Small Extracellular Vesicles Released from miR-211-5p-Overexpressed Bone Marrow Mesenchymal Stem Cells Ameliorate Spinal Cord Injuries in Rats

Animals

Male Wistar rats were purchased from Shanghai Model Organisms. Ethical approval for the research protocol was obtained from the Ethics Committee of the First Affiliated Hospital of Anhui Medical University. Throughout the breeding period, all rats were provided with unrestricted access to food and water. The study employed a total of 32 rats, with eight rats allocated to each experimental group.

BMSCs isolation

Four-week-old Wistar rats’ hindlimbs were stripped of the skin and muscle tissue attached to it to expose the rat's femur. The metaphyses at both ends of the femur and tibia were clipped to reveal the bone marrow cavity. DMEM/F12 medium (containing 10% fetal bovine serum and 1% double antibody) was used to flush out the cells in the medullary cavity of the bone. The marrow cavity cells were collected by centrifugation and planted into culture flasks and placed in an incubator for culture (37°C, 5% CO₂ saturated humidity). Flow cytometry was used to select and identify BMSCs at passages 3–5. CD29, CD105, CD34, and CD45 were makers. Cells negatively expressed CD34 and CD45 and positively expressed CD29 and CD 105 were regarded as BMSCs.

MicroRNAs

The miR-211-5p and relative control miRNA (NC) used in this study was synthesized by RiboBio Biotechnology as follows:

miR-211-5p: 5′-UUCCCUUUGUCAUCCUUUGCCU-3′;

5′-AGGCAAAGGAUGACAAAGGGAA-3′.

Collection of EVs from BMSCs

Lipofectamine 2000 was used for the transfection of miRNAs to BMSCs at 3–5 passages for 48 h. The fetal bovine serum was centrifuged at 1,00,000 × g ultra-high speed, and the bottom sediment was discarded to obtain serum without EVs. Phosphate-buffered saline (PBS) was used to wash the BMSCs three times, and the complete medium containing the fetal bovine serum was replaced to culture the cells. After 36 h of culture, the cell supernatant was collected. The cell supernatant was centrifuged at 8,000 r/min for half an hour at 4°C to remove dead cells and cell debris. A 0.22 µm filter was used to filter out smaller cell debris. The obtained supernatant was sucked into the ultrafiltration tube and centrifuged at 6,000 × g for half an hour at 4°C to concentrate the supernatant to ∼200 μl. qEV kit was used to purify EVs according to the operating instructions. Electron microscopy was used for the detection of EVs.

Dual-luciferase reporter assay

Luciferase Reporter Assay Substrate Kit - Firefly (ab228530; Abcam) was used for the dual-luciferase reporter assay in this study following the operating instructions.

Osteogenic and adipogenic differentiation of BMSCs

Invitrogen-Mesenchymal Stem Cell Differentiation Medium (Thermo Fisher Scientific) was used to induce osteogenic differentiation. Invitrogen-Mesenchymal Stem Cell Differentiation Medium (Thermo Fisher Scientific) was used to induce adipogenic differentiation. Alizarin red S and oil red O are used to stain mesenchymal stem cells after osteogenic differentiation and adipogenic differentiation.

Quantitative real-time PCR

qRT-PCR analysis was performed using a FastKing One Step RT-qPCR Kit (SYBR Green) purchased from Tiangen Biotech following the directions. β-Actin was used as an internal reference. The primers were as follows:

miR-211-5p: F: 5′-TTCCCTTTGTCATCCTTTG-3′,

R: 5′-GCAGGGTCCGAGGTATTC-3′;

COX2: F: 5′-CCATGTCAAAACCGTGGTGAATG-3′,

R: 5′-ATGGGAGTTGGGCAGTCATCAG-3′);

U6: F: 5′-CCTGCTTCGGCAGCACA-3′,

R: 5′-AACGCTTCACGAATTTGCGT-3′.

β-Actin F: 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′,

R: 5′-GGCCGGACTCATCGTACTCCTGCTT-3′.

Western blotting assay

Cell lysis buffer (Thermo Fisher Scientific) was used for the extraction of proteins. The protein of each mouse was separated and transferred to a PVDF membrane. The primary antibody with working solution including anti-CD9 (1:1,000), anti-CD63 (1:1,000), anti-Alix (1:1,000), anti-COX2 (1:1,000), and anti-GAPDH (1:1,000) were incubated with membranes at 6°C for 12 h.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was performed to evaluate the expression of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in the spinal cords of rats by Rat IL-6 ELISA Kit (#ERA31RBX5, Thermo Fisher Scientific), Rat IL-6 ELISA Kit (#ERA31RBX5, Thermo Fisher Scientific), Rat IL-1 beta Uncoated ELISA Kit with Plates (#88-7013-22, Thermo Fisher Scientific) and Invitrogen TNF alpha Rat ELISA Kit (#BMS622, Thermo Fisher Scientific) following the instructions.

The establishment of SCI model

In this study, we used spinal cord hemisection to establish a SCI model. A 3.5 vol% isoflurane was used to induce anesthesia in rats. The rat's back was depilated and thoroughly disinfected with Aner Iodine. A longitudinal incision along the midline of the back was made ∼2 cm centered on the T10 spinous process of the thoracic vertebra to expose the T10 spinous process and lamina. T9–T10 lamina was opened to reveal the white spinal cord and the posterior median vein of the spinal cord. The ophthalmic iris knife was inserted vertically from the posterior median vein and cut the right spinal cord to remove residual fibrous tissue. After the rat woke up, the right hindlimb could be seen paralyzed, and the left hindlimb was normal or slightly restricted, indicating that the spinal cord hemisection was successful. After the rats in the sham operation group were anesthetized, the skin and muscle were sutured after T9–T10 laminectomy, and the spinal cord was not hemisectioned.

EV intervention

EVs (50 μg in 50 μl PBS) were intravenously injected into rats once a day for a total of three times from the first to the third day after SCI surgery.

Basso–Beattie–Bresnahan (BBB) scoring

The BBB scores of rats were assessed by two professional researchers who were unaware of the grouping. The total score of BBB used in this study was 21. The rats without any motor function had a BBB score of 0.

A von Frey filament test

Semmes–Weinstein monofilaments (Stoelting) was used for the von Frey filament test. The fore used in this study was 0.008–300 N. A von Frey filament test was performed 10 min before the SCI modeling, and it was performed on days 1, 4, 7-, 14-, 21- and 28 post SCI.

Motor evoked potential

The American AXON evoked potential meter was used for the detection of rat motor evoked potential. The stimulating electrode is a self-made pair of needle electrodes, and the distance between the cathode and the anode is ∼0.3 cm. The muscle action potential was recorded on the tibialis anterior muscle of the calf, and the distance between the positive and negative electrodes of the recording electrode was ∼0.5 cm. Another silver needle electrode was pierced under the skin of the tail to ground. The stimulation method was a single direct current pulse stimulation. The stimulation intensity was 10 mA. The frequency was 5 Hz, the wave width was 0.1 ms, and the filter bandpass was 30–1,500 Hz.

Statistical analysis

The statistical analyses were conducted utilizing one-/two-way analysis of variance (ANOVA), followed by post hoc testing for relevant comparisons. The presented data are expressed as mean ± standard deviation (SD). A significance level of p < 0.05 was employed to determine statistical differences. Prior to analysis, the normality of the data was assessed using Anderson–Darling test, D’Agostino and Pearson’s test, Shapiro–Wilk test, and Kolmogorov–Smirnov test. These tests were applied to ensure the assumptions of normality were met before proceeding with the statistical analyses.