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Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia
Published on: December 28, 2014
Flow cytometric analysis of inflammatory cells in ischemic rat brain
Marilena Campanella1, Clara Sciorati, Glauco Tarozzo
1Schering-Plough Research Institute, Milan, Italy.
Insights
Researchers developed a flow cytometry method to quantify inflammatory cells in ischemic rat brains. This technique reliably measures neuroinflammation in experimental models of cerebral ischemia.
Area of Science:
- Neuroscience
- Immunology
- Biomedical Engineering
Background:
- Cerebral ischemia involves inflammation driven by microglia activation and leukocyte infiltration.
- Flow cytometry is ideal for analyzing inflammatory cells but hasn't been applied to ischemic brain tissue.
Purpose of the Study:
- Establish a flow cytometry method for measuring inflammatory cells in ischemic brain tissue.
- Quantify inflammatory cell changes in a rat model of cerebral ischemia.
Main Methods:
- Developed two cell-isolation techniques using mechanical dissociation and Percoll gradient separation.
- Applied methods to a rat model of permanent middle cerebral artery occlusion.
- Used morphological and immunophenotypic analyses (CD11b, CD45, T-cell receptor antibodies) to identify and quantify cells.
Main Results:
- Both isolation methods yielded consistent and reproducible results.
- Identified a cell-scatter gate (R1a) enriched in inflammatory cells (granulocytes, macrophages, lymphocytes).
- Observed significantly higher numbers of inflammatory cells in the ischemic hemisphere compared to the non-ischemic hemisphere.
Conclusions:
- Quantitative flow cytometry is a feasible and reliable method for analyzing neuroinflammation in experimental cerebral ischemia.
- This technique offers a rapid assay for assessing inflammatory responses in ischemic brain models.
Background And Purpose:
Inflammation plays a key role in cerebral ischemia through activation of microglia and infiltration by leukocytes. Flow cytometry is a well-established method for quantitative and qualitative analysis of inflammatory cells. However, this technique has not been applied to the study of cerebral ischemia inflammation. The aim of this study was to establish a flow cytometric method to measure inflammatory cells in ischemic brain.
Methods:
To perform flow cytometry on brain tissue, we developed 2 cell-isolation methods based on different mechanical dissociation and Percoll gradient separation techniques. The methods were tested on a rat model of permanent middle cerebral artery occlusion. Morphological and immunophenotypic analyses, with the use of anti-CD11b, anti-CD45, and alphabeta T-cell receptor antibodies, were employed to identify and quantify inflammatory cells.
Results:
Both methods gave consistent results in terms of yield and reproducibility. The cell suspension contained granulocytes, macrophages, lymphocytes, and neural cells. Morphological and immunophenotypic analyses enabled the identification of a cell-scatter gate (R1a) enriched in inflammatory cells. With both methods, a higher number of events in R1a were recorded in the ischemic hemisphere than in the nonischemic hemisphere (P< or =0.001). CD11b, CD45, and alphabeta T-cell receptor staining confirmed that this augmentation was a reflection of the increase in the number of granulocytes, cells of the monocytic lineage, and lymphocytes.
Conclusions:
Quantitative flow cytometric analysis of ischemic rat brain is feasible and provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia.
