ExTaq kit (TaKaRa). The relative expression of genes was normalized to the expression of the housekeeping gene GAPDH. The primer sequences were as follows: GAPDH forward, 5′-GTTCCTACCCCCAA TGTGTCC-3′ and reverse, 5′-TAGCCCAAGA TGCCCTTCAGT-3′; CD36 forward, 5′- ACCTTTTGTTGAGAAGTCTCGAAC-3′ and reverse, 5′- CTTTTTCAGTGCAGAAACAGTGG-3′; LRP1 forward, 5′- CTGGTCGATAGCAAGATTGTATTTC-3′ and reverse, 5′- GTGGCGTAGAGGTAGTTCTCAAAC -3′; HSP60 forward, 5′- CAGTCCTTCGCCAGATGAGAC -3′ and reverse, 5′- GGGACTTCCCCAACTCTGTTC -3′; and TIM23 forward, 5′- ACTGGTATGAACCCCCTGTCTC -3′ and reverse: 5′- CTGAGTTTCCTTCAATCCTAAACG -3′. Analysis was performed with three biological replicates and three independent technical replicates for each sample, with the average value taken and normalized to GAPDH gene levels to give the threshold cycle (ΔCT) values.
All data are expressed as the mean ± standard deviation, and SPSS 23.0 software (SPSS, Inc., Chicago, IL, USA) was used for the analysis of significant differences. GraphPad Prism 8.0 software was used for graphing. T tests were used for comparisons between two groups, one-way analysis of variance was used to compare multiple groups, and Student-Newman–Keuls (SNK) tests were used for comparisons between groups. For repeated measurement data, repeated measurement analysis of variance was used. For the comparison of data between two groups, Student’s t test was used when the variance was uniform, and Welch’s t test was used when it was not uniform. P < 0.05 was considered to indicate a significant difference.