Isolation, cell culture, and identification of bone marrow derived macrophages (BMMs) and endothelial progenitor cells (EPCs)
The primary bone marrow-derived macrophages (BMMs) were extracted as previously documented [39]. Briefly, three C57BL/6 mice were executed via cervical dissection. Under aseptic circumstances, muscle and connective tissue were taken from mouse bones, as well as the extremities of the tibia and fibula. A syringe is used to remove bone marrow, which is then combined with phosphate-buffered saline (PBS) to create a bone marrow stem cell suspension. After centrifugation, the cell precipitate was resuspended with BMMs growth medium (IMDM (Gibco, Waltham, MA), 10% FBS (Gibco), 1% Antibiotic-Antimycotic (Gibco), 10ng/ml macrophage colony-stimulating factor (M-CSF, PeproTech)). The aforesaid cell suspensions were planted in six-well tissue culture plates, with the initial fluid change on the third day and every other day after that, yielding mature BMMs in roughly a week. Immunofluorescence labeling was used to identify expression markers such as F4/80 and CD68 (Abcam, Cambridge, UK).
Primary bone marrow endothelial progenitor cells (EPCs) were extracted as described previously [40]. Similar to the techniques described above, we acquire bone marrow cell suspensions from C57BL/6 mice, the suspensions were progressively put on top of the Lymphatic Separation Medium (MultiSciences; Hangzhou; China), and mononuclear cells were recovered following centrifugation at 2500g × 30min at 4°C. They were gently aspirated from the centrifuge tubes and centrifuged at 500g for 5 minutes before being resuspended in EPCs growth medium (Endothelial Cell Growth Basal Medium-2 (EBM™-2, LONZA), Endothelial Cell Growth Medium-2 BulletKit™ (EGM™-2, LONZA)). The cell suspension was seeded onto 6-well plates that had already been treated with Human Fibronectin (Sigma), and it was then continuously incubated at 37°C in a humid environment with 5% CO2. After cell attachment, the media was replaced every two days, and EPCs were collected after around one week. The traditional approach for identifying endothelial progenitor cells uses dual fluorescent labeling (Dil-ac-LDL and FITC-UAE-1 (Solarbio)), and the unique antigens CD133 and VEGFR2 (Cell Signaling Technology, CST) set them apart from regular endothelial cells and other heterogeneous cells.
Isolation Of Endothelial Progenitor Cells-derived Exosomes (Epc-exos) And Content Analysis
Endothelial progenitor cells were incubated in the complete culture medium with exosome-depleted FBS (160,000 g, 16 hours) for 48 hours after being grown in the regular medium until 80–90% confluent. EPC-EXOs were extracted from the finished medium utilizing several techniques, according to the manufacturer's protocol, which can be obtained in one of two ways: either by centrifuging the exosomes in an ultrahigh speed centrifuge (120000g, 3h) or by extracting the exosomes using Amicon® Ultra Centrifugal Filters (Millipore) with ExoQuick-TC for Tissue Culture Media and Urine (System Biosciences, SBI, USA). This is done after the cell supernatant collect- ed and the cell debris removed by preliminary centrifugation (3000g, 30min). The exosomes were dissolved in PBS and kept at -80°C for storage. To further describe the morphology of EPC-EXOs, transmission electron microscopy (TEM, FEI company, USA) examination was employed. The diameter and particle count of EPC-EXOs were measured using nanoparticle tracking analysis (NTA, ZetaView,Germany) Immunoblot analysis was used to find the exosome markers CD9, CD63, and TSG101 (Abcam) as well as the cellular marker Calnexin (Abcam).
Exosomes Labeling And Uptake Assay By Bmms
We labeled the exosomes using a red fluorescent lipophilic dye Di (Sigma). After being cultured with 100µg/µl of tagged exosomes for 12 hours, BMMS were rinsed with PBS, fixed for 20 minutes in 4% paraformaldehyde, and stained with Prolong Mounting Media Containing DAPI (GeneTex Inc, Irvine, CA). To ascertain if exosomes are being picked up or not, images were acquired using the Zeiss Apotome.
Spinal Cord Injury (Sci) Model And Treatments
According to our earlier work [41], male C57BL/6 wild-type mice were profoundly anesthetized and the spinal cord was moderately contused at the T10 level using a modified Allen weight descender (10g weight with a vertical height of 20 mm). Following surgery, daily bladder massages were given until full spontaneous or voluntary urination, and daily antibiotics were given for three days. For the SCI model consistency, the following criteria were required for inclusion [42]: Ⅰ) mice weighing between 20 and 25g; Ⅱ) 10g weight and 20mm vertical height requirements for weight reduction devices; Ⅲ) tail wagging, hind limb twitching right after SCI, and flaccid paralysis of the hind limbs when awake. Exclusion criteria included: Ⅰ) The animals' weight and age were below average. Ⅱ) During a laminectomy, the spinal cord unintentionally suffered damage. Ⅲ) The mice's hindlimbs could still move after they emerged from anesthesia. Following SCI, 100µl of the EPC-EXOs (10µg/µl) solution was progressively administered into the tail vein, and samples were taken at 7, and 14 days after SCI.
The Tracking Tests Of Epc-exos In Vivo
For in vivo tracking tests, EPC-EXOs were tagged with 5µM of the fluorescent lipophilic dye Dil (Yeasen, Shanghai, China), resuspended in PBS at a concentration of 10µg/µl, and kept out of the light. Following SCI, the administration of the suspension of Dil-labeled EPC-EXOs was performed via tail vein injection as previously mentione. Samples were taken seven days after the injury, dehydrated, cut into frozen sections, and examined by immunofluorescence at 594 nm for the absorption of Dil-labeled exosomes by macrophages in the injury region.
Evaluation Of The Locomotive Behavior Function
The BMS (Basso Mouse Scale) method was used to evaluate motor function in the hind limbs before surgery and 1, 3, 7, 14, 21, 28, and 56 days following spinal cord damage. From 0 (totally paralyzed) to 9, the BMS scale measures pain (normal movement). The mean of the BMS primary score and the BMS subscale score were recorded, and the mean value was calculated as the final score value, in order to prevent bias in the BMS scale data. The BMS subscale system was also combined with the assessment by two researchers who were trained well but had no prior knowledge of the experimental design.
Footprint Analysis
Four weeks after SCI, mice were evaluated for their gait and motor coordination. Different colors were used to paint the front and back paws (red or blue). The mice were put on a straight track with a piece of white paper at the bottom, and the animals were prompted to go in a straight line. The footprint pattern was scanned after being captured in photographs so that the recovery of motor function could be evaluated.
Neuroelectrophysiology
The mice were given effective anesthesia before the motor evoked potentials (MEPs) were measured. Before surgery (baseline) and 28 days after SCI, the mean MEP values (including amplitude and latency duration) in various intervention groups were obtained. The electrodes were implanted as reported in our prior research [39]. For each mouse, experimental MEP (mV) reflected the relative level of mobility recovery.
Hematoxylin-eosin Staining (He Staining)
After cardiac perfusion 28 days after surgery, mice were sedated with 0.3% pentobarbital, and the spinal cord tissue was carefully removed. The samples were first dehydrated with varying levels of alcohol, made transparent with xylene (Macklin), and then embedded in paraffin wax. Once the wax blocks had fully hardened, the samples were divided into sections. The sections were sealed with neutral gum after being stained with hematoxylin and eosin (LEAGENE, Beijing). An N2-Ni-U ortho-fluorescence microscope (Nikon, Shanghai) was used to capture the images.
Extraction Of Rna And Quantitative Real-time Pcr (Rt-qpcr) Analysis
The miRNA First-Strand cDNA Synthesis Kit 2.0 and miRNA QPCR mix (GeneCopoeia) were used to synthesize cDNA by miRNA reverse transcription and to detect the expression level of the contained miRNAs with a series of miRNA primers (mmu-miR-21a-5p, mmu-miR-222-3p, mmu-miR-221-3p, mmu-miR-155-3p, mmu-miR-29a-3p, mmu-miR-199a-3p, mmu-miR-146a-5p, mmu-let-7i-5p and RNU6) provided by GeneCopoeia, and the above kits contain universal downstream primers. RNU6 was used as an endogenous control to normalize the results.
The target genes were reversely transcribed into first-strand cDNA and amplified using the Promega Reverse Transcription System and GoScript™ QPCR Master Mix (Promega) with BRYT Green master mix. The outcomes were calibrated using GAPDH to acquire a more precise reading of the target gene's expression level. Table S1 displays the primer sequences.
All reactions were run through a real-time PCR system (FTC-3000, Funglyn Biotech Inc., Toronto, Canada) for processing and analysis, and relative gene expressions were calculated using the 2 −ΔΔCT method.
Mir-222-3p Transfection Via Mir-222-3p Mimic, Inhibitor, And Plasmids
BMMS were seeded in six-well plates, and when their growth was fused to 60%-80%, they were washed three times with PBS. MiR-222-3p mimic, mimic NC, inhibitor, inhibitor NC (Ribobio, Guangzhou, China) working solution and Lipofectamine™ 3000 (ThermoFisher) suspension were prepared with Opti-MEM (Gibco), respectively. After standing at room temperature for 10 min, the corresponding suspensions were mixed with each other and then stood at room temperature for another 10 min. BMMs complete medium was added proportionally, and the mixed suspensions were added to the cells and the mixed system was reacted in an incubator at 37 degrees for 8 h. After that, the cells were washed 3 times with PBS, and then the complete medium was added and the incubation was continued for 48 hours. The whole length of the SOCS3 ORF sequence was cloned into the pcDNA 3.1(+) vector using plasmids expressing SOCS3 that were purchased from Changsha Youbio Co, Ltd. In accordance with the manufacturer's instructions, LipofectamineTM 3000 (ThermoFisher) was used to transfect the mimic, inhibitor, and plasmid. The transfected cells were saved for later research.
Western Blot Assays
BMMs and spinal cord tissue was lysed using RIPA (Solarbio) with 1% protease and 2% phosphatase inhibitor (Sigma). Protein concentration was measured using a BCA Protein Assay Kit (Solarbio, Beijing, China). And then the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) after being separated using SDS-PAGE gels (Millipore, Billerica, MA). The membrane was incubated with primary antibodies at 4°C overnight after being blocked for 60 min with 5% non-fat powdered milk (Sangon Biotech) or 5% BSA (Biofroxx) in TBST (Solarbio) at room temperature. The following primary antibodies and dilutions were utilized: rabbit anti-SOCS3 (1:500; Proteintech), rabbit anti-iNOS (1:1000; Proteintech), rabbit anti-Arg-1 (1:5000; Proteintech), rabbit anti-β-actin (1:5000; CST), rabbit anti-JAK2 (1:1000; CST), rabbit anti-p-JAK2 (1:1000; CST), rabbit anti-STAT3 (1:1000; Proteintech), rabbit anti-p-STAT3 (1:1000; CST), rabbit anti-Calnexin (1:1000; Proteintech), rabbit anti-CD9 (1:1000; Abcam), rabbit anti-CD63 (1:1000; Abcam) and rabbit anti-TSG 101 (1:1000; Abcam). The membrane was then treated with peroxidase-conjugated anti-rabbit after being cleaned three times with TBST (1:5000; CST). The β-actin was used to evaluate equal protein loadings. The enhanced chemiluminescence reagent (ThermoFisher, Waltham, MA) and ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad) were used to examine the data.
Immunofluorescence Staining
Slices of the spinal cord were permeabilized with 0.3% Triton X-100 in PBS for 30 minutes and blocked with 5% BSA (Biofroxx) or Animal Free Blocker (Vectorlabs) for 1 hour at room temperature for the spinal cord samples. For the cell samples, cells were permeabilized with 0.2% Triton X-100 for 15 min, fixed with 4% PFA in PBS for 20 min, and blocked with 5% BSA for 30 min. Following an overnight incubation at 4°C, the samples were incubated with the primary antibodies F4/80 (1:100, Santa Cruze), CD68 (1:100, Abcam), FITC-UEA-1 (MKbio, USA), Dil-Ac-LDL (MKbio, USA), CD86 (1:200, Abcam), CD206 (1:200, Abcam), VEGFR2 (1:200, CST), and CD133 (1:200, Abcam), followed by an incubation with the corresponding secondary antibodies (1:400, Abcam) for 1.5 hours at room temperature. The slices were mounted using Prolong Mounting Media Containing DAPI after being cleaned three times with PBS (GeneTex Inc, Irvine, CA). Using the Zeiss Apotome, variations in the fluorescence intensities of F4/80, CD86, and CD206 were computed for the quantitative investigation of the M1 and M2 phenotypes. In order to measure the area of F4/80+ cells, four identically sized areas were randomly chosen from the injury center. In each area, the green fluorescent area in response to macrophage aggregation was measured in each group, and the value of this area was used to calculate the area of F4/80+ cells. Calculating the number of F4/80+CD86+ cells or the proportion of F4/80+CD206+ cells to F4/80+ cells in an area of a certain size allowed researchers to study the polarization of macrophages (Image-Pro Plus 6.0).
Luciferase Reporter Assay
24-well plates were planted with HEK 293T cells. Mus musculus wild-type (WT) or mutant (MUT) SOCS3 3'UTR segments were cloned into the pmiR-RB-Report™ plasmid vector for the creation of plasmids (Ribobio, Guangzhou, China). After that, HEK 293T cells were transfected with the plasmids (miR-222-3p mimic, inhibitor, mimic NC, and inhibitor NC). After 48 hours, a 1 × PLB cell lysis solution was added, and the cells were agitated for 15 minutes at room temperature. Cell scrapers were then used to collect the cell lysate. A total of 20µl of cell lysate and 100µl of each of the following solutions were applied to each well of a 96-well plate: 100µl of Luciferase Assay Buffer II and 100 mL of Stop & Glo® Substrate, respectively. Using the Promega Dual-Luciferase system, we accomplished this experiment (Promega, USA). The Firefly Luciferase and Renilla Luciferase activity were detected using the Centro XS3 LB 960 (Berthold, Germany) and MikroWin software, and their differences were quantified and examined.
Statistical Assay
Software for statistical analysis included GraphPad Prism 8.0 (La Jolla, CA, USA) and SPSS 22.0 (SPSS, Inc.). All data were displayed as means ± standard error of measurement (SEM). Researchers who carried out statistical analysis were treated in a blind manner. Two groups were compared using the unpaired t-test, and pre- and post-treatment data were compared using the paired t-test. One-way or two-way ANOVA was used with the Tukey's post hoc test to analyze the differences between three or more groups or between the groups over time. Statistical significance was defined as p less than 0.05.