Expression and detrimental role of hematopoietic prostaglandin D synthase in spinal cord contusion injury

Prostaglandin D(2) (PGD(2) ) is a potent inflammatory mediator, which is implicated in both the initiation and resolution of inflammation in peripheral non-neural tissues. Its role in the central nervous system has not been fully elucidated. Spinal cord injury (SCI) is associated with an acute inflammatory response, which contributes to secondary tissue damage that worsens functional loss. We show here, with the use of hematopoietic prostaglandin D synthase (HPGDS) deficient mice and a HPGDS selective inhibitor (HQL-79), that PGD(2) plays a detrimental role after SCI. We also show that HPGDS is expressed in macrophages in the injured mouse spinal cord and contributes to the increase in PGD(2) in the contused spinal cord. HPGDS(-/-) mice also show reduced secondary tissue damage and reduced expression of the proinflammatory chemokine CXCL10 as well as an increase in IL-6 and TGFβ-1 expression in the injured spinal cord. This was accompanied by a reduction in the expression of the microglia/macrophage activation marker Mac-2 and an increase in the antioxidant metallothionein III. Importantly, HPGDS deficient mice exhibit significantly better locomotor recovery after spinal cord contusion injury than wild-type (Wt) mice. In addition, systemically administered HPGDS inhibitor (HQL-79) also enhanced locomotor recovery after SCI in Wt mice. These data suggest that PGD(2) generated via HPGDS has detrimental effects after SCI and that blocking the activity of this enzyme can be beneficial.     

Inhibition of the Ca2?-dependent K? channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K? channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca2? signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.     

Macrophages in spinal cord injury: Phenotypic and functional change from exposure to myelin debris

Macrophage activation and persistent inflammation contribute to the pathological process of spinal cord injury (SCI). It was reported that M2 macrophages were induced at 3–7 days after SCI but M2 markers were reduced or eliminated after 1 week. By contrast, M1 macrophage response is rapidly induced and then maintained at injured spinal cord. However, factors that modulate macrophage phenotype and function are poorly understood. We developed a model to distinguish bone‐marrow derived macrophages (BMDMs) from residential microglia and explored how BMDMs change their phenotype and functions in response to the lesion‐related factors in injured spinal cord. Infiltrating BMDMs expressing higher Mac‐2 and lower CX3CR1 migrate to the epicenter of injury, while microglia expressing lower Mac‐2 but higher CX3CR1 distribute to the edges of lesion. Myelin debris at the lesion site switches BMDMs from M2 phenotype towards M1‐like phenotype. Myelin debris activates ATP‐binding cassette transporter A1 (ABCA1) for cholesterol efflux in response to myelin debris loading in vitro. However, this homeostatic mechanism in injured site is overwhelmed, leading to the development of foamy macrophages and lipid plaque in the lesion site. The persistence of these cells indicates a pro‐inflammatory environment, associated with enhanced neurotoxicity and impaired wound healing. These foamy macrophages have poor capacity to phagocytose apoptotic neutrophils resulting in uningested neutrophils releasing their toxic contents and further tissue damage. In conclusion, these data demonstrate for the first time that myelin debris generated in injured spinal cord modulates macrophage activation. Lipid accumulation following macrophage phenotype switch contributes to SCI pathology. GLIA 2015;63:635–651     

Cell proliferation and replacement following contusive spinal cord injury

After spinal cord injury (SCI), about 50% of the oligodendrocytes and astrocytes in the residual white matter at the injury site are lost by 24 h. However, chronically after SCI, the density of oligodendrocytes is normal. Previous studies have shown that the adult rat spinal cord contains a pool of proliferating glial progenitors whose progeny could help restore cell density after injury. To study proliferation in response to injury, we performed SCI on adult female rats at the T8 level, using a standardized contusion model. Animals received bromodeoxyuridine (BrdU) injections during the first week after SCI, and were perfused within 2 h for acute studies, and at 6 weeks for chronic studies. The tissue was analyzed using immunohistochemical detection of BrdU and cell marker antigens. We demonstrate that cell proliferation in the residual white matter is increased at 1-7 days after SCI, peaking on day 3. Dividing cells include oligodendrocytes, astrocytes, microglia/macrophages, and a high proportion of NG2(+) glial precursors. By 6 weeks, some cells that had been labeled 2-4 days after SCI were still present. Double immunohistochemistry showed that while very few of these cells expressed NG2 or the microglia/macrophage marker OX42, about 50% expressed CC1 or glial fibrillary acidic protein (GFAP), markers of mature oligodendrocytes and astrocytes, respectively. The post-injury environment represented by residual white matter is thus permissive to the differentiation of glial precursors. Cells that are stimulated to divide during the first week after SCI develop chronically into mature phenotypes that replace macroglia lost after injury.     

Valproic acid attenuates microgliosis in injured spinal cord and purinergic P2X4 receptor expression in activated microglia

Peripheral injection with a high dose of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, into animals with mild or moderate spinal cord injury (SCI) for 1 week can reduce spinal cord tissue loss and promote hindlimb locomotor recovery. A purinergic adenosine triphosphate (ATP) receptor subtype, P2X₄ receptor (P2X₄R), has been considered as a potential target to diminish SCI‐associated inflammatory responses. In this study, using a minipump‐based infusion system, we found that intraspinal infusion with VPA for 3 days into injured spinal cord significantly improved hindlimb locomotion of rats with severe SCI induced by a 10‐g NYU impactor dropping from the height of 50 mm onto the spinal T9/10 segment. The neuronal fibers in the injured spinal cord tissues were significantly preserved in VPA‐treated rats compared with those observed in vehicle‐treated animals. Moreover, the accumulation of microglia/macrophages and astrocytes in the injured spinal cord was attenuated in the animal group receiving VPA infusion. VPA also significantly reduced P2X₄R expression post‐SCI. Furthermore, in vitro study indicated that VPA, but not the other HDAC inhibitors, sodium butyrate and trichostatin A (TSA), caused downregulation of P2X₄R in microglia activated with lipopolysaccharide (LPS). Moreover, p38 mitogen‐activated protein kinase (MAPK)‐triggered signaling was involved in the effect of VPA on the inhibition of P2X₄R gene expression. In addition to the findings from others, our results also provide important evidence to show the inhibitory effect of VPA on P2X₄R expression in activated microglia, which may contribute to reduction of SCI‐induced gliosis and subsequently preservation of spinal cord tissues. © 2013 Wiley Periodicals, Inc.     

Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat.

The post-traumatic inflammatory response in acute spinal cord contusion injury was studied in the rat. Mild and severe spinal cord injury (SCI) was produced by dropping a 10 g weight from 3 and 12 cm at the T12 vertebral level. Increased immunoreactivity of TNF-alpha in mild and severe SCI was detected in neurons at 1 h post-injury, and in neurons and microglia at 6 h post-injury, with a less significant increase in mild SCI. Expression was short-lived and declined sharply by 1 d post-injury. RT-PCR showed an early significant up-regulation of IL-1 beta, IL-6 and TNF-alpha mRNAs, maximal at 6 h post-injury with return to control levels by 24 h post-injury, the changes being less statistically significantly in mild SCI. Western blot showed early transient increases of IL-1 beta, IL-6 and TNF-alpha proteins in severe SCI but not mild SCI. Immunocytochemical, western blotting and RT-PCR analyses suggest that endogenous cells (neurons and microglia) in the spinal cord, not blood-borne leucocytes, contribute to IL-1 beta, IL-6 and TNF-alpha production in the post-traumatic inflammatory response and that their up-regulation is greater in severe than mild SCI.     

Endogenous expression of interleukin‐4 regulates macrophage activation and confines cavity formation after traumatic spinal cord injury

Traumatic spinal cord injury (SCI) triggers inflammatory reactions in which various types of cells and cytokines are involved. Several proinflammatory cytokines are up‐regulated after SCI and play crucial roles in determining the extent of secondary tissue damage. However, relatively little is known about antiinflammatory cytokines and their roles in spinal cord trauma. Recent studies have shown that an antiinflammatory cytokine, interleukin‐4 (IL‐4), is expressed and exerts various modulatory effects in CNS inflammation. We found in the present study that IL‐4 was highly expressed at 24 hr after contusive SCI in rats and declined thereafter, with concurrent up‐regulation of IL‐4 receptor subunit IL‐4α. The majority of IL‐4‐producing cells were myeloperoxidase‐positive neutrophils. Injection of neutralizing antibody against IL‐4 into the contused spinal cord did not significantly affect the expression levels of proinflammatory cytokines such as IL‐1β, IL‐6, and tumor necrosis factor‐α or other antiinflammatory cytokines such as IL‐10 and transforming growth factor‐β. Instead, attenuation of IL‐4 activity led to a marked increase in the extent of ED1‐positive macrophage activation along the rostrocaudal extent at 7 days after injury. The enhanced macrophage activation was preceded by an increase in the level of monocyte chemoattractant protein‐1 (MCP‐1/CCL2). Finally, IL‐4 neutralization resulted in more extensive cavitation at 4 weeks after injury. These results suggest that endogenous expression of antiinflammatory cytokine IL‐4 regulates the extent of acute macrophage activation and confines the ensuing secondary cavity formation after spinal cord trauma. © 2010 Wiley‐Liss, Inc     

Acute Inflammatory Response in Spinal Cord Following Impact Injury

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral–caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.     

Proinflammatory cytokine regulation of cyclic AMP‐phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β on cyclic AMP‐phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF‐α or IL‐1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL‐1β but remained suppressed with TNF‐α through 24 h. Cyclic AMP was also reduced in TNF‐α‐stimulated primary microglia, albeit to a lesser extent. Accompanying TNF‐α‐induced cyclic AMP reductions, but not IL‐1β, was increased cyclic AMP‐PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF‐α; IL‐1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF‐α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF‐α but not IL‐1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX‐42⁺ microglia; PDE4B was absent in OX‐42⁺ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up‐regulation from 24 h to 1 wk post‐SCI, the peak of microglia activation. These studies show that TNF‐α and IL‐1β differentially affect cyclic AMP‐PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases. © 2012 Wiley Periodicals, Inc.     

Anti‐inflammatory effects of ADAMTS‐4 in a mouse model of ischemic stroke

ADAMTS‐4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS‐4 is an inflammatory‐regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS‐4 in neuroinflammation. First, we evidenced an increase of ADAMTS‐4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS‐4 has predominantly an anti‐inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS‐4 prior to LPS exposure decreased NO production and the synthesis/release of pro‐inflammatory cytokines including NOS2, CCL2, TNF‐α, IL‐1β and MMP‐9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS‐4 prior to LPS exposure, the production of NO and the synthesis/release of pro‐inflammatory cytokines were increased. Finally, the feasibility of ADAMTS‐4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS‐4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase‐1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS‐4 increased the production of IL‐10 and IL‐6 in the peri‐ischemic area. By having anti‐inflammatory and neuroregenerative roles, ADAMTS‐4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492–1507     

Microglia and macrophages differentially modulate cell death after brain injury caused by oxygen‐glucose deprivation in organotypic brain slices

Macrophage can adopt several phenotypes, process call polarization, which is crucial for shaping inflammatory responses to injury. It is not known if microglia, a resident brain macrophage population, polarizes in a similar way, and whether specific microglial phenotypes modulate cell death in response to brain injury. In this study, we show that both BV2‐microglia and mouse bone marrow derived macrophages (BMDMs) were able to adopt different phenotypes after LPS (M1) or IL‐4 (M2) treatment in vitro, but regulated cell death differently when added to mouse organotypic hippocampal brain slices. BMDMs induced cell death when added to control slices and exacerbated damage when combined with oxygen–glucose deprivation (OGD), independently of their phenotype. In contrast, vehicle‐ and M2‐BV2‐microglia were protective against OGD‐induced death. Direct treatment of brain slices with IL‐4 (without cell addition) was protective against OGD and induced an M2 phenotype in the slice. In vivo, intracerebral injection of LPS or IL‐4 in mice induced microglial phenotypes similar to the phenotypes observed in brain slices and in cultured cells. After injury induced by middle cerebral artery occlusion, microglial cells did not adopt classical M1/M2 phenotypes, suggesting that another subtype of regulatory phenotype was induced. This study highlights functional differences between macrophages and microglia, in response to brain injury with fundamentally different outcomes, even if both populations were able to adopt M1 or M2 phenotypes. These data suggest that macrophages infiltrating the brain from the periphery after an injury may be cytotoxic, independently of their phenotype, while microglia may be protective.     

Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist.Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI.     

Characterization of the early neuroinflammation after spinal cord injury in mice

The occurrence of neuroinflammation after spinal cord injury (SCI) is well established, but its function is debated, with both beneficial and detrimental consequences ascribed. A discriminate of the role of neuroinflammation may be the time period after SCI, and there is evidence to favor early neuroinflammation being undesirable, whereas the later evolving phase may have useful roles. Here, we have focused on the inflammatory response in the first 24 hours of SCI in mice. We found elevation of interleukin (IL)-1beta and other cytokines and chemokines within 15 minutes to 3 hours of injury. The early neuroinflammation in SCI is likely to be CNS-derived and involves microglia, as demonstrated by in situ hybridization for IL-1beta in microglia, by an in vitro model of SCI in which elevation of inflammatory cytokines occurs in the absence of a dynamic source of infiltrating leukocytes, and by the correlation of decreased levels of inflammatory molecules and microglia activity in IL-1beta-null mice. Nonetheless, as there are no specific immunohistochemical markers that clearly differentiate microglia from their peripheral counterparts, macrophages, the latter cannot be definitively excluded as participants in early neuroinflammation in mouse SCI. These results of an instantaneous inflammatory response validate approaches to modulate microglia/macrophage activity to improve recovery from SCI.     

An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Na_v1.1, Na_v1.2, and Na_v1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821     

Lipocalin 2 plays an immunomodulatory role and has detrimental effects after spinal cord injury

Lipocalin 2 (Lcn2) plays an important role in defense against bacterial infection by interfering with bacterial iron acquisition. Although Lcn2 is expressed in a number of aseptic inflammatory conditions, its role in these conditions remains unclear. We examined the expression and role of Lcn2 after spinal cord injury (SCI) in adult mice by using a contusion injury model. Lcn2 expression at the protein level is rapidly increased 12-fold at 1 d after SCI and decreases gradually thereafter, being three times as high as control levels at 21 d after injury. Lcn2 expression is strongly induced after contusion injury in astrocytes, neurons, and neutrophils. The Lcn2 receptor (Lcn2R), which has been shown to influence cell survival, is also expressed after SCI in the same cell types. Lcn2-deficient (Lcn2?/?) mice showed significantly better locomotor recovery after spinal cord contusion injury than wild-type (Lcn2?/?) mice. Histological assessments indicate improved neuronal and tissue survival and greater sparing of myelin in Lcn2?/? mice after contusion injury. Flow cytometry showed a decrease in neutrophil influx and a small increase in the monocyte population in Lcn2?/? injured spinal cords. This change was accompanied by a reduction in the expression of several pro-inflammatory chemokines and cytokines as well as inducible nitric oxide synthase early after SCI in Lcn2?/? mice compared with wild-type animals. Our results, therefore, suggest a role for Lcn2 in regulating inflammation in the injured spinal cord and that lack of Lcn2 reduces secondary damage and improves locomotor recovery after spinal cord contusion injury.     

Temporal and Spatial Expression of KIF3B after Acute Spinal Cord Injury in Adult Rats

The KIF3 subunit KIF3B was proved to be associated with mitosis. It has been known to be engaged in intracellular transport of neurons. To elucidate the certain expression and biological function in central nervous system, we performed an acute spinal cord contusion injury model in adult rats. Western blot analysis indicated a marked upregulation of KIF3B after spinal cord injury (SCI). Immunohistochemistry revealed wide distribution of KIF3B in spinal cord, including neurons and glial cells. Double immunofluorescent staining for proliferating cell nuclear antigen and phenotype-specific markers showed increases of KIF3B expression in proliferating microglia and astrocytes. Our data suggest that KIF3B may be implicated in the proliferation of microglia and astrocytes after SCI.     

NADPH oxidase isoform expression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury

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.