Background: Spinal cord injury (SCI) results in the activation of the NADPH oxidase (NOX) enzyme, inducing production of reactive oxygen species (ROS). We hypothesized that the NOX2 isoform plays an integral role in post-SCI inflammation and functional deficits. Methods: Moderate spinal cord contusion injury was performed in adult male mice, and flow cytometry, western blot, and immunohistochemistry were used to assess NOX2 activity and expression, inflammation, and M1/M2 microglia/macrophage polarization from 1 to 28 days after injury. The NOX2-specific inhibitor, gp91ds-tat, was injected into the intrathecal space immediately after impact. The Basso Mouse Scale (BMS) was used to assess locomotor function at 24 h post-injury and weekly thereafter. Results: Our findings show that gp91ds-tat treatment significantly improved functional recovery through 28 days post-injury and reduced inflammatory cell concentrations in the injured spinal cord at 24 h and 7 days post-injury. In addition, a number of oxidative stress markers were reduced in expression at 24 h after gp91ds-tat treatment, which was accompanied by a reduction in M1 polarization marker expression. Conclusion: Based on our findings, we now conclude that inhibition of NOX2 significantly improves outcome after SCI, most likely via acute reductions in oxidative stress and inflammation. NOX2 inhibition may therefore have true potential as a therapy after SCI.

Injury to the central nervous system (CNS) includes both traumatic brain and spinal cord injury (TBI and SCI, respectively). These injuries, which are heterogeneous and, therefore, difficult to treat, result in long-lasting functional, cognitive, and behavioral deficits. Severity of injury is determined by multiple factors, and is largely mediated by the activity of the CNS inflammatory system, including the primary CNS immune cells, microglia. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family of enzymes is a primary source of reactive oxygen species (ROS), key inflammatory mediators after CNS injury. ROS play a central role in inflammation, contributing to cytokine translation and release, microglial polarization and activation, and clearance of damaged tissue. NOX has been suggested as a potential therapeutic target in CNS trauma, as inhibition of this enzyme family modulates inflammatory cell response and ROS production. The purpose of this review is to understand how the different NOX enzymes function and what role they play in the scope of CNS trauma.

BackgroundSpinal cord injury (SCI) among people over age 40 has been steadily increasing since the 1980s and is associated with worsened outcome than injuries in young people. Age-related increases in reactive oxygen species (ROS) are suggested to lead to chronic inflammation. The NADPH oxidase 2 (NOX2) enzyme is expressed by microglia and is a primary source of ROS. This study aimed to determine the effect of age on inflammation, oxidative damage, NOX2 gene expression, and functional performance with and without SCI in young adult (3 months) and middle-aged (12 months) male rats.MethodsYoung adult and middle-aged rats were assessed in two groups—naïve and moderate contusion SCI. Functional recovery was determined by weekly assessment with the Basso, Beattie, and Breshnahan general motor score (analyzed two-way ANOVA) and footprint analysis (analyzed by Chi-square analysis). Tissue was analyzed for markers of oxidative damage (8-OHdG, Oxyblot, and 3-NT), microglial-related inflammation (Iba1), NOX2 component (p47PHOX, p22PHOX, and gp91PHOX), and inflammatory (CD86, CD206, TNFα, and NFκB) gene expression (all analyzed by unpaired Student’s t test).ResultsIn both naïve and injured aged rats, compared to young rats, tissue analysis revealed significant increases in 8-OHdG and Iba1, as well as inflammatory and NOX2 component gene expression. Further, injured aged rats showed greater lesion volume rostral and caudal to the injury epicenter. Finally, injured aged rats showed significantly reduced Basso–Beattie–Bresnahan (BBB) scores and stride length after SCI.ConclusionsThese results show that middle-aged rats demonstrate increased microglial activation, oxidative stress, and inflammatory gene expression, which may be related to elevated NOX2 expression, and contribute to worsened functional outcome following injury. These findings are essential to elucidating the mechanisms of age-related differences in response to SCI and developing age-appropriate therapeutics.

Microglia are the macrophages of the central nervous system (CNS), which function to monitor and maintain homeostasis. Microglial activation occurs after CNS injury, infection or disease. Prolonged microglial activation is detrimental to the CNS as they produce nitric oxide (NO), reactive oxygen species (ROS) and pro-inflammatory cytokines, resulting in neuronal cell dysfunction and death. Microglial activation is implicated in the neurological deficits following traumatic brain injury (TBI) and Alzheimer’s disease. Intranasal insulin administration is a promising treatment of Alzheimer’s disease and TBI. However, the exact effect of insulin on microglia is currently unclear. The goal of this study was therefore to examine the effect of insulin administration on activated microglia. The microglial cell line BV2 were exposed to a pro-inflammatory stimulus, lipopolysaccharide (LPS), followed by insulin administration. Outcome measures were conducted at 24 hours after treatment. In vitro assays quantified NO and ROS production. Western blot, immunocytochemistry and phagocytosis assay further examined the effect of insulin on microglial activity. Insulin treatment significantly reduced NO, ROS and TNFα production and increased phagocytic activity. Insulin treatment also significantly reduced iNOS expression, but had no significant effect on any other M1 or M2 macrophage polarization marker examined. These data suggest that insulin has very specific effects to reduce pro-inflammatory or chemoattractant properties of microglia, and this may be one mechanism by which insulin has beneficial effects in CNS injury or neurodegenerative conditions.

Repeated mild traumatic brain injury (rmTBI) results in worsened outcomes, compared with a single injury, but the mechanism of this phenomenon is unclear. We have previously shown that mild TBI in a rat lateral fluid percussion model results in globally depressed glucose uptake, with a peak depression at 24 h that resolves by 16 days post-injury. The current study investigated the outcomes of a repeat injury conducted at various times during this period of depressed glucose uptake. Adult male rats were therefore subjected to rmTBI with a latency of 24 h, 5 days, or 15 days between injuries, followed by assessment of motor function, histopathology, and glucose uptake using positron emission tomography (PET). Rats that received a 24 h rmTBI showed significant deficits in motor function tasks, as well as significant increases in lesion volume and neuronal damage. The level of microglial and astrocytic activation also was associated with the timing of the second impact. Finally, rmTBI with latencies of 24 h and 5 days showed significant alterations in [(18)F]fluorodeoxyglucose uptake, compared with baseline scans. Therefore, we conclude that the state of the metabolic environment, as indicated by FDG-PET at the time of the repeat injury, significantly influences neurological outcomes.

Spinal cord injury (SCI) results in both acute and chronic inflammation, as a result of activation of microglia, invasion of macrophages and activation of the NADPH oxidase (NOX) enzyme. The NOX enzyme is a primary source of reactive oxygen species (ROS) and is expressed by microglia and macrophages after SCI. These cells can assume either a pro- (M1) or anti-inflammatory (M2) polarization phenotype and contribute to tissue response to SCI. However, the contribution of NOX expression and ROS production to this polarization and vice versa is currently undefined. We therefore investigated the impact of SCI on NOX expression and microglial/macrophage polarization over time in a mouse model of contusion injury. Adult C57Bl/6 mice were exposed to a moderate T9 contusion SCI and tissue was assessed at acute, sub-acute and chronic time points for NOX isoform expression and co-expression with M1 and M2 microglia/macrophage polarization markers. Two NOX isoforms were increased after injury and were associated with both M1 and M2 markers, with an M1 preference for NOX2 acutely and NOX4 chronically. M2 cells were primarily found at acute time points only; the peak of NOX2 expression was associated with the decline in M2 polarization. In vitro, NOX2 inhibition shifted microglial polarization toward the M2 phenotype. These results now show that microglial/macrophage expression of NOX isoforms is independent of polarization state, but that NOX activity can influence subsequent polarization. These data can contribute to the therapeutic targeting of NOX as a therapy for SCI.

Spinal cord injury (SCI) results in an acute reduction in neuronal and glial cell viability, disruption in axonal tract integrity, and prolonged increases in glial activity and inflammation, all of which can influence regional metabolism and glucose utilization. To date, the understanding of glucose uptake and utilization in the injured spinal cord is limited. Positron emission tomography (PET)-based measurements of glucose uptake may therefore serve as a novel bio-marker for SCI. This study aimed to determine the acute and sub-acute glucose uptake pattern after SCI to determine its potential as a novel non-invasive tool for injury assessment and to begin to understand the glucose uptake pattern following acute SCI. Briefly, adult male Sprague-Dawley rats were subjected to moderate contusion SCI, confirmed by locomotor function and histology. PET imaging with [18F]Fluorodeoxyglucose (FDG) was performed prior to injury and at 6 and 24 hours and 15 days post-injury (dpi). FDG-PET imaging revealed significantly depressed glucose uptake at 6 hours post-injury at the lesion epicenter that returned to sham/naïve levels at 24 hours and 15 dpi after moderate injury. FDG uptake at 15 dpi was likely influenced by a combination of elevated glial presence and reduced neuronal viability. These results show that moderate SCI results in acute depression in glucose uptake followed by an increase in glucose uptake that may be related to neuroinflammation. This acute and sub-acute uptake, which is dependent on cellular responses, may represent a therapeutic target.

Background Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs). Methods Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1β (interleukin-1 beta). Results We show TNFα and IL-1β induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1β effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity. Conclusion Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.