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	Figure 1. Therapeutic effects of ASC secretome on Xenopus laevis tadpoles after complete transection on swimming recovery, axonal growth and regeneration, in the refractory stage of development. (a and b) Swimming pattern and quantification of the distance traveled by the refractory animals after SCI, at 2, 3, and 5 days after treatment with ASC secretome (CM group) or neurobasal medium (NB). Tadpoles with no SCI and treated with neurobasal medium (SH group) were used as controls for healthy animals. ASC secretome promoted significant functional recovery (868.861 ± 223.901 mm) of Xenopus laevis tadpoles after SCI from the refractory period, 5 days post-treatment, compared to neurobasal-treated animals (NB group; 283.015 ± 88.471 mm). (c) Representative confocal images of longitudinal cross sections of Xenopus laevis spinal cord after immunostaining for βIII-tubulin (axonal sprouting) and GAP-43 (axonal regeneration), at the refractory stage. Quantification of the percentage of (d) βIII-tubulin and (e) GAP-43 immunoreactivity in the spinal cord of Xenopus laevis tadpoles, at the refractory stage. At this stage, the ASC secretome group (CM) showed a clear gap closure and the formation of a robust axonal bridge at the lesion core (LC), between the two stumps of the spinal cord, 5 days post-treatment. This was confirmed by elevated, but not statistically significant, expression of βIII-tubulin in CM group (81.171 ± 1.679) when compared to NB group (54.529 ± 6.015). GAP-43 positive regenerating cells were present in the spinal cord tissue of these animals, with significant differences observed at 2 days post-treatment (CM group: 10.879 ± 1.071 vs NB group: 0.219 ± 0.037). Mean ± SEM; n = 12 for locomotor assessment; n = 5 for histological evaluation *p < 0.05; **p < 0.01; ***p < 0.001.
	Figure 2. Recovery of motor and sensorial function of mice with complete spinal cord transection after ASC secretome treatment. (a) BMS test was performed up to 6 weeks after treatment. ASC secretome treatment (CM) significantly improved the locomotor function of the transected animals, when compared to NB treatment CM-group at 6 weeks (3.778 ± 0619) versus NB-group (0.357 ± 0.210). Animals with no SCI treated with NB medium (SH group) showed completely normal locomotor performance. (b) Von-Frey Trial was performed at 2 and 6-week after ASC secretome or NB treatment as measure of sensitivity regain. Although no statistical differences were found between groups at both time-points, CM group show a trend of recovery of the sensorial function (0.237 ± 0.351) early at 2 weeks post-injury, when compared to NB group (0.022 ± 0.025). Data is presented as Mean ± SEM; n = 8 (SH), n = 7 (NB), n = 9 (CM); **p < 0.01; ***p < 0.001; ****p < 0.0001.
	Figure 3. Therapeutic effects of ASC secretome in mice spinal cord 6 weeks after complete transection on neuroinflammation and white matter injury. (a–d) Representative confocal images of longitudinal cross sections of mice spinal cord after immunostaining for Iba-1 ((a), neuroinflammation), fluoromyelin (b), white matter injury. Quantification of the (c) percentage of Iba-1 reactivity, (d) area of injured white matter. ASC secretome group (CM) presented statistically significant effect in the modulation of microglial response, decreasing the area of inflammatory microglial cells (21.124 ± 2.060) while increasing the area of homeostatic ones (96.396 ± 7.370) when compare to NB group (39.554 ± 4.536) and (61.105 ± 5.944), respectively. Similar effects were produced toward the area of injured white matter as CM-group had lower (23.933 ± 2.764), in comparison to the (NB) (39.554 ± 4.536) white matter degeneration. Dashed Lines denotes area coverage of ameboid microglia in (a) and epicenter regions in (b). Data is presented as Mean ± SEM; n = 5; *p < 0.05; **p < 0.01; ***p < 0.001.
	Figure 4. Therapeutic effects of ASC secretome in mice spinal cord 6 weeks after complete transection on axonal growth and regeneration. (a–e) Representative confocal images of longitudinal cross sections of mice spinal cord after immunostaining for ßIII-tubulin (axonal growth) and GAP-43 (axonal regeneration). Axonal outgrowth at the lesion site, as measured by a significant increase of ßIII-tubulin for ASC secretome group (69.106 ± 19.013), in comparison to NB group (22.677 ± 5.535) (e). Regeneration fibers were also clearly observed in CM group, as denoted by a significant increase GAP-43 expression at the lesion site (65.695 ± 16.990), when compared to NB group (23.193 ± 4.423) (f). Data is presented as Mean ± SEM; n = 5; ****p < 0.0001. White arrows point to ßIII-tubulin and GAP-43 immuno reactive fibers in (b and d) single channel images respectively. Scale bars are 200 and 50 µm in (a, c) and (b, d) respectively.
	Figure 5. Concentration of cytokines in the blood serum of mice at 6 weeks post-injury, and their interaction with other molecules present in ASC secretome. (a) The concentration (pg/ml) of pro-inflammatory—IL-1β, IL-6, IFN-γ and anti-inflammatory—IL-4 cytokines in the blood serum of SCI mice was assessed using multiplex-based ELISA. Secretome group (CM) showed decreased levels of pro-inflammatory cytokines (IL1-β: 6.463 ± 2.383; IL-6: 2.286 ± 1.460; IFN-γ: 1.573 ± 0.704), and increased levels of the anti-inflammatory cytokine (IL-4: 0.696 ± 0.247), when compared to NB- (IL-4: 0.390 ± 0.256; IL1-β: 10.864 ± 1.026; IL-6: 84.702 ± 38.767; IFN-γ: 9.976 ± 3.174) and SH- (IL-4: 0.606 ± 0.363; IL1-β: 4.232 ± 1.106; IL-6: 1.143 ± 1.143; IFN-γ: 2.871 ± 1.521) treated groups. (b) Representative networks of molecules present in ASC secretome, and respective receptors and signaling molecules, based on their role on inflammation and immune response (in green), and neurogenesis and axonogenesis (in blue), identified using STRINGS bioinformatics tools. in Mean ± SEM; n = 7 for SH and CM groups; n = 4 for NB group; *p < 0.05
	Figure 6. Schematics of the experimental paradigm employed for the Xenopus laevis model of transection SCI.
	Figure 7. Schematics of the experimental paradigm employed for the mouse model of transection SCI.5.5. Secretome injection in the Xenopus tadpole’s spinal cord after injury.

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