Additional file 2: Figure S2. of Anticoagulation with warfarin and rivaroxaban ameliorates experimental autoimmune encephalomyelitis

Effects of rivaroxaban on the coagulation status of SJL/J mice. For anticoagulation with rivaroxaban, SJL/J mice were treated with 30 mg/kg rivaroxaban for 3 days every 12 h via oral gavage. One hour after the last application, the rivaroxaban plasma concentration (a) and the thrombin activity (b) were determined in treated animals and controls (n = 5 per group). To monitor the thrombin activity over time, 0, 1, 2, 3, and 4 h after the application, the thrombin activity was measured (c, n = 5 per time point). (DOC 68 kb

β-catenin transcriptional activity improves impedance of bEnd5 cells A–D.

<p>Transendothelial electrical resistance (TEER, top panels) and the corresponding capacitance (Ccl, bottom panels) of the endothelial monolayers at various times after they reached confluence. <b>A.</b> Representative TEER/Ccl measurement comparing MBMECs, bEnd5 and bEnd5 infected with LEFΔN-βCTA (βCTA-bEnd5), indicating that β-catenin transcriptional activation leads to increased electrical resistance in bEnd5 cells. Vertical line at 125 hours indicates media exchange and boxed insert shows magnification of the Ccl curves after the media exchange, highlighting pronounced lower values for the βCTA-bEnd5 compared to bEnd5 controls. <b>B.</b> Parental bEnd5 cells in comparison to the primary mouse MBMECs cells (n = 3). <b>C.</b> bEnd5 cells infected with lenti-LEFΔN-βCTA in comparison to the lenti-GFP control. <b>D.</b> bEnd5 cells treated with the GSK3α/β inhibitor 6-BIO (2.5 µM) in comparison to the DMSO-treated cells. <b>E.</b> bEnd5 cells co-cultured with astrocytes (+AC) in comparison to bEnd5 cell monocultures (−AC).</p

β-catenin transcriptional activity reduces dextran permeability of bEnd5 cells.

<p><b>A,B.</b> Endothelial monolayer permeability to FITC-labeled 38-kDa dextran, was measured as percentage (%) of relative fluorescence units (RFUs). <b>A.</b> bEnd5 cells infected with LEFΔN-βCTA in comparison to GFP as control. <b>B.</b> bEnd5 cells treated with Wnt3a conditioned medium (Wnt3aCM) in comparison to the control medium (controlCM). p values were obtained by a 2-tailed paired t-test (Graphpad Prism 5.0), using values from n = 3 independent experiments, and pairing for time points.</p

BBB endothelial signature genes.

<p>BBB endothelial signature genes.</p

LiCl treatment improves the BBB-specific phenotype of hCMEC/D3 cells.

<p><b>A.</b> Basal mRNA expression of the BBB endothelial cell-related genes in hCMEC/D3 cells (as indicated; +++: 20<Δ²-Ct<25; ++: 25<Δ²-Ct<30; +: 30<Δ²-Ct<35). (For details see statistical analysis paragraph in Materials and Methods). <b>B.</b> qRT-PCR analysis from hCMEC/D3 cells treated with 10 mM LiCl compared with untreated cells. The RNA level obtained from untreated cells was set to 1 and the ratio LiCl treated versus control is shown for each gene. * p<0.05. Cldn, Claudins; VE-cad, VE-cadherin; Abcb1b, multidrug resistance protein 1; Abcg2, ATP-binding cassette transporter G2; Slc2a1, Solute carrier family 2 (facilitated glucose transporter) 1. <b>C.</b> hCMEC/D3 cell permeability (Pe) to Lucifer Yellow. Cells were untreated (EBM2) or treated with controlCM, 50% Wnt 3aCM or 10 mM LiCl (Pe values normalized to EBM2 Pe = 1.7×10⁻³ cm/min). <b>D.</b> hCMEC/D3 cell permeability (Pe) to Lucifer Yellow. Cells were incubated with DMSO as control or 20 µM XAV939 (XAV), 10 mM LiCl, or 10 mM LiCl plus 20 µM XAV939 (LiCl+XAV; Pe values normalized to DMSO Pe = 1.65×10⁻³ cm/min). All cell treatments were performed for 6 days.</p

Box plots showing the genetic comparisons between immortalized mouse endothelial cell lines and primary brain microvascular endothelium as reference.

<p><b>A.</b> Global distribution of the BBB-specific genes in the bEnd5, H5V and lung cells in comparison with MBMECs (set to 1; dotted line). A non-parametric test (i.e., Wilcox test, alpha value set at 0.05) was used to determine whether or not the differences between these cell types are significant. *p<0.1; **p<0.05; n.s., not significant. <b>B.</b> Global distribution of the BBB-specific genes in the bEnd5 cell systems, compared with MBMECs (set to 1; dotted line). Various conditions were tested: conditioned medium from Wnt3a-transfected L-cells (Wnt3aCM) undiluted (pure) or diluted 1 to 3 in growing medium; two different commercial recombinant Wnt3a preparations (100 ng/ml; Peprotech and R&D); BIO and 6-BIO (2.5 µM). Undiluted Wnt3aCM treatment was for 3 days (first boxplot from the left) or 24 hours (second boxplot from the left), as all the other cell activations. β-CTA (cells infected with LEFΔN-βCTA) and +Astros (co-culture with astrocytes). No significance differences were detected between these conditions, although a negative trend was seen for R&D Wnt3a and BIO conditions, as median values are lower than 1.</p

Reporter-gene–positive neurons can be found in multiple areas of the brain.

<p>(A and B) β-galactosidase–positive cells in the SN/VTA that can be TH-positive dopaminergic neurons as well as TH-negative cells with neuronal morphology (C). (D–F) β-galactosidase–positive cells in the cortex are also positive for NeuN. (G–I) Overview of the dentate gyrus in the hippocampus with recombined neurons in the granular cell layer that are positive for the neuronal marker NeuN. (J and K) Recombined neurons after ECL in hippocampal areas CA2 and CA3 and nonneuronal GFAP-negative recombined cells at the lesion site (L). Scale bar, 100 µm (D, G, H, and J), 50 µm (A, E, K, and L), and 10 µm (B, C, F, and I).</p

Peripheral inflammation increases the number of recombined Purkinje neurons.

<p>The number of recombined Purkinje neurons is low in healthy animals (A) but increases dramatically after peripheral inflammatory conditions (B). Inflammatory injuries were induced by subcutaneous injection of LLC2s or peritonitis. Animals were analyzed 12 d after injection when tumors were formed. Peritonitis was induced by a single i.p. injection of thioglycolate broth (1 ml in 3% PBS). Mice with peritonitis and ECL were analyzed 4 d after injection. (C) Filled bars represent results from Vav-iCre and empty bars from Tie2-Cre reporter mice. The <i>p</i> values were calculated by two-tailed <i>t</i> test for groups with unequal variance. (D and E) We did not observe any recombined Purkinje neurons that were binucleated in either transgenic mouse line after induction of an inflammation. (F and G) Microglia (white arrows) were always negative for the marker gene in healthy animals as well as after an inflammation. (H and I) Transendothelial electrical resistance (TEER, top panel) decreases and the corresponding capacitance (Ccl, bottom panel) of the bEnd5 endothelial monolayers increases significantly 24 h and 48 h after addition of bone-marrow-derived EVs compared to conditioned medium supernatant after ultracentrifugation. Vertical line at 0 h indicates media exchange. Scale bar, 100 µm (A and B), 50 µm (D and F), 10 µm (E), and 5 µm (G).</p

Model for EV transfer of RNA from immune cells to the brain.

<p>Possible modes of RNA transfer from blood to brain: (A) EVs are released from blood cells into the blood stream, where they can cross the BBB and fuse with neurons. (B) Alternatively, leukocytes enter the brain and only EVs released in short distance to the target cell are able to bind and release their content. This direct signaling of immune cells to the brain is independent of microglia.</p

EVs containing Cre mRNA are sufficient to induce recombination in Purkinje neurons after intracerebellar injection <i>in vivo</i>.

<p>(A) EV preparations enriched for exosomes prepared from the peripheral blood and bone marrow of Vav-iCre mice were brought into the circulation by tail vein injection or were directly injected into the cerebellum. Injection of Cre RNA-containing EVs into tail veins did not lead to recombination events in the brain (<i>n</i> = 4). (B) β-galactosidase–positive Purkinje neuron in the cerebellum of a reporter mouse 4 d after intracerebellar injection of EVs. (C) Other reporter-gene–positive cells with a shape and size reminiscent of glial cells in proximity to the Purkinje cell layer. (D) Reporter-gene–positive cells displaying a microglia-like morphology. (E) Quantification of reporter-gene–expressing Purkinje neurons after intracerebellar injection of vesicle preparations from Vav-iCre–positive peripheral blood. Control mice (shaded part) were injected with 1 µl purified Cre-recombinase protein at 1 U/µl (light grey) or lysate prepared from Vav-iCre bone marrow (dark grey) and never showed any recombined cells. Scale bar, 50 µm (B and C) and 25 µm (D).</p