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. 2021 May 29;8(15):2100805. doi: 10.1002/advs.202100805

Figure 3.

Figure 3

Administration of Axon miRs in the presence of methylprednisolone synergistically enhanced functional recovery without alteration in axon regeneration. A,B) Representative fluorescent images of NF200 staining in Axon miRs + Mp‐ and Neg miR + Mp‐treated rats at 4 and 12 weeks post SCI. Images shown in the second row are the enlarged images of the boxed areas from the first row. C) Quantification analysis of area percent occupied by NF200+ signals within the scaffold region in Axon miRs + Mp‐ and Neg miR + Mp‐treated rats at weeks 4 and 12. Results indicate that in the presence of methylprednisolone, Axon miRs treatment significantly enhanced nerve regeneration versus Neg miR. *p < 0.05; **p < 0.01, Shapiro‐Wilk normality test followed by Kruskal–Wallis test and Mann–Whitney post hoc test. These results, however, are not significantly different versus corresponding samples without methylprednisolone (Figure 2B). N = 4 in Axon miRs + Mp and Neg miR + Mp groups at week 4; N = 6 in Axon miRs + Mp group; and N = 8 in Neg miR + Mp group at week 12. D) Quantification analysis of cyst size (labelled as yellow) at rostral and caudal regions of the injury site at week 12, suggesting that in the presence of methylprednisolone, Axon miRs treatment decreased cavity size at rostral region versus Neg miR. *: p < 0.05, Student's t‐test between Axon miRs + Mp and Neg miR + Mp group for all regions. These results, however, are not significantly different versus corresponding samples without methylprednisolone (Figure 2C). N = 4 in each group at week 4; N = 6 in Axon miRs + Mp group; and N = 8 in Neg miR + Mp group at week 12. E,F) Representative fluorescent images of Tuj1 staining at 12 weeks post SCI. Quantification analysis of area percent occupied by Tuj1+ signals within the scaffold region, indicating that Axon miRs did not affect immature neurons even in the presence of methylprednisolone. Student's t‐test. These results are also not significantly different versus corresponding samples without methylprednisolone (Figure 2E). N = 6 in Axon miRs + Mp group and N = 8 in Neg miR + Mp group. G–I) Schematic diagram and representative fluorescent images of BDA+ signals inside the scaffold. Quantification analysis of area percent occupied by BDA+ signals once again suggests that Axon miRs did not significantly enhance the regeneration of propriospinal axons even in the presence of methylprednisolone. Student's t‐test. These results are also not significantly different versus corresponding samples without methylprednisolone (Figure 2G,H). N = 3 in each group for (G); N = 3 in Axon miRs + Mp group; and N = 5 in Neg miR + Mp group for (H). J,K) Representative fluorescent images of 5‐HT staining in Axon miRs + Mp‐ and Neg miR + Mp‐treated rats at 12 weeks post SCI. Quantification analysis of area percent occupied by 5‐HT+ serotonergic axons at rostral region, within the scaffold and caudal region. Results suggest that in the presence of methylprednisolone, Axon miRs did not enhance the regeneration of 5‐HT+ serotonergic axons versus Neg miR. Student's t‐test between Axon miRs + Mp and Neg miR + Mp group for all regions. These results are also not significantly different versus corresponding samples without methylprednisolone (Figure 2J). N = 3 in Axon miRs + Mp group and N = 5 in Neg miR + Mp group. L–N) Representative fluorescent images of CGRP staining in Axon miRs + Mp‐ and Neg miR + Mp‐treated rats at 4 and 12 weeks post SCI. Quantification analysis of area percent occupied by CGRP+ sensory axons at rostral region, within the scaffold and caudal region. Results suggest that in the presence of methylprednisolone, as compared to Neg miR treatment, Axon miRs enhanced the regeneration of CGRP+ sensory axons within the scaffold at week 4, but had no significant effect at week 12. *p < 0.05, Student's t‐test between Axon miRs + Mp and Neg miR + Mp group for all regions. These results are also not significantly different versus corresponding samples without methylprednisolone (Figure 2L). N = 3 in Axon miRs + Mp group and N = 5 in Neg miR + Mp group. O) BBB scores obtained at bi‐weekly intervals from weeks 1 to 12 indicate that in the presence of methylprednisolone, Axon miR treatment enhanced motor recovery at week 12. *p < 0.05; **p < 0.01, Student's t‐test between Axon miRs + Mp and Neg miR + Mp group for each time point. N = 6 in Axon miRs + Mp group and N = 8 in Neg miR + Mp group. P) Von Frey Hair test, which was indicated by the paw withdrawal threshold as measured at weekly intervals from weeks 1 to 12, indicate that in the presence of methylprednisolone, Axon miRs enhanced rate of sensory function recovery. *p < 0.05, Student's t‐test between Axon miRs and Neg miR group for each time point. N = 6 in Axon miRs + Mp group and N = 8 in Neg miR + Mp group. All data were represented as mean ± SD, except for (O) and (P) which are shown in mean ± SEM.