2.1 Cell culture
Human bone marrow mesenchymal stem cells (BMSCs; ScienCell) were cultured in MEM (Minimum Essential Medium) Alpha Medium (Corning) and 10% fetal bovine serum (FBS, Gibco, NY, USA). BMSCs between passage 4 were used for subsequent experiments. Human brain microvascular endothelial cells (HBMECs) were purchased from ScienCell Research Laboratories (ScienCell Research Laboratories, San Diego, CA, USA). Cells were cultured in endothelial cell medium (ScienCell Research Laboratories) and incubated in a humidified atmosphere at 5% CO2 and 37°C.
2.2 Inhibition of TIMP2
After the harvest of BMSC-conditioned medium, BMSCs were divided into four groups and transfected with siRNA (small interfering RNA). Three siRNAs (siTIMP2 #1, siTIMP2#2 and siTIMP2 #3) and the scramble control siRNA (Con siRNA) were obtained from Asia-Vector Biotechnology (Shanghai, China), which also performed virus packaging. Cell transfection was performed following the manufacturer’s instructions. In brief, cells were inoculated in 24-well culture plates, 1×105 target cells were inoculated in each well, and the cells were cultured overnight. When the cell density was 30–50%, 50 µl serum-free medium and 1.25 µl siRNA were to a sterilized EP tube, mixed gently and incubated at room temperature for 5 minutes (the final concentration of siRNA was 50 nM). Then, in a sterilized EP tube, 3 µl liposome transfection reagent (please refer to the corresponding transfection reagent manual) was dissolved in 50 µl serum-free medium, mixed gently and incubated for 5 min at room temperature. SiRNA and liposome solution were gently mixed and incubated for 10–20 min at room temperature. The mixed solution was added to the 24-well plate cells, and the cells were collected for 24–72 hours for determination. The siRNA sequences used in this study were as follows: siTIMP2#1: GAGATCAAGCAGATAAAGA; siTIMP2#2: GGAAAGAAGGAATATCTCA; and siTIMP2#3: GGAAGTGGACTCTGGAAAC.
2.3 Real-time PCR
Four groups of BMSCs were tested by PCR after transfection. TRIzol reagent was used to isolate total RNA from cells, while exosomal miRNAs were isolated using an Exosome RNA Purification Kit (Qiagen). For the miRNA analysis, reverse transcription reactions of miRNA were performed using a 4× Reverse Transcription Master Mix kit (EZBioscience). qRT-PCR miRNA analyses were carried out with FastStart Universal SYBR Green Master Mix (Roche). U6 and GAPDH were used as internal controls. The primer sequences were as follows: TIMP2 Forward 5`-GAACATCAACGGGCACCAG-3` and Reverse 5`-TCCCTCCAGAACCCACAACC-3`; U6 Forward 5`-CTCGCTTCGGCAGCACA-3` and Reverse 5`-AACGCTTCACGAATTTGCGT-3`; and GAPDH Forward 5`-CTCTGATTTGGTCGTATTGGG-3` and Reverse 5`-TGGAAGATGGTGATGGGATT-3`.
2.4 Exosome isolation
BMSCs and siTIMP2-BMSCs were separated for the exosome analysis. After harvesting BMSC- and siTIMP2-BMSC-conditioned media, the cells were removed by centrifuging for 10 min at 500 g. Next, the apoptosis vesicles and debris were removed by centrifugation of the supernatant for 20 min at 12,000 g and filtering through a 0.22 µm filter. Then, ultracentrifugation was performed for 70 min at 110,000 g (Beckman Optima XPN, 45Ti) to collect the exosomes in the form of a pellet, which was further resuspended in phosphate-buffered saline (PBS) for further purification by ultracentrifugation for 70 min at 110,000 g to remove the contaminating protein. Finally, exosomes were stored at -80°C after being resuspended in PBS. The protein content of the exosomes was determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA).
2.5 Exosome characterization and internalization
The qNano platform (iZON Science, UK) was used to analyze the distribution and absolute size of the exosomes. Morphological examination of isolated exosomes was performed through transmission electron microscopy (TEM; HT 00, Hitachi, Japan). The expression of CD9, CD63, and TSG101 in exosomes was evaluated by Western blot analysis, and the following primary antibodies were used: CD9, CD63, and TSG101 (1:1000; rabbit IgG, Proteintech). A green fluorescent dye (DIO; Life Technologies) was utilized to label the exosomes in accordance with the manufacturer’s instructions. Ultracentrifugation at 110,000 g at 4°C for 70 min was performed to remove excess dye. HBMECs were incubated with DIO-labeled exosomes at a concentration of 50 µg/ml for 8 hours, followed by the use of 4,6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA) to stain nuclei, which were then analyzed by fluorescence microscopy.
2.6 Cell viability assay
Cell viability was determined using the cell counting kit-8 (CCK-8) assay. HBMECs were plated into 96-well plates and treated with endothelial cell medium. After 1, 2, 3, 4, and 5 days of incubation, 10 µl CCK-8 reagent (Dojindo, Japan) was added to the culture medium and the absorbance of each well was observed at 450 nm by a microplate reader (Bio-Rad 680, Hercules, USA).
2.7 Spinal cord injury
Adult female Sprague–Dawley rats (220–250 g) were obtained from the Animal Room of Shanghai Sixth People's Hospital. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, and all procedures were approved by the Animal Research Committee of the Sixth People's Hospital at Shanghai Jiao Tong University. All animals were housed under standard temperature conditions with a 12 h light/dark cycle and provided food and water. Rats were anaesthetized with 10% chloral hydrate (3.5 ml/kg, i.p.), and a laminectomy was performed at the T9 level to expose the underlying cord without disrupting the dura. The exposed spinal cord was subjected to moderate contusion injury (150 kdyn force with no dwell time) using an Infinite Horizon Impact Device. The sham-operated group rats underwent a T9 laminectomy without contusion injury. Postoperative care included manual urinary bladder emptying every 12 h until the return of bladder function and the administration of cefazolin sodium (50 mg/kg, i.p.).
2.8 Exosome treatment
BMSC exosomes and siTIMP2-BMSC exosomes were injected subcutaneously near the back wound after SCI and administered once a day for 1 week for behavioral tests or for the indicated time points for other experiments. The sham-operated group rats received no exosome treatment.
2.9 Behavioral tests
Functional deficits after SCI were examined as previously described. The Basso, Beattie, and Bresnahan (BBB) scores were assessed on an open field scale by two blinded independent examiners at 1, 3, 5, 7, and 14 days postoperation. Briefly, the BBB locomotion rating scale scores ranged from 0 points (complete paralysis) to 21 points (normal locomotion). The scale was based on the natural progression of locomotion recovery in rats with thoracic SCI.
2.10 Western blot analysis
For protein analysis, the protein extract was homogenized in modified RIPA buffer (50 mM Tris–HCl, 1% NP-40, 20 mM DTT, 150 mM NaCl, pH = 7.4) containing protease inhibitor cocktail (10 µl/ml; GE Healthcare Biosciences, PA, Little Chalfont, UK). The complex was then centrifuged at 11,792 g, and the supernatant was obtained for the protein assay. For protein analysis in vitro, HBMECs were lysed in RIPA buffer [25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate and 0.1% SDS] with protease and phosphatase inhibitors. The extracts above were quantified with bicinchoninic acid reagents (Thermo, Rockford, IL, USA). Equivalent amounts of 50 µg protein were separated using a 12% gel and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% nonfat milk in TBS with 0.05% Tween 20 for 1 h and then incubated with the following antibody solutions: MMP-2, MMP-9, beta-catenin, occludin, claudin-5, zo-1, and GAPDH. The membranes were washed with TBS three times and incubated with secondary antibodies for 2 h at room temperature. Signals were visualized using the ChemiDicTM XRS + Imaging System (Bio-Rad), and band densities were quantified with ImageJ software. The results are expressed as a relative density ratio normalized to the value of the Sham or Control group. Anti-beta-catenin, occludin, claudin-5, zo-1, and GAPDH were purchased from Abcam (Cambridge, UK).
2.11 Hematoxylin and eosin staining
The rats were anesthetized with 10% chloral hydrate (3.5 ml/kg, i.p.) and then perfused with 0.9% NaCl, followed by 4% paraformaldehyde in 0.01 M PBS (pH = 7.4) at 7 days after surgery. The spinal cords from the T7–T10 level around the lesion epicenter were excised, and transverse paraffin sections (5 mm thick) were mounted on poly-L-lysine-coated slides for histopathological examination by hematoxylin and eosin staining.
2.12 Immunofluorescence staining
The sections were incubated with 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies in blocking buffer (zo-1, occludin, MMP-2, and MMP-9, Santa Cruz Biotechnology). Then, the cords were separately incubated with secondary antibody (Alexa Fluor 488-conjugated anti-IgG, Abcam; Texas red-conjugated anti-IgG, Santa Cruz Biotechnology). The nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China). Cells grown on 14×14 mm microscopic glass were washed with ice-cold PBS, fixed with 4% paraformaldehyde for 30 min, washed with ice-cold PBS, and blocked in 5% BSA for 1 hr. Then, the cells were incubated with anti-zo-1 (Abcam) and anti-Occludin (Abcam) diluted in 1% BSA at 4°C overnight. Cells were washed with PBS followed by incubation with Alexa Fluor 488-conjugated anti-IgG or Texas red-conjugated anti-IgG secondary antibodies for 1 hr at room temperature. After washing with PBS, the nuclei were stained with DAPI for 7 min and washed with PBS. Finally, Antifade Mounting Medium (Beyotime Institute of Biotechnology) was added to the cells.
2.13 Evaluation of BSCB permeability
2.13.1 Evans blue dye assays
At 1 day after SCI, the rats were intravenously injected with 2% Evans blue dye (EB; Sigma-Aldrich, 2 ml/kg) solution in saline into the tail vein. Two hours after injection, the rats were anesthetized with 10% chloral hydrate (3.5 ml/kg, i.p.) and then perfused with 0.9% normal saline. The injured spinal cord tissues of EB were weighed and immersed in N,N0-dimethylforma-mide (Shanghai, China) at 50°C for 72 h. The optical density of the supernatant was examined with an enzyme-labeled meter (at an excitation wavelength of 620 nm and an emission wavelength of 680 nm). Dye in the samples was determined as lg/g of tissue from a standard curve plotted using known amounts of dye.
2.13.2 FITC-dextran assays
At 1 day after SCI, the rats were injected with 2% FITC-dextran (MW 70 kDa, 4 mg/kg; Sigma-Aldrich) solution in PBS intravenously into the tail vein. Two hours after injection, the rats were anesthetized with 10% chloral hydrate (3.5 ml/kg, i.p.) and then perfused with 0.9% normal saline. The injured spinal cord tissues with FITC-dextran were weighed and homogenized in PBS and centrifuged. The optical density of the supernatant was examined (at an excitation wavelength of 493 nm and an emission wavelength of 517 nm).
2.13.3 Paracellular permeability assay
Human brain microvascular endothelial cells were seeded on Transwell permeable supports (PET membrane 24-well cell culture inserts with 0.4 µm pore size; Corelle; Corning Life Sciences, Corning, New York, USA) at a density of 1×105 cells/well in 200 µl medium overnight and subjected to oxygen-glucose deprivation (OGD) for 10 h. Then, the cells were incubated with FITC-dextran (1 mg/ml) in medium for another 2 h OGD. Thereafter, FITC-dextran passing through the Transwell (in the lower chambers) was determined by using an enzyme-labeled meter at an excitation wavelength of 493 nm and an emission wavelength of 517 nm.
2.14 Statistical analysis
Means ± SEM were used to express the data. Student’s t-test was used to perform single comparisons, and one-way analysis of variance (ANOVA) was used to perform multiple comparisons. A value of P < 0.05 was used as a benchmark for statistical significance.