Table 1.
Inflammatory mediators associated with gene signaling in endothelial cells and brain capillaries exposed to tau proteins.
Transcriptomic analysis of endothelial cells from in vitro BBB model and isolated capillaries from brainstem of transgenic rats (SHR72) and control animals. RT-PCR reactions were run in triplicate with Actb and Rplp1 used as the reference genes. Minimum fold change was set at ≥ 2, ≤ -2.
Fig 1.
CD3+ T-cell and CD4+ T-cell occurrence in SHR-72 transgenic rat model for tauopathies compare to control animals.
(A) CD4+ T-cell in SHR rats. (B) CD4+ T-cell in SHR-72 rats. (C) Quantification of CD4+ T-cell in control and transgenic animals. (D) CD3+ T-cell in SHR rats. (E) CD3+ T-cell in SHR-72 rats. (F) Quantification of CD3+ T-cell in control and transgenic animals. Scale bar 20 μm, n = 5.
Fig 2.
Increased CD3+ and CD4+ T-cell occurrence in the brainstem of control animals.
(A-D; white arrow) Immunofluorescence staining showed more parenchymal than perivascular CD4+ T-cells in control animals. (E- H; white arrow) Immunofluorescence staining showed brain parenchyma infiltrating CD4+ T-cells in control animals, high magnification. (I-L) Immunofluorescence staining did not show the presence of parenchymal CD3+ T-cells in control animals. (M-P) Immunofluorescence staining did not show the presence of parenchymal CD3+ T-cells in control animals, high magnification. (R) Quantification of intravascular, extravascular and parenchymal CD4+ and CD3+ T-cells. Scale bar 50 μm, (DAPI: blue color; pan- laminin: red color; CD4+marker: green, CD3+ marker: green), n = 7.
Fig 3.
Increased CD3+ and CD4+ T-cell occurrence in the brainstem of SHR-72 transgenic rat model for tauopathies.
(A-D) Immunofluorescence staining showed CD4+ T-cells in SHR-72 transgenic animals. (E- H) Immunofluorescence staining showed more perivascular than brain parenchyma infiltrating CD4+ T-cells in SHR-72 transgenic animals. (I-L) Immunofluorescence staining showed CD3+ T-cells in SHR-72 transgenic animals. (M-P; white arrow) Immunofluorescence staining showed mainly intravascular CD3+ T-cells in SHR-72 transgenic animals. (R; white arrow) Presence of CD4+ extravascular and parenchymal T- cells, high magnification. (S) Presence of CD3+ intravascular T- cells, high magnification. (T) Quantification of intravascular, extravascular and parenchymal CD4+ and CD3+ T-cells. Scale bar 50 μm, (DAPI: blue color; pan- laminin: red color; CD4+marker: green, CD3+ marker: green), n = 7.
Fig 4.
Active infiltration of CFDA—labeled PB-MoM cells into the area with neurofibrillary pathology.
(A) CFDA- labeled PB-MoM cells in control SHR brain tissue (brainstem) after 48 hours. (B) CFDA- labeled PB-MoM cells in transgenic SHR-72 brain tissue (brainstem) after 48 hours. (C) CFDA- labeled PB-MoM cells in control SHR brain tissue after 72 hours. (D) CFDA- labeled PB-MoM cells in transgenic SHR-72 brain tissue after 72 hours. (E) Quantification of CFDA- labeled PB-MoM cells in the brainstem of control and transgenic rats after 48 and 72 hours. (F, G) Double immunostaining with pan-laminin showed the presence of intravascular CFDA- labeled cells. Scale bar: 100 μm and 20 μm, n = 4.
Fig 5.
Distribution of CFDA-positive cells.
CFDA-positive cells were distributed predominantly in the brainstem and middle brain of transgenic rats, areas with neurofibrillary pathology. (A, B) CFDA-positive PB-MoM cells in control and transgenic rats. In SHR72 animals CFDA-positive cells colocalized with CD11b. (C, D) Infiltration of CFDA positive PB-MoM cells in areas with increased immunostaining for ICAM-1. Scale bar: 100 μm.
Fig 6.
Expression of adhesion molecules is localized in the brain area affected by neurofibrillary pathology.
Immunostaining of adhesion proteins ICAM-1, VCAM-1 and selectins in transgenic and control rats. Confocal microscopy showed that brain capillaries stained with ICAM-1, VCAM-1 and selectins antibodies (red color) were distributed throughout the brain affected by neurofibrillary lesions immunolabelled with pThr212 and pSer202/pThr205 (AT8, green color). Low or no signal was detected in the brainstem of control rats and frontal cortex of control and transgenic rats. (A) Presence of ICAM-1 in the brainstem of a control subject, (B) increased expression of ICAM-1 in the brainstem of transgenic rats, (C) presence of ICAM-1 in the frontal cortex of control subject, (D) expression of ICAM-1 in the frontal cortex of transgenic rats. (R) Quantification of ICAM-1 expression. (E) Presence of VCAM-1 in the brainstem of control animal, (F) in the brainstem of the transgenic animal, (G) in the frontal cortex of control animals and (H) in the frontal cortex of transgenic rats (S) with the following quantification. (I) Presence of Selp in the brainstem of controls and (J) in the brainstem of transgenic rats. (K) Presence of selp in the frontal cortex of control and (L) transgenic rats. (T) Quantification of selp immunoreactivity in control and transgenic animals. (M) Presence of Sele in the brainstem of controls and (N) transgenic rats. (O) Presence of Sele in the frontal cortex of controls and (P) transgenic animals. (U) Quantification of Sele immunoreactivity in control and transgenic animals. Scale bar 20 μm, n = 10.
Fig 7.
Expression of adhesions molecules correlates with the presence of neurofibrillary pathology.
(A) Correlation between ICAM-1 immunoreactivity and the presence of neurofibrillary pathology (pThr212). (B) Correlation between VCAM-1 immunoreactivity and the presence of neurofibrillary pathology (pThr212). (C) Correlation between Sele immunoreactivity and neurofibrillary pathology (AT8) and (D) Selp immunoreactivity and neurofibrillary pathology (AT8).
Fig 8.
Ultrastructural analysis of brain microvessels from control and SHR-72 transgenic animals.
(A) Representative brain capillaries from the brainstem of SHR control animals were characterized by normal ultrastructural appearance consisting of smooth luminal surface of endothelial cells (EC), long tight junctions (TJ) and continuous basement membrane (BM). (B) Pericyte (P) with well-defined endothelial cell nuclei surrounded by BM and normal astrocyte. (C) Represented a detailed picture of a continuous BM and (D) showed EC, closed TJ and axons. (E-H) Representative brain capillaries from the brainstem of SHR-72 transgenic animals were characterized by the wavy shape of the luminal surface with numerous processes, which clearly distinguish EC from a transgenic animal from the EC in control animals. (E) The BM is uneven (thicker/thinner), and the shape of TJ is irregular. (F) thick and uneven BM. (G) Represented a detailed picture of the discontinuity of BM and deformed TJ. (H) Electron micrograph showing oedematous astrocyte (black arrow). Scale bar 1 μm.
Fig 9.
Oligomeric tau and PHF-tau stimulate PB-MoM transmigration across the in vitro BBB model.
Effect of the interaction of oligomeric tau and PHF-tau with rat primary mixed glial cells on the transmigration of peripheral blood cells across in vitro BBB model. (A) After 24 hours, CD45 positive transmigrated peripheral blood cells were stained and examined by immunocytochemistry in control conditions, (B) after stimulation with oligomeric tau, (C) rat PHF-tau and (D) human PHF-tau and (E) quantified. Scale bar 20 μm. Data are means ± SD of individual experiments run in sixplicate, n = 30. Differences at p<0.05 were accepted as statistically significant.
Fig 10.
Involvement of adhesion molecules ICAM-1 and VCAM-1 in tau-mediated transendothelial migration of PB-MoM.
We test the effect of antibodies ICAM-1 and VCAM-1 on transendothelial migration of PB-MoM cells across in vitro BBB model. The graph showed a decline in amount-percentage of transmigrated cells after antibody inhibition. Data are means ± SD of individual experiments run in six plicate, n = 30. Differences at p<0.05 were accepted as statistically significant.