Cellular meningeal inflammation. (A) Images were taken from sagittal midline spinal cord sections and are oriented with the dorsal aspect of the cord at the top, and the rostral end of the epicenter to the left. (B) At 2 days post-PTS (2d-PTS) injury there was evidence of both microglia/macrophages (left panel; Iba-1), and neutrophils (right panel; PMN) in the meningeal layers surrounding the kaolin. (C) At 7 days post-injury (7d-PTS) some neutrophils (PMN) remained around the injury epicenter, whereas there was little evidence of these cells surrounding the kaolin directly. (D) Microglia/macrophages (Iba-1) completely surrounded the kaolin, and were even found in the meninges at very remote sites from the injury epicenter (right panel; scale bar=1 mm in low-power images and=100 μm in high-power images; PTS, post-traumatic syringomyelia; PMN, polymorphonuclear leukocyte; GFAP, glial fibrillary acidic protein). Color image is available online at www.liebertonline.com/neu

Spatial cytokine and chemokine expression. The dorsal meninges were removed from the parenchyma and each fraction was analyzed for cytokine/chemokine expression with multiplex ELISA. A schematic of the sectioning is shown at center. Non-injured sham animals are represented with hatched lines on the graphs. (A) Meningeal cytokines/chemokines. All cytokines and chemokines in the dorsal spinal cord meningeal fractions of the arachnoiditis group were significantly increased relative to sham animals at 1 day post-injury. There were significant increases in meningeal IL-6, MCP-1, and GRO/KC at 1 day in SCI animals compared to sham animals (p<0.05). Relative to sham animals, PTS animals had significant increases in all of the meningeal cytokines and chemokines at 1 day post-injury (p<0.05). Relative to the SCI group, the PTS group had a significant increase in all inflammatory mediators at 1 day post-injury (p<0.05). Relative to the arachnoiditis group, the PTS group had a significant increase only in MCP-1 at 1 day post-injury (p<0.05). (B) Parenchymal cytokines/chemokines. The spinal cord fractions free of dorsal meninges from arachnoiditis animals contained significantly more MIP-1α and GRO/KC at 1 day post-injury compared with sham animals. SCI significantly increased IL-6, MCP-1, MIP-1α, and GRO/KC at 1 day post-injury, and IL-1α, MCP-1, MIP-1α, and GRO/KC at 3 days post-injury, compared to non-injured sham animals. Similarly, PTS animals contained significantly higher parenchymal cytokine and chemokine levels at 1 day and 3 days post-injury compared to shams. Relative to SCI alone, PTS significantly increased IL-1α and IL-1β at 1 day post-injury, and significantly increased each chemokine at 3 days post-injury. Relative to arachnoiditis alone, PTS animals had a significant increase in all inflammatory mediators reported at 1 and 3 days post-injury (n=3 per group; p<0.05 by two-way ANOVA for each molecule; *p<0.05 by Bonferroni post-hoc testing; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; MCP-1, monocyte chemotactic protein-1; MIP-1α, macrophage inflammatory protein-1α; IL-6, interleukin-6; SCI, spinal cord injury; IL-1α, interleukin-1α; IL-1β, interleukin-1β). Color image is available online at www.liebertonline.com/neu

Neutrophil extravasation. (A) Neutrophil extravasation was determined by assessing MPO activity in granular fractions at 1 and 3 days post-injury (measured in units per gram of tissue). There was a significant increase in MPO activity in SCI and PTS animals compared to sham animals and arachnoiditis animals at 1 and 3 days post-injury. At 3 days following injury, there was a significant increase in MPO activity in PTS animals compared to SCI animals (n=3 per group; p<0.05 by two-way ANOVA and Bonferroni post-hoc). (B) Longitudinal immunohistochemical images taken at 2 days post-injury show neutrophils stained with a PMN antibody (green) in arachnoiditis, SCI, and PTS animals. The images were taken from sagittal midline spinal cord sections, and are oriented with the dorsal aspect of the cord at the top and the rostral side of the epicenter to the left. Note the increased number of labeled cells in PTS animals (scale bars=1 mm in low-power images and=100 μm in high-power images; PTS, post-traumatic syringomyelia; PMN, polymorphonuclear leukocyte; MPO, myeloperoxidase; ANOVA, analysis of variance; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury; DAPI, 4,6-diamino-2-phenylindole). Color image is available online at www.liebertonline.com/neu

Blood–spinal cord barrier permeability. (A) Evans blue (EB) extravasation into the spinal cord was determined in injured animals. Representative EB (red) fluorescence images from arachnoiditis, SCI, and PTS animals 2 days following injury show a larger area of extravasation in PTS animals. Note the intense fluorescence in the meninges of PTS animals. The slides were counterstained with DAPI (blue). (B) EB extravasation was measured in homogenized tissue by absorbance spectrophotometry. There was a significant increase in the amount of EB in SCI and PTS animals compared to sham and arachnoiditis animals at 1 day post-injury. PTS animals contained significantly more EB relative to SCI animals at 3 days post-injury (n=3 per group; p<0.05 by two-way ANOVA and Bonferroni post-hoc test). (C) PTS animals exhibited an increase in matrix metalloproteinase-9 (MMP-9) activity in both parenchymal and meningeal fractions at 2 days post-injury. (D) In situ zymography was used to determine spatial MMP-9 activity at 2 days following injury. Note the increase in fluorescence in the parenchyma and meninges in PTS sections compared to SCI and arachnoiditis animals. Images were taken from sagittal midline spinal cord sections, and are oriented with the dorsal aspect of the cord at the top and the rostral side of the epicenter to the left (scale bar=1 mm; PTS, post-traumatic syringomyelia; ANOVA, analysis of variance; SCI, spinal cord injury; DAPI, 4,6-diamino-2-phenylindole). Color image is available online at www.liebertonline.com/neu

Fibrous scarring. Shown are longitudinal fluorescence immunohistochemical images from SCI and PTS animals at 7 days post-injury labeled for collagen IV (red, Col IV) and CSPG (green). Note the extensive scarring in the meninges caudal to and surrounding the kaolin. Additionally, this image shows an increase in parenchymal CSPG and collagen IV immunoreactivity (scale bar=1 mm; PTS, post-traumatic syringomyelia; SCI, spinal cord injury; CSPG, chondroitin sulfate proteoglycan). Color image is available online at www.liebertonline.com/neu