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Updated: Oct 18, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion
R Gregersen1, K Lambertsen, B Finsen
1Department of Anatomy and Neurobiology, University of Southern Denmark/Odense University.
Abstract:
The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57x129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 +/- 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 +/- 4.6 and 9.2 +/- 3.4 cells/mm2, respectively) and <2 cells/mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57B1/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.
Insights
Tumor necrosis factor (TNF) production in brain ischemia is primarily by microglia/macrophages. This occurs within a specific time frame after stroke, peaking at 12 hours post-ischemia.
Area of Science:
- Neuroscience
- Immunology
Background:
- Brain ischemia triggers inflammation, with tumor necrosis factor (TNF) implicated.
- The precise cellular sources and timing of TNF expression in ischemic brain tissue remain unclear.
Purpose of the Study:
- To investigate the cellular localization and temporal dynamics of TNF mRNA expression following focal cerebral ischemia in mice.
- To determine the cell types responsible for TNF production in the ischemic brain.
Main Methods:
- Nonradioactive in situ hybridization for TNF mRNA on mouse brain sections at multiple time points post-ischemia.
- Quantification of TNF mRNA-expressing cells and infarct volume using Cast-Grid analysis.
- Colocalization studies using immunohistochemistry for microglia/macrophages (Mac-1), neutrophils (Ly-6G), and astrocytes (GFAP).
Main Results:
- TNF mRNA expression peaked at 12 hours post-ischemia (19.2 cells/mm2), significantly decreasing thereafter.
- TNF mRNA was predominantly found in microglia/macrophages (Mac-1 positive) within the infarct and surrounding areas.
- No TNF mRNA expression was detected in neutrophils or astrocytes at the examined time points.
- Bioactive TNF protein was confirmed in the ischemic neocortex.
Conclusions:
- Microglia/macrophages are the primary source of TNF production in the early stages of focal cerebral ischemia in mice.
- TNF expression is transient, with a distinct temporal window of production peaking around 12 hours post-ischemia.
