Extended Data Fig. 8: Inflammatory changes to the brain tissue during acute disease induced by transfer of T_β-syn cells.

a–d, T_β-syn cell infiltration into the brain leptomeninges is associated with BBB leakage. a, Representative T1-weighted MRI images highlighting the areas of Gd leakage in meninges and parietal cortex over an EAE course induced by T_β-syn or T_MBP cell transfer. b–d, MRI, intravital TPLSM and immunohistochemistry were performed in the same rat at the onset of T_β-syn-cell-induced disease. b, T1-weighted MRI images before and after Gd administration. c, TPLSM images. Left, infiltration pattern of T_β-syn cells (green) into the leptomeninges. Red, Texas red dextran-labelled vessels, macrophages. Right, colour-coded (HiLo LUT) images highlighting the areas of dye leakage (arrows). Saturated signal, red; no signal, blue; circles, meningeal phagocytes that took up the dye during the recording time. Shown are tile-scan overview images (top) and magnified views of selected areas (bottom). d, Overview fluorescence image of a sagittal brain section (top) and magnified view of a selected cortical area (bottom) stained for ED1. Note the presence of high numbers of recruited ED1⁺ myeloid cells (purple) in meninges and adjacent cortex. e–h, Migration behaviour of T_β-syn cells in the cortical grey matter. Intravital TPLSM recording performed at the peak of T_β-syn cell cortical infiltration reveals a vigorous migratory activity of the T_β-syn cells within the compact grey matter. e, The 30-min time-lapse trajectories. f, Number of infiltrating T_β-syn cells. Data are mean + s.e.m., n = 3. g, T_β-syn cell velocity (n = 340 cells). h, Superimposed trajectories over a 30-min time span recording. Σ, sum of all cell trajectory vectors divided by the number of cells (n = 30 cells, each line represents a cell). i–k, T_β-syn cell infiltration induces neuronal damage. Histological and electron microscope analyses at the peak of T_β-syn cell infiltration. i, Relative changes in spine density on apical dendrites (cortical layer 2/3) measured by confocal microscopy in brain slices of rats transferred with the indicated antigen-specific T cells. Each dot represents a separate rat. Data are mean + s.e.m. j, Electron micrograph of an apoptotic neuron (highlighted in red, magnified view shown on the right) with a pyknotic and fragmented nucleus (asterisks) next to an intact neuron (green) with euchromatic nucleus. k, Apoptotic pyramidal neuron (N) with numerous apoptotic bodies (i) and pyknotic nucleus (ii) shown at higher magnification. Note the dendritic process (D) that emerges from the strongly altered cell body. l, m, T_β-syn cell infiltration induces microglial activation. l, Anti-IBA1 staining of cortical tissue performed at the indicated time points after T_β-syn cell transfer. Overviews pictures with magnified areas and representative 3D-reconstructed images of IBA1⁺ microglial cells (shown in greyscale). m, Quantification of the morphological parameters of IBA1⁺ microglial cells. Data are mean + s.d. i, Mann–Whitney U-test. m, One-way ANOVA with Dunnett’s multiple comparison correction for process length and segments and two-way ANOVA with Sidak correction for multiple comparisons for number of intersections. **P < 0.01, ***P < 0.001, ****P < 0.0001.