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Delayed inflammatory mRNA and protein expression after spinal cord injury.

Byrnes KR, Washington PM, Knoblach SM, Hoffman E, Faden AI - J Neuroinflammation (2011)

Bottom Line: As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury.Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma.These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Georgetown University Medical Center, NW, Washington, DC (20057), USA. kbyrnes@usuhs.mil

ABSTRACT

Background: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI.

Methods: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression.

Results: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma.

Conclusions: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

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The effect of DPI on inflammatory markers after spinal cord injury. Inflammatory markers were compared in Vehicle (left image) and DPI-treated (right image) spinal cord injured tissue at 28 days post-injury. DPI resulted in significant reductions in p22PHOX (red, A, B), progranulin (red, D, E) and galectin-3 (green, G, H) immunolabeling. DAPI-labeled nuclei are shown for contrast in G, H. Immunolabeling was quantified and graphs are shown (C, F, I). Western blotting for progranulin was performed at 28 days post-injury, and revealed a significant decrease in progranulin protein expression in the DPI treated group (J). Representative samples of the western blot are shown. Size bar = 500 μM. Bars represent mean +/- SEM; *p < 0.05.
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Figure 9: The effect of DPI on inflammatory markers after spinal cord injury. Inflammatory markers were compared in Vehicle (left image) and DPI-treated (right image) spinal cord injured tissue at 28 days post-injury. DPI resulted in significant reductions in p22PHOX (red, A, B), progranulin (red, D, E) and galectin-3 (green, G, H) immunolabeling. DAPI-labeled nuclei are shown for contrast in G, H. Immunolabeling was quantified and graphs are shown (C, F, I). Western blotting for progranulin was performed at 28 days post-injury, and revealed a significant decrease in progranulin protein expression in the DPI treated group (J). Representative samples of the western blot are shown. Size bar = 500 μM. Bars represent mean +/- SEM; *p < 0.05.

Mentions: Tissue was then assessed for multiple markers of chronic inflammation 28 days after injury. Immunohistochemistry was performed for the NADPH oxidase component p22PHOX and the chronically expressed inflammatory proteins galectin-3 and progranulin. Immunolabeling for p22PHOX, progranulin and galectin-3 was significantly reduced by DPI treatment (Figure 9). Western blotting further confirmed the reduction of chronically expressed progranulin after DPI treatment, with a significant reduction in expression at 28 days after injury in the treated group in contrast to the vehicle group (Figure 9J).


Delayed inflammatory mRNA and protein expression after spinal cord injury.

Byrnes KR, Washington PM, Knoblach SM, Hoffman E, Faden AI - J Neuroinflammation (2011)

The effect of DPI on inflammatory markers after spinal cord injury. Inflammatory markers were compared in Vehicle (left image) and DPI-treated (right image) spinal cord injured tissue at 28 days post-injury. DPI resulted in significant reductions in p22PHOX (red, A, B), progranulin (red, D, E) and galectin-3 (green, G, H) immunolabeling. DAPI-labeled nuclei are shown for contrast in G, H. Immunolabeling was quantified and graphs are shown (C, F, I). Western blotting for progranulin was performed at 28 days post-injury, and revealed a significant decrease in progranulin protein expression in the DPI treated group (J). Representative samples of the western blot are shown. Size bar = 500 μM. Bars represent mean +/- SEM; *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198932&req=5

Figure 9: The effect of DPI on inflammatory markers after spinal cord injury. Inflammatory markers were compared in Vehicle (left image) and DPI-treated (right image) spinal cord injured tissue at 28 days post-injury. DPI resulted in significant reductions in p22PHOX (red, A, B), progranulin (red, D, E) and galectin-3 (green, G, H) immunolabeling. DAPI-labeled nuclei are shown for contrast in G, H. Immunolabeling was quantified and graphs are shown (C, F, I). Western blotting for progranulin was performed at 28 days post-injury, and revealed a significant decrease in progranulin protein expression in the DPI treated group (J). Representative samples of the western blot are shown. Size bar = 500 μM. Bars represent mean +/- SEM; *p < 0.05.
Mentions: Tissue was then assessed for multiple markers of chronic inflammation 28 days after injury. Immunohistochemistry was performed for the NADPH oxidase component p22PHOX and the chronically expressed inflammatory proteins galectin-3 and progranulin. Immunolabeling for p22PHOX, progranulin and galectin-3 was significantly reduced by DPI treatment (Figure 9). Western blotting further confirmed the reduction of chronically expressed progranulin after DPI treatment, with a significant reduction in expression at 28 days after injury in the treated group in contrast to the vehicle group (Figure 9J).

Bottom Line: As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury.Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma.These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Georgetown University Medical Center, NW, Washington, DC (20057), USA. kbyrnes@usuhs.mil

ABSTRACT

Background: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI.

Methods: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression.

Results: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma.

Conclusions: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

Show MeSH
Related in: MedlinePlus