Methylprednisolone-encapsulating PLGA nanoparticles (MP-NP). A) An SEM image of the lyophilized MP-NPs. Scale bar = 1μm. B) Schematic of topical and local delivery of the MP-NPs onto dorsal over-hemisection lesioned spinal cord (see Methods section). C) MP-NP release profile over 4 days at 37°C in saline. D) Quantitative comparison of amount of released nitrite from reactive microglia when exposed to MP-NPs and Saline-NPs. The co-incubation of MP-NPs with reactive microglia significantly suppresses nitric oxide production by reactive microglia. Error bars represent s.e.m. *P< 0.05.

Acute therapeutic effects of MP-NPs on hemisectioned spinal cord, 24 hours after treatment. A, B) Semi-quantitative immunofluorescence analysis used for the semi-quantitative analysis of the lesioned spinal cord reaction (see Methods section). (A) shows representative longitudinally sectioned and Calpain-immunostained spinal cord tissue. The immunofluorescence images (i.e., white boxes) were taken from around the incision site, indicated by a white arrow. (B) shows one of images from (A). For each image, forty intensity line profiles were generated and averaged to create an average intensity profile (inset). The average intensity profile was then averaged again to generate a representative quantitative intensity per image. C, D) Comparison of GFAP, a marker for astrocytes, within an immunostained spinal cord tissue between 24 hours (C) and 2 weeks (D) after injury. These two representative figures were from the Saline-NP-treated animals. The arrow indicates the incision site. E) shows the results of Western blot analysis for Calpain, iNOS, and Bax between experimental groups. Abbreviation: Single=Single-MP and Saline=Saline-NP. F–J) show representative (longitudinally sectioned) Calpain immunostained spinal cord tissue at 24 hours after injury: (F) MP-NP, (G) MP-sys, (H) Single-MP, and (I) Saline-NP-treated animal. White arrows indicate incision sites. (J) shows a quantitative comparison of the intensity of Calpain at 24 hours after injury. K–O) show representative iNOS immunostained spinal cord tissue at 24 hours after injury: (K) MP-NP, (L) MP-sys, (M) Single-MP, and (N) Saline-NP. (O) shows a quantitative comparison of the intensity of iNOS at 24 hours after injury. P–T) show representative Bcl-2 immunostained spinal cord tissue at 24 hours after injury: (P) MP-NP, (Q) MP-sys, (R) Single-MP, and (S) Saline-NP. (T) shows a quantitative comparison of the intensity of Bcl-2 at 24 hours after injury. Inset figures in (P) are double immunostained for Bcl-2 (red) and for NeuN (green), showing the highly increased expression of the Bcl-2 around Neurons. U–Y) show representative Bax immunostained spinal cord tissue at 24 hours after injury: (U) MP-NP, (V) MP-sys, (W) Single-MP, and (X) Saline-NP. (Y) shows a quantitative comparison of the intensity of Bax at 24 hours after injury. Error bars represent s.e.m. ^#P<0.05 between normal and others. *P<0.05 between MP-NP and others. ^%P<0.05 between MP-sys and others (i.e., Single-MP and Saline-NP).

Significantly reduced lesion volume from MP-NP local treatment. A–D) show GFAP (a marker for astrocytes, red) and NF160 (a marker for axons, green) double immunostained (longitudinally sectioned) spinal cord tissue at the lesion site 2 weeks after injury: (A) MP-NP, (B) MP-sys, (C) Saline-NP, and (D) Single-NP-treated animals. Scale bar = 500μm. White line in (B) shows the lesion area. E–G) show GFAP and NF160 double immunostained spinal cord tissue at the lesion site 4 weeks after injury: (E) MP-NP, (F) MP-sys, and (G) Saline-NP-treated animals. Numbered images (1–5) are magnified and from (E–G). The arrow in (E) indicates NF160⁺ axons observed at the dorsal surface of the lesion site in MP-NP-treated animals. The arrow in (1) indicates the NF160⁺ axons observed at the dorsal rostral to the lesion site. Image (4) shows the fragmented NF160⁺ axons in the dorsal rostral to the lesion site (Saline-NP-treated animal, G). The arrow in (E) indicates NF160⁺ axons observed at the dorsal surface of the lesion site in MP-NP-treated animals. H) shows a quantitative comparison of the lesion volume (delineated by GFAP⁺ astroglial scar, see Methods section) at two different timepoints: 2 weeks and 4 weeks after injury. Compared to Saline-NP-treated animals, the lesion volume is significantly reduced at both 2 weeks and 4 weeks after injury by MP-NP local treatment, but not by either a systemically delivered high dose of MP (MP-sys) or a single local injection of unencapsulated MP (Single-MP). Error bars represent s.e.m. *P< 0.05 between MP-NP and others. Abbreviation: r = rostral and c = caudal stump.

Semi-quantification of injury-related cellular markers. A and D) show a quantitative comparison of the averaged intensity of GFAP immunoreactivity between experimental groups shown as the average intensity (thick line) ± s.e.m. (thin line) at 2 weeks (A) and 4 weeks (D) after injury. The Y-axis represents relative GFAP intensity and the X-axis represents a distance from interface between GFAP⁺ astroglial scar and lesion core (indicated by the white box in (Fig. 4B) and the arrow in (Fig. 4C)). Background subtraction using intact GFAP immunoreactivity was performed. The statistical average intensity difference (summation of the area under the curve) was determined for three different distances: 0–100μm, 100–300μm, and 300–500μm. B and E) show a quantitative comparison of the averaged intensity of CSPG immunoreactivity between experimental groups at 2 weeks (B) and 4 weeks (E) after injury. Background subtraction using secondary antibody controls was performed. C and F) show a quantitative comparison of the averaged ED-1⁺ cells/mm² between experimental groups at 2 weeks (C) and 4 weeks after injury (F). *P<0.05 between MP-NP and others. ^%P<0.05 between MP-sys and Saline-NP.

Local MP-NP treatment significantly reduces injury-related cellular markers. A–C) show representative GFAP immunostained (longitudinally sectioned) spinal cord tissue: (A) MP-NP, (B) MP-sys, and (C) Saline-NP-treated animals. Scale bar = 500μm. D–F) show representative CSPG immunostained spinal cord tissue: (D) MP-NP, (E) MP-sys, and (F) Saline-NP-treated animals. G–I) show representative ED-1 (a marker for macrophages/reactive microglia, green) and GFAP (red) double immunostained spinal cord tissue: (G) MP-NP, (H) MP-sys, and (I) Saline-NP-treated animals.

Behavioral assessment. A and B) show a quantitative comparison of two different behavioral assessments between treatment groups at three different timepoints: 1, 2, and 4 weeks after injury: Grid walking (A) and Beam walking (B). Error bars represent s.e.m. ^#P<0.05 between normal and others. *P<0.05 between MP-NP and others. ^%P<0.05 between MP-sys and Saline-NP.