Characterization of the expression and inflammatory activity of NADPH oxidase after spinal cord injury

Abstract

Reactive oxygen species (ROS) and the NADPH oxidase (NOX) enzyme are both up-regulated after spinal cord injury (SCI) and play significant roles in promoting post-injury inflammation. However, the cellular and temporal expression profile of NOX isotypes, including NOX2, 3, and 4, after SCI is currently unclear. The purpose of this study was to resolve this expression profile and examine the effect of inhibition of NOX on inflammation after SCI. Briefly, adult male rats were subjected to moderate contusion SCI. Double immunofluorescence for NOX isotypes and CNS cellular types was performed at 24 h, 7 days, and 28 days post-injury. NOX isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on injury status. NOX2 and 4 were found in all cell types assessed, while NOX3 was positively identified in neurons only. NOX2 was the most responsive to injury, increasing in both microglia and astrocytes. The biggest increases in expression were observed at 7 days post-injury and increased expression was maintained through 28 days. NOX2 inhibition by systemic administration of gp91ds-tat at 15 min, 6 h or 7 days after injury reduced both pro-inflammatory cytokine expression and evidence of oxidative stress in the injured spinal cord. This study therefore illustrates the regional and temporal influence on NOX isotype expression and the importance of NOX activation in SCI. This information will be useful in future studies of understanding ROS production after injury and therapeutic potentials.

Keywords: NOX2; animal studies; cytokines; gp91ds-tat; oxidative stress.

Figure 1

Evolution of the spinal cord injury. H&E stained spinal cord tissue prior to injury (A) and at 24 hours (B), 7 days (C) and 28 days (D) after a moderate contusion injury in the adult male Sprague Dawley rat. Areas assessed are outlined in the dashed line. Panel E demonstrates a longitudinal section of injured spinal cord to indicate ‘lesion’ (dotted line) and ‘perilesional’ (solid line) regions. Bar = 1mm (A – D); 2mm (E).

Figure 2

Microglia/macrophages expressed NOX2 in the spinal cord at the lesion epicenter, within regions formerly occupied by white matter. Cells labeled with the microglial marker Iba1 (green) were positive for NOX2 (red, arrows) in injured spinal cord tissue. NOX2 staining increased by 7 days-post injury, but decreased by 28 days. Bar=300μm.

Figure 3

Microglia/macrophages expressed NOX4 in the spinal cord at the lesionepicenter, within regions formerly occupied by white matter. Both naïve and injured animals had CD11b positive (green) and NOX4 positive (red) cells (arrows). When merged, colabeled cells were yellow. To demonstrate consistent microglia/macrophage labeling with CD11b, adjacent sections were immunolabeled with Iba-1 and imaged at the same magnification, demonstrating similar labeling patterns. Bar= 50μm.

Figure 4

Neurons in the spinal cord expressed NOX2 and NOX3 in the dorsal horns 3mm caudalto the injury site. NOX2 (red) positive cells were also NeuN+ (green, arrows) in naïve and 28 day injured spinal cord tissue (A; bar = 50μm). NeuN positive cells (red) were found to express NOX3 (green) at all time points post-injury, with no obvious change in expression profile. Image B shows the gray matter 3mm caudal to the injury site at 28 days post-injury; bar = 1mm).

Figure 5

Astrocytes in the spinal cord were NOX2 positive in the spinal cord 3mm rostral to the lesion site, in white matter regions. GFAP (green) positive cells were also stained with an antibody against NOX2 (red; arrows) in naïve tissue and at 24 hours, 7 days and 28 days post-injury. Qualitatively, there appeared to be an increase in GFAP+/NOX2+ cells by 24 hours and 7 days post-injury that decreased by 28 days post-injury. Bar=20μm.

Figure 6

Astrocytes in the spinal cord were NOX4 positive in the spinal cord 3mm rostral to the lesion site, in white matter regions. GFAP+ (green) and NOX4+ (red; arrows) cell presence was consistent in injured and uninjured spinal cord tissue. Bar=20μm.

Figure 7

NOX2 inhibition alters acute microglial response without significantly altering other cellular presence. Western blot analysis of neuronal (A), astrocytic (B) and microglial (C) markers at 24 hours after gp91ds-tat treatment at 6 hours post-injury shows no significant effect of treatment on neuronal or astrocyte presence, but a significant inhibition of microglial markers. Bars represent mean +/- SEM. Blots are representative of an n of 4/group.

Figure 8

Quantitative analysis of relative cytokine expression in rats treated with 1 mg/kg gp91ds-tat or scrambled peptide 15 minutes following moderate contusion SCI. Two groups of cytokines were found: (A) high-expressing proteins, with more than 15pg/ml concentrations, and (B) low-expressing proteins, with expression of less than 5pg/ml. Significant reductions in several cytokines were observed, particularly in the low expression group (B); overall 13 cytokines were found reduced with gp91ds-tat treatment. N = 4/group, *p < 0.05.

Figure 9

Quantitative analysis and representative blot of carbonylation modification via oxyblot of rats treated with gp91ds-tat or scrambled peptide at 15 minutes, 6 hours, and 7 days following a moderate contusion SCI. Injury demonstrated a marked increase in oxidative damage in comparison to naïve tissue. Gp91ds-tat induced a trend for decreased oxidative modification in all three groups. A significant (*) reduction can be observed in the 6 hours group. N = 4/treatment/group. *p<0.05.