Measurement and Characterization of Superoxide Generation in Microglial Cells: Evidence for an NADPH Oxidase-Dependent Pathway
Open accessAbstract
While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.
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Microglia release O2− or O2−-derived hydrogen peroxide when exposed to LPS, opsonized zymosan, or aggregated proteins that accumulate in the brain during neurodegenerative diseases.
“release O 2 ⅐Ϫ or O 2 ⅐Ϫ -derived hydrogen peroxide when exposed to LPS, opsonized zymosan, or aggregated proteins that accumulate in the brain during neurodegenerative diseases (Sankarapandi et al., 1998;Zhang et al., 2005;B. Qin et al., 2006;Wilkinson et al., 2006).”
From Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase (2008) · cites this paper as Sankarapandi et al., 1998
The Abstract and Discussion report that microglia generate superoxide when stimulated with opsonified zymosan. The paper does not establish the same response to LPS or to aggregated proteins associated with neurodegenerative disease, and it does not directly establish hydrogen peroxide release under those conditions. (LLM, conf=0.88)
Experimental — accuracy not yet measuredMicroglia express the phagocyte NADPH oxidase.
“Like other phagocytes, microglia express the phagocyte NADPH oxidase (Sankarapandi et al., 1998;Bianca et al., 1999)”
From Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase (2008) · cites this paper as Sankarapandi et al., 1998
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Cites
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Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase
Chéret, C., Gervais, A., Lelli, A., Colin, C., Amar, L., Ravassard, P., Mallet, J., Cumano, A., Krause, K.-H., & Mallat, M. (2008) · The Journal of Neuroscience · doi:10.1523/jneurosci.3568-08.2008 · Cited by 225 · see citing works
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Key Inflammatory Biomarkers in Perinatal Asphyxia
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- Authors
- Sankarapandi, S., Zweier, J. L., Mukherjee, G., Quinn, M. T., & Huso, D. L.
- Affiliations
- Johns Hopkins University (US)
- Montana State University (US)
- Corresponding author
- Zweier, Jay L.
- Venue
- Archives of Biochemistry and Biophysics
- Publisher
- Elsevier BV
- Published
- 1998-05
- Volume
- 353
- Issue
- 2
- Pages
- 312-321
- ISSN
- 0003-9861 (ISSN-L)
- OpenAlex
- W1972807774
- Cited by
- 136
- References
- 4
- Citations by year
- 2024: 1 · 2022: 1 · 2021: 1 · 2020: 2 · 2019: 1 · 2018: 3 · 2017: 2 · 2016: 4 · 2015: 4 · 2014: 3 · 2013: 6 · 2012: 5
- FWCI
- 2.47
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Archives of Biochemistry and Biophysics (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- U.S. Department of Agriculture
- American Heart Association
- National Institutes of Health (HL38324)
- National Heart, Lung, and Blood Institute (HL-38324, HL52315, P50HL052315)
- National Institute of Neurological Disorders and Stroke (NS35693, R01NS035693)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR042426)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Keywords
- microglia, superoxide production, NADPH oxidase, oxygen free radicals, phorbol ester stimulation, opsonized zymosan, diphenyleneiodonium, gp91phox, p22phox, brain injury, superoxide dismutase, catalase, deferoxamine
- MeSH terms
- Animals, Cattle, Cells, Cultured, Cyclic N-Oxides, Electron Spin Resonance Spectroscopy, Free Radicals, Sheep, Superoxides, Microglia, Fluorescent Antibody Technique, Indirect, NADPH Oxidases
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