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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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Confirmation of components of the NADPH oxidase enzyme. NADPH oxidase components gp91PHOX (A) and p22PHOX (B) were confirmed using western blotting at 28 days and 6 months post-injury. Representative western blots and graphical representation are shown. Bars represent mean +/- SEM. *p < 0.05. p22PHOX expression was also confirmed with immunohistochemistry at 28 days post-injury in injured (C) and sham-injured (D) tissue. No immunolabeling was observed in negative controls where primary antibody was excluded (E), and positive labeling had typical ramified or 'bushy' appearance of microglia/macrophages. Size bar = 100 μM.
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Figure 6: Confirmation of components of the NADPH oxidase enzyme. NADPH oxidase components gp91PHOX (A) and p22PHOX (B) were confirmed using western blotting at 28 days and 6 months post-injury. Representative western blots and graphical representation are shown. Bars represent mean +/- SEM. *p < 0.05. p22PHOX expression was also confirmed with immunohistochemistry at 28 days post-injury in injured (C) and sham-injured (D) tissue. No immunolabeling was observed in negative controls where primary antibody was excluded (E), and positive labeling had typical ramified or 'bushy' appearance of microglia/macrophages. Size bar = 100 μM.

Mentions: The NADPH oxidase enzyme components p22PHOX and gp91PHOX were both identified in the 'delayed expression' cluster. The protein products of these genes make up the membrane-bound components of NADPH oxidase enzyme, which is involved in ROS production by phagocytic cells. To confirm that the increase in gene expression is accompanied by an increase in protein expression and functional activity, western blotting, immunohistochemistry and function assays were performed for p22PHOX and gp91PHOX. Western blot analysis of gp91PHOX protein expression at 28 days post-injury indicated an increase in expression in injured tissue compared to sham tissue (Figure 6A). At 6 months post-injury, western blotting demonstrated a significant increase in p22PHOX protein compared to sham, confirming the delayed expression suggested by the mRNA data (Figure 6B). Immunohistochemistry at 28 days post-injury demonstrated an increase in p22PHOX immunolabeling in contrast to sham-injured tissue (Figure 6C).


Delayed inflammatory mRNA and protein expression after spinal cord injury.

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

Confirmation of components of the NADPH oxidase enzyme. NADPH oxidase components gp91PHOX (A) and p22PHOX (B) were confirmed using western blotting at 28 days and 6 months post-injury. Representative western blots and graphical representation are shown. Bars represent mean +/- SEM. *p < 0.05. p22PHOX expression was also confirmed with immunohistochemistry at 28 days post-injury in injured (C) and sham-injured (D) tissue. No immunolabeling was observed in negative controls where primary antibody was excluded (E), and positive labeling had typical ramified or 'bushy' appearance of microglia/macrophages. Size bar = 100 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Confirmation of components of the NADPH oxidase enzyme. NADPH oxidase components gp91PHOX (A) and p22PHOX (B) were confirmed using western blotting at 28 days and 6 months post-injury. Representative western blots and graphical representation are shown. Bars represent mean +/- SEM. *p < 0.05. p22PHOX expression was also confirmed with immunohistochemistry at 28 days post-injury in injured (C) and sham-injured (D) tissue. No immunolabeling was observed in negative controls where primary antibody was excluded (E), and positive labeling had typical ramified or 'bushy' appearance of microglia/macrophages. Size bar = 100 μM.
Mentions: The NADPH oxidase enzyme components p22PHOX and gp91PHOX were both identified in the 'delayed expression' cluster. The protein products of these genes make up the membrane-bound components of NADPH oxidase enzyme, which is involved in ROS production by phagocytic cells. To confirm that the increase in gene expression is accompanied by an increase in protein expression and functional activity, western blotting, immunohistochemistry and function assays were performed for p22PHOX and gp91PHOX. Western blot analysis of gp91PHOX protein expression at 28 days post-injury indicated an increase in expression in injured tissue compared to sham tissue (Figure 6A). At 6 months post-injury, western blotting demonstrated a significant increase in p22PHOX protein compared to sham, confirming the delayed expression suggested by the mRNA data (Figure 6B). Immunohistochemistry at 28 days post-injury demonstrated an increase in p22PHOX immunolabeling in contrast to sham-injured tissue (Figure 6C).

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