Fig. 9: CNS border reporter mice allow for precise assignment of 2D2 CD4 T cells to distinct CNS compartments during EAE.
From: In vivo imaging of the barrier properties of the glia limitans during health and neuroinflammation
a Female CNS border reporter mice received an intravenous injection of 1 × 105, 2 × 105, 0.5 × 106 or 1 × 106 naïve GFP+ 2D2 CD4 T cells at day −1 prior to induction of aEAE at day 0. 2P-IVM of the spinal cord was performed at peak of aEAE (15-22 days p.i.). b–f Representative images of 2P-IVM of the cervical spinal cord of mice following the protocol in (a). Data from overlayed images acquired at 920 and 1045 nm. The AQP4-mRuby3+ glia limitans is visible in red, endothelial and leptomeningeal adherens junctions are visible in green. 2D2 CD4 T cells are visualized by their GFP signal (green). SHG in blue from dura mater, trabeculae of the SAS and subpial collagen. b–f Overview of the spinal cord window of mice at peak of EAE (score 1) injected with 2-5 × 105 2D2 CD4 T cells (n = 2). c, d YZ MIP zoom-in depicting localization of CD4 T cells from (b), above (c) or below (d) the glia limitans. e YZ MIP of region from (b) showing CD4 T cells in the dura mater (triangle) or the SAS (arrows). f YZ MIP of a FOV including the dorsal vein (DV), showing CD4 T cells in the subpial space (triangle) and in the parenchyma (arrowheads). g Quantification of CD4 T cells per FOV in the parenchyma, blood vessels, subpial space, SAS or dura mater. One mouse each was injected at day −1 with 1 × 105, 2 × 105, and 1 × 106 GFP+ 2D2 CD4 T cells, respectively. Data imaged at day 16-18 p.i. are shown for each mouse, from 1 aEAE induction, as mean ± SD, where each point represents a FOV (n = 1). h Violin plots showing displacement, crawling speed, and directionality of CD4 T cells during the observation period of 10 minutes. A minimum of 30 cells per condition were analyzed from 3 mice at the peak of aEAE (score 2, day 15-22 p.i.) injected at day −1 with 0.5 × 106 GFP+ 2D2 CD4 T cells, from 1 aEAE induction. Manual cell tracking was performed with Imaris 9.8 software. Data were analyzed using a two-sided unpaired parametric t-test. Source data are provided as a Source Data file. i Confocal imaging of 20 µm thick skull cryosections from CNS border reporter mice at the peak of aEAE (n = 3) counterstained for laminin (magenta). Top panel shows the surface of the brain, where cells are found in the subpial space (arrowheads) or parenchyma (arrow). Bottom panel shows CD4 T cells within the CNS parenchyma (arrow) or in the perivascular space (triangle). PTx pertussis toxin, MIP maximum intensity projection, A.M arachnoid mater, SAS subarachnoid space, GL glia limitans.
