Generation of miR-132-3p enriched or knock down MSC exosomes
Mesenchymal stem cells (MSC) were cultured from bone marrow of C57BL/6 mice as we previously described [16]. Control MSC exosomes (EXCon), miR-132-3p enriched MSC exosomes (EXmiR-132-3p), and miR-132-3p knock down MSC exosomes (EXantagomiR-132-3p) were collected from MSC transfected with scrambled control (MSCCon), lentivirus carrying the murine miR-132-3p (MSCmiR-132-3p), or levtivirus carrying the murine antago miR-132-3p (MSCantagomiR-132-3p), respectively.
Animals
Adult (8–12 weeks old) male C57BL/6 mice were purchased from the Animal Experiment Center of Guangdong Province (Guangzhou, China) and raised in the animal care facility at the Guangdong Medical University. The mice were housed in a pathogen-free environment. Surgeries were performed under 2.5% isoflurane anesthesia. All experimental procedures were approved by the laboratory animal care and use committees at Guangdong Medical University.
EX infusion of mouse VD model
The VD mouse model was generated as previously described [17]. Briefly, mice were anesthetized with isoflurane, fixed on a stereotaxic instrument, and body temperature was maintained at 37°C with a feedback-regulated water heating system. A skin incision was made in the neck area, the bilateral carotid arteries were separated and locked by ligation for 10 min and then released for 10 min. This process was repeated three times. The sham-operated mice (Control) underwent the same procedure except the bilateral carotid arteries were locked. Twenty-four hours after VD, the mice were administered PBS (vehicle), EXCon, EXmiR-132-3p, or EXantagomiR-132-3p (1 × 1010 particles/100 μl) via the tail vein once every 7 days for 21 days. This dose was selected based on previous studies on MSC EX administration for treating CNS diseases[18]. Memory and cognitive performance were measured by the Morris water maze (MWM) test on the last 5 days of the experiment. Immediately after the MWM test, the mice were sacrificed at day 21 following MSC EX infusion, and the cortex and hippocampus were collected and used for various measurements including NTA analysis, immunofluorescence, immunohistochemistry, and Golgi staining.
Detection of MSC EX in brain tissue
MSC EX were isolated from the cortex and hippocampus of Sham (MSC EXSham) or VD (MSC EXVD) mice as described previously with some modification[19]. Briefly, the cortex and hippocampus were separated and finely minced with small, sharp scissors in 100 μl of papain solution (20 units/ml). Brains in solution were pipetted into a 15 ml conical tube containing 3.5 ml papain solution and incubated for 20 min at 37°C to dissociate the tissue and free the extracellular space. Then, the total brain EXs were isolated using sequential centrifugation at 300 x g for 10 min, 2000 x g for 20 min, 20,000 x g for 90 min, and 100,000 x g for 2 h after the supernatant was passed through a 0.22 μm syringe filter (Millex-GP, Millipore). The pelleted EXs were incubated with 10 μl of biotin-conjugated anti-CD29 antibody (Miltenyi Biotec) in a 100 μl reaction volume for 2 h, followed by the addition of 10 μl of anti-biotin microbeads for 15 min. The microbeads labeled EX from the total brain EX suspension were separated with a DynaMag-2 magnet (Life technology). After an overnight magnetic separation, the microbead-bound EXs were collected and resuspended in 100 μl of particle-free PBS. The suspension was incubated with 10 μl multisort release reagent (Miltenyi Biotec) to cleave off the microbeads. The EX in the fluid were collected and considered MSC EX (CD29+ EXs). The pelleted MSC EX were resuspended in phosphate-buffered saline (PBS) and aliquoted for nanoparticle tract analysis (NTA), transmission electron microscopy, and EX-specific marker CD63 and MSC-specific marker CD29 measurement using western blot analysis.
Detection of MSC EX interacting with neurons in the cortex and hippocampus
MSC EX were labeled with PKH26 (Sigma) and resuspended in PBS for infusion [8]. After 24 h of infusion, the brains were dissected from mice, frozen in liquid nitrogen, and cut into 20-μm-thick sections. The brain sections were incubated with rabbit monoclonal anti-NeuN antibody (1:300, Abcam) at 4°C overnight and the sections were then incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody for 1 h. After rinsing with wash solution, the interaction of MSC EX with neurons in the cortex and hippocampus were detected by confocal microscopy (Olympus Corporation, Japan).
Quantitative RT-PCR
The levels of miR-132-3p in MSC, MSC EX, neurons, cortices, and the hippocampus were measured by quantitative real-time PCR (qRT-PCR). Total miRs from mice and cells were isolated using the miRNeasy Mini kit (QIAGEN) based on the manufacturer’s instructions and miR-132-3p cDNA was generated for qRT-PCR analysis (Roche 480). Samples were analyzed in triplicate and U6 was used as an internal control. The primer sequences used are listed below: 5-CCAGCATAACAGTCTACAGCCA-3 and 5-AACGCTTCACGAATTTGCGT-3 for miR-132-3p, and 5-CTCGCTTCGGCAGCACA-3 and 5-AACGCTTCACGAATTTGCGT-3 for U6. A total reaction mixture of 10 μl was amplified in a 96-well PCR plate (BIOplastics, Netherlands) using the following cycling conditions: 95°C for 3 min followed by 40 cycles of 95°C for 12 s and 60°C for 40 s.
Morris water maze test
The labyrinth was composed of a barrel water tank (120 cm in diameter with a platform filled with tap water at a temperature of 22 ± 2°C). An escape platform with a diameter of 10 cm was placed 1–2 cm below the water surface, mice were place into the maze at one of four points (N, S, E, W) facing the wall of the tank. Over a period of five days, the mice were allowed to find the platform for 90 s. If the mice failed to find the platform, it was guided to the platform and allowed to rest for 15 s. On day five, the platform was removed and a probe test was conducted. The percentage time spent in each of the four quadrants and the number of target area crossings, total distance, and mean speed were recorded.
Immunohistochemistry
The number and arrangement of neurons in hippocampal CA1 region were detected by immunohistochemical staining. Each group of brain tissue was fixed in 4% formalin for 24 h, and then the samples were dehydrated in serially graded ethanol solutions, defatted in methanol and embedded in paraffin. The brain tissues were coronally sectioned at a thickness of 6 μm, and deparaffinized in xylene, rehydrated in descending concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% H2O2 (v/v) for 20 min. The sections were placed in 0.01 M citrate buffer and heated in a microwave oven at 95°C for 20 min after washed with PBS. Brain sections were stained with hematoxylin and eosin (H&E), followed by dehydration, hyalinization, fixation, and observed under a high-magnification optical microscope (Olympus, Japan). Neuronal damage was evaluated on a scale of 0 = normal, 1 = a few (< 30%) neurons damaged, 2 = many (30 to 70 %) neurons damaged, and 3 = majority of neurons (> 70%) damaged (ref: Strain related differences in susceptibility to transient forebrain ischemia in SV-129 and C57 Black/6 mice).
Immunofluorescence staining
Mouse brain sections or cultured neurons were fixed in 4% paraformaldehyde and washed with PBS three times. Antigen retrieval was performed with citrate buffer (pH 7.0). The samples were then permeabilized with 0.1% Triton X-100 for 5 min on ice, blocked in PBS containing 10% normal goat serum at room temperature for one hour, and washed three times with PBS. Brain sections and cultured neurons were incubated with primary antibodies overnight at 4°C. After washing with PBS, the samples were incubated with secondary antibodies in blocking buffer for 1 h. Subsequently, the cellular nucleus was stained with DAPI (1:1000, Abcam) for 7 min at room temperature. Samples were washed with wash buffer three times and images were acquired using a confocal microscope (Olympus, Japan).
The primary antibodies used for immunostaining included NeuN (rabbit, 1:300, Abcam), MAP2 (rabbit, 1:500, Abcam), Tau46 (mouse, 1:200, Millipore), Aβ (rabbit, 1:300, Abcam), PSD95 (1:500, Abcam), and synaptophysin (1:500, Abcam). The samples were then incubated with 488/564/647 goat anti-rabbit/mouse secondary antibodies (1:250, Invitrogen).
Golgi staining
Golgi staining was performed using the FD Rapid Golgi Stain system (FD Neuro Technologies) according to the manufacturer’s instructions. Briefly, freshly dissected brains were immersed in the impregnation solution and stored at room temperature in the dark for two weeks. Brains were then transferred to solution C and stored at 4°C for 48 h in the dark. Brains were coronally sectioned at a thickness of 200 μm with a freezing microtome (Thermo Scientific, USA). Brain sections were stained with solution D and E. Golgi-stained neurons and dendritic segments in the cortex and hippocampal regions were observed using an Olympus confocal microscope. Dendritic branching and spine density was detected using NIH Image J software.
Neuron OGD model and functional analysis
Isolation of primary neurons
Primary neurons were cultured from C57BL/6 mouse embryos (aged 5–8 days). Briefly, brain tissues were dissected from mouse pups and the meninges were removed from the brain tissue and dissociated by enzymatic digestion. Isolated primary neurons were plated onto poly-D-lysine-coated dishes and cultured in Neurobasal medium supplemented with 2% B27 (Gibco), 0.5 mM of glutamine, 10% fetal bovine serum (Gibco), and 1% penicillin/streptomycin (Sigma) and maintained in a 5% CO2 incubator at 37°C. The media was changed every three days.
OGD
The neuron oxygen-glucose deprivation (OGD) model was established according to a previously publication with minor modification[20]. Briefly, the cultured primary neurons were washed and incubated with glucose-free DMEM (Gibco, USA) and incubated in an anaerobic chamber (Thermo Fisher Scientific, USA) containing 1% O2, 5% CO2, and 94% N2 for 2 h. After incubation, the culture medium was replaced with normal medium in a normoxic condition. During reoxygenation time, neurons were co-cultured with various MSC EX (EXCon, EXmiR-132-3p) or culture medium (vehicle). To explore the underlying mechanism, neurons were pretreated with a Ras inhibitor (NSC 23766, 100 mM, Sellectkchem) for 2 h.
Neuron functional analysis
Neuron viability was measured using the CCK-8 assay kit according to the manufacturer’s instructions. Neuronal apoptosis was analyzed using the Annexin V-PE/7-AAD apoptosis detection kit (BD Bioscience, USA) based on the manufacturer’s instructions followed by flow cytometric analysis. Intracellular ROS production by neurons was measured by dihydroenthidium (DHE, Beyontime, China) staining and visualized under a confocal microscope (Olympus, Japan).
Western blot analyses
The neurons were homogenized in lysis buffer including proteinase and phosphate kinase inhibitors (Sigma, USA). Proteins (30 μg) were mixed with loading buffer and electrophoresed through SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF, Millipore Corporation, USA). After blocking non-specific antigens in 5% non-fat milk, the membranes were incubated with primary antibodies against RASA1 (1:1000, CST, USA), Ras (1:1000, Abcam, USA), Akt and phospho-Akt (1:500, CST), GSK-3β and phospho-GSK-3βser9 (1:1000, Abcam), p-Tau (1:1000, Abcam), and CD63 (1:400, Abcam). The bands were visualized using an ECL kit (Amersham, Sweden).