Transplantation of neural precursors generated from spinal progenitor cells reduces inflammation in spinal cord injury via NF-κB pathway inhibition

Abstract

Background: Traumatic spinal cord injury (SCI) triggers a chain of events that is accompanied by an inflammatory reaction leading to necrotic cell death at the core of the injury site, which is restricted by astrogliosis and apoptotic cell death in the surrounding areas. Activation of nuclear factor-κB (NF-κB) signaling pathway has been shown to be associated with inflammatory response induced by SCI. Here, we elucidate the pattern of activation of NF-κB in the pathology of SCI in rats and investigate the effect of transplantation of spinal neural precursors (SPC-01) on its activity and related astrogliosis.

Methods: Using a rat compression model of SCI, we transplanted SPC-01 cells or injected saline into the lesion 7 days after SCI induction. Paraffin-embedded sections were used to assess p65 NF-κB nuclear translocation at days 1, 3, 7, 10, 14, and 28 and to determine levels of glial scaring, white and gray matter preservation, and cavity size at day 28 after SCI. Additionally, levels of p65 phosphorylated at Serine536 were determined 10, 14, and 28 days after SCI as well as levels of locally secreted TNF-α.

Results: We determined a bimodal activation pattern of canonical p65 NF-κB signaling pathway in the pathology of SCI with peaks at 3 and 28 days after injury induction. Transplantation of SCI-01 cells resulted in significant downregulation of TNF-α production at 10 and 14 days after SCI and in strong inhibition of p65 NF-κB activity at 28 days after SCI, mainly in the gray matter. Moreover, reduced formation of glial scar was found in SPC-01-transplanted rats along with enhanced gray matter preservation and reduced cavity size.

Conclusions: The results of this study demonstrate strong immunomodulatory properties of SPC-01 cells based on inhibition of a major signaling pathway. Canonical NF-κB pathway activation underlines much of the immune response after SCI including cytokine, chemokine, and apoptosis-related factor production as well as immune cell activation and infiltration. Reduced inflammation may have led to observed tissue sparing. Additionally, such immune response modulation could have impacted astrocyte activation resulting in a reduced glial scar.

Keywords: Inflammation; NF-κB; Spinal cord injury; Stem cells transplantation; TNF-α; p65.

Fig. 1

Pattern of NF-κB activity in the lesion center and adjacent cranial and caudal areas at 1, 3, 7, 10, 14, and 28 days after SCI, presented as a number of NF-kB p65⁺ nuclei per square millimeter (a). The most pronounced levels of NF-kB p65 nuclear translocation were detected in the lesion centers at day 28 after SCI and were found to be significantly higher than all other time points except for values from day 3 after SCI. In addition, NF-κB-related transcription was significantly higher 3 days after injury when compared with 1-day interval (b). Data are shown as the mean and SEM, *p ≤ 0.05, **p ≤ 0.01

Fig. 2

Immunohistochemical staining with hematoxylin and DAB (NF-kB p65) of the spinal cord from uninjured rats (a; A1, A2), 28 days after injury from rats treated with saline (b; B1, B2), or 28 days from rats transplanted with SPC-01 (c; C1, C2). Black arrows point to the nuclei void of NF-kB p65 expression, red arrows highlight the nuclei with translocated p65, and green arrows indicate cells with nuclei negative to p65 surrounded by p65⁺ cytoplasm (A1, A2, B1, B2, C1, C2). NF-κB nuclear translocation in the cranial and caudal areas did not differ significantly between the treatment groups (d, e). In the lesion center of the spinal cords, a significantly lower NF-κB p65 activity was observed at 28 days in the group treated with SPC-01 cells (f). A comparison of nuclear expression of NF-κB in the entire studied areas (gray and white matter) of injured spinal cords at day 28 after SCI showed lower p65 nuclear translocation in SPC-01-treated rats, which was more prominent in the gray matter of the injured spinal cords (g). Data are shown as the mean and SEM, *p ≤ 0.05, ** p ≤ 0.01. Scale bars, 200 μm

Fig. 3

The ratio of phosphorylated NF-κB p65ser536 to total p65 was measured at 10, 14, and 28 days after SCI. No significant change in the activity between the treatment groups was detected. Data are shown as mean and SEM

Fig. 4

Levels of cytokine (TNF-α) were determined at 10, 14, and 28 days after SCI in animals receiving SPC-01 or saline. Transplantation of SPC-01 cells resulted in marked reduction of TNF-α levels 10 and 14 days after injury when compared with animals injected with saline only. Results are represented as a percentage relative to uninjured controls. Data are shown as mean and SEM, *p ≤ 0.05, ***p ≤ 0.001

Fig. 5

The spinal cord sections of rats at day 28 after SCI treated either with saline or SPC-01 were used to assess the gray (a) and white (b) matter tissue sparing. No local significant differences were found, but paired t test and two-way ANOVA revealed a significant difference in gray matter preservation between the treatment groups with p values of 0.0189 and 0.05, respectively (a). Rats transplanted with SPC-01 cells displayed a significantly smaller cavity size than rats treated with saline (c). Data are shown as mean and SEM, *p ≤ 0.05

Fig. 6

Glial scar distribution was assessed at day 28 after SCI using rat anti-GFAP-Cy3. Reduction in gliosis was observed in the central parts of the spinal cord lesion in rats transplanted with SPC-01 (a). Representative images of GFAP expression in saline-treated (b) or SPC-01 transplanted (c) rats with depicted glial scarring. Paired t test and two-way ANOVA revealed a strong significant difference between the treatment groups with p = 0.0068 and 0.0439, respectively. Data are shown as mean and SEM. Scale bars, 500 μm