The emerging role of neutrophils as modifiers of recovery after traumatic injury to the developing brain.

The innate immune response plays a critical role in traumatic brain injury (TBI), contributing to ongoing pathogenesis and worsening long-term outcomes. Here we focus on neutrophils, one of the "first responders" to TBI. These leukocytes are recruited to the injured brain where they release a host of toxic molecules including free radicals, proteases, and pro-inflammatory cytokines, all of which promote secondary tissue damage. There is mounting evidence that the developing brain is more vulnerable to injury that the adult brain. This vulnerability to greater damage from TBI is, in part, attributed to relatively low antioxidant reserves coupled with an early robust immune response. The latter is reflected in enhanced sensitivity to cytokines and a prolonged recruitment of neutrophils into both cortical and subcortical regions. This review considers the contribution of neutrophils to early secondary pathogenesis in the injured developing brain and raises the distinct possibility that these leukocytes, which exhibit phenotypic plasticity, may also be poised to support wound healing. We provide a basic review of the development, life cycle, and granular contents of neutrophils and evaluate their potential as therapeutic targets for early neuroprotection and functional recovery after injury at early age. While neutrophils have been broadly studied in neurotrauma, we are only beginning to appreciate their diverse roles in the developing brain and the extent to which their acute manipulation may result in enduring neurological recovery when TBI is superimposed upon brain development.

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.

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.

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.

(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.

Reduced activity-dependent protein levels in a mouse model of the fragile X premutation.

Environmental enrichment results in increased levels of Fmrp in brain and increased dendritic complexity. The present experiment evaluated activity-dependent increases in Fmrp levels in the motor cortex in response to training on a skilled forelimb reaching task in the CGG KI mouse model of the fragile X premutation. Fmrp, Arc, and c-Fos protein levels were quantified by Western blot in the contralateral motor cortex of mice following training to reach for sucrose pellets with a non-preferred paw and compared to levels in the ipsilateral motor cortex. After training, all mice showed increases in Fmrp, Arc, and c-Fos protein levels in the contralateral compared to the ipsilateral hemisphere; however, the increase in CGG KI mice was less than wildtype mice. Increases in Fmrp and Arc proteins scaled with learning, whereas this relationship was not observed with the c-Fos levels. These data suggest the possibility that reduced levels of activity-dependent proteins associated with synaptic plasticity such as Fmrp and Arc may contribute to the neurocognitive phenotype reported in the CGG KI mice and the fragile X premutation.

808 nm wavelength light induces a dose-dependent alteration in microglial polarization and resultant microglial induced neurite growth.

BACKGROUND AND OBJECTIVE: Despite the success of using photobiomodulation (PBM), also known as low level light therapy, in promoting recovery after central nervous system (CNS) injury, the effect of PBM on microglia, the primary mediators of immune and inflammatory response in the CNS, remains unclear. Microglia exhibit a spectrum of responses to injury, with partial or full polarization into pro- and anti-inflammatory phenotypes. Pro-inflammatory (M1 or classically activated) microglia contribute to chronic inflammation and neuronal toxicity, while anti-inflammatory (M2 or alternatively activated) microglia play a role in wound healing and tissue repair; microglia can fall anywhere along this spectrum in response to stimulation. MATERIALS AND METHODS: The effect of PBM on microglial polarization therefore was investigated using colorimetric assays, immunocytochemistry, proteomic profiling and RT-PCR in vitro after exposure of primary microglia or BV2 microglial cell line to PBM of differing energy densities (0.2, 4, 10, and 30 J/cm(2) , 808 nm wavelength, 50 mW output power). RESULTS: PBM has a dose-dependent effect on the spectrum of microglial M1 and M2 polarization. Specifically, PBM with energy densities between 4 and 30 J/cm(2) induced expression of M1 markers in microglia. Markers of the M2 phenotype, including CD206 and TIMP1, were observed at lower energy densities of 0.2-10 J/cm(2) . In addition, co-culture of PBM or control-treated microglia with primary neuronal cultures demonstrated a dose-dependent effect of PBM on microglial-induced neuronal growth and neurite extension. CONCLUSION: These data suggest that the Arndt-Schulz law as applied to PBM for a specific bioassay does not hold true in cells with a spectrum of responses, and that PBM can alter microglial phenotype across this spectrum in a dose-dependent manner. These data are therefore of important relevance to not only therapies in the CNS but also to understanding of PBM effects and mechanisms.

Motor deficits on a ladder rung task in male and female adolescent and adult CGG knock-in mice.

The fragile X premutation is a tandem CGG trinucleotide repeat expansion on the FMR1 gene between 55 and 200 repeats in length. A CGG knock-in (CGG KI) mouse with CGG trinucleotide repeat lengths between 70 and 350 has been developed and used to model the histopathology and cognitive deficits reported in carriers of the fragile X premutation. Previous studies have shown that CGG KI mice show progressive deficits in processing spatial and temporal information. To characterize the motor deficits associated with the fragile X premutation, male and female CGG KI mice ranging from 2 to 16 months of age with trinucleotide repeats ranging from 72 to 240 CGG in length were tested for their ability to perform a skilled ladder rung walking test. The results demonstrate that both male and female CGG KI mice showed a greater number of foot slips as a function of increased CGG repeat length, independent of the age of the animal or general activity level.