Fig. 10: Aqp4-mRuby3 reporter mice allow distinction between CNS border patrolling from CNS infiltrating CD8 T cells in CNS immune surveillance and neuroinflammation.
From: In vivo imaging of the barrier properties of the glia limitans during health and neuroinflammation
a Female mice received an intravenous injection of 2 × 105 naïve GFP+ OT-I T cells followed one day later by a peripheral infection with LCMV-OVA. On day 7 after infection, 2P-IVM of cervical spinal cord window was performed. Aqp4-mRuby3 reporter mice (b–d) or CNS border reporter; ODC-OVA mice (e–g) were used. b–g Representative XY MIP (b, e) and YZ MIP (c, f) images of the spinal cord surface are shown. CD8 T cells (green) can be distinguished above the glia limitans (arrowheads) and below (arrows). 4 mice were analyzed per condition from 1 CD8 T cell-mediated model induction. d, g YZ MIP zoom-ins of yellow boxed areas in c, f showing the fluorescence signals (top panel) and their segmented surfaces (bottom panel) rendered with Imaris 9.8 software. b–d Data from overlaying images acquired with 2P excitation wavelengths of 980 nm and 860 nm. The dura mater and subpial collagen are visible in blue due to SHG of the collagen type I fibers. The glia limitans is visible in red, vessels were labeled by intra-carotid injection of an Alexa Fluor (AF)−633-conjugated anti-endoglin antibody (white), GFP+ OT-I CD8 T cells are visible in green. e–g Data acquired with 2P excitation wavelengths of 920 nm and 1045 nm. The glia limitans is visible in red. h Quantification of cells per FOV above or below the glia limitans during immune surveillance (b–d) or neuroinflammation (e–g) conditions. Data representative of 4 mice are shown as mean ± SD and were analyzed using a two-way ANOVA with Fisher´s LSD multiple comparisons. i–k Violin plots of the displacement (i), crawling speed (j), and directionality (k) of OT-I CD8 T cells. A minimum of 30 cells per compartment were analyzed from 4 CNS border reporter (immune surveillance) and 3 CNS border reporter; ODC-OVA mice (neuroinflammation). Manual cell tracking was performed with Imaris 9.8 software. Data were analyzed using a two-way ANOVA with Fisher´s LSD multiple comparisons. Source data are provided as a Source Data file. DV dorsal vein, MIP maximum intensity projection, SHG second harmonic generation, SAS subarachnoid space, GL glia limitans.
