Neural crest stem cells protect spinal cord neurons from excitotoxic damage and inhibit glial activation by secretion of brain-derived neurotrophic factor

Abstract

The acute phase of spinal cord injury is characterized by excitotoxic and inflammatory events that mediate extensive neuronal loss in the gray matter. Neural crest stem cells (NCSCs) can exert neuroprotective and anti-inflammatory effects that may be mediated by soluble factors. We therefore hypothesize that transplantation of NCSCs to acutely injured spinal cord slice cultures (SCSCs) can prevent neuronal loss after excitotoxic injury. NCSCs were applied onto SCSCs previously subjected to N-methyl-D-aspartate (NMDA)-induced injury. Immunohistochemistry and TUNEL staining were used to quantitatively study cell populations and apoptosis. Concentrations of neurotrophic factors were measured by ELISA. Migration and differentiation properties of NCSCs on SCSCs, laminin, or hyaluronic acid hydrogel were separately studied. NCSCs counteracted the loss of NeuN-positive neurons that was otherwise observed after NMDA-induced excitotoxicity, partly by inhibiting neuronal apoptosis. They also reduced activation of both microglial cells and astrocytes. The concentration of brain-derived neurotrophic factor (BDNF) was increased in supernatants from SCSCs cultured with NCSCs compared to SCSCs alone and BDNF alone mimicked the effects of NCSC application on SCSCs. NCSCs migrated superficially across the surface of SCSCs and showed no signs of neuronal or glial differentiation but preserved their expression of SOX2 and Krox20. In conclusion, NCSCs exert neuroprotective, anti-apoptotic and glia-inhibitory effects on excitotoxically injured spinal cord tissue, some of these effects mediated by secretion of BDNF. However, the investigated NCSCs seem not to undergo neuronal or glial differentiation in the short term since markers indicative of an undifferentiated state were expressed during the entire observation period.

Keywords: Apoptosis; Excitotoxicity; Neuroprotection; Secretion of soluble factors; Suppressed glial activation.

Fig. 1

a–f Micrographs obtained through the ventral horn of SCSCs (a no-NMDA, b NMDA, c NMDA + NCSC, d NMDA + IL1RA) 10 days after the onset of the experiments using confocal microscopy after staining against NeuN. The diagram at the bottom (f) shows the timeline of the experiment. Graph (e) shows the number of counted neurons within the ventral horn in two different timepoints (bars denote means and spreads denote SEM)

Fig. 2

a–e Micrographs of sections through the gray matter of SCSCs (a no-NMDA, b NMDA, c NMDA + NCSC, d NMDA + IL1RA) 6 days post-NCSC application stained for NeuN (red), TUNEL (green) and DAPI (blue). The inner parts of the sections were devoid of e-GFP-positive NCSCs since the latter remained on the surface of the slice cultures. The above sections were obtained from the inner parts of the cultures, an area that NCSCs did not reach. Apoptotic cells were identified by co-localization of TUNEL and DAPI (arrows) and apoptotic neurons were identified as co-localization of TUNEL, DAPI and NeuN (arrowheads, shown also in higher magnification in the right top corner in c). The proportion of apoptotic cells in relation to the number of neurons was significantly higher in the NMDA group (b) compared to the other groups. Graph (e) shows the apoptotic index of cells in different groups (statistical analysis was performed by non-parametric Kruskal–Wallis followed by Mann–Whitney U tests; solid bars indicate means and error bars denote SEM, p ≤ 0.05)

Fig. 3

The graph shows the number of activated microglial cells within the white matter of SCSCs in two different timepoints (statistical analysis performed by ANOVA with planned contrasts; solid bars indicate means and error bars denote SEM, p ≤ 0.05)

Fig. 4

a–e Micrographs obtained through the gray matter of SCSCs (a no-NMDA, b NMDA, c NMDA + NCSC, d NMDA + IL1RA) 10 days after the onset of the experiments using confocal microscopy after staining against GFAP. In order to examine the effect of NCSCs on astroglial activation, non-ramified GFAP-positive astrocytes were counted within the gray matter. Arrows denote non-ramified activated astrocytes, while arrowheads denote astrocytes in a resting state. Graph (e) shows the number of non-ramified GFAP positive astrocytes within the gray matter of SCSCs in two different timepoints (statistical analysis was performed by ANOVA followed by Bonferroni correction; solid bars indicate means and error bars denote SEM, p ≤ 0.05)

Fig. 5

The graph shows the concentrations of BDNF and NGF in supernatants from co-cultures (SCSCs and NCSCs) and SCSCs alone after a 24-h incubation. The concentration of BDNF was significantly higher in the SCSC–NCSC group compared to SCSC alone (statistical analysis was performed by ANOVA; solid bars indicate means and error bars denote SEM, p ≤ 0.05)

Fig. 6

a–j Images of SCSCs maintained for 6 days in vitro after culture preparation and immediate application of NCSCs. Images of unsectioned cultures and neurospheres after light and confocal microscopy (a–c): black arrowheads in image of light microscopy (a) show neurospheres of NCSCs with no signs of migration. Confocal laser scanning images (b) and (c) show NCSCs marked with eGFP (green) and NeuN (DyLight-549, red) after immunohistochemistry. NCSCs migrated on top of SCSCs (c) but remained in spheres when there was no contact with the cultures (b). Images of longitudinal sections through SCSCs and NCSCs (e, f, g, h). Diagram (d) shows how the cultures were sectioned longitudinally. NCSCs were applied on SCSCs directly after culture preparation in order to avoid scar formation that occurs on top of the cultures and maintained for 6 days in vitro. Images (g) and (h) represent parts of (e) and (f) in a higher magnification indicated by dashed lines. The majority of NCSCs migrated on the surface of the culture and some NCSC appeared to migrate through the culture (white arrows). The spheres stained against GFAP showed signs of GFAP immunoreactivity in the edges (white arrowheads in h) but no signs of co-localization between GFAP and eGFP autofluorescence were observed, suggesting astrocytic migration from SCSCs through the neurospheres. Staining of longitudinal sections against SOX2 and Krox20 (i, j). Few cells showed colocalization with SOX2 (small arrows in i) and Krox20 (large arrows in j), markers of undifferentiated neural crest stem cells. The small boxes in the upper right-hand corner of (i) and (j) represent areas of co-localization between eGFP and SOX2 or Krox20, respectively, in higher magnification

Fig. 7

a, b Overview images of neurospheres maintained on laminin-coated coverslips (a) and Healon 5® (b) after 3 div in the presence of NCSC-differentiation medium. NCSCs from all the neurospheres maintained on standard laminin-coated coverslips migrated over the area of the coverslip after 3 div in contrast to neurospheres maintained on Healon 5®. Migration of NCSCs from neurospheres maintained on Healon 5® was observed in only 12% of neurospheres after 3 div and in 25% of neurospheres after 6 div