Cell culture medium
BMSCs complete medium consists of α-MEM basic medium (BL306A, biosharp) plus 10% fetal bovine serum (04-001-1ACS, Biological Industries), 1% penicillin-streptomycin solution (C0222, Beyotime), 20ng/mL EGF (50482-MNCH LC13JA1403, Sino Biological), and 20ng/mL bFGF (50177-M08H LC12SE1702, Sino Biological).
Isolation, culture and purification of primary BMSCs
The adult male C57BL/6 mice were sacrificed by cervical dislocation and then sterilized and immersed in 75% ethanol for 5-10 minutes. The femur and tibia were separated under aseptic conditions and cut into pieces with a volume less than 3mm3. Red blood cells were lysed with red blood cell lysate (C3702-120mL, Beyotime), and after centrifugation, the fragments were re-suspended in culture medium and inoculated in a culture flask, and placed in an incubator at 37°C, 5% CO2 and 95% O2 with saturated humidity. Cells were passaged each time when the cells form a certain number of colonies in the culture flask. The third-generation cells which were incubated with APC-CD44 (103012, Biolegend), FITC-CD45 (103107, Biolegend) and PE-CD11b (101207, Biolegend) were used for flow sorting, and the cells of CD44+/CD45-/CD11b- were sorted by Beckman flow cytometry sorting system (Moflo-XDP, Backman). The sorted cells were collected in a sterile flow tube, transferred to a cell culture flask to continue culturing, and replaced the culture medium every 48h.
Extraction, separation, and fluorescent labeling of BMSC-exos
After the sorted cell culture reaches a certain density, the complete culture medium was replaced by an exosomal-free serum, and the cell supernatant was collected every 48h. The exosomes were extracted using the cell supernatant exosome isolation kit (4478359, Thermo Fisher Scientific), resuspended with PBS, and freezed at -20°C for later use. The BCA protein quantification method was used to quantify the extracted exosomes, the expression of exosomal marker proteins was detected by western blot, and the PKH26 staining kit (MX4021-100UL, Maokangbio) was used to stain and label the exosomes. In preparation for injection via lateral ventricle or caudal vein, the exosomes were resuspended and collected with an appropriate amount of pre-chilled artificial cerebrospinal fluid (ACSF).
Animals
Twenty four male C57BL/6 mice aging 4-weeks were purchased from the Anhui Animal Center. The breeding conditions were 12h alternating light and dark, free to food and water, with ambient temperature 20±2℃ and humidity 50±5%. When the mice grew to 12 weeks of age, they were randomly divided into a control group (6 animals) and an STZ group (18 animals), with three animals in each cage. All animal manipulation procedures have been approved by the Experimental Animal Ethics Committee of Anhui Medical University and comply with the "Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health" (NIH Publication No. 85-23, revised in 1985).
Establishment of animal model and experimental design
All mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1mL/10g), their heads were skin-prepared and disinfected, and then fixed on a stereotaxic apparatus. A 0.8-1cm incision was made in the center of the scalp. Suitable 3% H2O2 was used to digest and erase the mucosa on the surface of the skull until the Bregma was exposed, then the surface of the skull was wiped with normal saline, so that the surface of the skull was clearly exposed. The surface of the mouse skull was adjusted to a level, with fontanelle as the origin of the coordinates, and located the bilateral ventricles (the coordinates are 1.0mm left and right of the Bregma, 0.5mm back, and 2.5mm deep). The electric cranial drill was used to drill the hole, then the STZ was injected into the lateral ventricle of mice by the autosampler in the STZ group (the injection dose was 0.3mg/kg, the injection speed was 0.5μL/min, the injection volume was 1μL/side, and the STZ was dissolved in ACSF, and it was prepared for current use). Mice in the control group were given ACSF with an equal volume. At the end of the injection, the needle was stopped for 5 minutes and then the needle was withdrawn, the mouse scalp was sutured, and an appropriate amount of normal saline was injected into the intraperitoneal cavity, and then transferred to an electric blanket until awakened and placed in a squirrel cage.
After a one month recovery period for all the mice, the STZ-injected mice were divided into 3 groups including a model group, an exosomes lateral ventricle injection (Lv) group, and an exosomal caudal vein injection (Cv) group, with 6 mice in each group. The right cerebral ventricle of all mice was intubated using a stereotaxic instrument and a drug delivery catheter (62003, 62102, RWD). The mice in the Lv group were injected with 0.5μg BMSC-exos (dissolved in ACSF) per day in the lateral ventricle, the control group and the model group were injected with equal volumes of ACSF, and the mice in the Cv group were injected with 25μg BMSC-exos (dissolved in PBS) per day in the tail vein. All mice were administered for 5 days. After the administration, a series of behavioral tests were performed on all mice. The experimental time and steps are shown in Figure. 1.
Behavioral tests
All behavioral tests were performed under dark, sound-proof conditions. All mice were given 30 minutes to adapt to the environment before the behavioral experiments. All of the tests were carried out between 08:30 and 12:30, with matching between the groups. The observers were blind to the treatment. The behavioral tests were monitored and recorded by a digital camera interfaced to a computer running the ANY-maze video imaging software (Stoelting Co, Wood Dale, American).
Open field test (OFT)
The experimental apparatus of the OFT is a cube white opaque box with a size of 45cm×45cm×45cm. Each mouse was put into the box with its back facing one side of the box wall and allowed to explore freely for 5 min.
Elevated plus maze test (EPM)
The EPM apparatus consists of two opposite open arms (50×10×0.5cm), two opposite closed arms (50×10×20cm), and a central open platform (10×10cm). Each mouse was placed on the central platform facing one of the open arms, and allowed to explore the instrument freely for 5 minutes. The moving distance of each mouse in the closed arm is counted.
Y maze test (Y-maze)
The Y-maze apparatus is composed of three white opaque plexiglass arms. The size of each arm is 40cm×20cm×10cm. This task consisted of 2 sessions (training and test) and conducted on 2 successive days. In the training session, the novel arm was closed, and the mice were placed at the end of the starting arm facing the wall and allowed to explore in the maze for 10 min. 24 h later, the test session was performed. The novel arm was opened, and the mice were permitted to explore within the three arms for 5 min. The ambulatory distance in each arm was recorded and the ratio of moving distance in the novel arm to that in the total arm was taken as the preference index of the novel arm.
Novel object recognition test (NOR)
The NOR apparatus is a cube white opaque box with a size of 45cm×45cm×45cm. The experiment is divided into two stages. The first three days are the first part (adaptation period). Each mouse was put into the apparatus with facing one side wall every day, explored and adapted freely for 10 minutes. The fourth day is the second part (the inspection period). The inspection period is divided into two stages. The first stage: two objects of the same shape, size, and color were placed on the two thirds of the diagonal of the bottom of the box. Each mouse was put into the instrument facing one side, explored freely for 10 minutes, and entered the second stage after 1 hour. The second stage: one of the objects was replaced by a novel object with different shapes and colors, each mouse was put back into the apparatus and explored freely for 5 minutes. The behavioral software was used to record the mouse's behavior on the novel and old objects within 5 minutes. Calculating the mouse's preference for novel and old objects, that is, the novel object preference index: time to explore novel objects/total time to explore novel and old objects, and perform statistical analysis.
Tail suspension test (TST)
The TST apparatus is a 40 cm high white open box. The 1.5cm part of the tail end of the mouse is fixed with paper tape and hung on a crossbar directly above the box. The experiment process was recorded by an automatic camera. Each mouse was hung for 6 minutes. The first two minutes is the adaptation period, and the last 4 minutes is the official tail suspension time. The immobility time of the mice in the last 4 minutes was observed and recorded through video observation. The judgement was based on keeping the two front paws of the mouse as the standard, and all the results were recorded by the same person.
Transmission electron microscope observation of BMSC-exos morphology
A small amount of exosomal suspension was put on the parafilm, and the exosomes in the suspension was absorbed with the sample-loading copper mesh for 3 minutes, then it was stained with 2% phosphotungstic acid negative staining solution for 3 minutes. At last, the sample was transferred to a transmission electron microscope for observation and image collection.
Immunofluorescence staining (IF)
Three mice in each group were randomly selected for cardiac perfusion, perfused with PBS until there was no more blood outflow, replaced with 4% paraformaldehyde perfusion, and reperfused for 3 minutes after the systemic spasm. The whole brain of each mouse was separated completely and fixed overnight in 4% paraformaldehyde solution at room temperature, then immersed in 30% sucrose solution and dehydrated to the bottom. The whole brain was filled with a disposable embedding box, filled with OCT embedding solution and frozen at -80℃overnight. Slice with 30μm-thick were prepared with a cryostat, attached to a glass slide, then returned to room temperature and dried in a fume hood for 10 minutes. The tissue was surrounded with an immunohistochemistry pen, and the goat serum (ZLI-9022, ZSGB-BIO) was used to seal the sections at room temperature for 30 minutes. After the liquid was cleaned, the sections were incubated with the diluted rabbit anti-GFAP (1∶100, 16825-1-AP, Proteintech), rabbit anti-DCX (1∶100, 13925-1-AP, Proteintech), or rabbit anti-Iba-1 (1:100, DF6442, Affinity) overnight at 4°C in the dark. After washing with PBST, the sections were incubated with the goat anti-rabbit FITC or goat anti-rabbit Cy3 at 37°C for 1 hour. After washing with PBST, the tablets were sealed with anti-Fluorescence quenching mounting tablets containing DAPI (S2110, Solarbio), then the plates were mounted with a laser scanning confocal fluorescence microscope (LSM800, ZEISS) for imaging.
Nissl stain
The slices of the ventricles were degreased with xylene, stained with Nissan staining solution to dark blue, washed with double distilled water, and an appropriate amount of differentiation solution was added dropwise for differentiation. Then dehydrated with gradient alcohol, degreased again with xylene, and finally sealed with neutral gum. The slices were placed in a fume hood to dry for 2 hours, then observed and collected images with a microscope.
Western blot assays
Exosomal marker protein detection
The exosomal suspension was mixed with SDS protein loading buffer for Western blot experiments to detect the surface marker protein anti-CD63 (1:1000; sc-5275, Santa Cruz), anti-TSG101 (1:1000; WL05130, Wanleibio), anti-HSP70 (1:1000; WL01019, Wanleibio) expression, with cell culture supernatant as a control.
Detection of protein expression in mouse hippocampus
In each group, 3 mice were selected immediately after cervical dislocation and sacrificed. The brains were dissected immediately, and hippocampal tissues were isolated and stored in liquid nitrogen. The Western blot method is consistent with our previous research, using RIPA (P0013B, Beyotime) lysate containing protease inhibitors and phosphatase inhibitors to lyse the tissues, and using the BCA protein quantification kit (P0010, Beyotime) to determine the protein concentration in each sample. The protein loading buffer and protein sample were mixed in boiling water and heated for 10 minutes. After cooling, the sample was added to 12.5% SDS-PAGE. After electrophoresis, the membrane was transferred and the protein was quickly blocked. The primary antibody was incubated overnight at 4°C. The main antibody dilution ratios are as follows: anti-Aβ1−42 (1:1,000; 25524-1-AP, Proteintech), anti-Tau5 (1:1000; sc-58860, Santa Cruz), anti-p-Tau (ser396) (1:1,000; sc-32275, Santa Cruz), anti-BACE (1:500; WL02795, Wanleibio), anti-GFAP (1:1000; 16825-1-AP, Proteintech), anti-Synaptotagmin-1 (1:1000; YT4484, Immuno Way), anti-Synapsin-1 (1:1000; BS3667, Bioworld), anti-BDNF (1:1000; ab108319, Abcam), anti-IL-1β (1:500; WL00891, Wanleibio), anti-IL-6 (1:1000; WL02841, Wanleibio), anti-TNF-α (1:1000; WL01581, Wanleibio), and anti-β-actin (1:1000; TA-09, ZSGB-BIO). The membranes were then processed with appropriate HRP-conjugated secondary antibodies with regard to the proteins of interest. The protein levels were analysed using ImageJ (Wayne Rasband, National Institutes of Health, USA) and normalized relative to that of the internal control β-actin.
Quantitative Real-time PCR (QPCR)
Quantitative real-time polymerase chain reaction (QPCR) was performed to determine IL-1βmRNA, IL-6mRNA and TNF-αmRNA levels in all experimental groups. Part of the hippocampus from three mice in each group was used to extract RNA. The extraction steps are as follows. In short, the Trizol (15596026, ambion) was used to lyse the tissues and add chloroform to incubate and then centrifuge, then the supernatant was mixed well with isopropanol, and precipitated overnight at -20°C. The supernatant was discarded after centrifugation and the precipitate was resuspended with 75% ethanol. The supernatant was discarded after centrifugation, and the precipitate was resuspended in enzyme-free water, and performed RNA quantification. After quantification is completed, the Evo M-MLV RT Premix (AG11706, Accurate Biology) was used to reverse transcription, and then use cDNA, upstream primers, downstream primers, enzyme-free water and SYBR Green (AG11701, Accurate Biology) for real-time fluorescence quantification. PCR was performed in a thermal cycler as follows: 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s, and 55 ℃ for 30 s. IL-1β-F primer sequence was 5′CTTTGAAGTTGACGGACCC3′, IL-1β-R primer sequence was 5′TGAGTGATACTGCCTGCCTG3′, IL-6-F primer sequence was 5′AGTCCGGAGAGGAGACTTCA3′, IL-6-R primer sequence was 5′ATTTCCACGATTTCCCAGAG3′, TNF-α-F primer sequence was 5′CACCACCATCAAGGACTCAA3′, TNF-α-R primer sequence was 5′AGGCAACCTGACCACTCTCC3′, β-actin-F primer sequence was 5′AGTGTGACGTTGACATCCGT3′, β-actin-R primer sequence was 5′TGCTAGGAGCCAGAGCAGTA′. The experimental results are calculated using the 2∆∆CT method and normalized analysis.
Statistical Analysis
All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences, version 17.0). Data are expressed as the mean ± S.E.M., and p < 0.05 was considered statistically significant. Intergroup statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the LSD post hoc test. All statistical charts are drawn using Graphpad Prism 7.0 and Microsoft Powerpoint 16.0.