Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model
© The Author(s). 2017
Received: 12 October 2017
Accepted: 17 November 2017
Published: 1 December 2017
In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.
The cerebral vasculature controls and restricts the transport of biomolecules between blood and the CNS parenchyma by means of the blood-brain barrier (BBB) [1, 37]. While specialized brain endothelial cells are physically connected via unique belt-like tight junctions that mediate BBB tightness, perivascular cells and astrocytes also contribute to BBB physiology, collectively forming the neurovascular unit (NVU). In a wide range of neurological disorders including multiple sclerosis (MS) increased vascular permeability has been observed  but the primary cause for the pathophysiology of the NVU and the relation to disease specific pathomechanisms remains unclear.
MS is an acquired inflammatory demyelinating disease of the CNS in which BBB permeability is increased in both newly forming demyelinating lesions and even in normal appearing white matter [18, 59]. BBB impairment is observed at the onset of clinical symptoms in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, coinciding with initial immune cell infiltration and glial activation [19, 51]. In this model, pro-inflammatory cytokines/chemokines produced by activated immune cells on the peripheral side of the barrier, or by glial cells in the CNS, contribute to BBB hyperpermeability [4, 42]. Thus, BBB disruption could potentially be secondary to pathology. However, it is unclear whether demyelination or other disease factors cause BBB disturbances.
We previously showed in the non-inflammatory cuprizone model of demyelination , that BBB permeability is increased at the peak of demyelinating disease, and that this BBB dysfunction can be utilized for CNS delivery of therapeutics . However, it is unclear which pathomechanism triggers the BBB breach in the cuprizone model. Here, we further characterize these BBB disturbances, relating BBB pathophysiology to histopathology in different brain regions and at different stages of disease progression. We demonstrate that early disease processes are associated with elevated levels of several pro-inflammatory mediators of predominantly astroglial origin. This local inflammatory milieu, together with a primary effect of cuprizone on endothelial cells, leads to the downregulation of BBB maintenance factors, endothelial efflux transporters, and tight junction proteins resulting in morphological disruption of tight junctions. These endothelial disturbances are associated with local hyperpermeability of the BBB and edema, even before the onset of demyelination.
Materials and methods
All animal studies were performed in compliance with the animal policies of the Max Planck Institute of Experimental Medicine, and were approved by the German Federal State of Lower Saxony. Adult male C57BL/6N mice or CX3CR1GFP/GFP mice  were taken at 8–10 weeks of age. Animals were randomly assigned to an experimental group. Cuprizone (0.2% w/w, Sigma) was mixed into powder chow (V1124 ssniff).
Histological analyses were done as described [11, 49] with minor modifications. Anesthetized mice were perfused with paraformaldehyde (PFA) and brains were cut by vibratome (40 μm, Leica VT1200)) or embedded in paraffin. Brain sections (HMP 110, MICROM) at Bregma −1.58 were taken for histological characterization using standard protocols using LSAB2 (Dako) or Vector Elite ABC (Vector Labs). For occludin and ZO1 staining, animals were perfused with PFA containing 0.2% glutaraldehyde and cut on a vibratome. Tissue sections or fixed endothelial cells were processed for immunolabeling by permeabilization (0.4% Triton X-100 in PBS), blocking (4% horse serum, 2% BSA, 0.2% Triton X-100 in PBS) and incubation with first antibody (1% HS, 0.05% Triton X-100 in PBS). Incubation with fluorophore coupled secondary antibodies (ThermoFisher) and DAPI (4′,6-diamidino-2 phenylindole) were done in 1.5% HS in PBS, after which sections were mounted in AquaPolymount (Polysciences). Gallyas silver impregnation was done as described . Specimens were analyzed on an Axio Imager.Z1 (Zeiss) equipped with an AxioCam MRc3, × 0.63 Camera Adaptor and the ZEN 2012 blue edition software using ×10 objective (Plan Apochromat × 10/0.45 M27) or × 20 objective (Plan-Apochromat × 20/0.8) or by confocal laser scanning microscopy (Leica SP5 HCX PL APO CS 63×/1.20) using the Leica Confocal Software (Leica Microsystems). Quantification of positive areas in the corpus callosum above the fornix were done by semi-automated analysis with ImageJ software macro and color deconvolution plug-in. Vessel paint was performed as described  with minor modifications. Mice were intravenously injected with 200 μl of 20 mg/ml FITC-Dextran (46,945, Sigma-Aldrich Inc., Germany). After 30 min circulation time animals were anaesthetized, flushed, stained with DID (ThermoFischer, D7757) and fixed with PFA before sectioning with a vibratome (100 μm). All Images were processed with NIH ImageJ and Adobe Photoshop CS5.1 software. Electron microscopic analysis was done as previously described . Briefly, tissue was fixed in 4% PFA, 2.5% glutaraldehyde, 0.1 M Phosphate buffer and sagittal sections were cut on a vibratome (Leica VT1200). The corpus callosum with adjacent tissue was punched and embedded in epon (LYNXII, EMS). Ultrathin uranyl acetate contrasted sections were imaged with a LEO EM912 AB (Zeiss) equipped with a 2 k–CCD camera (TRS, Moorenweis).
Blood-brain barrier permeability
Tracer injections were done as described [11, 49] with minor modifications. For measurement of BBB permeability, tracers were i.v. injected (Evans blue 50 mg g-1 body weight; sodium fluorescein 200 mg g-1 body weight). After 4 h incubation, animals were perfused with PBS to remove tracer from the circulatory system. The region of interest was dissected, and tissue samples were weighed. For lyophilization, tissues were exposed to a shelf temperature of −56 °C for 24 h under vacuum of 0.2 mBar (Christ LMC-1 BETA 1–16). Samples were weighed for calculation of water content and edema. Lyophilized tissue samples were extracted with formamide at 57 °C for 24 h on a shaker at 300 rpm (Eppendorf Thermomixer). Integrated density of tracer fluorescence was determined in triplicates after 1:3 ethanol dilution to increase sensitivity. Tracer concentration was calculated using a standard curve of tracer spiked brain samples.
Brain tissue samples were lysed in sucrose buffer (18% sucrose, 10 mM Tris/HCl pH 7.4, 1 mM sodium hydrogen carbonate, 1 mM magnesium chloride, 0.1% Triton, 0.2% lithiumdodecyl sulphate, 0.025% sodium deoxycholate) with protease inhibition (Roche) using a Precellys 24 homogenizer (Bertin technologies). Detection of immunolabeled proteins was performed with ECL detection reagent (Perkin Elmer) using ChemoCam Imager (Intas).
Magnetic cell isolation
Glial cells were isolated according to the adult brain dissociation protocol (Miltenyi biotec). Corpus callosum and cortex were isolated using a brain matrix from Bregma +1.10 to −2.46. Antibody labeling steps were done according to the respective antibody Microbead kit protocol (Miltenyi biotec), oligodendrocytes (O4, 130–096-670); astrocytes (ACSA-2, 130–097-679), microglia (CD11b, 130–093-636), and endothelial cells (CD31, 130–091-935). Purity of cell populations was routinely determined by qPCR on extracted and reverse transcribed RNA (see below) and revealed only minimal contamination by other cell types.
Primary mouse brain endothelial cell cultures were established from 7 days old mice or rats. Briefly, cortices were digested with 1 mg/ml collagenase/dispase and 2.5 μg/ml DNAse (Roche) in dissection buffer (HBSS, 10 mM HEPES, 0.5% BSA, 5000 U/ml penicillin/streptomycin) for 45 min at 37 °C. After trituration, cells were resuspended in 25% BSA and centrifuged at 1000 g for 20 min to pellet microvessels. Isolated microvessels from individual mice were plated in Endobasal Medium (Promocell) with 0.4% puromycin for positive selection on coverslips or polyester transwell inserts (Corning). Primary astrocyte cultures were prepared from 0 to 2 days old mice as previously described . Primary microglia cultures were prepared from P0-P2 old mice by differential shaking of mixed glial cultures as described . Cell purity was routinely determined by immune stainings and always exceeded 95%. For coculture experiments, endothelial cells cultured in transwell inserts above astrocytes plated on the bottom of the well plate. Confluent cells cultures were treated with a final concentration of 250 μM cuprizone in 0.125% DMSO or in 0.125% DMSO alone for up to 72 h. An epithelial Voltohmmeter (EVOM2, World Precision Instruments) equipped with Endohm-12 chamber electrodes was used to measure transendothelial electrical resistance (TEER). Metabolic activity was determined using a WST1 assay (Cayman) according to the manufactures protocol, after exposure to increasing concentrations of cuprizone (0–250 μM) for up to 72 h, or 20 μM peroxide as positive control.
Expression analyses were done as described . For tissue expression analyses, corpus callosum and cortex was dissected from Bregma +1.10 to −2.46. RNA was extracted using QIAshredder and RNeasy protocols (Qiagen). Concentration and quality of RNA was evaluated using a NanoDrop spectrophotometer and RNA Nano (Agilent). cDNA was synthesized with Superscript III (Invitrogen) and quantitative PCRs were done in triplicates with the GoTaq pPCR Master Mix (Promega) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression values were normalized to the mean of two housekeeping genes, HPRT (Hypoxanthin-Phosphoribosyl-Transferase 1) and Rplp0 (60S acidic ribosomal protein P), and quantification was done by applying the ΔΔCt method, normalized to age matched untreated controls (set to 1). All primers (Additional file 1: Table S1) were intron-spanning.
CAII (Said Ghandour), Olig2 (Charles Stiles/ John Alberta), GFAP (Chemicon), MAC3 (Pharmigen), Iba1 (Wako), PECAM1 (dianova), AQP4 (Santa Cruz), occludin, ZO-1 and claudin-5 (Thermo Fisher), GAPDH (Enzo). For generation of GLUT1 antisera, rabbits were immunized with the C-terminal intracellular peptide (CDKTPEELFHPLGADSQV). Anti-GLUT1 antibody was purified by affinity chromatography.
Statistical analysis was performed using Prism software (GraphPad Software), and results are presented as the mean ± s.e.m.. Two-way ANOVA, one-way ANOVA and two-tailed unpaired Student’s t tests were performed as appropriate. Only P values <0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Vascular permeability is increased at the peak of cuprizone induced demyelination
When we examined the morphology of the BBB by transmission electron microscopy, cuprizone fed animals often displayed a discontinuous electron-dense junctional area with focally increased junctional width (Fig. 1c, Additional file 2: Figure S2), implying that the physical barrier of the BBB mediated by endothelial tight and adherens junctions could be altered. This observation was confirmed by immunofluorescence analysis of selected tight junction proteins that showed strongly reduced staining intensity in cuprizone fed animals (Fig. 1d–g). Increased BBB permeability was demonstrated by extravasation of tracers such as FITC-dextran (Fig. 1f, see also below). By electron microscopy we also observed locally split endothelial and astroglial basement membranes, hypertrophic astrocytes, and sporadic endothelial cells with atypical ultrastructure in cuprizone fed animals (Fig. 1c, Additional file 2: Figure S2). Moreover, while the number of blood vessels in the entire cross-sectional area of the corpus callosum remained unchanged, vessel density was strongly reduced (Fig. 1h), likely caused by the tissue swelling due to the dramatic gliosis at this time point (Additional file 2: Figure S1). Together, these data show that the increased BBB permeability at the peak of cuprizone induced demyelination coincides with substantial upregulation of BBB-disrupting pro-inflammatory mediators. This is associated with strong downregulation of endothelial tight junction proteins and morphological changes at the endothelial barrier.
Cuprizone directly affects BBB permeability in vitro
We next explored whether cuprizone directly damages cellular constituents of the NVU, namely mouse brain endothelial cells or astrocytes, as observed for mature oligodendrocytes . Using primary cultures of either cell type in a WST1 assay that measures the activity of cellular dehydrogenases, cuprizone did not affect metabolic activity within 24 h, even when administered in concentrations up to 500 μM (Additional file 2: Figure S3a, b). However, after 72 h incubation with 250 μM cuprizone, the WST1 signal in endothelial cells decreased by about 14% compared to vehicle treated cultures (Additional file 2: Figure S3c, d), which were not caused by cell death but rather reflected metabolic adaptations to the toxic cuprizone insult.
BBB dysfunction depends on local pathology
To substantiate the heterogeneity of BBB pathology with another marker for NVU integrity, we compared Aquaporin 4 (AQP4) distribution in corpus callosum and cortex by immunostaining. AQP4 localization was restricted to astrocyte endfeet in control animals and the cortex of mice up to 5 weeks cuprizone (Fig. 3e, f), suggesting this brain region intact with respect to BBB integrity. In contrast, in the corpus callosum of these mice, AQP4 displayed a diffuse staining pattern, and Aqp4 mRNA expression was significantly upregulated (2.71 ± 0.01 fold, n = 4 animals per group, P < 0.0001, Student’s t-test), a condition also found in MS lesions  and typical for cerebral edema in EAE. Together these findings suggest that the morphological disturbances at the BBB are associated with local active disease in cuprizone fed animals. This prompted us to investigate at which disease state BBB dysfunction develops.
BBB impairment and edema are very early disease processes
To assess how this regional heterogeneity of edema and BBB permeability correlates with local pathology and gliosis in this early disease phase, we performed a series of quantitative expression analyses on dissected corpus callosum and cortex together with histopathological evaluation of tissue sections, after 5 days of cuprizone. Expression of marker proteins for astrocytes and microglia reflected the mild histopathological changes (Fig. 4h, Additional file 1: Table S3, Additional file 2: Figure S1). Despite reduced Plp1 and Olig2 expression, mice lacked signs of demyelination and loss of oligodendrocytes, as reported previously [13, 24]. The abundance of mRNAs of endothelial tight junction proteins were only moderately downregulated in cortex compared to corpus callosum, mirroring the immunostaining data at 5 weeks of cuprizone exposure (Fig. 4h, compare Figs. 1d-e, 3a). Expression of the ABCB1 transporter and associated caveolins as well as of the BBB maintenance factor sonic hedgehog remained almost unaffected in the cortex compared to untreated wild type mice. It is possible that a direct effect of cuprizone contributed to the residual downregulation of endothelial markers and to the slight BBB hyperpermeability (Fig. 4g), similar to our observations in vitro (compare Fig. 2). In contrast, all markers for NVU integrity were strongly downregulated in corpus callosum at this early time point of cuprizone induced pathology.
Levels of the inflammatory mediators Il6, Il1b, Tnf, and Ccl2 were strongly elevated in the corpus callosum but only moderately increased in the cortex after 5 days of cuprizone (Fig. 4i). These data demonstrate that the BBB integrity is already affected within the first days of cuprizone exposure, coinciding with elevated levels of inflammatory mediators but preceding overt demyelination and oligodendrocyte loss.
Reduced inflammation ameliorates BBB pathology
These findings prompted us to test directly whether demyelination and oligodendrocyte loss or local gliosis and the secretion of inflammatory mediators correlate with BBB dysfunction. Therefore, we used type 3 CXC chemokine receptor (CXCR3) deficient mice  that develop demyelination in response to cuprizone as wild type mice but show strongly reduced reactive gliosis and expression of pro-inflammatory cytokines and chemokines such as TNF, IL6 and CCL2 .
Although CXCR3 is mainly expressed by microglial cells in untreated mice , and also when mice are challenged with cuprizone (Additional file 2: Figure S4), the cell type responsible for establishing the cytokine milieu during initial cuprizone pathology that contributes to BBB dysfunction is unknown. Therefore, we acutely isolated microglia, astrocytes, oligodendrocytes, and endothelial cells from wild type and CXCR3 deficient mice after 5 days of cuprizone treatment and from untreated wild type control animals, and analyzed mRNA abundance of Tnf, Il1b, Il6, and Ccl2. We chose these inflammatory mediators because their expression pattern correlates with the extent of BBB disturbances: after 5 days of cuprizone, their expression levels were most strongly increased in corpus callosum of wild type mice, moderately increased in the corpus callosum of CXCR3 mutant animals, and only weakly upregulated in the cortex of wild type animals compared to untreated wild type controls (compare Figs. 4i and 5b). Oligodendroglia did not significantly contribute to the cytokine and chemokine profile after 5 days of cuprizone and surprisingly, neither did microglia (Fig. 5g, Additional file 1: Table S4). Endothelial cells showed moderate upregulation of cytokine and chemokine expression. In contrast, we identified astrocytes as the major source of all tested pro-inflammatory mediators at this early disease phase (Fig. 5g). Further, the increased expression of inflammatory molecules was completely abolished in astroglia of CXCR3 deficient mice, suggesting that microglial CXCR3 signaling induces astroglial upregulation of cytokines and chemokines in response to cuprizone.
BBB impairment is considered as an important feature in MS pathologies, but the causal relation to disease processes during initial lesion formation and its impact on disease activity is unknown. In the current study we analyzed the temporal and spatial relationship of BBB dysfunction to gliosis, expression of inflammatory mediators, and demyelinating pathology in the cuprizone mouse model of demyelination. By using wild type and CXCR3 deficient mice, we demonstrate that BBB impairment is most pronounced in the corpus callosum of wild type animals, to a lesser extent in the corpus callosum of CXCR3 deficient mice, and only minimally in the cortex of wild type mice in the initial disease phase before the onset of demyelination. Our data indicate that IL6, IL1β, TNF, and CCL2 are the most likely candidates to contribute to BBB dysfunction in the cuprizone model.
It is well known that TNF, IL6, IL1β and CCL2 can each induce downregulation of endothelial tight junction proteins in vitro and induce BBB hyperpermeability in vivo [14, 20, 43, 47, 53, 58]. Enhanced paracellular leakage is mediated by downregulation of mRNA of tight junction proteins as shown in our study and in a model of bacterial infection . Pro-inflammatory cytokines induce signaling cascades that can lead to downregulation of stabilizing factors such as sonic hedgehog [3, 58], to activation of effector proteins such as matrix metalloproteinases , and to the formation of reactive oxygen species . In our experimental paradigm, increased expression of nitric oxide synthases and downregulated sonic hedgehog signaling likely contributed to tight junction disruption. In addition, all three cytokines have the ability to reduce ABCB1 mediated efflux, facilitating transendothelial passage . We found downregulation of ABCB1 not only in cuprizone fed mice but also in endothelial cultures in the absence of inflammatory mediators, suggesting that cuprizone directly affected transendothelial passage.
Although it is generally assumed that microglia secrete the majority of effector molecules, our data show that in the initial disease phase of the cuprizone model microglia do not themselves contribute to the upregulation of IL6, IL1β, TNF or CCL2; rather astrocytes (with a moderate participation by endothelial cells) are the main source of these pro-inflammatory cytokines and chemokines. At later disease stages, however, microglia substantially add to the production of inflammatory factors . Astrocytes participate in recruiting microglia as shown in mouse mutants with acutely depleted astrocytes . They promote recovery and repair in mouse models of remyelination but can also facilitate demyelination in acute active lesions of MS patients [11, 31, 35]. Whether astrocytes are the main source of disease promoting factors in presymptomatic MS patients before the onset of demyelination is unknown. We demonstrate that astrocytes, which are intimately involved in regulating BBB function via their endfeet, create a local inflammatory milieu that likely participates in destabilizing BBB integrity. Although cuprizone by itself does not induce astroglial, endothelial, or microglial expression of any of the tested pro-inflammatory mediators in vitro, it mildly affects metabolic activity in endothelial cells (this study) in addition to mature oligodendrocytes . Death of oligodendrocytes in vivo is enforced by local glial activation . We hypothesize that also vascular cells contribute to this complex crosstalk that lead to BBB impairment and demyelination in the cuprizone model.
Paracellular influx of fluid because of BBB disruption is the leading cause of vasogenic edema , also found in MS and inflammatory models of MS [9, 48, 60]. Edema correlates with increased AQP4 abundance and its mislocalization from (frequently hypertrophic) astroglial endfeet that is associated with altered basement membrane morphology in EAE [2, 60]. We also observed loosening of the astrocytic and endothelial basement membranes in the cuprizone model, emphasizing similar pathogenic processes in these disparate models of MS. Although it is well-known that cuprizone intoxication causes spongiform degeneration of the CNS , edema has been largely ignored, potentially because of the absence of massive BBB disruption in this model [8, 12, 30]. For the first time, to our knowledge, we quantified edema in the cuprizone model and found that increased brain water content was most pronounced during overt demyelination. Importantly, edema was already obvious before the onset of demyelination and oligodendrocyte loss.
In agreement with others [13, 23], we observed marked regional differences in disease manifestation in the cuprizone model. Interestingly, similar to our observation in the cortex of cuprizone treated animals, cortical pathology in MS also differs substantially from white matter lesions; the former comprising only mild gliosis and modest alteration of tight junctions [21, 56]. In addition to the differences in tissue architecture and expression profiles of neural cells [22, 38], the regional heterogeneity of pathology could also be influenced by differences in vasculature. As we show here in accordance with previous studies, vessel density in gray matter is about double of that in white matter, and steady state levels of tight junction proteins are lower . Together, these differences likely modify BBB properties, which might play a role in rendering cortical MS lesions less susceptible to disease exacerbation.
Does an impaired BBB directly affect the course of demyelinating disease? Chronic upregulation of inflammatory mediators directly impairs BBB integrity and can induce demyelinating pathology ; conversely, their genetic or pharmacological reduction can improve BBB function but their role in modulating severity of demyelinating disease in the context of EAE or cuprizone is less clear [7, 16, 36, 39]. The induction of an inflammatory milieu and BBB impairment have been uncoupled by inhibition of nitric oxide synthesis in the cuprizone model  or by targeted overexpression of claudin-1 in EAE  that both support BBB tightness but presumably do not (directly) affect expression of pro-inflammatory molecules. In these experimental paradigms, clinical symptoms of treated/transgenic mice were ameliorated, suggesting that endothelial integrity might contribute to disease expression. In a study with 39 patients with neuromyelitis optica, BBB leakage in normal appearing white matter correlated with progression to MS pathology . We show here that BBB dysfunction and edema occurred before demyelination, suggesting that demyelination itself does not cause BBB damage. We speculate that BBB dysfunction might serve as a predictive marker for local disease activity.
In summary, our data show that in vitro cuprizone directly increases BBB permeability mediated by downregulation of tight junction proteins. In vivo, astroglial derived pro-inflammatory cytokines create a local inflammatory milieu that likely further destabilizes local BBB integrity and induces edema. We envision that glial activation and production of pro-inflammatory mediators in any neurological disorder destabilizes BBB integrity. In demyelinating disease, these presymptomatic disease processes might have prospective value for future disease activity and disease progression.
We are grateful to Klaus-Armin Nave for constant support. We thank Silvia Thüne, Annette Fahrenholz, Martin Meschkat, and Beatriz Salas Vegue for technical support. We thank Iva Tzetanova for providing primary microglial cells. We cordially thank Julia Edgar for editing and critical discussions. This study was funded by the Deutsche Forschungsgemeinschaft (SA 2014/2-1 to GS). WM was funded by an ERC Advanced grant awarded to Klaus-Armin Nave. All authors read and approved the final manuscript.
GS, and SAB conceptualized the study and designed the experiments, SAB, TD, JW, SKS, NG, LS, TR, BHC, WM performed experiments. KK generated the GLUT1 antibody. SAB and GS wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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