Related Experiment Video
Updated: May 3, 2026
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke
Zhiwei Tang, Yan Gan, Qiang Liu
1Department of Neurology and the BNI-ASU Center for Preclinical Imaging, Barrow Neurological Institute, St, Joseph's Hospital and Medical Center, 350 West Thomas Road, Phoenix, Arizona 85013, USA. Fu-Dong.Shi@dignityhealth.org.
Insights
Inhibition of CX3C chemokine receptor 1 (CX3CR1) signaling protects the brain from ischemic injury. This occurs by reducing peripheral macrophage recruitment and CNS microglia/macrophage activation, leading to improved neurological function.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Chemokine (C-X3-C motif) ligand 1 (CX3CL1)/CX3C chemokine receptor 1 (CX3CR1) signaling is crucial for neuron-microglia/macrophage communication in the central nervous system (CNS).
- CX3CR1 deficiency is linked to better outcomes after ischemic brain injury, but the underlying mechanisms remain unclear.
Purpose of the Study:
- To investigate how CX3CR1 deficiency influences microglia/macrophage functions in the context of protection following brain ischemia.
- To elucidate the role of CX3CR1 signaling in the cellular and inflammatory responses to ischemic stroke.
Main Methods:
- Transient middle cerebral artery occlusion (MCAO) model in wild-type (WT) and CX3CR1-deficient (CX3CR1⁻/⁻) mice.
- Assessment of infarct volume, neurological deficits, neuronal apoptosis, and reactive oxygen species (ROS) production.
- Analysis of microglia/macrophage activation, proliferation, polarization, and inflammatory cytokine release using immunohistochemistry, flow cytometry, and molecular techniques.
Main Results:
- CX3CR1⁻/⁻ mice showed significantly smaller infarcts and reduced neurological deficits post-MCAO compared to WT mice.
- Reduced neuronal apoptosis and lower ROS levels were observed in CX3CR1⁻/⁻ MCAO mice.
- Impaired CX3CR1 signaling led to decreased peripheral macrophage recruitment, suppressed microglia/macrophage proliferation, promoted M2 polarization, and attenuated inflammatory cytokine production.
Conclusions:
- Inhibition of CX3CR1 signaling offers neuroprotection in ischemic brain injury.
- Therapeutic strategies targeting CX3CR1 could reduce peripheral macrophage infiltration and CNS glial cell activation.
- Modulating CX3CR1 signaling presents a potential therapeutic approach for improving neurological function after stroke.
Background:
Chemokine (C-X3-C motif) ligand 1 (CX3CL1)/ CX3C chemokine receptor 1 (CX3CR1) signaling is important in modulating the communication between neurons and resident microglia/migrated macrophages in the central nervous system (CNS). Although CX3CR1 deficiency is associated with an improved outcome following ischemic brain injury, the mechanism of this observation is largely unknown. The aim of this study was to investigate how CX3CR1 deficiency influences microglia/macrophage functions in the context of its protection following brain ischemia.
Methods:
Wild-type (WT) and CX3CR1-deficient (CX3CR1⁻/⁻) mice were subjected to transient middle cerebral artery occlusion (MCAO) and reperfusion. The ischemic brain damage was monitored by rodent high-field magnetic resonance imaging. Neurological deficit was assessed daily. Neuronal apoptotic death and reactive oxygen species (ROS) production were analyzed by immunostaining and live imaging. Activation/inflammatory response of microglia/macrophage were assessed using immunohistochemistry, flow cytometry, 5-bromo-2-deoxyuridine labeling, cytokine ELISA, and real-time PCR.
Results:
CX3CR1⁻/⁻ mice displayed significantly smaller infarcts and less severe neurological deficits compared to WT controls, following MCAO. In addition, CX3CR1⁻/⁻ MCAO mice displayed fewer apoptotic neurons and reduced ROS levels. Impaired CX3CR1 signaling abrogated the recruitment of monocyte-derived macrophages from the periphery, suppressed the proliferation of CNS microglia and infiltrated macrophage, facilitated the alternative activation (M2 state) of microglia/macrophages, and attenuated their ability to synthesize and release inflammatory cytokines.
Conclusion:
Our results suggest that inhibition of CX3CR1 signaling could function as a therapeutic modality in ischemic brain injury, by reducing recruitment of peripheral macrophages and expansion/activation of CNS microglia and macrophages, resulting in protection of neurological function.
