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Updated: May 13, 2026
Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Published on: February 12, 2016
CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice
Stefano Fumagalli1, Carlo Perego, Fabrizio Ortolano
1Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy.
Abstract:
The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30' MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1(-/-) (26.42 ± 7.41 mm(3) , mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1(-/+) (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two-photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1(-/-) and cx3cr1(-/+) mice throughout the study. In cx3cr1(-/-) mice, they displayed a significantly higher number of ramifications >10 μm at baseline and at 24 h after ischemia compared to cx3cr1(-/+) mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1(-/-) immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45(high) (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1(-/-) mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.
Insights
Deleting the fractalkine receptor CX3CR1 reduced brain infarct size in mice after stroke. This CX3CR1 deletion also altered microglia morphology and promoted a protective M2 polarization, suggesting a potential therapeutic target for stroke.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Fractalkine (CX3CL1) and its receptor CX3CR1 play complex roles in neurological disorders.
- Previous studies on CX3CR1 in neurological conditions have produced conflicting findings.
- Understanding CX3CR1's role in ischemic stroke is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the impact of CX3CR1 deletion on brain injury following ischemic stroke.
- To analyze microglia dynamics, morphology, and activation states in the absence of CX3CR1.
- To determine the effect of CX3CR1 deficiency on microglia/macrophage polarization after stroke.
Main Methods:
- Used a transient middle cerebral artery occlusion (MCAo) model in mice lacking CX3CR1 (cx3cr1(-/-)) and compared them to wild-type and heterozygous mice.
- Employed in vivo two-photon microscopy for longitudinal analysis of microglia behavior before and after ischemia.
- Conducted post-mortem immunohistochemistry to quantify markers of microglia/macrophage activation and polarization.
Main Results:
- Mice lacking CX3CR1 exhibited significantly smaller brain infarct volumes compared to control groups.
- CX3CR1 deficiency altered microglia morphology, with fewer hypertrophic/amoeboid forms observed.
- Post-ischemic analysis revealed decreased markers of M/M activation (CD11b, CD68) and M1 polarization (iNOS), alongside increased M2 polarization markers (Ym1) and leukocyte recruitment (CD45high) in cx3cr1(-/-) mice.
Conclusions:
- CX3CR1 deletion confers protection against ischemic brain injury in the early stages post-stroke.
- The protective effect is associated with altered microglia morphology and a shift towards an anti-inflammatory M2 polarization profile.
- Targeting the CX3CR1 pathway may represent a novel therapeutic strategy for mitigating ischemic stroke damage.
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