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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 lesion volume after spinal cord injury. Lesion volume in vehicle and DPI-treated injured spinal cords was measured at 28 days post-injury using T2-weighted MRI and histology. Representative MRI images of the lesion (hyperintense region, arrow) are shown for vehicle (A) and DPI-treated (B) spinal cords (outlined with dotted line). Representative cresyl violet images are shown for vehicle (D) and DPI-treated (E) spinal cords as well. Quantitation of MRI (C) and histology (F) based measurements are shown. Bar size = 0.25 cm (A, B); 200 μm (D, E). Bars represent mean +/- SEM. *p < 0.05. N = 3/group.
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Figure 8: The effect of DPI on lesion volume after spinal cord injury. Lesion volume in vehicle and DPI-treated injured spinal cords was measured at 28 days post-injury using T2-weighted MRI and histology. Representative MRI images of the lesion (hyperintense region, arrow) are shown for vehicle (A) and DPI-treated (B) spinal cords (outlined with dotted line). Representative cresyl violet images are shown for vehicle (D) and DPI-treated (E) spinal cords as well. Quantitation of MRI (C) and histology (F) based measurements are shown. Bar size = 0.25 cm (A, B); 200 μm (D, E). Bars represent mean +/- SEM. *p < 0.05. N = 3/group.

Mentions: Our data has demonstrated that NADPH oxidase expression and activity is chronically up-regulated after SCI. To determine if this enzyme plays a role in chronic expression of microglial-related inflammatory proteins after SCI, particularly those in the 'delayed expression cluster', an inhibitor of NADPH oxidase, DPI [37] was administered continuously for 7 days starting 30 minutes after injury (n = 3/group). At 28 days post-injury, all rats underwent T2-weighted MRI for assessment of post-injury lesion volume, which we have previously shown to correlate with histological measurements of lesion volume [38]. Vehicle-treated rats had a lesion volume of 0.16 +/- 0.04 cm3 (Figure 8). A significant reduction in lesion volume was found in rats that received DPI treatment (Figure 8; 0.05 +/- 0.01 cm3). This reduction was confirmed with histology. The day after MRI, tissue was dissected and lesion volume measured in cresyl violet stained sections. Based on histological measurements, DPI-treated spinal cord had significantly (p < 0.05) smaller lesion volume than vehicle-treated spinal cord tissue (Figure 8).


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 lesion volume after spinal cord injury. Lesion volume in vehicle and DPI-treated injured spinal cords was measured at 28 days post-injury using T2-weighted MRI and histology. Representative MRI images of the lesion (hyperintense region, arrow) are shown for vehicle (A) and DPI-treated (B) spinal cords (outlined with dotted line). Representative cresyl violet images are shown for vehicle (D) and DPI-treated (E) spinal cords as well. Quantitation of MRI (C) and histology (F) based measurements are shown. Bar size = 0.25 cm (A, B); 200 μm (D, E). Bars represent mean +/- SEM. *p < 0.05. N = 3/group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: The effect of DPI on lesion volume after spinal cord injury. Lesion volume in vehicle and DPI-treated injured spinal cords was measured at 28 days post-injury using T2-weighted MRI and histology. Representative MRI images of the lesion (hyperintense region, arrow) are shown for vehicle (A) and DPI-treated (B) spinal cords (outlined with dotted line). Representative cresyl violet images are shown for vehicle (D) and DPI-treated (E) spinal cords as well. Quantitation of MRI (C) and histology (F) based measurements are shown. Bar size = 0.25 cm (A, B); 200 μm (D, E). Bars represent mean +/- SEM. *p < 0.05. N = 3/group.
Mentions: Our data has demonstrated that NADPH oxidase expression and activity is chronically up-regulated after SCI. To determine if this enzyme plays a role in chronic expression of microglial-related inflammatory proteins after SCI, particularly those in the 'delayed expression cluster', an inhibitor of NADPH oxidase, DPI [37] was administered continuously for 7 days starting 30 minutes after injury (n = 3/group). At 28 days post-injury, all rats underwent T2-weighted MRI for assessment of post-injury lesion volume, which we have previously shown to correlate with histological measurements of lesion volume [38]. Vehicle-treated rats had a lesion volume of 0.16 +/- 0.04 cm3 (Figure 8). A significant reduction in lesion volume was found in rats that received DPI treatment (Figure 8; 0.05 +/- 0.01 cm3). This reduction was confirmed with histology. The day after MRI, tissue was dissected and lesion volume measured in cresyl violet stained sections. Based on histological measurements, DPI-treated spinal cord had significantly (p < 0.05) smaller lesion volume than vehicle-treated spinal cord tissue (Figure 8).

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