Animal and SCI models
A cohort of 54 adult female Sprague–Dawley (SD) rats weighing around 220 g was acquired via Zhejiang Viton Lever Co. All procedures involving the animals adhered to the regulatory framework outlined in the Standards for National Management and Utilisation of Experimental Rats in China. Subsequently, the animals underwent random allocation to three distinct experimental cohorts: Sham operation (Sham group), SCI induced by extrusion (SCI group), and SCI intervention with BMMSCs (BMMSCs group). The Sham group was left untreated except for laminectomy; the BMMSCs group was injected with 0.5 ml (1 × 107 ml) of third-generation (P3) BMMSCs on days 1, 2, and 3 after injury, and the SCI group was injected with an equal volume of PBS. Considering that local injections and direct implantation of BMMSCs may lead to secondary spinal cord injuries, we injected BMMSCs into the tail vein to facilitate their wider distribution throughout the body, thereby improving their efficacy in SCI treatment. Prior to the surgical intervention, anaesthesia was induced through intraperitoneal administration of a 1% (w/v) solution of pentobarbital sodium. The dose was 40 mg/kg. Following successful anesthesia and positioning in the prone orientation, the dorsal region was shaved to expose the spinous processes and vertebral segments within the thoracic spine, specifically at the levels of T9 through T11, and the surgical site was aseptically prepared. The spinal cord was exposed by removing the vertebral plate centered on T10, followed by a 10-s compression using vascular clamps to establish the SCI model (Fig. 1A). Postoperatively, upon the rats regaining consciousness, intraperitoneal administration of 50,000 U/kg penicillin was carried out to prevent infections, fluid loss was replenished with 5 ml saline over three days. Twice daily, Expressing the bladder manually was conducted until bladder voiding occurring naturally occurred.
Isolation of spinal cord tissue and identification of BMMSCs. (A) A vascular clip was employed to apply pressure to the exposed spinal cord. (B) Twenty-eight days post-injury, spinal cord tissue specimens were harvested from each experimental group of rats for subsequent analysis. (C) Bone marrow mesenchymal stem cells (P3, 500 pixels). (D) Detection of surface antigens on P3 BMMSCs by flow cytometry.
Behavioural assessment
Functional recovery following SCI was evaluated utilizing the Basso, Beattie, and Bresnahan (BBB) assessment and Footprinting. The BBB scale evaluations took place in an unconfined setting at postoperative days 1, 3, 7, 14, 21, and 28, with sensory and motor functions evaluated by three blinded investigators. The assessment scale, spanning from 0 (representing paralysis of the whole body) to 21 (representing full functional recovery)27, was recorded independently by each investigator, and the composite score was derived by computing the mean of the evaluations. Following this, at the 28-day juncture, rats’ forelimbs and hindlimbs were delineated with red and blue ink correspondingly, before allowing the animals to traverse a white paper-padded pathway to document their footprints. Two impartial observers independently examined the footprints for the experimental parameters without prior knowledge.
Tissue section preparation and HE and Nissl staining
Rats were sedated with 1% (w/v) solution of pentobarbital sodium and then perfused via the left ventricle with 0.9% sodium chloride solution. Following perfusion, the affected spinal cord tissues (as depicted in Fig. 1B) were extracted and immersed in 4% paraformaldehyde (w/v) solution for fixation over a period of 24 h. Subsequently, the tissues underwent dehydration, embedding, and sectioning into longitudinal and transverse slices measuring 4–5 μm in thickness. Nissl staining and haematoxylin and eosin (HE) staining were performed on these sections. Pathological alterations, including changes in the spinal cord tissue cavity area, inflammatory cell infiltration, and neuronal apoptosis, were evaluated through image acquisition using a pathology section scanner for further detailed analysis.
Tissue immunofluorescence
Paraffin-embedded spinal cord tissue sections, measuring 5 μm in thickness, were prepared in both longitudinal and transverse orientations. After antigen retrieval, the sections were treated with a 5% bovine serum albumin (BSA) solution for 2 h at room temperature to block non-specific binding. Subsequently, they were incubated overnight at 4 °C with the primary antibody to facilitate specific binding to the target antigens, including anti-GFAP (1:300; CST), anti-Neun (1:500; CST), anti-LC3II/I (1:300; CST), anti-p62 (1:400; ABMART), anti-MAP2 (1:100; CST), anti-Bcl-xL (1:300; CST) and anti-GAP43 (1:200; CST). Following this, the sections underwent incubation with a secondary antibody for 1 h at room temperature. To visualize the nuclei, counterstaining was conducted using a solution containing 4’,6-diamidino-2- phenylindole (DAPI).
TUNEL staining
TUNEL stands for Terminal deoxynucleotidyl transferase dUTP Nick End Labelling, which was employed to detect apoptotic cells in 5-μm-thick longitudinal paraffin sections following established protocols. The sections underwent deparaffinization and antigen retrieval procedures. Subsequently, cell permeabilization was achieved by treating the sections with Triton X-100 for 12 min at 37 °C. The sections were exposed to the experimental reagent mixtures for 0.9 h at room temperature. Following the incubation period, DAPI was used to stain nuclei for 5 min. Before coverslipping, an anti-fade mounting medium was applied dropwise. Upon UV excitation, DAPI-labeled nuclei emitted blue fluorescence, while TUNEL-positive apoptotic nuclei exhibited red fluorescence.
Transmission electron microscopy
Autophagic processes within spinal cord tissues were visualized through transmission electron microscopy (TEM) analysis. The spinal cord tissues were immersed at room temperature to achieve fixation. Subsequently, the sections underwent dehydration, embedding, fixation, and staining with conventional peroxynitrite acetate. TEM imaging was conducted utilizing an HT 7700 transmission electron microscope manufactured by HITACHI.
Cell culture
PC-12 cells and BMMSCs were procured from Shanghai Anwei Biotechnology Co Ltd. Culturing of PC-12 cells involved maintenance in high-glucose Dulbecco’s Modified Eagle Medium (DMEM), enriched with 10% fetal bovine serum (FBS), and supplemented with 100 units per milliliter of penicillin and 100 µg per milliliter of streptomycin. This was performed in a 37 °C humidified incubator with 5% CO2, and passage occurred once cells reached approximately 80% confluence. Regular medium changes every 2 days were performed, and cell morphology and growth were monitored using an inverted microscope in preparation for subsequent transfection experiments. Primary BMMSCs were cultured in specialized stem cell medium with medium changes every 2–3 days. Following passage, cells were expanded to the third cell culture passage (P3) was used for further experiments, and flow cytometry was utilized for the characterization of the cellular phenotype of P3 BMMSCs.
Transfection and grouping of cells
The experimental setup included multiple groups: the control group, the miR-202-3p mimics group, the miR-202-3p inhibitor group, and the miR-202-3p negative control (NC) group. PC-12 cells were distributed into 6-well plates with each well harboring a population of 6 × 105 cells. When 50%-80% confluence is reached, the cells underwent transfection with Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) along with the respective miRNAs. Following a 6-h transfection period, the medium was replaced, and incubation with Rapamycin (0.5 μmol/L) was sustained for 48 h. Subsequently, samples were collected for Western blot analysis to assess miR-202-3p influence on proteins associated with cellular autophagy and apoptosis. Additionally, miR-202-3p expression levels were assessed through qPCR both in vivo and in vitro.
Western blotting
RIPA lysis buffer was utilized for extract total protein from both spinal cord tissue samples and cellular specimens. Protein quantification was carried out employing a BCA protein kit (Solarbio). Subsequently, protein samples of equal quantity underwent SDS‒PAGE separation, transfer to nitrocellulose (NC) membranes, and incubation with a 5% skim milk powder solution for a duration of 2 h. Following blocking, primary antibodies were applied to the membranes and then incubated at 4 °C, targeting Beclin1 (1:1000), p62 (1:3000), Erk (1:1000), p-Erk (1:1000), mTOR (1:1000), p-mTOR (1:1000), CTSD (1:1000), VPS34 (1:1000), NeuN (1:1000), Bcl-xL (1:1000), GAP43 (1:1000), MAP2 (1:1000), and GAPDH (1:5000). Subsequent to primary antibody incubation, the membranes underwent a 2-h incubation period at 37 °C with secondary antibodies. Following washing steps, protein bands on the membrane were visualized using fluorescent reagents and captured with Image Lab software (Bio-Rad). ImageJ software was employed for quantitative analysis of protein expression levels.
qPCR
For the evaluation of miRNA-202-3p expression levels, total RNA extraction was performed from PC-12 cells and spinal cord tissues employing a specialized miRNA extraction kit (UElandy). The utilization of a NanoDrop 2000 (Thermo Scientific, USA) spectrophotometer enabled the determination of both the quality and quantity of miRNAs, followed by cDNA synthesis through reverse transcription. Subsequently, U6 was employed as an internal reference for qPCR analysis utilizing SYBR Premix Ex Taq (Bio-Rad Laboratories, Inc.).
Statistical analysis
Statistical analyses were performed using GraphPad Prism 10.0 software. Data distribution normality was evaluated utilizing the Shapiro–Wilk test. For normally distributed data, one-way analysis of variance (ANOVA) was followed by Tukey’s post hoc test for further analysis. In this context, we defined statistical significance as follows: *p < 0.05 indicated significance, **p < 0.01 signified a higher level of significance, ***p < 0.001 represented even greater significance, and ****p < 0.0001 was considered highly significant.
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- Source: https://www.nature.com/articles/s41598-024-81332-y