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Updated: May 5, 2026
Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Published on: February 12, 2016
Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia
Hannah X Chu1, Hyun Ah Kim1, Seyoung Lee1
1Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Abstract:
We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45(+high) cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8(+) T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4(+) T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.
Insights
Significant leukocyte infiltration occurs early in the brain after permanent focal ischemia. Neutrophils and other inflammatory cells rapidly enter the brain parenchyma, especially when reperfusion is absent.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Focal cerebral ischemia triggers inflammatory responses.
- Understanding leukocyte infiltration is crucial for developing stroke therapies.
Purpose of the Study:
- To investigate the extent and timing of leukocyte infiltration in a mouse model of permanent cerebral ischemia.
- To compare leukocyte infiltration in permanent versus transient middle cerebral artery occlusion (MCAO).
Main Methods:
- Permanent and transient middle cerebral artery occlusion (MCAO) models in C57BL6/J male mice.
- Flow cytometry and immunohistochemical analysis to quantify leukocyte populations (CD45(+high) cells) and their distribution.
- Assessment of various immune cell subtypes including neutrophils, monocytes, macrophages, and lymphocytes.
Main Results:
- Profound leukocyte infiltration (∼15,000 cells) observed in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO).
- Neutrophils were the predominant infiltrating cell type, with significant increases in lymphoid and myeloid cells.
- Transient MCAO (tMCAO) showed approximately 50% fewer infiltrating leukocytes compared to pMCAO at 24 hours.
- Leukocytes dispersed throughout the infarct parenchyma within 3 hours of pMCAO.
Conclusions:
- Early and significant infiltration of inflammatory cells occurs in the brain following focal ischemia, particularly in the absence of reperfusion.
- The timing and extent of leukocyte infiltration differ between permanent and transient ischemic events.
- These findings highlight the early inflammatory component of ischemic stroke.
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