The Integrin Pathway Partially Mediates Stretch-Induced Deficits in Primary Rat Microglia.

Stretch-injured microglia display significantly altered morphology, function and inflammatory-associated gene expression when cultured on a synthetic fibronectin substrate. However, the mechanism by which stretch induces these changes is unknown. Integrins, such as α5ß1, mediate microglial attachment to fibronectin via the RGD binding peptide; following integrin ligation the integrin-associated signaling enzyme, focal adhesion kinase (FAK), autophosphorylates tyrosine residue 397 and mediates multiple downstream cellular processes. We therefore hypothesize that blocking the RGD binding/integrin pathway with a commercially available RGD peptide will mimic the stretch-induced morphological alterations and functional deficits in microglia. Further, we hypothesize that upregulation of stretch-inhibited downstream integrin signaling will reverse these effects. Using primary rat microglia, we tested the effects of RGD binding peptide and a FAK activator on cellular function and structure and response to stretch-injury. Similar to injured cells, RGD peptide administration significantly decreases media nitric oxide (NO) levels and iNOS expression and induced morphological alterations and migratory deficits. While stretch-injury and RGD peptide administration decreased phosphorylation of the tyrosine 397 residue on FAK, 20 nM of ZINC 40099027, an activator specific to the tyrosine 397 residue, rescued the stretch-induced decrease in FAK phosphorylation and ameliorated the injury-induced decrease in media NO levels, iNOS expression and inflammatory associated gene expression. Additionally, treatment alleviated morphological changes observed after stretch-injury and restored normal migratory behavior to control levels. Taken together, these data suggest that the integrin/FAK pathway partially mediates the stretch-injured phenotype in microglia, and may serve as a pathway to modulate microglial responses.

Differential effects of NOX2 and NOX4 inhibition after rodent spinal cord injury.

Reactive oxygen species (ROS) are a contributing factor to impaired function and pathology after spinal cord injury (SCI). The NADPH oxidase (NOX) enzyme is a key source of ROS; there are several NOX family members, including NOX2 and NOX4, that may play a role in ROS production after SCI. Previously, we showed that a temporary inhibition of NOX2 by intrathecal administration of gp91ds-tat immediately after injury improved recovery in a mouse SCI model. However, chronic inflammation was not affected by this single acute treatment, and other NOX family members were not assessed. Therefore, we aimed to explore the effect of genetic knockout (KO) of NOX2 or acute inhibition of NOX4 with GKT137831. A moderate SCI contusion injury was performed in 3 month old NOX2 KO and wild-type (WT) mice, who received no treatment or GKT137831/vehicle 30 minutes post-injury. Motor function was assessed using the Basso Mouse Scale (BMS), followed by evaluation of inflammation and oxidative stress markers. NOX2 KO mice, but not GKT137831 treated mice, demonstrated significantly improved BMS scores at 7, 14, and 28 days post injury (DPI) in comparison to WT mice. However, both NOX2 KO and GKT137831 significantly reduced ROS production and oxidative stress markers. Furthermore, a shift in microglial activation toward a more neuroprotective, anti-inflammatory state was observed in KO mice at 7 DPI and a reduction of microglial markers at 28 days. While acute alterations in inflammation were noted with GKT137831 administration, this was not sustained through 28 days. In vitro analysis also showed that while GKT137831 reduced ROS production by microglia, it did not translate to changes in pro-inflammatory marker expression within these cells. These data demonstrate that NOX2 and NOX4 play a role in post-injury ROS, but a single dose of NOX4 inhibitor fails to enhance long-term recovery.

Therapeutic targeting of microglia mediated oxidative stress after neurotrauma.

Inflammation is a primary component of the central nervous system injury response. Traumatic brain and spinal cord injury are characterized by a pronounced microglial response to damage, including alterations in microglial morphology and increased production of reactive oxygen species (ROS). The acute activity of microglia may be beneficial to recovery, but continued inflammation and ROS production is deleterious to the health and function of other cells. Microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), mitochondria, and changes in iron levels are three of the most common sources of ROS. All three play a significant role in post-traumatic brain and spinal cord injury ROS production and the resultant oxidative stress. This review will evaluate the current state of therapeutics used to target these avenues of microglia-mediated oxidative stress after injury and suggest avenues for future research.

Spinal cord injury chronically depresses glucose uptake in the rodent model.

The pathophysiology following spinal cord injury (SCI) progresses from its lesion epicenter resulting in cellular and systemic changes acutely, sub-acutely and chronically. The symptoms of the SCI depend upon the severity of the injury and its location in the spinal cord. However, there is lack of studies that have longitudinally assessed acute through chronic in vivo changes following SCI. In this combinatorial study we fill this gap by evaluating acute to chronic effects of moderate SCI in rats. We have used fluorodeoxyglucose (FDG) imaging with positron emission tomography (PET) as a marker to assess glucose metabolism, motor function, and immunohistochemistry to examine changes following moderate SCI. Our results demonstrate decreased FDG uptake at the injury site chronically at days 28 and 90 post injury compared to baseline. This alteration in glucose uptake was not restricted to the lesion site, showing depressed FDG uptake in non-injured areas (cervical spinal cord and cerebellum). The alteration in glucose uptake was correlated with reductions in neuronal cell viability and increases in glial cell activation at 90 days at the lesion site, as well as chronic impairments in motor function. These data demonstrate the chronic effects of SCI on glucose metabolism both within the lesion and distally within the spinal cord and brain.

Gait analysis in a rat model of traumatic brain injury.

Gait disruptions following traumatic brain injury (TBI) are noted in the clinical population. To date, thorough analysis of gait changes in animal models of TBI to allow for correlation of pathological alterations and utilization of this as a therapeutic outcome have been limited. We therefore assessed gait using the DigiGait analysis system as well as overall locomotion using the Beam Walk test in adult male Sprague-Dawley rats following a commonly used model of TBI, parietal lobe controlled cortical impact (CCI). Rats underwent DigiGait baseline analysis 24 h prior to injury, followed by a moderate CCI in the left parietal lobe. Performance on the DigiGait was then assessed at 1, 3, 7, and 14 days post-injury, followed by histological analysis of brain tissue. Beam walk analysis showed a transient but significant impairment acutely after injury. Despite observance of gait disturbance in the clinical population, TBI in the parietal lobe of rats resulted in limited alterations in hind or forelimb function. General hindlimb locomotion showed significant but transient impairment. Significant changes in gait were observed to last through the sub-acute period, including right hindpaw angle of rotation and left forelimb and right hindlimb swing phase duration. Slight changes that did not reach statistical significant but may reflect subtle impacts of TBI on gait were reflected in several other measures, such as stride duration, stance duration and stance width. These results demonstrate that moderate-severe injury to the parietal cortex and underlying structures including corpus callosum, hippocampus, thalamus and basal ganglia result in slight changes to gait that can be detected using the Digigait analysis system.

Role of Insulin in Neurotrauma and Neurodegeneration: A Review.

Insulin is a hormone typically associated with pancreatic release and blood sugar regulation. The brain was long thought to be "insulin-independent," but research has shown that insulin receptors (IR) are expressed on neurons, microglia and astrocytes, among other cells. The effects of insulin on cells within the central nervous system are varied, and can include both metabolic and non-metabolic functions. Emerging data suggests that insulin can improve neuronal survival or recovery after trauma or during neurodegenerative diseases. Further, data suggests a strong anti-inflammatory component of insulin, which may also play a role in both neurotrauma and neurodegeneration. As a result, administration of exogenous insulin, either via systemic or intranasal routes, is an increasing area of focus in research in neurotrauma and neurodegenerative disorders. This review will explore the literature to date on the role of insulin in neurotrauma and neurodegeneration, with a focus on traumatic brain injury (TBI), spinal cord injury (SCI), Alzheimer's disease (AD) and Parkinson's disease (PD).

Iron accentuated reactive oxygen species release by NADPH oxidase in activated microglia contributes to oxidative stress in vitro.

BACKGROUND: Excessive iron contributes to oxidative stress after central nervous system injury. NADPH oxidase (NOX) enzymes are upregulated in microglia after pro-inflammatory activation and contribute to oxidative stress. The relationship between iron, microglia, NOX, and oxidative stress is currently unclear. METHODS: We evaluated the effects of iron on lipopolysaccharide (LPS)-activated microglia and its secondary effect within neuronal co-cultures. Further, NOX2 and four specific inhibitors were tested to evaluate the relationship with the reactive oxygen species (ROS)-producing enzymes. RESULTS: An iron dose-dependent increase in ROS production among microglia treated with LPS was identified. Interestingly, despite this increase in ROS, inflammatory polarization alterations were not detected among the microglia after exposure to iron and LPS. Co-culture experimentation between primary neurons and exposed microglia (iron and LPS) significantly reduced neuronal cell number at 24 h, suggesting a profound neurotoxic effect despite the lack of a change in polarization phenotype. NOX2 and NOX4 inhibition significantly reduced ROS production among microglia exposed to iron and LPS and reduced neuronal damage and death in response to microglial co-culture. CONCLUSIONS: In conclusion, iron significantly increased ROS production and neurotoxicity without exacerbating LP-activated microglia phenotype in vitro, suggesting that iron contributes to microglia-related oxidative stress, and this may be a viable therapeutic target for injury or neurodegeneration. Further, this study highlights both NOX2 and NOX4 as potential therapeutic targets in the treatment of iron-induced microglia-related inflammation and neurotoxicity.

Aging alters glucose uptake in the naïve and injured rodent spinal cord.

Aging results in increased activation of inflammatory glial cells and decreased neuronal viability following spinal cord injury (SCI). Metabolism and transport of glucose is also decreased with age, although the influence of age on glucose transporter (GLUT) expression or glucose uptake in SCI is currently unknown. We therefore performed [18F]Fluorodeoxyglucose (FDG) PET imaging of young (3 month) and middle-aged (12 month) rats. Glucose uptake in middle-aged rats was decreased compared to young rats at baseline, followed by increased uptake 14 days post contusion SCI. qRT-PCR and protein analysis revealed an association between 14 day glucose uptake and 14 day post-injury inflammation. Further, gene expression analysis of neuron-specific GLUT3 and non-specific GLUT4 (present on glial cells) revealed an inverse relationship between GLUT3/4 gene expression and glucose uptake patterns. Protein expression revealed increased GLUT3 in 3 month rats only, consistent with age related decreases in glucose uptake, and increased GLUT4 in 12 month rats only, consistent with age related increases in inflammatory activity and glucose uptake. Inconsistencies between gene and protein suggest an influence of age-related impairment of translation and/or protein degradation. Overall, our findings show that age alters glucose uptake and GLUT3/4 expression profiles before and after SCI, which may be dependent on level of inflammatory response, and may suggest a therapeutic avenue in addressing glucose uptake in the aging population.

Alteration of FDG uptake by performing novel object recognition task in a rat model of Traumatic Brain Injury.

Traumatic Brain Injury (TBI) affects approximately 2.5 million people in the United States, of which 80% are considered to be mild (mTBI). Previous studies have shown that cerebral glucose uptake and metabolism are altered after brain trauma and functional metabolic deficits observed following mTBI are associated with changes in cognitive performance. Imaging of glucose uptake using [18F] Fluorodeoxyglucose (FDG) based Positron Emission Tomography (PET) with anesthesia during the uptake period demonstrated limited variability in results, but may have depressed uptake. Anesthesia has been found to interfere with blood glucose levels, and hence, FDG uptake. Conversely, forced cognitive testing during uptake may increase glucose demand in targeted regions, such as hippocampus, allowing for better differentiation of outcomes. Therefore, the objective of this study was to investigate the influence of a directed cognitive function task during the FDG uptake period on uptake measurements both in naïve rats and at 2 days after mild lateral fluid percussion (mLFP) TBI. Adult male Sprague Dawley rats underwent FDG uptake with either cognitive testing with the Novel Object Recognition (NOR) test or No Novel Object (NNO), followed by PET scans at baseline (prior to injury) and at 2days post mLFP. At baseline, FDG uptake in the right hippocampus was elevated in rats completing the NOR in comparison to the NNO (control group). Further, the NNO group rats demonstrated a greater fold change in the FDG uptake between baseline and post injury scans than the NOR group. Overall, these data suggest that cognitive activity during FDG uptake affects the regional uptake pattern in the brain, increasing uptake at baseline and suppressing the effects of injury.

The effects of insulin on the inflammatory activity of BV2 microglia.

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.

Age exacerbates microglial activation, oxidative stress, inflammatory and NOX2 gene expression, and delays functional recovery in a middle-aged rodent model of spinal cord injury.

BACKGROUND: Spinal 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. METHODS: Young 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). RESULTS: In 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. CONCLUSIONS: These 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.

Intranasal insulin treatment of an experimental model of moderate traumatic brain injury.

Traumatic brain injury (TBI) results in learning and memory dysfunction. Cognitive deficits result from cellular and metabolic dysfunction after injury, including decreased cerebral glucose uptake and inflammation. This study assessed the ability of intranasal insulin to increase cerebral glucose uptake after injury, reduce lesion volume, improve memory and learning function and reduce inflammation. Adult male rats received a controlled cortical impact (CCI) injury followed by intranasal insulin or saline treatment daily for 14 days. PET imaging of [18F]-FDG uptake was performed at baseline and at 48 h and 10 days post-injury and MRI on days three and nine post injury. Motor function was tested with the beam walking test. Memory function was assessed with Morris water maze. Intranasal insulin after CCI significantly improved several outcomes compared to saline. Insulin-treated animals performed better on beam walk and demonstrated significantly improved memory. A significant increase in [18F]-FDG uptake was observed in the hippocampus. Intranasal insulin also resulted in a significant decrease in hippocampus lesion volume and significantly less microglial immunolabeling in the hippocampus. These data show that intranasal insulin improves memory, increases cerebral glucose uptake and decreases neuroinflammation and hippocampal lesion volume, and may therefore be a viable therapy for TBI.

Central Nervous System Injury and Nicotinamide Adenine Dinucleotide Phosphate Oxidase: Oxidative Stress and Therapeutic Targets.

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.

[18F]FDG-PET Combined with MRI Elucidates the Pathophysiology of Traumatic Brain Injury in Rats.

Non-invasive measurements of brain metabolism using 18F-fluorodeoxyglucose (FDG) with positron emission tomography (PET) may provide important information about injury severity following traumatic brain injury (TBI). There is growing interest in the potential of combining functional PET imaging with anatomical and functional magnetic resonance imaging (MRI). This study aimed to investigate the effectiveness of combining clinically available FDG-PET with T2 and diffusion MR imaging, with a particular focus on inflammation and the influence of glial alterations after injury. Adult male Sprague Dawley rats underwent a moderate controlled cortical impact (CCI) injury followed by FDG-PET, MRI, and histological evaluation. FDG uptake showed significant alterations in the corpus callosum, hippocampus, and amygdala after TBI, demonstrating that a relatively "focal" CCI injury can result in global alterations. Analysis of MRI T2 intensity and apparent diffusion coefficient (ADC) also showed significant alterations in these regions to include cytotoxic and vasogenic edema. Histology showed increased glial activation in the corpus callosum and hippocampus that was associated with increased FDG uptake at sub-acute time-points. Glial activation was not detected in the amygdala but neuronal damage was evident, as the amygdala was the only region to show a reduction in both FDG uptake and ADC at sub-acute time-points. Overall, FDG-PET detected glial activation but was confounded by the presence of cell damage, whereas MRI consistently detected cell damage but was confounded by glial activation. These results demonstrate that FDG-PET and MRI can be used together to improve our understanding of the complex alterations in the brain after TBI.

Characterization of Macrophage/Microglial Activation and Effect of Photobiomodulation in the Spared Nerve Injury Model of Neuropathic Pain.

Objective: Neuropathic pain is common and debilitating with limited effective treatments. Macrophage/microglial activation along ascending somatosensory pathways following peripheral nerve injury facilitates neuropathic pain. However, polarization of macrophages/microglia in neuropathic pain is not well understood. Photobiomodulation treatment has been used to decrease neuropathic pain, has anti-inflammatory effects in spinal injury and wound healing models, and modulates microglial polarization in vitro. Our aim was to characterize macrophage/microglia response after peripheral nerve injury and modulate the response with photobiomodulation. Methods: Adult male Sprague-Dawley rats were randomly assigned to sham (N = 13), spared nerve injury (N = 13), or injury + photobiomodulation treatment groups (N = 7). Mechanical hypersensitivity was assessed with electronic von Frey. Photobiomodulation (980 nm) was applied to affected hind paw (output power 1 W, 20 s, 41cm above skin, power density 43.25 mW/cm 2 , dose 20 J), dorsal root ganglia (output power 4.5W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 85.5 J), and spinal cord regions (output power 1.5 W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 28.5 J) every other day from day 7-30 post-operatively. Immunohistochemistry characterized macrophage/microglial activation. Results: Injured groups demonstrated mechanical hypersensitivity 1-30 days post-operatively. Photobiomodulation-treated animals began to recover after two treatments; at day 26, mechanical sensitivity reached baseline. Peripheral nerve injury caused region-specific macrophages/microglia activation along spinothalamic and dorsal-column medial lemniscus pathways. A pro-inflammatory microglial marker was expressed in the spinal cord of injured rats compared to photobiomodulation-treated and sham group. Photobiomodulation-treated dorsal root ganglion macrophages expressed anti-inflammatory markers. Conclusion: Photobiomodulation effectively reduced mechanical hypersensitivity, potentially through modulating macrophage/microglial activation to an anti-inflammatory phenotype.

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.

(18)F-FDG-PET imaging of rat spinal cord demonstrates altered glucose uptake acutely after contusion injury.

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 biomarker 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 [(18)F] Fluorodeoxyglucose (FDG) was performed prior to injury and at 6 and 24h and 15days post-injury (dpi). FDG-PET imaging revealed significantly depressed glucose uptake at 6h post-injury at the lesion epicenter that returned to sham/naïve levels at 24h 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.

Outcome after Repetitive Mild Traumatic Brain Injury Is Temporally Related to Glucose Uptake Profile at Time of Second Injury.

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.

Inhibition of NOX2 reduces locomotor impairment, inflammation, and oxidative stress after spinal cord injury.

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.

Temporal dynamics of cerebral blood flow, cortical damage, apoptosis, astrocyte-vasculature interaction and astrogliosis in the pericontusional region after traumatic brain injury.

Traumatic brain injury (TBI) results in a loss of brain tissue at the moment of impact in the cerebral cortex. Subsequent secondary injury involves the release of molecular signals with dramatic consequences for the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The mechanisms behind the progression of tissue loss remain under investigation. In this study, we analyzed the spatial-temporal profile of blood flow, apoptotic, and astrocytic-vascular events in the cortical regions around the impact site at time points ranging from 5 h to 2 months after TBI. We performed a mild-moderate controlled cortical impact injury in young adult mice and analyzed the glial and vascular response to injury. We observed a dramatic decrease in perilesional cerebral blood flow (CBF) immediately following the cortical impact that lasted until days later. CBF finally returned to baseline levels by 30 days post-injury (dpi). The initial impact also resulted in an immediate loss of tissue and cavity formation that gradually increased in size until 3 dpi. An increase in dying cells localized in the pericontusional region and a robust astrogliosis were also observed at 3 dpi. A strong vasculature interaction with astrocytes was established at 7 dpi. Glial scar formation began at 7 dpi and seemed to be compact by 60 dpi. Altogether, these results suggest that TBI results in a progression from acute neurodegeneration that precedes astrocytic activation, reformation of the neurovascular unit to glial scar formation. Understanding the multiple processes occurring after TBI is critical to the ability to develop neuroprotective therapeutics to ameliorate the short and long-term consequences of brain injury.