Isolation, culture, and characterization of hPMSCs
The human placenta was transferred to a clean laboratory under completely sterile conditions and underwent the stem cell extraction process(22). Pieces of placental tissue were placed in dishes containing PBS and 1% penicillin/streptomycin. Vessels and blood clots were removed from the tissue and washed repeatedly with PBS, then mechanically divided into small pieces. Next, 3 mL of fragmented tissue was placed in a 15 mL centrifuge tube to which 1mL collagenase IV was added and incubated at 37 °C, with agitation for 90 min, until the tissue was digested to a sticky state. The samples were shaken for 10 min and centrifuged for 5 min at 1250 rpm, the supernatant was removed, and the samples were transferred to the T75 flask containing DMEM/F12 (Dulbecco’s Modified Eagle's Medium F12), 10% fetal bovine serum, and 1% penicillin-streptomycin under standard culture conditions and placed in a 5% CO2 incubator for 3–5 days. The cells were attached and every day thereafter, the cells were observed and passaged according to their growth. In the third passage of hPMSCs, flow cytometry was employed to characterize the surface markers including, CD34, CD29, CD44, CD45, and CD90. The multipotency of hPMSCs was evaluated by osteogenic and adipogenic media.
Isolation and characterization of hPMSCs-Exosome
In order to extract the exosomes(23), the third passage of hPMSCs with approximately 80% confluence was washed with PBS and incubated in a serum-free culture medium for 72 hours. The conditioned medium was collected from all flasks and centrifuged at 300×g, 10 min at 4 °C to remove cell, followed by centrifugation at 2000×g for 10 min at 4 °C in order to remove dead cells and 10000×g for 30 min cell debris. The supernatant was passed through a 0.22-μm filter and ultracentrifuge at 100,000 × g for 90 min at 4 °C was also implied. The supernatant was discarded, loaded upon 5 ml PBS and then ultracentrifuged at 100,000 × g for 90 min at 4 °C. Finally, the purified exosomes were collected in 500 μl isotonic serum and stored at -80 °C for further studies. To quantitate the exosomes, total protein concentration was measured using a Micro BCA Protein Assay kit. Dynamic light scattering (DLS) measurements were also performed with a Zetasizer 3000-HA (Malvern Instruments, UK) to measure the size distribution. The shape and morphology of exosomes were evaluated by the TEM. Exosome surface markers, including CD81 and CD9, were detected by Western blot analysis.
Transmission Electron Microscopy
Morphology and size of exosomes were determined using Transmission Electron Microscopy (TEM)(24). Briefly, 20 μL of isolated exosomes were placed on 300 mesh carbon-coated TEM grids for 2 min, negatively stained with 2% aqueous uranyl acetate for 1 min. The grid was allowed to air dry and was examined on a Zeiss EM10C TEM operating at an accelerating voltage of 100 kV.
The typical exosome markers CD81 and CD9 were assessed by Western bloting. Exosomes were lysed with RIPA buffer and Protease inhibitor. The total protein concentration was determined using the BCA assay kit. After SDS-PAGE electrophoresis, proteins were transferred to 0.45 μm PVDF membranes for 2 h at room temperature. The PVDF membranes were then washed by TBST and incubated overnight with the primary antibodies at 4 °C. Primary antibodies included CD81 (GTX101766), CD9 (GTX76184). After being washed with TBST for three times, the PVDF membranes were incubated for 2 h with the secondary antibody at room temperature. Blots were detected using enhanced Electro chemiluminescence (ECL).
Animals and experimental groups
Total 12 experimental rats were randomly divided into two groups: (1) control group (the rats received SCI and were treated with normal saline, n=6), and (2) experimental group (the SCI rats that treated with 30µg/300µl exosomes, n=6).
Balloon Compression Spinal Cord Injury
Adult specific pathogen-free grade Wistar female rats (n = 12, aged 8 weeks and weight 270-300 g) were used as an experimental model obtained from the Pasteur Institute of Iran. Female rats were anesthetized by medetomidine (5mg/kg)/ketamine (0.05mg/kg). The dorsal fascia was cut bilaterally. Following the dissection of the paraspinal muscles, a laminotomy from T13 was performed using a micromotor high-speed drill (up to 30,000 rpm). Fogarty catheter 2F was used to cause severe damage to the spinal cord, which was firmly attached to a Hamilton syringe and filled with saline. The catheter was passed through the laminotomy site (T13) in the epidural space and placed in the T11 (Fig. 1A, B). The CT image confirmed the presence of the balloon catheter in the epidural space (Fig. 1C). the balloon was then rapidly inflated with 15 μl Distilled water for 5 min(25). After removing the catheter, the balloon filling was rechecked. Next, muscles and skin were closed in layers. The rats had their bladders emptied manually after surgery until reflex and were given the Enrofloxacin antibiotic (10 mg/kg (every 24 hours for 5 days.
Exosome lumbar puncture injection and myelogram to check the accuracy of the injection
After surgery and creating a severe model of spinal cord injury, the SCI rats were intrathecally treated with exosomes. Insulin syringe 28 G needle was used to intrathecal space injection at L5–L6 via LP (Fig. 2A, B, and C). The rat spinal cord begins in the lower cervical region and continues to the level of the intervertebral disc between the third and fourth lumbar vertebrae. Therefore, intrathecal injection between L5 and L6 does not damage the spinal cord tissue. The location of the needle tip inside the subarachnoid space was confirmed using CT scan images. For myelogram, 300 µl of contrast medium and serum were injected as LP and one minute later, under CT scan, the movement of the injected solution towards the spinal cord was observed (Fig. 2D).
Post-injury function recovery and locomotors testing was assessed via the Basso, Beattie and Bresnahan (BBB) motor scale method(26). The scale (complete paralysis (score 0) to normal gait (score 21)) shows the successive recovery stages and the classification of the rat joint movement, hindlimb movements, stepping, forelimb and hindlimb coordination, trunk position and stability, paw placement and tail position. The BBB test was performed on days 1, 3, 7, 14, 21, 28, 35 and 42 after surgery. Locomotor function was scored by two independent investigators.
Animal perfusion fixation
Animal perfusion fixation was performed six weeks after surgery(27). The animals were firstly Intraperitoneal injected with ketamine (70 mg/kg) and xylazine (10 mg/kg) and were checked for the toe-pinch reflex to check for pain reflex before any further action. Following complete anesthesia, the animal was cut open below the diaphragm and the rib cage was cut on the lateral edges to expose the heart. After thoracotomy, the animal was transcardially perfused with PBS for 4 minutes and was cleared of blood and perfused with Paraformaldehyde (PFA 4%, in 0.1 M PBS, pH 7.4) for 4 minutes. The animal’s extremities were visualized for evidence of tremors resulting from the aldehyde-crosslinking of nerves and muscle as an indication of fixation is taking place. The spinal cord segments containing the injury epicenter were removed and postfixed overnight in 4% PFA at 4 ◦C.
Tissue Processing and Hematoxylin–Eosin (HE) Staining
Tissues were fixed and collected, they were typically dehydrated and embedded in melted paraffin wax; the resulting block was mounted on a microtome and cut into 10 and 20 µm slices. The nucleus and cytosol were respectively stained with hematoxylin and eosin. The sections were dried in the ethanol gradient (70% to 100%) and cleared with xylene. Finally, the samples were observed under a light microscope. The extent of the cavity created following spinal cord injury was tracked and quantified using Image J software(28).
Cell apoptosis assays with TUNEL staining
Apoptotic positive cells in the injury site at week 6 post-injury were identified and quantified by terminal deoxynucleotidyl transferase-mediated dUTp nick end-labeling (TUNEL) assay (Roche, Mannheim, Germany) according to the manufacturer’s protocols.
For immunohistochemistry staining, briefly, cross-sections containing the lesion area were deparaffinized with xylene and hydrated in graded alcohol containing 100%, 95%, 70%, and 50%, each for 3 minutes. To inactivate, the sections were immersed in 10% hydrogen peroxide for 10 minutes and then washed with PBS (PH=7.4) buffer for 10 minutes. Antigen retrieval was performed using citrate buffer (pH 6.0) for 5 minutes at 95 ° C. The sections were incubated in the IHC jar with the primary antibody including GFAP (MAD-000716QD) and NF200 (MAD-000384QD), for 50 minutes at room temperature. After washing in double-distilled water for 5 minutes, incubation with secondary antibody was performed for 40 minutes at room temperature. After washing with distilled water for 5 minutes, the sections were incubated in an IHC jar with DAB chromogen for 10 minutes at room temperature. Hematoxylin was used to stain cell nuclei. Finally, we used alcohol in a concentration gradient from dilute to concentrate for dehydration and were xylene treated to see better transparency and separation.
Statistical analyses were performed using SPSS Statistics 22.0. All data were presented as the means ± SD. TO analyze the data of BBB scores repeated measures followed by the Bonferroni post-test were used for comparing the groups. Levene's test was used to test if k samples have equal variances (homogeneity of variance).The Kolmogorov-Smirnov test was used to decide if a sample comes from a population with a specific distribution. The independent-samples- T Test was used to compare control and treatment values and p < 0.05 was considered significant.