Abstract
BackgroundThe 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.MethodsWe 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.ResultsDendra2-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.ConclusionsThe 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.
Highlights
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
Damage or disease can cause the recruitment of bone-marrow derived monocytes across the blood–retinal barrier, and these monocytes can assume morphologies and expression patterns that are similar to the native microglia yet have distinct cellular functions or susceptibilities to subsequent insults [1, 8,9,10,11]
Dendra2‐expressing retinal microglia can be efficiently photoconverted in vivo Cx3cr1D2/D2 mice were generated using CRIPSR/Cas9 to knock-in the Dendra2 (D2) coding sequence behind the Cx3cr1 promoter at the same location previously used to generate Cx3cr1GFP/Green fluorescent protein (GFP) mice [25]
Summary
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. Microglia are the long-lived, yolk-sac derived resident immune cells of the central nervous system (CNS) that prune developing circuits, maintain healthy synaptic contacts in adulthood, and phagocytose debris and pathogens [1,2,3]. Damage or disease can cause the recruitment of bone-marrow derived monocytes across the blood–retinal barrier, and these monocytes can assume morphologies and expression patterns that are similar to the native microglia yet have distinct cellular functions or susceptibilities to subsequent insults [1, 8,9,10,11]. D2 fluorescence acts as a stable, long-lived marker that allows cells to be noninvasively tagged and tracked over space and time
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