EPO-releasing neural precursor cells promote axonal regeneration and recovery of function in spinal cord traumatic injury

Abstract

Background: Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells.

Objective: The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury.

Methods and results: We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals.

Conclusion: Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI.

Keywords: Spinal cord injury; animal behavior; neural stem cells; regenerative medicine; transplantation.

Fig.1

Er-NPCs transplanted cells differentiate into cholinergic neurons. At 30 days after i.v. injection, several PKH26-labeled Er-NPCs (red) were accumulated at the edges of the lesion. Most Er-NPCs were positive for ChAT immunostaining (green; stars) (Scale bar = 50 μm).

Fig.2

Er-NPCs transplantation improves neural markers expression in injured spinal cord. Pictures represent confocal acquisitions of coronal sections taken at the edges of the lesion at 30 days after i.v. administration of PKH26 – labeled Er-NPCs (showed in red) (panel A). Sections were immunostained for β tubulin III and MAP-2 (showed in green). PKH26-labeled cells were positive for β-tubulin III and MAP-2 (white stars) (Scale bar = 75 and 100 μm) (panel A). Graphs reported in panel B show the quantification of immunoreactivity in sections taken at the lesion epicenter, 2 mm rostral or caudal to the lesion epicenter (please see schematic representation). Values represent average±SD. We determined the statistical differences by means of ANOVA test followed by Bonferroni’s post-test. ***p < 0.001 vs LAM; ^$$p < 0.01 vs PBS.

Fig.3

Mice locomotor activity evaluation after i.v. Er-NPCs infusion. The evaluation of motor function recovery of hind limb was determined by the open field locomotion test (Basso et al., 2006). During the observation, performed in double blind fashion, animals from the different groups were randomized. Values represent average±SD. We determined the statistical differences by means of two way ANOVA test followed by Bonferroni’s post-test.

Fig.4

Er-NPCs promote serotoninergic fibres sprouting through the lesion site. Serotoninergic (5-HT) fibers were investigated at the lesion site (panel A) and 2 mm away from the lesion (panel B) 4 weeks after Er-NPCs infusion in lesioned mice. In panel A, Er-NPCs are shown in red (PKH26) and 5-HT is shown in green. Nuclei were counterstained with DAPI (blue). In panel B, 5-HT staining is showed in red and neuronal fibers were identified with beta-tubulin III (green). Er-NPCs were labelled with Hoechst 33342 before the infusion (blue). Scale bars = 50 and 100 μm. Quantification was performed 2 mm caudally to the lesion epicenter at 10 and 30 days after Er-NPCs transplantation in lesioned animals (panel C). The quantification was performed in spinal cord coronal sections (n = 3 for each animal; at least 6 animal per group) as described in detail in M&M in intermediolateral and ventral horns (please see the representation). Values represent average±SD. We determined the statistical differences by means of an ANOVA test followed by Bonferroni’s post-test. ***p < 0.001 vs LAM; °p < 0.05 vs LES+PBS.

Fig.5

Er-NPCs promote chatecolaminergic fibres sprouting through the lesion site. Chatecolaminergic (TH) fibers were investigated at the lesion site (panel A) and 2 mm caudal from the lesion (panel B) 4 weeks after Er-NPCs infusion in lesioned mice. In panel A, Er-NPCs are shown in red and TH is shown in green. Nuclei were counterstained with DAPI (blue). In panel B, chatecolaminergic terminals are shown in red and neuronal fibers were identified with beta-tubulin III (green). Er-NPCs were labelled with Hoechst 33342 before the infusion (blue). Scale bars = 25 μm. Quantification of TH fibers quantification was performed 2 mm caudally to the lesion epicenter at 10 and 30 days after Er-NPCs transplantation in lesioned animals. The quantification was performed in spinal cord coronal sections (n = 3 for each animal; at least 6 animals per group) as described in detail in M&M in intermediolateral and ventral horns (please see the representation). Values represent average±SD. We determined the statistical differences by means of an ANOVA test followed by Bonferroni’s post-test. ***p < 0.001 vs LAM; °°p < 0.01 vs LES+PBS.

Fig.6

Myelin preservation in the injured cord of animals treated with Er-NPCs. Myelin preservation was evaluated by means of Fluoromyelin™ staining (green) performed in sections at the lesion epicenter, and 2 mm caudally to the lesion site (please see schematic representation).

Fig.7

GAP43 expression in Er-NPCS transplanted cord. Qualitative picture of GAP43 expression investigated 2 mm away (rostral and caudal) from the cord lesion site of lesioned animals transplanted with Er-NPCs and PBS. GAP 43 is showed in red and neuronal fibres were detected with beta-tubulin III staining (green). In order to perform double staining in these experiments Er-NPCs were labelled with Hoechst 33342 (blue). Scale bars = 75 μm. Pictures are representative of at least three different immunostaining experiments. The fluorescence intensity of GAP 43 is showed in the graph (below) and was performed in three animals per group. Statistical significance was determined by ANOVA test followed by Bonferroni’s post-test. ***p < 0.001 vs Les+Er-NPCs; °°°p < 0.001 vs Les+PBS rostral; ^$$$p < 0.001 vs Les+PBS caudal.

Fig.8

In vivo axonal transport recovery in spinal cord of animal transplanted or not with Er-NPCs. Panel A. Qualitative image of anterograde axonal transport at 25 days after lesion in PM-NPC treated animals. As described in material and methods section fluororuby was injected at T6/T7 at day 20 after lesioning and animal sacrificed five days later. Schematic reconstruction of spinal cord longitudinal sections of lesioned (below) and lesioned+Er-NPCs (above) treated animals. Er-NPCs were stained with Hoechst (blue). Panel B. Quantification of fluorescence 2 cm, 1 cm and 1 mm away from the lesion. Sections were taken from animals transplanted or not with Er-NPCs. Quantification was performed in three animals per group 25 days after lesion. We determined the statistical differences by means of an ANOVA test followed by Bonferroni’s post-test. **p < 0.01, ***p < 0.001 vs saline treatment; °°p < 0.01 vs 2 cm transplanted mice; ^{# # #}p < 0.001 vs 1 cm transplanted mice.

Fig.9

Detail of anterograde axonal labelling relative to Er-NPCs transplanted animals (at 30 days). The reconstruction is referring to longitudinal sections of lesion site epicenter (T9) and 1 mm away from the lesion. Scale bars = 100 μm.