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Published on: June 13, 2020
Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo
Eric B Miller1, Sarah J Karlen2, Kaitryn E Ronning1
1Center for Neuroscience, University of California, 1544 Newton Court, Davis, CA, 95618, USA.
Background:
The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging.
Methods:
We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo.
Results:
Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration.
Conclusions:
The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.
Insights
Researchers developed a new mouse model (Cx3cr1-Dendra2) to track immune cells like microglia and monocytes in the brain. This tool allows unambiguous, long-term in vivo tracking, aiding neuroinflammation research.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Tracking individual immune cells in the central nervous system (CNS) is crucial for understanding their roles in brain health and disease.
- Distinguishing between similar immune cell subpopulations in the dynamic CNS environment remains a significant challenge.
Purpose of the Study:
- To develop a novel method for unambiguous, long-term in vivo tracking of microglia and monocytes within the CNS.
- To create a tool that facilitates the study of distinct immune cell roles in neuroinflammation and neurodegenerative diseases.
Main Methods:
- Recombineering a photoconvertible fluorescent protein (Dendra2) downstream of the Cx3cr1 promoter in mice (Cx3cr1-Dendra2).
- Noninvasive in vivo imaging and photoconversion of Dendra2-expressing cells from green to red fluorescence.
- Utilizing Optical Coherence Tomography (OCT) for simultaneous imaging and light scattering measurements.
Main Results:
- Successful noninvasive photoconversion of Dendra2-expressing microglia in both ex vivo and in vivo settings.
- Robust D2 photoconversion enabling cell subpopulation quantification via flow cytometry and immunohistochemistry.
- Demonstrated ability to track microglial responses to neuronal damage and monocyte infiltration during neurodegeneration.
Conclusions:
- The Cx3cr1-Dendra2 mouse model provides a powerful new tool for in vivo tracking of microglia and monocytes.
- This model enables unambiguous spatial and temporal tracking of immune cells, crucial for studying neuroinflammation.
- Facilitates disentangling the roles of specific immune cell subpopulations in CNS disorders.
