Limits...
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.

Show MeSH

Related in: MedlinePlus

Confirmation of galectin-3 expression at the protein level using western blotting and immunohistochemistry. Western blotting was performed at 28 days and 6 months post-injury, and showed a significant increase in expression in moderately injured spinal cord over sham-injured tissue. Representative western blots and graphical representations are shown in A, B. Immunohistochemistry was performed at 28 days post-injury. Sham-injured tissue demonstrated no positive immunostaining for galectin-3 (C, D). Galectin-3 positive cells (labeled with a fluorescent red secondary antibody) were found throughout injured tissue at this time point, and labeled large, ameboid cells that are typical of macrophages and activated microglia (E, F). DAPI (blue) was used as a counterstain for cells. Insets show magnified areas from the white matter. Injured tissue double-labeled for microglia/macrophages (Iba-1) and galectin-3 (arrows) demonstrate correlation in expression in the merged image (G). Size bar = 500 μM (C, E); 100 μM (D, F); 75 μm (G). Bars represent mean +/- SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198932&req=5

Figure 4: Confirmation of galectin-3 expression at the protein level using western blotting and immunohistochemistry. Western blotting was performed at 28 days and 6 months post-injury, and showed a significant increase in expression in moderately injured spinal cord over sham-injured tissue. Representative western blots and graphical representations are shown in A, B. Immunohistochemistry was performed at 28 days post-injury. Sham-injured tissue demonstrated no positive immunostaining for galectin-3 (C, D). Galectin-3 positive cells (labeled with a fluorescent red secondary antibody) were found throughout injured tissue at this time point, and labeled large, ameboid cells that are typical of macrophages and activated microglia (E, F). DAPI (blue) was used as a counterstain for cells. Insets show magnified areas from the white matter. Injured tissue double-labeled for microglia/macrophages (Iba-1) and galectin-3 (arrows) demonstrate correlation in expression in the merged image (G). Size bar = 500 μM (C, E); 100 μM (D, F); 75 μm (G). Bars represent mean +/- SEM.

Mentions: Galectin-3 and progranulin were selected to confirm that mRNA up-regulation was paralleled by increases in protein expression using western blot and immunohistochemistry at time points from 28 days through 6 months post-injury. western blotting indicated that galectin-3 was significantly increased in injured tissue in comparison to sham-injured tissue at 28 days and 6 months post-injury (Figure 4A, B). This was confirmed with immunolabeling, which demonstrated that galectin-3 was expressed at high levels at 28 days post-injury in round/ameboid shaped cells, indicative of activated and phagocytic microglia/macrophages (Figure 4C - F). Double-label immunohistochemistry was used to confirm expression of proteins of interest in microglia/macrophages and other cell types. Galectin-3 was found to consistently label a large subset of Iba-1 positive cells, indicating microglial/macrophage expression (Figure 4F). No co-labeling was found with NeuN or GFAP, indicating that galectin-3 was not expressed in neurons or astrocytes, respectively, at 28 days after SCI (data not shown). While an assessment of the number of Iba-1/galectin-3 positive cells was not done in this study, it was clear that the majority of Iba-1 positive cells were also galectin-3 positive, and few galectin-3 positive cells were identified that were not Iba-1 positive, suggesting that galectin-3 was primarily expressed on microglia/macrophages.


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 galectin-3 expression at the protein level using western blotting and immunohistochemistry. Western blotting was performed at 28 days and 6 months post-injury, and showed a significant increase in expression in moderately injured spinal cord over sham-injured tissue. Representative western blots and graphical representations are shown in A, B. Immunohistochemistry was performed at 28 days post-injury. Sham-injured tissue demonstrated no positive immunostaining for galectin-3 (C, D). Galectin-3 positive cells (labeled with a fluorescent red secondary antibody) were found throughout injured tissue at this time point, and labeled large, ameboid cells that are typical of macrophages and activated microglia (E, F). DAPI (blue) was used as a counterstain for cells. Insets show magnified areas from the white matter. Injured tissue double-labeled for microglia/macrophages (Iba-1) and galectin-3 (arrows) demonstrate correlation in expression in the merged image (G). Size bar = 500 μM (C, E); 100 μM (D, F); 75 μm (G). Bars represent mean +/- SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Confirmation of galectin-3 expression at the protein level using western blotting and immunohistochemistry. Western blotting was performed at 28 days and 6 months post-injury, and showed a significant increase in expression in moderately injured spinal cord over sham-injured tissue. Representative western blots and graphical representations are shown in A, B. Immunohistochemistry was performed at 28 days post-injury. Sham-injured tissue demonstrated no positive immunostaining for galectin-3 (C, D). Galectin-3 positive cells (labeled with a fluorescent red secondary antibody) were found throughout injured tissue at this time point, and labeled large, ameboid cells that are typical of macrophages and activated microglia (E, F). DAPI (blue) was used as a counterstain for cells. Insets show magnified areas from the white matter. Injured tissue double-labeled for microglia/macrophages (Iba-1) and galectin-3 (arrows) demonstrate correlation in expression in the merged image (G). Size bar = 500 μM (C, E); 100 μM (D, F); 75 μm (G). Bars represent mean +/- SEM.
Mentions: Galectin-3 and progranulin were selected to confirm that mRNA up-regulation was paralleled by increases in protein expression using western blot and immunohistochemistry at time points from 28 days through 6 months post-injury. western blotting indicated that galectin-3 was significantly increased in injured tissue in comparison to sham-injured tissue at 28 days and 6 months post-injury (Figure 4A, B). This was confirmed with immunolabeling, which demonstrated that galectin-3 was expressed at high levels at 28 days post-injury in round/ameboid shaped cells, indicative of activated and phagocytic microglia/macrophages (Figure 4C - F). Double-label immunohistochemistry was used to confirm expression of proteins of interest in microglia/macrophages and other cell types. Galectin-3 was found to consistently label a large subset of Iba-1 positive cells, indicating microglial/macrophage expression (Figure 4F). No co-labeling was found with NeuN or GFAP, indicating that galectin-3 was not expressed in neurons or astrocytes, respectively, at 28 days after SCI (data not shown). While an assessment of the number of Iba-1/galectin-3 positive cells was not done in this study, it was clear that the majority of Iba-1 positive cells were also galectin-3 positive, and few galectin-3 positive cells were identified that were not Iba-1 positive, suggesting that galectin-3 was primarily expressed on microglia/macrophages.

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