Fig. 7: Visualization of CNS zonation created by leptomeningeal barriers in healthy VE-cadherin GFP reporter mice in vivo.

A Representative images of the spinal cord window 2P-IVM imaging of a healthy VE-cadherin-GFP knock-in reporter mice. Before the spinal cord window preparation, a tracer-filled cannula was implanted into the cisterna magna. During 2P-IVM, the mice were infused with 2.5 µl of either 3 kDa, 10 or 40 kDa TRITC dextran, or TRITC BSA at a rate of 1 µl/min using a syringe pump. XY time-lapse sequence of a 400 µm × 400 μm scan field at a depth of 160–220 µm and 81–111 z-projections with 2 µm spacing were acquired for 45 mins (spinal cord). The dura mater is visible in blue due to the second-harmonic generation of the collagen type 1 fibers in the dura. Arachnoid mater (A.M.) and pia mater are visible in green due to the VE-cadherin-GFP expression. Infused tracer is seen in red. YZ MIP of the meningeal layers of the spinal cord of a healthy VE-cadherin GFP knock-in reporter mouse is shown. At 0 min, no tracer (red) was seen. At 45 mins, the 3KDa, 10 kDa, and 40 kDa TRITC dextran and TRITC BSA (red) crossed the pia mater but not the arachnoid mater. Yellow arrowheads highlight phagocytic cells in SAS that have taken up BSA. Images are representative of three independent experiments per tracer. B YZ MIP of the spinal cord meningeal layers of a VE-cadherin GFP knock-in reporter mouse showing the segmented volumes for longitudinal quantification of the fluorescence intensity of the cisterna magna injected TRITC tracers (magenta). Dura mater (dark blue) and subpial compartment (light blue) are segmented based on the second-harmonic generation signals. SAS (red) and spinal cord parenchyma (dark yellow) are segmented as cubes distributed in between the two VE-cadherin-GFP layers (green) or 20–50 µm under the VE-cadherin-GFP⁺ pia mater, respectively. C VE-cadherin-GFP knock-in mice were infused with 3-, 40-kDa-TRITC, and TRITC-BSA tracers into the cisterna magna and 2P-IVM was performed over time. Graphs show the longitudinal quantification of the mean fluorescence intensity of the injected tracer in the segmented volumes from the spinal cord meningeal cross-section shown in B. Data are normalized to the highest MFI value detected in the SAS after background signal subtraction. Background signal was determined as the average fluorescence signal measured in all segmented volumes prior to tracer injection. Each graph shows the full quantification of one mouse. Source data are provided as a Source Data file. D VE-cadherin-GFP mice were injected via the cisterna magna with 10-kDa-TRITC tracers and 2P-IVM was performed over time. Graphs show the longitudinal quantification of the mean fluorescence intensity of the injected tracer in the segmented volumes from the spinal cord meningeal cross-section shown in B. Data are normalized to the highest MFI value detected in the SAS after background signal substruction. Background signal was determined as the average fluorescence signal measured in all segmented volumes prior to tracer injection. Each graph shows the quantification of one individual mouse. Source data are provided as a Source Data file. E, F Graphs in D were combined after matching the dynamics of tracer arrival in the SAS of the field of view (FOV) (E). Area under the curve (AUC) was calculated in the different segmented volumes for the first 12 min after tracer arrival in the FOV. Data were pooled from three independent experiments and are shown as mean ± SD. Source data are provided as a Source Data file. F Data were pooled from three independent experiments and analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SD. Source data are provided as a Source Data file. DV dorsal vein, A.M. arachnoid mater.