Nischarin-siRNA delivered by polyethylenimine-alginate nanoparticles accelerates motor function recovery after spinal cord injury

Abstract

A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2a cells and primary cortical neurons. In recent years, more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury. We proposed that small interfering RNA targeting nischarin (Nis-siRNA) delivered by polyethyleneimine-alginate (PEI-ALG) nanoparticles promoted motor function recovery in rats with spinal cord injury. Direct microinjection of 5 μL PEI-ALG/Nis-siRNA into the spinal cord lesion area of spinal cord injury rats was performed. From day 7 after surgery, Basso, Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-siRNA group compared with the spinal cord injury group and PEI-ALG/Control-siRNA group. On day 21 after injection, hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-siRNA group. Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-siRNA group. These findings suggest that a complex of PEI-ALG nanoparticles and Nis-siRNA effectively suppresses nischarin expression, induces expression of growth-associated protein-43, and accelerates motor function recovery after spinal cord injury.

Keywords: alginate; growth-associated protein-43; motor function; nanoparticles; necrotic area; nerve regeneration; neural regeneration; nischarin; polyethylenimine; small interfering RNA; spinal cord injury.

Figure 1

Nischarin protein expression in the injured spinal cord of rats. (A, B) Western blot assay shows markedly upregulated nischarin protein expression in SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§P < 0.05). However, nischarin expression was significantly decreased in the PEI-ALG/Nis-siRNA group compared with the SCI group (*P < 0.05), SCI + PEI-ALG group (#P < 0.05), and SCI + PEI-ALG/Ctl-siRNA group (†P < 0.05). (C–G) Immunofluorescence staining to examine nischarin expression. (C, D) Immunofluorescence intensity of nischarin in the lesion site center. The dotted white lines indicate the edges of cavities. (E, F) Immunofluorescence intensity of nischarin in the region 1 cm distal to the lesion site. Scale bar: 100 μm. Data are expressed as the mean ± SEM (n = 3, one-way analysis of variance followed by Scheffe's post hoc test). SCI: Spinal cord injury; PEI-ALG: polyethylenimine-alginate; Nis-siRNA: small interfering RNA targeting nischarin; Ctl: control.

Figure 2

BBB score in rats with SCI at different time points. Within 3 weeks after spinal cord hemisection, BBB scores increased gradually in rats from different groups, indicating gradual recovery of hindlimb locomotor function. From 7 days after injury, BBB scores were significantly greater in the PEI-ALG/Nis-siRNA group compared with the SCI group (*P < 0.05, **P < 0.01), SCI + PEI-ALG group (#P < 0.05), and SCI + PEI-ALG/Ctl-siRNA group (†P < 0.05). Data are expressed as the mean ± SEM (n = 6–8, two-way analysis of variance followed by Bonferroni multiple-comparison test). SCI: Spinal cord injury; PEI-ALG: polyethylenimine-alginate; Nis-siRNA: small interfering RNA targeting nischarin; Ctl: control; BBB: Basso, Beattie and Bresnahan locomotor rating.

Figure 3

Hematoxylin-eosin staining of damaged areas in the spinal cord of rats on day 21 after SCI. (A) Representative SCI images in the four groups (scale bar, 100 μm). Necrotic cells (arrowhead) and infiltrated inflammatory cells (arrows) were observed in necrotic areas. (B) Quantification of necrotic areas in the injured spinal cord region. Necrosis areas were significantly reduced in the PEI-ALG/Nis-siRNA group compared with the SCI group (*P < 0.05), SCI + PEI-ALG group (#P < 0.05), and SCI + PEI-ALG/Ctl-siRNA group (†P < 0.05). Data are expressed as the mean ± SEM (n = 5; one-way analysis of variance followed by Scheffe's post hoc test). SCI: Spinal cord injury; PEI-ALG: polyethylenimine-alginate; Nis-siRNA: small interfering RNA targeting nischarin; Ctl: control.

Figure 4

Effect of Nis-siRNA delivered by PEI-ALG nanoparticles on GAP-43 expression in the injured spinal cord of rats on day 21 after SCI. (A) At 21 days after SCI, immunofluorescence staining was performed to examine GAP-43 expression. Scale bars: 100 μm. GAP-43-positive structures (arrows) were observed in the injured spinal cord. (B) Quantification of GAP-43 relative fluorescence intensity. GAP-43 expression was increased in the PEI-ALG/Nis-siRNA group compared with the SCI group (**P < 0.01), SCI+PEI-ALG group (##P < 0.01), and SCI + PEI-ALG/Ctl-siRNA group (††P < 0.01). Data are expressed as the mean ± SEM (n = 3; one-way analysis of variance followed by post hoc test). SCI: Spinal cord injury; PEI-ALG: polyethylenimine-alginate; Nis-siRNA: small interfering RNA targeting nischarin; Ctl: control; GAP-43: growth-associated protein-43.

Figure 5

Effect of Nis-siRNA delivered by PEI-ALG nanoparticles on GAP-43 protein expression in the injured spinal cord of rats on day 21 after SCI. Protein expression of GAP-43 was markedly decreased in the SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§P < 0.05, §§P < 0.01). However, GAP-43 protein expression was increased in the PEI-ALG/Nis-siRNA group compared with the SCI group (**P < 0.01), SCI + PEI-ALG group (##P < 0.01), and SCI + PEI-ALG/Ctl-siRNA group (†P < 0.05). Data are expressed as the mean ± SEM (n = 3, one-way analysis of variance followed by Scheffe's post hoc test). SCI: Spinal cord injury; PEI-ALG: polyethylenimine-alginate; Nis-siRNA: small interfering RNA targeting nischarin; Ctl: control; GAP-43: growth-associated protein-43.