FIGURE 3.
Intravenous infusion of MSCs and fractionated dosing of MSC‐sEVs promote M2 macrophage polarization in the injured spinal cord. a1‐3 ‐d1‐3, Confocal micrographs of a representative region of a frozen sectioned contused spinal cord harvested 7 days after the treatment (14‐day post‐SCI), immunostained with antibodies directed against Type M1 macrophage marker iNOS (green), Type M2 macrophage marker CD206 (red), and counterstained with DAPI (blue). Scale bar in a indicates 50 μm. e, Representative images of western blot of the lesion site harvested at 7 days after the treatment (14‐day post‐SCI), immunostained with antibodies directed against Type M1 macrophage marker iNOS, Type M2 macrophage marker CD206, and GAPDH as a control. f and g, Graphs of quantitative density analysis for western blot results for iNOS (f) and CD 206 (g) in each treatment condition normalized to control spinal cords. h, M1/M2 macrophage protein expression ratios calculated using quantitative density analysis of western blot data for iNOS and CD206. i and j, qRT‐PCR analysis of relative mRNA expression levels for M1 macrophage marker CLL2 (i) and M2 macrophage marker CD206 (j). [ΔCT was calculated against the endogenous control (GAPDH), and ΔΔCT (mRNA level) was calculated against the ΔCT of the control.] k, M1/M2 macrophage mRNA ratios calculated by using qRT‐PCR results for CLL2 and CD206. Values are presented as means ± SEM. A 1‐way ANOVA followed by the Tukey‐Kramer test or the Kruskal‐Wallis test followed by the Steel‐Dwass test was conducted. *: P < .05, **: P < .01, SCI: spinal cord injury, MSC: mesenchymal stem/stromal cell, MSC‐sEVs: small extracellular vesicles derived from MSCs, iNOS: inducible nitric oxide synthase, DAPI: 4′,6‐diamidino‐2‐phenylindole, GAPDH: Glyceraldehyde 3‐phosphate dehydrogenase, CCL2: C–C motif chemokine ligand 2, ΔCT: delta‐cycle threshold, qRT‐PCR: quantitative reverse transcription‐polymerase chain reaction, FC: fold change