Blood—Brain Barrier Breakdown Involves Four Distinct Stages of Vascular Damage in Various Models of Experimental Focal Cerebral Ischemia

Abstract

Ischemic stroke not only impairs neuronal function but also affects the cerebral vasculature as indicated by loss of blood—brain barrier (BBB) integrity. Therefore, therapeutical recanalization includes an enhanced risk for hemorrhagic transformation and bleeding, traditionally attributed to a ‘reperfusion injury’. To investigate the mechanisms underlying ischemia-/reperfusion-related BBB opening, we applied multiple immunofluorescence labeling and electron microscopy in a rat model of thromboembolic stroke as well as mouse models of permanent and transient focal cerebral ischemia. In these models, areas exhibiting BBB breakdown were identified by extravasation of intravenously administered fluorescein isothiocyanate (FITC)-albumin. After 24 hours, expression of markers for tight and adherens junctions in areas of FITC-albumin leakage consistently remained unaltered in the applied models. However, lectin staining with isolectin B4 indicated structural alterations in the endothelium, which were confirmed by electron microscopy. While ultrastructural alterations in endothelial cells did not differ between the applied models including the reperfusion scenario, we regularly identified vascular alterations, which we propose to reflect four distinct stages of BBB breakdown with ultimate loss of endothelial cells. Therefore, our data strongly suggest that ischemia-related BBB failure is predominantly caused by endothelial degeneration. Thus, protecting endothelial cells may represent a promising therapeutical approach in addition to the established recanalizing strategies.

Keywords

blood—brain barrier brain ischemia focal ischemia intracerebral hemorrhage neurovascular unit

Introduction

Ischemic stroke represents one of the leading causes of death world-wide and surviving patients often experience long-lasting disabilities. During the last decades, intensified research provided an improved understanding on stroke pathophysiology, although therapeutical options remain limited.^{1, 2, 3} As neuronal damage is primarily attributed to an acute occlusion of cerebral vessels, it is reasonable that ischemia affects not only the neuronal milieu, but also the function of the vasculature itself as indicated by the associated loss of blood—brain barrier (BBB) integrity.

In fact, the barrier function of vessels within the central nervous system is vulnerable as it equally depends on different cell types and signaling cascades, which together orchestrate the maintenance of the sensitive neuronal milieu under physiologic conditions. These neurovascular interdependencies are therefore reflected by the term NVU (neurovascular unit).^{4, 5} As part of the NVU, the endothelial layer and interendothelial belts of tight junctions (TJs) have been considered to be primarily responsible to regulate the permeability toward blood-sourced hydrophilic molecules into the brain parenchyma.^{6, 7, 8} On the molecular level, TJs consist of three transmembrane protein families, which comprise junction-associated proteins, claudins, and occludin all of which are linked to the cytoskeleton via zonula occludens (ZO) proteins.^{5, 9} As these sealing structures are responsible for the maintenance of the BBB under physiologic conditions, loss of BBB function after stroke or associated with reperfusion is consequently attributed to dysfunctional TJs leading to an increased permeability of affected vessels, although respective data are often derived from in vitro models.^{10, 11, 12, 13} However, using the translationally relevant thromboembolic stroke model in rats, our group has recently shown that ischemia-related BBB breakdown is predominantly a result of an endothelial cell damage while TJs proteins remained detectable by immunofluorescence labeling.¹⁴ This concept is further supported by recent data from other groups suggesting that BBB opening may also occur independently of altered TJs by an opening of endothelial connexin-43 hemichannels, which mediates endothelial swelling and cellular damage, thereby increasing BBB permeability.^{15, 16}

Nevertheless, the detailed mechanism underlying ischemia-related BBB breakdown in the clinical setting is poorly understood. In fact, loss of the barrier function as indicated by hemorrhagic transformation or even intracranial bleeding is frequently observed after therapeutical recanalization via recombinant tissue plasminogen activator (rt-PA), which still represents the only FDA-approved drug-related therapy.^{17, 18} These complications were traditionally attributed to a reperfusion-mediated vascular damage,^{19, 20, 21} which is commonly addressed as ‘reperfusion injury’.^{22, 23} As clinical treatment strategies tend to apply recanalization via rt-PA more frequently and in a broader time window from ischemia onset, knowledge of the underlying mechanisms for ischemia-related BBB breakdown, especially in a clinically relevant setting of reperfusion, may be of particular interest for the development of neuroprotective strategies in combination with rt-PA.

Therefore, in the present study, we applied three different rodent models of focal cerebral ischemia including a reperfusion scenario to investigate ischemia-related effects on the vasculature using multiple fluorescence labeling and electron microscopy.

Materials and methods

Study Setup and Content

Experiments involving animals were performed according to the European Communities Council Directive (86/609/EEC) after protocol approval by local authorities (Landesdirektion Leipzig, Germany, reference numbers TVV 24-10 and 02/09). Generally, efforts were made to minimize the total number and suffering of animals, which were housed at a temperature (21°C to 22°C) and humidity (45% to 60%) controlled room with 12 hours of light/dark cycle and free access to food and water.

Adult 129Sv/B6 mice (weighing about 25 g, bred by the Medizinisch-Experimentelles Zentrum, Leipzig, Germany) were subjected to right-sided middle cerebral artery occlusion (MCAO), while 10 mice were subjected to transient ischemia for 90 minutes (tMCAO) and 9 mice to permanent ischemia (pMCAO), as described below. Sufficient cerebral affection was ensured by functional assessment on the following day using a score in adaption to Menzies et al,²⁴ basically ranging from 0 (no apparent deficit) to 4 (spontaneous contralateral circling), whereas animals had to show a relevant deficit as indicated by a score of at least 2. Twenty-four hours after ischemia onset, fluorescein isothiocyanate (FITC)-albumin (2 mg diluted in 0.1 ml saline; Sigma, Taufkirchen, Germany) was intravenously administered to allow fluorescence-based localization of BBB breakdown as indicated by FITC-albumin leakage.²⁵ After a circulation period of usually 1 hour, animals were deeply anesthetized using a mixture of ketamine (150 mg/kg body weight intraperitoneally; Ketamin-ratiopharm, ratiopharm, Ulm, Germany) and xylazine (15 mg/kg body weight intraperitoneally; Rompun, Bayer, Leverkusen, Germany), followed by transcardial perfusion with saline for immunohistologic analyses by fluorescence microscopy (tMCAO, n=5; pMCAO, n=5), or saline and 4% paraformaldehyde (PFA) containing 0.5% glutaraldehyde (PFA-GA) for ultrastructural analyses by electron microscopy (tMCAO, n=5; pMCAO, n=4).

We further analyzed brain tissue from the translational relevant embolic model of right-sided middle cerebral artery occlusion (eMCAO) in Wistar rats (weighing about 300 g, bred by Charles River, Sulzfeld, Germany) as described below. Analogous to mice, rats underwent intravenous administration of FITC-albumin (20 mg diluted in 1 ml saline; Sigma) at 24 hours after ischemia induction with an additional circulation period of 1 hour before killing. Animals were finally anesthetized using CO₂ and were transcardially perfused with either saline for fluorescence microscopy (n=5), or saline and PFA-GA for electron microscopy (n=4).

Surgical Procedures for Ischemia Induction

The models of tMCAO and pMCAO in mice were performed using a filament-based setup originally described by Longa et al.²⁶ with minor modifications as reported earlier.²⁷ Briefly, a standardized silicon-coated 6-0 monofilament (Doccol Corporation, Redlands, CA, USA) was inserted into the right external or common carotid artery and moved forward into the internal carotid artery until bending was observed or resistance was felt. After a period of 90 minutes the filament was removed in the tMCAO group, and was left in place in the pMCAO group. Surgical procedures were generally performed in anesthetized mice using etomidate (33 mg/kg body weight intraperitoneally; Hypnomidate, Janssen-Cilag, Neuss, Germany), added by half/full dose for filament removal.

The model of eMCAO has been performed as reported earlier.¹⁴ Briefly, a polyethylene tube was introduced into the external carotid artery and moved forward through the internal carotid artery up to the origin of the middle cerebral artery. A blood clot with a medium length of 50 mm, prepared from blood collected in a polyethylene tube on the previous day, was injected with a small volume of saline. Finally, the catheter was removed and the external carotid artery stump ligated. Surgical procedures were performed in anesthesia using 2.0% to 2.5% isoflurane (Isofluran Baxter, Baxter, Unterschleißheim, Germany; mixture: 70% N₂O/30% O₂; vaporisator: VIP 3000, Matrix, New York, NY, USA).

During anesthesia, the body temperature was generally adjusted to 37.0°C by a thermostatically controlled warming pad with rectal probe (Fine Science Tools, Heidelberg, Germany). After surgery, animals spent further time on a warming pad until recovery.

Fluorescence Microscopy and Quantification

For histologic analysis by fluorescence microscopy, brains were removed from the skull and immediately snap frozen in isopentane on dry ice. Cyrostat sections of 10 μm thickness were prepared using a cryostat (Leica, Wetzlar, Germany). Before incubation with antibodies, the tissue was postfixed with zinc-formalin for 5 minutes at room temperature followed by blocking of unspecific binding of the applied antibodies using phosphate-buffered saline (PBS) containing 5% of normal goat serum and 0.3% of Triton X-100 for 20 minutes. For visualization of the endothelium, Alexa647-conjugated isolectin B4 (IB4, Griffonia simplicifolia agglutinin I-B₄, 1:100, Invitrogen, Carlsbad, CA, USA) was applied as a common marker targeting glycoproteins on the endothelial surface. Immunolabeling for albumin (rabbit anti-albumin, 1:200, Synaptic Systems, Göttingen, Germany) was applied to exclude an artificial pattern of extravasation for FITC-albumin. Further, primary antibodies directed against TJ proteins, i.e., mouse anti-occludin (1:200, antibodies-online, Aachen, Germany), guinea pig anti-occludin (1:200, Acris, Herford, Germany), rabbit anti-claudin 5 (1:200, Abcam, Cambridge, UK), rabbit anti-claudin 3 (Abcam) and rabbit anti-ZO 1 (1:200, Abcam), as well as the basement membrane marker rabbit anti-pan-laminin (1:200, Dako, Hamburg, Germany) were incubated overnight at 4°C in PBS containing 0.5% normal goat serum. Adherens junctions (AJs) were addressed using rat anti VE-cadherin (1:200, Abcam). Mouse anti microtubule-associated protein 2 (MAP2, 1:200, Merck-Millipore, Schwalbach, Germany), mouse anti heat shock protein 70 (HSP70, 1:200, Stressgen Biotechnologies, San Diego, CA, USA), and rabbit anti neuronal nuclei (NeuN, 1:200, Merck-Millipore) were used to correlate the extravasation pattern of FITC-albumin with differences in the level of affection throughout the regions of BBB breakdown. Fluorochrome-conjugated goat antibodies directed against the corresponding primary antibodies were kept on the tissue for 2 hours at room temperature in PBS containing 0.5% normal goat serum. After thorough rinsing in PBS, the sections were coverslipped with fluorescence mounting medium (Dako). Omitting primary antibodies served as a control, which resulted in the absence of staining. Sections were analyzed with Olympus fluorescence microscopes and images were acquired with the cellSens software (each Olympus, Hamburg, Germany).

For quantitative analysis, we calculated the ratio of TJ-positive vessels and the total number of vessels in distinct brain areas on consecutive sections (usually 700 to 900 measuring points per hemisphere of each animal, n=4). Therefore, we applied double fluorescence labeling for pan-laminin as a general marker of cerebral vessels²⁸ and for occludin as a typical constituent of endothelial TJs. To confirm exclusive analysis of areas depicting BBB breakdown, we focused on areas showing clear FITC-albumin extravasation into the neuropil and their corresponding areas on the contralateral hemisphere. Using low power (10x objective) magnification, we counted and compared the number of vessels positive for occludin with respect to the total number of laminin-immunopositive vessels, resulting in a ratio of occludin-positive vessels between areas of BBB breakdown and their control areas. Further, the total length of depicted laminin-immunopositive vessels was measured via ImageJ 1.48v software (NIH, USA) and correlated with the total length of occludin-positive TJ strands covering respective vessels. Thus, the ratio of the TJ length to the total vessel length was calculated and compared between areas of FITC-albumin extravasation and the respective contralateral control areas in the models of eMCAO, pMCAO, and tMCAO. Overall, analysis included 100 to 120 vessels of each animal analyzed (n=4 for each group).

Ultrastructural Analysis and Quantification

Brains for electron microscopy were also removed from the skull, post-fixed using PFA-GA and consecutively cut on a vibrating microtome (Leica) to obtain sections of 60 μm thickness in cooled PBS. After rinsing in Tris-buffered saline, the sections were incubated with peroxidase-conjugated anti-FITC IgG (1:2,000=0.5 μg/ml, Dianova, Hamburg, Germany) in Tris-buffered saline containing 1% BSA for 16 hours at 4°C. Subsequently, the tissue was stained with diaminobenzidine (DAB) to achieve a reaction product suitable for light and electron microscopy. After thorough rinsing and transfer into PBS, the sections were stained with 0.5% osmium tetroxide for 30 minutes followed by dehydration of the tissue in graded ethanol and a further staining step using 1% uranyl acetate. Afterwards, the sections were incubated in resin (Durcupan, Sigma-Aldrich, Steinheim, Germany) and embedded between coated microscope slides and coverslips to allow evaluation of the tissue with subsequent identification of areas exhibiting BBB leakage at the light microscopic level before transfer of the tissue onto blocks of resin and polymerization at 60°C for 48 hours. Finally, the respective areas were identified on the blocks and trimmed using a razor blade to prepare ultrathin sections of 55 nm thickness with a Leica ultramicrotome (Leica). Ultrathin sections were then transferred on formvar-coated grids and stained with lead citrate for 6 minutes. Ultrastructural analysis was performed using a Zeiss EM900 transmission electron microscope and a Zeiss SIGMA electron microscope equipped with a STEM detector (each Zeiss NTS, Oberkochen, Germany).

For quantification, the relative frequency of vessels matching either a normal phenotype or the different stages of vascular damage (for details see below) was calculated in contralateral control areas, in the border zone of detectable FITC-albumin leakage and areas of light microscopically evident FITC-albumin extravasation. Therefore, an average number of 75 vessels per area of each animal (225 vessels per animal) was analyzed by electron microscopy (n=3 for each group, 1,360 vessels in total).

Statistical Analyses

The obtained data were processed with SPSS 20 (IBM Corp., New York, NY, USA) and Graph Pad Prism 5.01v (GraphPad Software Inc., La Jolla, CA, USA) while using Mann—Whitney U and Kruskal—Wallis tests followed by Dunn's Multiple Comparison post hoc test to check for statistical significance between groups. In general, a P<0.05 was considered as statistically significant.

Results

Detection of Blood—Brain Barrier Breakdown by Fluorescence and Light Microscopy

Application of FITC-albumin to demark sites of BBB breakdown resulted in extravasation of the tracer, which was exclusively confined to ischemic brain areas after transient, permanent, and thromboembolic MCAO. Visualization of the endothelium by IB4 clearly indicated that leakage of FITC-albumin reached the adjacent neuropil in respective areas (Figure 1A). To rule out that the applied FITC-tagged albumin exhibits an artificial leakage pattern, we also used indirect, immunofluorescence labeling of intrinsic albumin to demark the areas of BBB breakdown. As expected, the counterstaining constantly revealed a colocalization of immunolabeled albumin and the applied FITC-albumin (Figure 1A).

Figure 1.

Focal cerebral ischemia leads to regional blood—brain barrier (BBB) breakdown as indicated by extravasation of fluorescein isothiocyanate (FITC)-albumin in different rodent models of stroke. Using fluorescence microscopy in combination with the endothelial marker isolectin B4 (IB4) revealed extravasation of intravenously administered FITC-albumin after thromboembolic (embolic model of right-sided middle cerebral artery occlusion (eMCAO), rat) as well as permanent (pCMAO, mice) and transient middle cerebral artery occlusion (tMCAO, mice). To rule out an artificial pattern of FITC-albumin, we applied indirect red fluorescent immunolabeling to demark intrinsic albumin, which regularly resulted in colocalization with the FITC signal (A). To identify areas of tracer extravasation for electron microscopy, peroxidase-conjugated anti-FITC IgG was used for conversion into an electron-dense precipitate with diaminobenzidine (DAB) as chromogen. Extravasation of FITC-albumin was consistently not observed on the contralateral, non-affected hemisphere. For illustration, sections were counterstained with hemalaun (B). Scale bar: 50 μm.

Further, immunohistochemical conversion via DAB allows clear-cut distinction of the area exhibiting BBB breakdown as indicated by DAB staining (Figure 1B). Importantly, extravasation of FITC-albumin was exclusively detectable in the ischemia-affected hemisphere in each of the applied models as indicated on hemalaun-stained coronary sections, which were not further processed for electron microscopy (Figure 1B). In fact, this nearly background-free demarcation of the areas exhibiting tracer extravasation via DAB is a prerequisite for the identification of affected areas at the ultrastructural level using electron microscopy.

Immunohistochemical Staining Patterns of Tight Junction Proteins Fail to Capture Blood—Brain Barrier Breakdown

In the past, the expression patterns of critical TJ proteins such as claudin 5, ZO 1, and occludin were often used to evaluate the integrity of the BBB in various models of in vitro and in vivo hypoxia and stroke.^{12, 13, 29} Since we have previously shown the lack of immunohistologically detectable alterations of the typical TJ protein occludin in a rat model of thromboembolic focal cerebral ischemia,¹⁴ immunofluorescence labeling for the essential TJ protein occludin was applied in brain sections of animals that underwent eMCAO (rats), pMCAO (mice), and tMCAO (mice) to rule out any functional differences between these models. Notably, in each of the applied models, occludin-positive TJ strands remained detectable in areas of tracer extravasation (exemplarily shown in Figure 2A). Moreover, by immunofluorescence labeling of occludin and laminin, the latter of which demarks all cerebral vessels irrespective of their position in the vascular tree, we were able to calculate the ratio of occludin-positive vessels in areas showing FITC-albumin extravasation and respective control areas. Importantly, the proportion of occludin-positive vessels in areas showing FITC-albumin extravasation and their corresponding areas on the contralateral hemisphere displayed a nearly identical ratio in all three models (Figure 2B), clearly failing statistical significance (P=0.248 between tMCAO and pMCAO, Mann—Whitney U test, each n=4; P=0.083 between all three groups, Kruskal—Wallis test, eMCAO n=4, t/pMCAO each n=4). In addition, we analyzed the total length of TJ strands covering the total laminin-positive vessel length of the respective vessels in areas of tracer extravasation and their corresponding control areas on the contralateral hemisphere in each of the applied models (Figure 2C). Of note, the comparison of the relative occludin-positive TJ length in relation to the total vascular length did neither reveal significant differences between control areas and areas of tracer extravasation in the respective models, nor in between the models of eMCAO, pMCAO, and tMCAO (P=0.0606, Kruskal—Wallis test followed by Dunn's Multiple Comparison post hoc test, each n=4).

Figure 2.

Staining patterns of occludin do not correlate with barrier function in models of thromboembolic (embolic model of right-sided middle cerebral artery occlusion (eMCAO)), permanent (pMCAO), and transient (tMCAO) middle cerebral artery occlusion. Immunohistologic staining for the essential tight junction (TJ) protein occludin in combination with antibodies for laminin to demark vascular basement membranes reveals that occludin remains detectable in areas of tracer extravasation in all the applied models (A). For quantification, double immunofluorescence labeling of laminin to demark cerebral vessels and occludin was applied (B) to calculate an interhemispheric ratio of occludin-positive vessels in areas of tracer extravasation and their respective control areas on the contralateral hemisphere. Thereby, the ratio of occludin-positive vessels did neither differ in between the analyzed hemispheres nor did the interhemispheric ratio differ in between the applied stroke models (B). Further, the occludin-positive TJ length was correlated with the total vascular length as indicated by laminin staining. Thereby, the relative TJ length was calculated and compared between areas of tracer extravasation and their control areas in all the applied models. Importantly, comparison of the relative TJ length did neither reveal significant differences between the control areas and areas of detectable BBB breakdown nor between the models applied (C). Scale bar: 20 μm.

To exclude detectable differences for other critical TJ proteins, further immunofluorescence-based analyses were performed addressing the proteins ZO 1, claudin 3, and claudin 5 in combination with occludin, all of which remained detectable in areas of massive tracer extravasation in each of the addressed models (Figure 3A). As BBB permeability is further known to be regulated by AJ,³⁰ we applied immunofluorescence labeling of the AJ-associated protein VE-cadherin in combination with claudin 3 in the mouse models of pMCAO and tMCAO. In line with our previous findings, the junctional marker VE-cadherin was clearly detectable in areas exhibiting ischemia-related BBB breakdown (Figure 3B).

Figure 3.

Crucial tight and adherens junction proteins remain detectable in areas displaying blood—brain barrier (BBB) breakdown in models of thromboembolic (embolic model of right-sided middle cerebral artery occlusion (eMCAO)), permanent (pMCAO), and transient (tMCAO) middle cerebral artery occlusion. In none of the applied models changes in the expression pattern of the crucial tight junction (TJ) proteins occludin, claudin 3, claudin 5, and zonula occludens (ZO) 1 were detectable in areas of tracer extravasation (A). Further, we analyzed the models of pMCAO and tMCAO for differences in the expression of the adherens junction marker VE-cadherin. Similar to the applied TJ markers, VE-cadherin was regularly detectable in areas of fluorescein isothiocyanate (FITC)-albumin leakage (B). Scale bars: 10 μm.

Therefore, the staining patterns for essential junctional proteins impressively, and model overlapping, fail to reflect functional alterations in BBB integrity after experimental focal cerebral ischemia.

Discontinuous Endothelial Layer Contributes to Blood—Brain Barrier Breakdown in Diverse Models of Focal Cerebral Ischemia

Since immunofluorescence labeling of TJ proteins failed to reflect endothelial integrity, we applied lectin staining with the endothelial surface marker Griffonia simplicifolia agglutinin IB4 in combination with laminin to demark vascular basement membranes to investigate the fate of endothelial cells at the sites of FITC-albumin extravasation in all three models of focal cerebral ischemia. Using fluorescence microscopy, we were thus able to show discontinuities in the staining pattern of IB4 reflecting structural alterations of the endothelial surface in ischemia-affected vessels. In contralateral control areas, IB4 regularly exhibits a uniform staining pattern for endothelial cells (Figure 4A), whereas affected vessels showing leakage of the tracer often displayed ruptures or even loss of the IB4 staining (arrowheads, Figures 4B and 4C). However, the expression of occludin regularly remained detectable within respective vessels in the applied models (Figure 4C). Thus, the observed BBB breakdown in the model of eMCAO, pMCAO, and tMCAO may rather be a result of an ischemia-induced endothelial damage than due to specific junctional alterations. Interestingly, the typical IB4 parenchymal staining pattern, which also allows concomitant detection of microglia, showed only in the model of eMCAO an enhanced microgliosis in the parenchyma surrounding the affected vasculature, while this effect was nearly absent in the models of pMCAO and tMCAO (Figures 4B and 4C). Thus, in contrast to the commonly applied labeling of TJ proteins, structural alterations within endothelial cells of vessels showing BBB breakdown can be displayed by IB4 staining.

Figure 4.

Staining with Griffonia simplicifolia agglutinin (isolectin B4, IB4) reveals structural alterations in the endothelial surface after experimental focal cerebral ischemia. On contralateral areas serving as controls, the luminal endothelial surface as demarked with isolectin B4 (IB4) was found to be smooth and continuous throughout the analyzed sections irrespective of the applied model, while extravasation of fluorescein isothiocyanate (FITC)-albumin was absent (A). In contrast, at the sites of FITC-albumin leakage, the endothelial staining pattern of IB4 frequently appeared to be irregular and discontinuous, which was interpreted as structural alterations affecting the endothelial surface (arrowheads). To exclude artifacts from sectioning, the vascular circumference was demarked by staining for laminin (B). Moreover, expression of occludin was still detectable in vessels with apparently impaired blood—brain barrier (BBB) integrity and an altered staining pattern for IB4 in each of the applied models (C). Scale bars: 20 μm.

Electron Microscopy Reveals Four Distinct Stages of Ischemia-Related Blood—Brain Barrier Breakdown Including Complete Loss of Endothelial Cells

After demonstration of an altered staining pattern for endothelial cells using fluorescence microscopy, we applied electron microscopy to confirm that the observed alterations indeed represent structural damage of the affected endothelial layer. With regard to the typically increased risk of hemorrhagic transformation after reconstitution of the cerebral blood flow, we directly compared the models of pMCAO and tMCAO to analyze the influence of a putative reperfusion injury on endothelial ultrastructure by electron microscopy.

Notably, we were not able to find differences of ischemia-induced endothelial degeneration between the applied transient and permanent model. However, identical patterns of vascular damage were regularly observed after tMCAO and pMCAO, which could be assigned to four different levels of ischemia-related ultrastructural affection.

In contralateral control areas, the endothelial layer as well as the adjacent parenchyma regularly appeared to be intact and extravasation of the tracer indicated by DAB grains was not observed in each of the applied models (Figure 5A). In the ischemic tissue of both models, a first level of endothelial affection was regularly observed in the form of an endothelial edema leading to a pale and less electron-dense cytoplasm of respective cells. Here, the cellular integrity was found to be preserved. Although the cells appear to be swollen, no extravasation of the tracer was detected (Figure 5B). At the second stage, endothelial cells fail to maintain the barrier function toward hydrophilic molecules, which is shown by leakage of FITC-albumin into endothelial cells, thereby showing typical DAB grains inside the cytoplasm (Figure 5C). Importantly, at this stage, the leakage still appears to be limited to the endothelial layer, whereas an extravasation into the neuropil is not yet visible. At the third stage, the integrity of the endothelial layer is lost. Here, if the endothelial cell is still detectable at all, then the luminal cell membrane is found to be permeable toward FITC-albumin and furthermore often covers the cell only irregularly, thereby displaying ruptures. In other cases, the endothelial cell is lost completely, and thus the underlying basement membrane was often found to limit the lumen of the respective vessel. At this stage, after complete loss of the endothelial integrity, extravasation of FITC-albumin was regularly observed in the adjacent parenchyma as indicated by grains of DAB (Figure 5D). Of note, since especially this pattern of vascular damage was found to occur after reperfusion (tMCAO) and permanent ischemia (pMCAO) as well, endothelial cell death appears not to be pronounced or even restricted to the reperfusion scenario. Strikingly, at the fourth stage, also the basement membrane regularly appeared to be degraded, which then ultimately led to an extravasation of erythrocytes (Figure 6A) as a prerequisite for hemorrhagic transformation of the ischemic tissue.

Figure 5.

Ultrastructural analysis reveals four distinct stages of impaired vascular integrity in the model of permanent (pMCAO) and transient (tMCAO) middle cerebral artery occlusion. In control areas, ultrastructural analysis revealed a normal vascular structure showing an unaltered endothelial layer and intact basement membranes in all the applied models of ischemic stroke while extravasation of fluorescein isothiocyanate (FITC)-albumin was not observed (A). Ischemia-affected vessels showing no extravasation of FITC-albumin regularly exhibited signs of endothelial swelling in the sense of an intracellular edema (B). Thereby, the cytoplasm appeared to be less electron dense and swollen. Notably, tight junction complexes between neighboring cells regularly remained detectable (arrows). At this stage of blood—brain barrier (BBB) breakdown, the endothelial barrier as well as the basement membrane (asterisk) were always found to be intact (B). At the second stage (C), the apical endothelial plasma membrane became leaky which was associated with regressed endothelial edema. Here, FITC-albumin was found within the endothelial cytoplasm as indicated by typical grains of DAB. However, the signal of the tracer was confined to the endothelial cytoplasm and did not cross the basolateral plasma membrane or the basement membrane (asterisk). At the third stage, the cellular structure of the endothelial cell was lost completely (D). Consequently, extravasation of FITC-albumin was regularly found in the neuropil beyond the glial basement membrane (asterisk). Complete loss of endothelial cells was frequently observed thereby exposing the basement membrane to the vascular lumen (arrowheads). However, the endothelial basement membrane is found to be morphologically intact. The arrow points to remnants of the endothelial plasma membrane. L, vascular lumen; E, endothelial cells.

Figure 6.

Most severe stage of ischemia-related vascular damage and quantification of vascular damage in differently affected brain regions. At the fourth stage (A), once the endothelial cell is missing, the integrity of the endothelial basement membrane (asterisk) is lost, thereby giving way for blood cells to extravasate into the parenchyma (left). As a result, erythrocytes can enter the neuropil in the sense of a hemorrhagic transformation of the ischemic tissue (right). (B) Comparison of the relative frequency of the incidence of each of the described stages of vascular damage in contralateral areas, the border zone of fluorescein isothiocyanate (FITC)-albumin leakage and areas of light microscopically evident tracer extravasation reveals significant differences between differently affected areas in the model of permanent (pMCAO) and transient (tMCAO) middle cerebral artery occlusion. Error bars are given as standard deviation of the mean. Stages (s): s0 complies without ultrastructural alterations, whereas s1 to s4 comply with an increased vascular damage as described for stage 1 to stage 4 (details are given in the text).

On the basis of these findings, we hypothesized that the described stages of vascular damage may be related to differently affected vessels in the ischemic areas and thus might reflect distinct phases of ischemia-induced BBB breakdown. On the assumption that the maximum tissue damage is located in the center of the ischemic territory, we quantified the relative frequency of the here described stages for vascular damage in three different areas in the models of pMCAO and tMCAO. Further, areas of evident tracer extravasation (assumed to represent areas most affected by ischemia) and respective contralateral control areas were identified by light microscopy on resin-embedded sections and further processed for electron microscopy. In both models, the relative frequency of vessels exhibiting either a normal morphology or one of the described stages of vascular damage was calculated and compared between the control areas, the border zones of tracer extravasation, and areas showing evident FITC-albumin leakage. Noteworthy, the distribution of the described stages in the different areas was found to be nearly identical in both models (Figure 6B). While in the control areas almost all of the present vessels were found to exhibit a normal ultrastructure (stage 0, s0) with no apparent signs of degeneration, the relative frequency of vessels matching one of the four stages of vascular damage (s1 to s4) was found to be significantly increased in border zones and areas of evident FITC-albumin extravasation (P=0.0273, Kruskal—Wallis test, followed by Dunn's Multiple Comparison test, n=3 for each group and area). In border zones, the highest relative frequency for affected vessels was observed for vessels matching stage 1, while stage 4 was not observed at all. Further, in areas of light microscopically evident FITC-albumin extravasation, the highest percentage of affected vessels shifted to vessels matching stage 3, which represents the stage of FITC-albumin extravasation beyond the vascular and basement membranes. Most importantly, in the less affected border zones of FITC-albumin leakage, the highest percentage of affected vessels is matching stage 1, while the most severe of our observed stages of vascular damage (stage 4) is only present in areas of evident tracer extravasation. Therefore, we propose that stage 1 indeed represents the initial step of ischemia-related BBB breakdown.

To address differences in the levels of ischemic affection with regard to the widely accepted concept of an ischemic core and subsequent layers of a surrounding ‘penumbra’, we applied double fluorescence labeling of either MAP2 and laminin or NeuN and HSP70, the latter of which has been identified to demark the inner layer of a molecular ‘penumbra’.^{31, 32} Of note, the areas of tracer extravasation appeared to be neither limited to the zones of enhanced neuronal HSP70 expression, nor to the zones of decreased MAP2 immunoreactivity, but rather exceeded them on the outer border (Supplementary Figure 1). Therefore, the analyzed border zones of tracer extravasation presumably correspond to the outer layers of the ischemic ‘penumbra’, but not to the ischemic core.

On the basis of these quantitative data and the observed ultrastructural alterations of cerebral vessels in the absence of model-specific alterations when comparing permanent ischemia with the reperfusion scenario, we hypothesized that BBB breakdown after ischemic stroke can be assigned to a model-overlapping four-step process of vascular damage that is summarized in Figure 7.

Figure 7.

Schematic overview of the proposed four-step process of ischemia-related blood—brain barrier (BBB) breakdown. In the contralateral control areas, diaminobenzidine (DAB) deposits indicating the converted tracer fluorescein isothiocyanate (FITC)-albumin (black grains) are strongly confined to the vascular lumen (L) while the endothelial cell is found to be unaffected. At the first stage, endothelial cells show a swollen cytoplasm in the sense of an intracellular edema. However, the endothelial barrier remains intact and confines the signal of the tracer to the vascular lumen. At the second stage, the endothelial surface becomes permeable toward the applied tracer, whereas the tracer is still confined to the endothelial layer. Of note, endothelial tight junctions (TJs) (arrowhead) remain detectable and the basement membrane (BM) appears to be continuous. At the third stage, the tracer is found to extravasate into the adjacent neuropil beyond vascular and glial basement membranes. The endothelial integrity is lost completely. Concomitantly, cellular fragments disappear, thereby exposing the basement membrane to the vascular lumen. Again, even along endothelial debris, the TJ complexes are regularly detectable. At the fourth stage, endothelial cells often disappear completely. The integrity of the basement membrane is lost and erythrocytes are found to exit the vasculature thus indicating a hemorrhagic transformation of the ischemic tissue. L, vascular lumen; E, endothelial cells; BM, basement membrane; N, neuropil; black grains, BBB tracer; blue, astrocytes; red, erythrocytes.

Discussion

Although the mechanisms underlying BBB breakdown under pathologic conditions were recently rated as high priority for stroke research,¹⁸ related pathophysiologic processes associated with hemorrhagic transformation and cerebral hemorrhage, especially after reconstitution of cerebral blood flow, are still poorly understood. Given the fact that in recent years recanalization using rt-PA was applied more frequently and in a broader time window from ischemia onset, detailed knowledge on BBB breakdown in naive ischemia and in the setting of reperfusion becomes increasingly important to develop neuroprotective strategies that could be coadministered to rt-PA.³³

In line with our previous work that showed endothelial damage as a potential mechanism for BBB breakdown in a thromboembolic model of focal cerebral ischemia,¹⁴ we here investigated alterations in endothelial cells as well as associated TJs and AJ in diverse animal models of ischemic stroke including transient ischemia to evaluate putative effects of reperfusion on the vasculature. In contrast to mouse models using rather short periods of transient ischemia, we decided to extend the ischemic time frame to 90 minutes to better mirror the clinical situation, while longer periods of transient ischemia inherit the risk of an increased mortality and can thus hardly be established in this model. Further, we made use of two animal species (mice and rats) to meet current recommendations for preclinical stroke research.³⁴

Since earlier studies attributed loss of BBB function predominantly to dysfunctional TJs,^{12, 13, 29, 35} which was supported by altered staining patterns of typical TJ proteins such as occludin, ZO 1, and claudin 5 in association with an increased BBB permeability,^{12, 13} we also analyzed the expression patterns of TJ- and AJ-associated proteins by double fluorescence labeling. To ensure precise identification of ischemia-affected vessels at sites of BBB breakdown, we applied the established tracer FITC-albumin,²⁵ which can be detected using fluorescence microscopy and after immunohistochemical conversion via DAB also by light and electron microscopy as previously described.¹⁴ As FITC-albumin does not represent an intrinsic marker of BBB permeability, efforts were made to first confirm identical patterns of ischemia-related extravasation for native albumin and FITC-albumin by immunohistochemical labeling.

In the present study, we show that the ratio of occludin-positive vessels does not differ between the affected and nonaffected hemisphere, while the interhemispheric ratio of occludin-positive vessels was found to be nearly identical after thromboembolic as well as filament-based transient and permanent ischemia. Moreover, quantification of the relative length of occludin-positive TJ strands did neither differ between areas of tracer extravasation and their control areas, nor between the models applied. In fact, we did not observe any differences in the staining pattern between areas of BBB breakdown and the contralateral areas for any of the critical TJ proteins including occludin, ZO 1, the claudins 3 and 5 or the AJ marker VE-cadherin. However, there is a variety of data demonstrating modulation of junctional proteins under ischemic conditions, which in conjunction are shown to increase BBB permeability.^{11, 13, 29} Therefore, the proteins detected by immunofluorescence labeling are probably at least functionally impaired. However, the junctional complex is still found to be anchored to cellular debris and the basement membrane, thereby allowing recognition of respective proteins by antibodies. Thus, our results clearly show that, although frequently applied in the past, immunolabeling for detection of these antigens in models of hypoxia and stroke does not reliably allow evaluation of BBB integrity. In contrast, labeling of the endothelium via IB4 regularly reveals structural alterations affecting the surface of the endothelial layer, which is likely responsible for the observed breakdown of the BBB. Interestingly, this concept of a transendothelial extravasation has already been described in early studies of ischemia^{36, 37} and was later mostly neglected in the literature.

Using electron microscopy in combination with immunohistochemical conversion of intravenously administered FITC-albumin via DAB, our study provides novel evidence that BBB breakdown results from a stepwise pattern of vascular damage, which was found to occur irrespective of the nature of ischemia when comparing permanent ischemia with a reperfusion scenario. Thereby, four distinct stages of vascular damage were identified, which appeared to be equally distributed in the models of permanent and transient ischemia. Therefore, vascular alterations could be assigned to a model-overlapping process of ischemia-related BBB into breakdown involving four distinct stages of BBB damage, which ultimately lead to loss of the vascular structure and consequently to loss of barrier function after focal cerebral ischemia (summarized in Figure 7).

Although the described experiments on tMCAO and pMCAO in mice were limited to a single observation time point of 24 hours after ischemia induction, which might question the temporal nature of the suggested four-step model of BBB breakdown, we propose that our observations of differently affected vessels are likely relating to differences in the intensity of affection throughout the ischemic areas. This view is supported by the finding that the majority of affected vessels in the border zones of detectable tracer extravasation, i.e., in areas of a less impaired BBB, only showed signs of endothelial swelling, which is in line with studies demonstrating the critical role of activated connexin-43 hemichannels under ischemic conditions.^{15, 16} In these areas, extravasation of FITC-albumin was not observed, which corresponds to the lack of stages 3 and 4 in these areas. Of note, the analyzed border zones of tracer extravasation do not correlate with the inner layer of a molecular penumbra,³¹ but rather represent functionally affected regions, showing distinct signs of endothelial alterations. Thus, endothelial swelling is likely to represent the initial step of BBB breakdown, which leads to further endothelial damage and even loss of endothelial cells as we have shown in three models focal cerebral ischemia. Importantly, the role of ischemia-induced endothelial cell death in the pathogenesis of BBB breakdown has been shown before. While the underlying mechanism leading to BBB damage was shown to involve induction of apoptosis in endothelial cells, the application of antiapoptotic agents resulted in a reduction of the endothelial permeability.³⁸ Therefore, we propose that ischemia-related BBB breakdown is predominantly driven by structural alterations affecting the endothelial integrity. In the context of potentially deleterious events originating from abrupt reopening of cerebral vessels (reperfusion injury), it is important to note that we observed differences neither in regard to expression patterns of essential junctional proteins such as occludin, claudin 3, claudin 5, ZO 1, or VE-cadherin, nor in regard to ultrastructural alterations in the endothelial layer between permanent and transient ischemia. Thus, endothelial cell damage or even loss of the endothelial layer is not restricted to a putative ‘reperfusion injury’, which was addressed in the model of tMCAO. Of note, the per se detrimental nature of the ‘reperfusion injury’ is currently challenged by clinical studies demonstrating that hemorrhagic transformation is also associated with an improved clinical outcome,³⁹ and also by the fact that drug-related recanalization approaches have proven beneficial effects in large randomized trials.¹⁷

Further, after pMCAO as well as tMCAO structural alterations in the vascular basemement membrane were equally observed as a most severe stage of vascular damage in areas of FITC-albumin extravasation. Thus, after breaching of the endothelial barrier, loss of the underlying basement membrane ultimately leads to extravasation of erythrocytes, representing the complete loss of the vascular integrity as a prerequisite for hemorrhagic transformation in the course of ischemic stroke. This view is supported by studies showing an upregulation of matrix metalloproteinases, which has been shown in diverse models of ischemia.^{29, 40} Importantly, loss of the vascular basement membranes was not observed in less affected border zones of tracer extravasation.

Even though our previous work revealed identical alterations in the endothelial layer at 5 hours after ischemia onset in line with the current findings, future studies are required to explore endothelial damage at various time points after stroke onset to precisely address the phasic time course of BBB permeability.¹¹ Further, as this study was designed to reveal morphologic alterations between the applied models as captured by immunofluorescence labeling and electron microscopy, the underlying molecular mechanisms of observed endothelial damage need to be further elucidated, which would help to establish interventions specifically addressing endothelial cell death.

In summary, our data strongly suggest that ischemia-related BBB breakdown is predominantly caused by endothelial damage, a feature that was consistently observed after experimental thromboembolic as well as filament-based transient and permanent focal cerebral ischemia in mice and rats. On the basis of ultrastructural data, we further propose a four-step sequence of BBB damage that may offer novel strategies of therapeutic intervention. In conclusion, future research on stroke therapy is likely to provide novel tools for protection of endothelial cells, e.g., via drugs that could be effectively administered additionally to the widely accepted concept of early recanalization.

Footnotes

ACKNOWLEDGMENTS

The authors would like to thank Judith Craatz and Jana Brendler (Institute of Anatomy, University of Leipzig) for technical assistance in tissue preparation and Dr Martin Gericke (Institute of Anatomy) for fruitful discussions as well as Dr Petra Madaj-Sterba and Sigrid Weisheit (Medizinisch-Experimentelles Zentrum, University of Leipzig, Germany) for animal care. Funding was provided by Dr Carsten Hobohm (Department of Neurology, University of Leipzig), by the Formel.1 project (University of Leipzig to MK) and the Deutsche Forschungsgemeinschaft (DFG-FOR1336 to IB).

The authors declare no conflict of interest.

Notes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website ()

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