Figure 1.
Detection of FITC-albumin leakage following experimental ischemic stroke.
(A) Double fluorescence labeling of laminin (color-coded in blue) and GFAP (red) in combination with applied FITC-albumin (green) reveals areas of ischemia-related BBB breakdown. By application of both, an antibody detecting astrocytes (GFAP, red) and an antibody for pan-laminin (blue) which visualizes vascular as well as glial basement membranes [47] the observed leakage can clearly be demonstrated to reach the brain parenchyma proper. Therefore, FITC-albumin is detectable within all the three compartments of the neurovascular unit, represented by the vascular wall (1st compartment), the perivascular space (2nd compartment) and the adjacent neuropil (3rd compartment), delineated by astocytic endfeet (red) forming the glia limitans [54]. (B) To prove specificity of the applied reagent used for conversion of extravasated FITC-albumin into a permanent labeling by DAB for ultrastructural analysis, we exemplarily performed control stainings on vibratome sections, which were not further processed for electron microscopy. The low magnification of a coronary section clearly demonstrates the specificity of the applied reagent and its reaction product (DAB, brown). These sections were counterstained with hemalaun (blue). Areas of FITC-albumin leakage are clearly confined to the striatum. Higher magnification reveals a general leakage of FITC-albumin into the neuropil (brown background). In perivascular and juxtavascular areas the DAB staining appears to be more intense. (C) To confine ultrastructural analysis to areas with BBB breakdown, we identified areas of FITC-albumin extravasation after embedding in resin on coated microscope slides. These areas were selectively processed for ultrastructural analysis by electron microscopy. In corresponding control areas no FITC-albumin extravasation was observed. Please note, the general brown tissue background is a consequence of the embedding procedure using osmium tetroxide and uranyl acetate, which clearly can be distinguished from DAB staining as indicated on the left.
Figure 2.
Expression of claudin-5 and occludin in areas of FITC-albumin extravasation.
Double fluorescence labeling of claudin-5 (blue) and occludin (red), both being transmembrane proteins critical for tight junction formation, demonstrates extravasation of FITC-albumin (green) in the vicinity of vessels expressing both markers. Please also note the presence of discontinuities in the staining pattern of control vessels with an ‘intact’ endothelial barrier (arrow heads).
Figure 3.
Ratio of occludin-positive vessels in areas of FITC-albumin leakage.
(A) Quantitative analysis of differences in the expression of occludin in areas of FITC-albumin extravasation and their corresponding control areas was performed using low power (10× objective) magnification. Here, laminin-immunolabeling revealed the total number of vessels, whereas occludin-immunoreactivity visualized a critical tight junction constituent. The ratio of occludin-positive vessels to the total number of vessels was determined in 5 animals. (B) The ratio of occludin-positive vessels to the total number of vessels did not differ significantly in areas of FITC-albumin leakage and their corresponding control areas. Bars represent means and added lines indicate standard errors.
Figure 4.
Vascular leakage in areas of ultrastructurally intact tight junctions.
(A) Ultrastructural analysis of a control area located on the contralateral hemisphere shows a smooth endothelial layer (E) with an intact tight junction complex (arrow). The surrounding basement membrane is clearly visible and the adjacent neuropil does not show any structural alterations. (B–D) In areas of FITC-albumin extravasation the tight junction complexes (arrows) regularly appear to be established. The adjacent neuropil often displays cellular edema and cellular debris. Extravasated FITC-albumin and its product of conversion (black DAB grains, arrow heads) can constantly be found in the adjacent brain parenchyma proper. E = endothelial cells; L = vascular lumen.
Figure 5.
Ultrastructural evidence for transcellular, not paracellular leakage.
(A) Vessels in control areas on the contralateral hemisphere do not show signs of a transcellular, vesicle-mediated extravasation of FITC-albumin. (B–D) In contrast to alterations within the belts of tight junctions, ultrastructural examination regularly revealed signs for a transcellular leakage of the tracer. The endothelial cytoplasm exhibits a remarkable increase in vesicle density (white arrows) across the whole vascular circumference. Again, tight junctions (black arrow) are found to be intact. DAB grains are indicated by arrow heads. Control = contralateral hemisphere, E = endothelial cells; L = vascular lumen.
Figure 6.
Evidence for leakage across structurally altered endothelium.
In addition to the observation of a dramatic increase of the vesicle density, we often observed a disintegration of the whole endothelial layer. In areas exhibiting BBB breakdown the cellular surface of the endothelium was frequently found to be ruffled or discontinuous. Therefore, the vascular wall was often shown to consist of endothelial debris and basement membranes, only (brackets). Thus, in contradiction to a variety of studies our data strongly suggest a transendothelial leakage pattern of affected vessels. DAB grains indicating extravasation of FITC-albumin are demarked by arrow heads. E = endothelial cells; L = vascular lumen; asterisk = cellular debris in the lumen of the vessel; arrow heads = DAB grains; arrow = tight junction.
Figure 7.
Evidence for FITC-albumin leakage across disintegrated endothelium in the early stroke phase.
Finally, the same pattern of extravasation and endothelial damage was found at 5 hours after ischemia induction (A–D), as shown at 25 hours before. While the tight junctions remain, the rest of the endothelial cells may be constituted of debris, only (A and B). Often, the vascular basement membrane is exposed to the vascular lumen (B). Furthermore, electron dense vesicles carrying FITC-albumin can often be observed in the endothelium and the adjacent basement membrane (C) where the content is found to be deployed (D). E = endothelial cells; L = vascular lumen; arrow heads = DAB-filled vesicles; arrow = tight junction.