RESUMO
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.
Assuntos
Lesões Encefálicas Traumáticas/fisiopatologia , Lateralidade Funcional/fisiologia , Hipocampo/fisiopatologia , Reconhecimento Psicológico/fisiologia , Animais , Comportamento Animal/fisiologia , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Lesões Encefálicas Traumáticas/metabolismo , Fluordesoxiglucose F18 , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Humanos , Masculino , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Ferro/farmacologia , Microglia/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aminopiridinas/farmacologia , Animais , Animais Recém-Nascidos , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Inibidores Enzimáticos/farmacologia , Ferritinas/genética , Ferritinas/metabolismo , Ferro/metabolismo , Lipopolissacarídeos/farmacologia , Microglia/fisiologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirazóis/farmacologia , Pirazolonas , Piridinas/farmacologia , Piridonas , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
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.
Assuntos
Envelhecimento/metabolismo , Modelos Animais de Doenças , Microglia/metabolismo , NADPH Oxidase 2/biossíntese , Estresse Oxidativo/fisiologia , Traumatismos da Medula Espinal/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Expressão Gênica , Inflamação/metabolismo , Inflamação/patologia , Masculino , Microglia/patologia , Destreza Motora/fisiologia , NADPH Oxidase 2/genética , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Roedores , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Terapia com Luz de Baixa Intensidade/métodos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Microglia/imunologia , Neuralgia/imunologia , Neuralgia/terapia , Animais , Masculino , Neuralgia/patologia , Tratamentos com Preservação do Órgão , Medição da Dor , Traumatismos dos Nervos Periféricos/imunologia , Traumatismos dos Nervos Periféricos/terapia , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
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.
Assuntos
Macrófagos/metabolismo , Microglia/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/genética , NADPH Oxidase 4/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologiaRESUMO
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.
Assuntos
Inflamação/etiologia , Glicoproteínas de Membrana/metabolismo , Transtornos dos Movimentos/etiologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Traumatismos da Medula Espinal/complicações , Análise de Variância , Animais , Antígenos CD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Glicoproteínas/uso terapêutico , Macrófagos , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Transtornos dos Movimentos/tratamento farmacológico , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio , Fatores de TempoRESUMO
The microtubule cytoskeleton regulates microglial morphology, motility, and effector functions. The microtubule-severing enzyme, fidgetin-like 2 (FL2), negatively regulates cell motility and nerve regeneration, making it a promising therapeutic target for central nervous system injury. Microglia perform important functions in response to inflammation and injury, but how FL2 affects microglia is unclear. In this study, we investigated the role of FL2 in microglial morphology and injury responses in vitro. We first determined that the pro-inflammatory stimulus, lipopolysaccharide (LPS), induced a dose- and time-dependent reduction in FL2 expression associated with reduced microglial ramification. We then administered nanoparticle-encapuslated FL2 siRNA to knockdown FL2 and assess microglial functions compared to negative control siRNA and vehicle controls. Time-lapse live-cell microscopy showed that FL2 knockdown increased the velocity of microglial motility. After incubation with fluorescently labeled IgG-opsonized beads, FL2 knockdown increased phagocytosis. Microglia were exposed to low-dose LPS after nanoparticle treatment to model injury-induced cytokine secretion. FL2 knockdown enhanced LPS-induced cytokine secretion of IL-1α, IL-1ß, and TNFα. These results identify FL2 as a regulator of microglial morphology and suggest that FL2 can be targeted to increase or accelerate microglial injury responses.
RESUMO
BACKGROUND: Brain injury results in an increase in the activity of the reactive oxygen species generating NADPH oxidase (NOX) enzymes. Preliminary studies have shown that NOX2, NOX3, and NOX4 are the most prominently expressed NOX isotypes in the brain. However, the cellular and temporal expression profile of these isotypes in the injured and non-injured brain is currently unclear. METHODS: Double immunofluorescence for NOX isotypes and brain cell types was performed at acute (24 hours), sub-acute (7 days), and chronic (28 days) time points after controlled cortical impact-induced brain injury or sham-injury in rats. RESULTS: NOX2, NOX3, and NOX4 isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on both cellular source and post-injury time. NOX4 was found in all cell types assessed, while NOX3 was positively identified in neurons only, and NOX2 was identified in microglia and neurons. NOX2 was the most responsive to injury, increasing primarily in microglia in response to injury. Quantitation of this isotype showed a significant increase in NOX2 expression at 24 hours, with reduced expression at 7 days and 28 days post-injury, although expression remained above sham levels at later time points. Cellular confirmation using purified primary or cell line culture demonstrated similar patterns in microglia, astrocytes, and neurons. Further, inhibition of NOX, and more specifically NOX2, reduced pro-inflammatory activity in microglia, demonstrating that NOX is not only up-regulated after stimulation, but may also play a significant role in post-injury neuroinflammation. CONCLUSIONS: This study illustrates the expression profiles of NOX isotypes in the brain after injury, and demonstrates that NOX2, and to a lesser extent, NOX4, may be responsible for the majority of oxidative stress observed acutely after traumatic brain injury. These data may provide insight into the design of future therapeutic approaches.
Assuntos
Astrócitos/enzimologia , Lesões Encefálicas/enzimologia , Microglia/enzimologia , NADPH Oxidases/biossíntese , Neurônios/enzimologia , Animais , Imunofluorescência , Imuno-Histoquímica , Isoenzimas/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Raios Infravermelhos , Terapia com Luz de Baixa Intensidade , Microglia/efeitos da radiação , Neuritos/efeitos da radiação , Animais , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta à Radiação , Raios Infravermelhos/uso terapêutico , Microglia/metabolismo , Neuritos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Fibronectinas , Integrinas , Ratos , Animais , Integrinas/metabolismo , Fibronectinas/metabolismo , Microglia/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fosforilação , Tirosina/metabolismo , Oligopeptídeos/farmacologia , Oligopeptídeos/metabolismo , Peptídeos/metabolismoRESUMO
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.
Assuntos
Roedores , Traumatismos da Medula Espinal , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidases/metabolismo , Traumatismos da Medula Espinal/patologia , Camundongos Knockout , NADPH Oxidase 4/metabolismoRESUMO
BACKGROUND: Traumatic brain injury initiates biochemical processes that lead to secondary neurodegeneration. Imaging studies suggest that tissue loss may continue for months or years after traumatic brain injury in association with chronic microglial activation. Recently we found that metabotropic glutamate receptor 5 (mGluR5) activation by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) decreases microglial activation and release of associated pro-inflammatory factors in vitro, which is mediated in part through inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Here we examined whether delayed CHPG administration reduces chronic neuroinflammation and associated neurodegeneration after experimental traumatic brain injury in mice. METHODS: One month after controlled cortical impact traumatic brain injury, C57Bl/6 mice were randomly assigned to treatment with single dose intracerebroventricular CHPG, vehicle or CHPG plus a selective mGluR5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine. Lesion volume, white matter tract integrity and neurological recovery were assessed over the following three months. RESULTS: Traumatic brain injury resulted in mGluR5 expression in reactive microglia of the cortex and hippocampus at one month post-injury. Delayed CHPG treatment reduced expression of reactive microglia expressing NADPH oxidase subunits; decreased hippocampal neuronal loss; limited lesion progression, as measured by repeated T2-weighted magnetic resonance imaging (at one, two and three months) and white matter loss, as measured by high field ex vivo diffusion tensor imaging at four months; and significantly improved motor and cognitive recovery in comparison to the other treatment groups. CONCLUSION: Markedly delayed, single dose treatment with CHPG significantly improves functional recovery and limits lesion progression after experimental traumatic brain injury, likely in part through actions at mGluR5 receptors that modulate neuroinflammation.
Assuntos
Lesões Encefálicas/complicações , Encefalite/etiologia , Encefalite/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Transtornos Cognitivos/etiologia , Imagem de Tensor de Difusão , Modelos Animais de Doenças , Ectodisplasinas/metabolismo , Encefalite/tratamento farmacológico , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/uso terapêutico , Hipocampo/patologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Transtornos dos Movimentos/etiologia , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/patologia , Fenilacetatos/uso terapêutico , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5 , Receptores Imunológicos/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Estatísticas não Paramétricas , Tiazóis/farmacologia , Fatores de TempoRESUMO
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.
RESUMO
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.
Assuntos
Glucose/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Fluordesoxiglucose F18/farmacocinética , Masculino , Tomografia por Emissão de Pósitrons , Ratos , Ratos Sprague-Dawley , Medula Espinal/diagnóstico por imagem , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/diagnóstico por imagemRESUMO
BACKGROUND: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. METHODS: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. RESULTS: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. CONCLUSIONS: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.
Assuntos
Expressão Gênica , Inflamação/genética , Inflamação/imunologia , RNA Mensageiro/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Animais , Galectina 3/genética , Galectina 3/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Macrófagos/fisiologia , Imageamento por Ressonância Magnética , Masculino , Análise em Microsséries , Microglia/fisiologia , Família Multigênica , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologiaRESUMO
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.
Assuntos
Lesões Encefálicas Traumáticas/complicações , Análise da Marcha , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/fisiopatologia , Lobo Parietal/lesões , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Activation of metabotropic glutamate receptor 5 (mGluR5) has neuroprotective properties in vitro and has been reported to limit postischemic lesion volume in vivo. Previously, mGluR5 has been identified on microglia in vitro, but the effects of mGluR5 activation on inflammation in vivo or on recovery after spinal cord injury is unknown. METHODS: Rats received intrathecal infusion of the selective mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) for 7 days after moderate impact spinal cord injury at T9. Complementary studies examined CHPG effects on activated spinal microglia cultures. RESULTS: Functional motor recovery was significantly increased by CHPG treatment up to 28 days after injury, with improvements in weight bearing, step taking, and coordination of stepping behavior. CHPG treatment significantly reduced lesion volume and increased white matter sparing at 28 days after injury. Administration of CHPG attenuated microglial-associated inflammatory responses in a dose-dependent fashion, including expression of ED1, Iba-1, Galectin-3, NADPH oxidase components, tumor necrosis factor-alpha, and inducible nitric oxide synthase. Because mGluR5 is expressed by microglial cells in the rat spinal cord, such effects may be mediated by direct action on microglial cells. mGluR5 stimulation also reduced microglial activation and decreased microglial-induced neurotoxicity in spinal cord microglia cultures; the latter effects were blocked by the selective mGluR5 antagonist MTEP. INTERPRETATION: These data demonstrate that mGluR5 activation can reduce microglial-associated inflammation, suggesting that the protective effects of mGluR5 agonists may reflect this action. Ann Neurol 2009;66:63-74.
Assuntos
Regulação da Expressão Gênica/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral , Modelos Animais de Doenças , Ectodisplasinas/metabolismo , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Galectina 3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Lipopolissacarídeos/toxicidade , Imageamento por Ressonância Magnética/métodos , Masculino , Proteínas dos Microfilamentos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Fenilacetatos/farmacologia , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/agonistas , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: To investigate the utility of noninvasive magnetic resonance imaging (MRI) protocols to demonstrate pathological differences between rats and mice after spinal cord injury (SCI). Rats and mice are commonly used to model SCI; however, histology and immunohistochemistry have shown differences in neuropathology between the two species, including cavity formation and scar/inflammatory responses. MATERIALS AND METHODS: Moderate contusion SCI was performed on adult male rats and mice. At 28 days postinjury, animals underwent T1-weighted (T1W), with or without gadolinium contrast, or T2-weighted (T2W) magnetic resonance imaging (MRI), to be compared with histology at the same timepoint. RESULTS: In both species, all MRI methods demonstrated changes in spinal cord anatomy. Immunohistochemistry indicated that T2W accurately reflected areas of inflammation and glial scar formation in rats and mice. Quantitation of lesion volume by histology and functional performance correlated best with T2W measurements in both species. Gadolinium contrast accurately reflected the blood-spinal cord-barrier permeability in both species, which appeared greater in rats than in mice. CONCLUSION: These data demonstrate that MRI, with either a T1W or T2W protocol, can effectively distinguish pathological differences between rats and mice.
Assuntos
Gadolínio/farmacologia , Imageamento por Ressonância Magnética/métodos , Traumatismos da Medula Espinal/fisiopatologia , Animais , Meios de Contraste/farmacologia , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Medula Espinal/patologia , Traumatismos da Medula Espinal/diagnóstico , Fatores de TempoRESUMO
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).
RESUMO
The Group I metabotropic glutamate receptor 5 (mGluR5) can modulate addiction, pain, and neuronal cell death. Expression of some mGluRs, such as Group II and III mGluRs, has been reported in microglia and may affect their activation. However, the expression and role of mGluR5 in microglia is unclear. Using immunocytochemistry and Western blot, we demonstrate that mGluR5 protein is expressed in primary microglial cultures. Activation of mGluR5 using the selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) significantly reduces microglial activation in response to lipopolysaccharide, as indicated by a reduction in nitric oxide, reactive oxygen species, and TNFalpha production. Microglial induced neurotoxicity is also markedly reduced by CHPG treatment. The anti-inflammatory effects of CHPG are not observed in microglial cultures from mGluR5 knockout mice and are blocked by selective mGluR5 antagonists, suggesting that these actions are mediated by the mGluR5 receptor. Anti-inflammatory actions of mGluR5 activation are attenuated by phospholipase C and protein kinase C inhibitors, as well as by calcium chelators, suggesting that the mGluR5 activation in microglia involves the G(alphaq)-protein signal transduction pathway. These data indicate that microglial mGluR5 may represent a novel target for modulating neuroinflammation, an important component of both acute and chronic neurodegenerative disorders.