Fig. 8: Visualization of CNS zonation in CNS border reporter mice in vivo.
From: In vivo imaging of the barrier properties of the glia limitans during health and neuroinflammation
Representative 2P-IVM images of the brain surface through a thinned skull of Aqp4-mRuby3 (a) and CNS border reporter mice (b, c), and of the spinal cord in CNS border reporter mice (d–g). a During 2P-IVM 10 kDa-FITC dextran (green) was infused into the cisterna magna (i.c.m) and an anti-endoglin-AF633 antibody (white) injected via a carotid artery catheter to label the vasculature lumen. AQP4-mRuby3 depicting the glia limitans visible in red. Data were obtained by overlaying images with λex1 = 860 nm and λex2 = 980 nm. XY MIP at 24 minutes after i.c.m tracer infusion. YZ MIP from area between dashed lines in XY MIP. Data is representative of 2 mice. b–h A mix of 2.5 µL of 10 kDa dextran-FITC and PEGylated Flash Red beads (1 µm) was injected i.c.m at 1 µL/min during 2P-IVM. Time-lapse sequences were acquired for at least 60 min with λex = 920 nm and 1045 nm. SHG showing the dura mater and skull bone is visible in blue (λex1 = 920 nm) or green (λex2 = 1045 nm). b, c YZ MIP of a mouse skull thinning preparation, b before infusion, c: after infusion: visualization of beads (white arrowheads) and tracer (green arrowheads) arriving in the subarachnoid space (SAS) at indicated times. d–f YZ MIP of a spinal cord window preparation, d before infusion, e after infusion, visualization of the beads and tracer arriving in the SAS at indicated times. f Zoomed views from areas outlined in (e). g, h Longitudinal quantification of tracer mean fluorescence intensity (MFI) in the spinal cord. g shows volumes used for MFI measurement within the SAS (green squares) and parenchyma (white squares) in e. Graphs in (h) show the MFI of insets in g. Data from three mice were analyzed by a two-sided mixed-effects analysis with Šidák’s multiple comparisons and are shown as mean ± SD. P values denoted as * = 0.0163, ** = 0.004, *** = 0.0004, ****<0.0001. Source data provided as a Source Data file, including exact p-values. SAS subarachnoid space, GL glia limitans, Lepto leptomeninges, MIP maximum intensity projection, i.c.m intra-cisterna magna, SHG second harmonic generation.
