Extravascular CX3CR1+ cells extend intravascular dendritic processes into intact central nervous system vessel lumen

Abstract

Within the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+ APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+ cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.

Figure 1

Intravital microscopy reveals persistent intravascular CX3CR1⁺ dendritic projections into intact CNS vessels. A–C: A low-power snapshot from Supplementary Movie 1 is shown in image A, demonstrating the overall distribution and ramified cellular morphology of CX3CR1⁺ cells (green) in the CNS parenchyma of a Cx3cr1^+/GFP mouse through a cranial window. Scale bar = 100 pun. A few CX3CR1⁺ cells are found in close proximity to the blood vessel (red) (B–D, Supplementary Movie 2). B: Fluorescence and 3D surface rendering of a snapshot from inset in image A at time = 0 min of Supplementary Movie 2, demonstrating intravascular dendritic insertion (white arrows) of extravascular CX3CR1⁺ cells (green) through an intact CNS vessel (red). Scale bar = 15 µm. C: Fluorescence and 3D surface rendering of the same cells as in image B at time = 45 min, showing persistence of intraluminal dendritic insertions (white arrows) over the duration of the imaging session. Projections from two extravascular CX3CR1 cells are seen touching each other inside the blood lumen. Scale bar = 15 µm. D: Extravascular CX3CR1⁺ cells with ramified morphology displayed projections (white arrows) into the vessels (red) on day 12 after EAE induction, while a nonramified, elongated perivascular CX3CR1⁺ cell nearby did not (asterisk) and were not included in the final analysis. Scale bar = 10 µm. E: The number of intraluminal projections (white arrows) by extracellular CX3CR1⁺ cells increases in the CNS of Cx3cr1^+/GFP mouse on day 5 after EAE induction. Scale bar = 10 µm. Only CX3CR1⁺ cells in the parenchyma were analyzed in images A–E (Supplementary Fig. 2).

Figure 2

IHC and EM confirmation of intravascular dendritic projections by CX3CR1⁺ cells into intact CNS vessels. A, B: IHC of fixed, noninflamed naive Cx3cr1^+/GFP brain tissues confirmed the presence of dendritic extensions (green) flanked by GFAP⁺ astrocyte end feet (pink) next to vessel lumen as outlined by laminin (blue). Scale bar = 5 µm. C, D: IHC of fixed, EAE-induced Cx3cr1^+/GFP tissues confirmed the presence of dendritic extensions (arrows) by extracellular parenchyma (P) CX3CR1⁺ cells (green) into the vessel lumen (L) as outlined by anti-CD31 (C, red) and tomato-lectin (D, red) staining. Scale bar = 10 µm. E, F: EM of fixed tissue sections of a noninflamed, naive brain from a Cx3cr1^+/GFP mouse confirmed the presence of CX3CR1⁺ dendritic processes (green, P) with intact endothelial endothelium (red, En) and basement membrane (yellow, B). The CX3CR1⁺ cell is in close contact with axons (purple, Ax) and astrocyte end feet (blue, A), demonstrating its parenchymal location in the brain. Gold particle: immuno-gold labeling for GFP (arrows, G). Magnification = 18,500×.

Figure 3

Increasing number of extravascular CX3CR1⁺ cells with intravascular dendritic projections in the brain during early EAE induction. A: A snapshot of vessel (red) within the CNS of a Thy-1-YFP-H × Cx3cr1^+/GFP mouse taken 4 days after cranial window implantation shows the steady-state distribution of CX3CR1⁺ cells (green) and intact neurons (yellow) within the CNS parenchyma, with the CX3CR1⁺ cells remaining in a ramified state. Scale bar = 50 µm. B, C: Snapshots of the mouse brain on days 0 (B) and 12 (C) after EAE induction show morphologic changes and an increase in the number of CX3CR1⁺ microglia (green). The blood vessels are outlined by the TRITC-dextran dye (red). Intraluminal portions of the CX3CR1⁺ cells are highlighted in gray. Projections (arrows) occur in both the large and small vessels but are more common in the smaller vessels. YFP axon signal is removed for ease of visualizing microglia. Scale bar = 50 µm. D, E: Coronal fluorescence (D) and surface rendering (E) view of a blood vessel (red) in image C demonstrates an intraluminal dendritic projection (white arrows) by an extravascular CX3CR1⁺ cell (green). Scale bar = 15 µm. F: The number of intraluminal projections is quantified and normalized to total vessel surface area (#Projections/mm²) over the course of EAE induction, showing a twofold increase in projection frequency over the first 12 days. Only CX3CR1⁺ cells in the parenchyma were analyzed (Supplementary Fig. 2). n.s. = not significant.

Figure 4

Increasing number of extravascular CX3CR1⁺ cells with intravascular dendritic projections in the spine during early EAE induction. A: A snapshot of the central dorsal spinal vein of a Thy-1-YFP-H × Cx3cr1^+/GFP mouse taken immediately after T10 laminectomy shows the steady-state distribution of CX3CR1⁺ cells (green) and intact axons in the spinal parenchyma (yellow), with the CX3CR1⁺ cells remaining in a ramified state. Scale bar = 50 µm. B, C: Snapshots of the spine on days 0 (B) and 12 (C) after EAE induction show morphologic changes and an increase in the number of CX3CR1⁺ cells (green). The blood vessels are outlined by TRITC-dextran dye (red). Intraluminal portions of the CX3CR1⁺ cells are highlighted in gray. Projections (arrows) occur in both the large and small vessels but are more common in the smaller vessels. YFP axon signal is removed for ease of visualizing microglia. Scale bar = 50 µm. D, E: Coronal fluorescence (D) and surface rendering (E) view of a blood vessel (red) in image C demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1⁺ cells (green). Scale bar = 15 µm. F: The number of intraluminal projections is quantified and normalized to total vessel surface area (# projections/mm²) over the course of EAE induction, showing a twofold increase in projection frequency over the first 12 days. Only CX3CR1⁺ cells in the parenchyma were analyzed (Supplementary Fig. 2).

Figure 5

Intravascular projections decrease and then recover in aseptic traumatic spinal cord injury and do not increase in the CNS tumor microenvironment. A, B: Snapshots from tile scan of the spinal cord dorsal columns at T10 on days 0 (A) and 8 (B) after crush injury (dashed box) shows intact dorsal vein (red) and CX3CR1⁺ cell (green) distribution. Intraluminal portions of the CX3CR1⁺ cells are highlighted in gray. The morphology of CX3CR1⁺ cells on day 8 appeared to be more rounded and less ramified. C, D: Coronal fluorescence (C) and surface rendering (D) view of a blood vessel (red) in image B demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1⁺ cell (green). E: Quantification of intravascular projections post crush injury showing a near-full recovery in projection numbers in 8 days. n.s. = not significant. F: A snapshot of the CNS tumor microenvironment 7 days after inoculation of MMl-DsRed into a Cx3cr1^+/GFP mouse shows local accumulation of CX3CR1⁺ cells and development of neo-vasculature. Intraluminal portions of the CX3CR1⁺ cells are highlighted in gray. MMl-DsRed signals are removed in images F–H for ease of visualizing CX3CR1⁺ cells. G, H: Fluorescence (G) and surface rendering (H) view of a blood vessel (red) in image F demonstrates intravascular dendritic projections (white arrows) by extravascular CX3CR1⁺ cells (green). I: The number of intravascular projections in the CNS tumor microenvironment is quantified from three tumor-bearing mice and normalized to total vessel surface area, showing an average number of 72.9 ± 6.3 projections/mm² (same as baseline in Figs. 3F and 4F). Only CX3CR1⁺ cells in the parenchyma were analyzed (Supplementary Fig. 2).