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

Abstract

Reactive oxygen species 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 hours, 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 minutes, 6 hours 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 reactive oxygen species production after injury and therapeutic potentials.

Age increases reactive oxygen species production in macrophages and potentiates oxidative damage after spinal cord injury

“…Through co-labeling of ox-DHE and gp91 phox we observed a significant increase in NOX2 activation in macrophage/microglia at the lesion epicenter of 14 MO vs. 4 MO SCI mice. Low levels of gp91 phox expression have also been shown in neurons and astrocytes following traumatic brain injury (TBI) and SCI (Cooney et al, 2014; Dohi et al, 2010), and we confirmed expression of gp91 phox in these cells is unaffected by age. We also observed that other unidentified cells also account for ~20% ROS production at 7 dpi.…”

“…Although there was no significant difference in NOX2 gene expression or protein level due to age (Supplementary Fig. 3A&D-D’), NOX2 is the main superoxide-generating enzyme found in macrophages and has been detected in microglia/macrophages following rat contusion SCI (Cooney et al, 2014). To further investigate whether age alters macrophage-specific NOX2 expression and ROS production, we then performed triple-labeling with TomL, DHE, and the anti-gp91 phox antibody, which identifies the membrane component of NOX2 in cells.…”

“…Increased carbonylation of protein, an oxidative stress marker, is detectable in rat spinal cord homogenates 24 hours post contusion injury (Cooney et al, 2014). In addition, superoxide generation occurs in neurons 1 hour after incomplete SCI (Aoyama et al, 2008).…”

“…This elevation peaks at 7 dpi and maintained up to 28 dpi (Cooney et al, 2014). Moreover, chronically expressed NOX2 in highly activated microglia has been observed 1 year after TBI (Loane et al, 2014).…”

Neuroprotective effects of Ganoderma lucidum polysaccharides against traumatic spinal cord injury in rats

“…Since the central nervous system consists largely of lipids, neuronal tissues such as the spinal cord are highly vulnerable to oxidative injury. Oxidative stress leads to oxidation of polyunsaturated fatty acids and proteins in the neurons, damages the DNA helix, and causes cell death and consumption of antioxidant enzymes that are capable of scavenging reactive oxygen species [54]. Due to elevated oxidative stress in the spinal cord, SOD levels were shown to decrease [23].…”