Animals and surgery
Pregnant Sprague-Dawley rats, 1-day-old neonatal Sprague-Dawley rats, and adult Sprague-Dawley rats (weighing 220–250 g) were obtained from the Experimental Animal Center of Nantong University (Certificate No: SYXK (SU) 2012-0031). All experimental procedures were approved by the local Animal Care Committee and were conducted in accordance with the guidelines of the National Institutes of Health (NIH) on animal care and with other relevant the ethical guidelines.
FF transections were performed as described by Hefti (Hefti, 1986). Briefly, after chlorpent anesthesia (2 mL/kg body weight, intraperitoneal), adult SD rats were transferred to the stereotaxic apparatus, and then FF transection was performed with a wire knife at the CA1 layer of the dorsal hippocampus, at coordinates of bregma: AP = 1.4, ML = 1.0 and AP = 1.4, ML = 4.0, depth 5.6 mm. There were no restrictions on the sex of the experimental animals.
Exosome isolation
Seven days following FF transection, deafferented and normal hippocampi were quickly dissected, trypsinized, and homogenized into ice-cold phosphate-buffered saline (PBS). Exosomes were precipitated using Total Exosome Isolation reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Homogenates were centrifuged at 2,000 × g at 4 °C for 30 min to remove cells and debris, and then supernatants were passed through a 0.22-µm filter to remove extracellular vesicles larger than exosomes. The supernatants were transferred to a new tube without disturbing the pellet and mixed with 0.5 volumes of Total Exosome Isolation reagent and incubated overnight at 4 °C. The mixture was then centrifuged at 10,000 × g for 30 min, and the supernatant was decanted, while the exosome pellet was resuspended into 100 µL PBS.
Cell culture
The isolation, culture, and differentiation of NSCs were performed as previously described with some modifications (He et al., 2018). Briefly, pregnant SD rats were anesthetized, and the embryos were removed by cesarean section. Hippocampi were dissected from embryonic day 14.5 (E14.5) embryos, and were then mechanically dissociated into a single-cell suspension. After centrifugation and resuspension, the cell suspensions were plated into flasks with a 1:1 Dulbecco’s Modified Eagle Medium (DMEM) and Ham F-12 mixture (both, Gibco, Grand Island, NY, USA) containing 2% B27 (Gibco), 20 ng/mL epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO, USA), and 20 ng/mL fibroblast growth factor 2 (bFGF; Sigma-Aldrich). Cells were passaged every 6 d to obtain neurospheres that originated from a single primary cell. For in vitro differentiation, cell suspensions were plated with DMEM/F-12 medium supplemented with 2% B27 and 2% fetal bovine serum (FBS, Gibco). For the mixed co-culture experiments, isolated exosomes were mixed with NSCs and processed in different ways after cocultivation.
Primary neurons were isolated using standard methods, as previously described (Xing et al., 2014). Briefly, hippocampi were dissected from E14.5 embryos, and the resultant single cell suspensions were diluted in serum-free neurobasal medium (Gibco) containing 2% B27 and 0.5 mM L-glutamine (Gibco). The cells were then seeded onto plates precoated with poly-D-lysine. Half of the medium was replaced every 3 d.
Primary astrocytes were derived from cerebral cortices of 1-day-old neonatal rats as previously described (Xing et al., 2014). Briefly, dissociated cortical cells were suspended in DMEM/F-12 containing 10% FBS and plated in flasks. After 3–4 d, the heterogenous primary cells were orbitally shaken to remove microglia and oligodendrocytes. Astrocytes were dissociated by trypsinization and then replated into flasks.
Transfection, lentiviral transduction, and injection
Prior to transfection or transduction, cells were cultured in plates overnight. Cells were transfected with the miR-107-3p/NC mimic or miR-107-3p/NC inhibitor (Ribobio, Guangzhou, China) using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Cells were transduced with lentivirus that was constructed by Genechem Company (Shanghai, China), including lentiviral vectors Nedl1 (abbreviated as LV-Nedl1 and LV-Ndel1i), corresponding to the negative control lentiviruses (LV-NC and LV-NCi) following the manufacturer’s instructions. Green fluorescence expression was then observed under a fluorescence microscope (Axio Scope A1, Zeiss, Oberkochen, Germany). The cells were cultured with lentivirus for 12 h to obtain the best infection complex value, after which the lentivirus was removed and replaced with fresh medium.
In total, 60 SD rats were used for lentivirus injections into the hippocampus. Briefly, after chlorpent anesthesia, adult SD rats were transferred to the stereotaxic apparatus. On day 7 after FF transection, injections of virus into the left and right hippocampal DG at two points were performed at the following coordinates: 3.6 mm to bregma, 1.39 mm to the right or left of the midline, and 3.9 mm in depth. Five µL of virus were loaded into an internal cannula needle with cannula tubing connected to a Hamilton syringe mounted onto a microinjection pump (Harvard Apparatus, Dover, MA, USA). The speed of the injection was 0.5 µL/min. The needle was kept in position for an additional 10 min after completing the injection, and then was slowly retrieved from the brain.
RNA preparation and qRT-PCR
Isolation of total RNA from tissues and cells was performed using TRIzol reagent (Vazyme Biotech, Nanjing, China) according to manufacturer’s instructions. For mRNA expression analysis, 1 µg of RNA was reverse transcribed into cDNA using the HiScript Q RT SuperMix for qPCR (+ gDNA wiper) Kit (Vazyme Biotech). The SYBR green (Roche, Basel, Switzerland) method was performed using a StepOnePlus RealTime PCR system (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. The sequences of primers used for qRT-PCR are displayed in Table S1.
For miRNA expression analysis, the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China) and the miRcute miRNA qPCR Detection Kit (SYBR Green; Tiagen Biotech) were used. According to the manufacturer’s protocol, 1 µg of total RNA was used. Forward primers for miRNAs were obtained from Ribobio (Guangzhou, China), and the reverse primer was commercially available and supplied in the miRcute miRNA qPCR Detection Kit. The 2−ΔΔCT method was used to calculate expression levels from qPCR data.
Western blot analysis
Briefly, proteins were extracted, quantified, isolated by 10% SDS-PAGE, transferred to 0.2 mm polyvinylidene fluoride membranes, and then blocked with 5% skim milk for 2 h. After incubating with primary antibodies overnight at 4 °C, the membranes were incubated with HRP-linked secondary antibodies for 2 h. Immunoreactive bands were viewed by enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). The primary antibodies used included anti-Tuj1 (1:1,000; Millipore, Billerica, MA), anti-Nedl1 (1:1,000; Abcam, Cambridge, UK), and anti-β-actin (1:1,000; Abcam).
Immunofluorescence and immunohistochemistry
Cells and tissues were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times, permeabilized and blocked with 10% normal goat serum containing 0.3% Triton X-100 and 1% BSA for 2 h, and then incubated with primary antibody overnight at 4 °C. For immunofluorescence, cells and tissues were washed three times with PBS and incubated with the corresponding fluorescent secondary antibody at room temperature for 2 h. Nuclei were counterstained with Hoechst 33342 (1:1,000; Pierce, Rockford, IL, USA). Primary antibodies included anti-Tuj1 (1:1000; Millipore) and anti-GFAP (1:1,000; Millipore). Images were captured by using a fluorescence microscope.
Immunohistochemistry was performed using a Super-Sensitive Horseradish Peroxidase Immunohistochemistry Kit (rabbit; Sangon Biotech, Shanghai, China). Sections were incubated with rabbit anti-Ndel1 antibody (1:1,000, Abcam) at 4 °C overnight followed by incubation with poly-HRP-conjugated anti-rabbit IgG. After rinsing in PBS, sections were detected using a DAB working solution.
Flow cytometry
Cells were fixed in a 1 × Fix/Perm Buffer working solution at 4 °C for 40 min. After washing with 1 × Perm/Wash Buffer, the cell samples were mixed with 80–100 µL of 1 × Perm/Wash Buffer and incubated with APC-conjugated anti-Tuj1 antibody and PE-conjugated anti-GFAP antibody (BD Biosciences) at 4 °C for 2 h. Cells were centrifuged and resuspended in flow cytometry stain buffer, and then analyzed using a flow cytometer.
Luciferase reporter assay
The luciferase reporter vectors were constructed by Genechem. For the luciferase reporter assays, HEK-293 cells plated in a 24-well plate were co-transfected with 100 ng plasmid and 100 ng luciferase construct. Luciferase and Renilla signals were measured 72 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA ) according to a protocol provided by the manufacturer.
Statistical analysis
Statistical analyses were mainly conducted using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Differences between two groups were compared using an unpaired Student two-tailed t-test, and differences among multiple groups were analyzed by one-way ANOVA. The results were considered statistically significant when *P < 0.05; **P < 0.01; ***P < 0.001.