Self-assembling peptide gels promote angiogenesis and functional recovery after spinal cord injury in rats

Abstract

Spinal cord injury (SCI) leads to disruption of the blood-spinal cord barrier, hemorrhage, and tissue edema, which impair blood circulation and induce ischemia. Angiogenesis after SCI is an important step in the repair of damaged tissues, and the extent of angiogenesis strongly correlates with the neural regeneration. Various biomaterials have been developed to promote angiogenesis signaling pathways, and angiogenic self-assembling peptides are useful for producing diverse supramolecular structures with tunable functionality. RADA16 (Ac-RARADADARARADADA-NH2), which forms nanofiber networks under physiological conditions, is a self-assembling peptide that can provide mechanical support for tissue regeneration and reportedly has diverse roles in wound healing. In this study, we applied an injectable form of RADA16 with or without the neuropeptide substance P to the contused spinal cords of rats and examined angiogenesis within the damaged spinal cord and subsequent functional improvement. Histological and immunohistochemical analyses revealed that the inflammatory cell population in the lesion cavity was decreased, the vessel number and density around the damaged spinal cord were increased, and the levels of neurofilaments within the lesion cavity were increased in SCI rats that received RADA16 and RADA16 with substance P (rats in the RADA16/SP group). Moreover, real-time PCR analysis of damaged spinal cord tissues showed that IL-10 expression was increased and that locomotor function (as assessed by the Basso, Beattie, and Bresnahan (BBB) scale and the horizontal ladder test) was significantly improved in the RADA16/SP group compared to the control group. Our findings indicate that RADA16 modified with substance P effectively stimulates angiogenesis within the damaged spinal cord and is a candidate agent for promoting functional recovery post-SCI.

Keywords: Spinal cord injury; angiogenesis; functional recovery; self-assembling peptide; substance P.

Figure 1.

Structural and rheological characterization of bioactive peptide gels. Optical examination of the peptides RADA16 (R), RADA16-SP (R-SP), and RADA + RADA16-SP (R + R-SP) on the horizontal plane (a) and a 45° inclined plane (b). EM images of the peptides RADA16 (c), RADA16-SP (d), and RADA16/SP (RADA16 + RADA16-SP) (e). White scale bar = 100 μm. (f) CD of RADA16 (blue), RADA16-SP (black), and RADA16/SP (red). (g) Storage modulus and (h) complex viscosity of RADA16 (blue), RADA16-SP (black), and RADA16/SP (red) before and after gelation. * p < 0.05, the Mann–Whitney U test.

Figure 2.

Representative images of live and dead staining of cortical neurons in the collagen (a), RADA16 (b) and RADA16/SP (c) groups on day 3 and the ratio of live cells to total stained cells (n = 4 per group) (d). Yellow scale bar = 50 μm. *p < 0.05, one-way ANOVA followed by the Games-Howell post hoc test.

Figure 3.

Representative images of H&E staining (a) and immunohistochemistry (b) for ED1 (green) and GFAP (red) in the injured spinal cord 8 weeks after injection of control, RADA16 or RADA16/SP. For low magnification images, a 10× objective was used; white scale bar, 1 mm. The yellow boxes are magnified on the right side; yellow scale bar, 50 µm. The cavity of the lesion was measured from sagittal H&E staining images (n = 4 per group) (c), and the total number of ED1+ inflammatory cells was counted within the lesion epicenter (1 mm2) (n = 4 per group) (d). The data are expressed as the mean ± SD. *p < 0.05, one-way ANOVA followed by the Games-Howell post hoc test.

Figure 4.

Spinal cord tissues immunostained with CD31 and VEGF antibodies and with DAPI from the control (a and b), RADA16 (c and d), and RADA16/SP (e and f) groups 8 weeks after injection. A 20× objective was used to obtain these images; yellow scale bar, 500 µm. The yellow boxes are magnified on the right side; white scale bar, 50 µm. The mean diameter (g and j), density (h and k), and number per field (i and l) of CD31+ or VEGF+ vessels within the lesion epicenter were quantified in 3 20× fields (n = 4 per group). *p < 0.05, one-way ANOVA followed by the Games-Howell post hoc test.

Figure 5.

Representative NF200- and DAB-stained images of sagittal spinal cord sections from the control (a), RADA16 (b) and RADA16/SP (c) groups 8 weeks after injection within the lesion cavities of contused spinal cords (outlined by black dashed lines). Injected hydrogels are remained within the lesion cavity in RADA16 and RADA16/SP groups (outlined by red dashed lines). A 10× objective was used to obtain these images; black scale bar, 500 µm. The black boxes are magnified on the right side; red scale bar, 50 µm. The mean density (d) and percentage of NF200+ axons within the lesion epicenter (e) were quantified in 3 20× fields (n = 4 per group). *p < 0.05, one-way ANOVA followed by the Games-Howell post hoc test.

Figure 6.

The relative mRNA expression levels of genes associated with inflammation and angiogenesis in the control, RADA16 and RADA16/SP groups 8 weeks after injection (n = 3 per group). (a) TNF-α, (b) IL-6, (c) IL-1β, (d) caspase 3, (e) IL-10, and (f) vWF levels. *p < 0.05, one-way ANOVA followed by the Games-Howell post hoc test.

Figure 7.

The hindlimb movement of the control, RADA16 and RADA16/SP groups was assessed with the BBB scale (a) and horizontal ladder test (b) over an 8-week period postinjury (n = 10 in the control and RADA16 groups, n = 9 in the RADA16/SP group). The data are expressed as the mean ± SD. *p < 0.05 between the control and RADA16/SP groups, two-way ANOVA followed by the Bonferroni post hoc test.