Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Mar:86:1-10.
doi: 10.1016/j.mvr.2012.12.001. Epub 2012 Dec 20.

Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels

Debayon Paul  1 , Ann E Cowan, Shujun Ge, Joel S Pachter
Affiliations

Novel 3D analysis of Claudin-5 reveals significant endothelial heterogeneity among CNS microvessels

Debayon Paul et al. Microvasc Res. 2013 Mar.

Abstract

Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. 3D Contour-based quantification of junctional CLN-5 in spinal cord microvessels
(a) CNS venule from a naïve mouse detailing CLN-5 (Green) staining at intercellular junctions. The image shows microvascular tributaries (e.g. capillaries, post-capillary venules) emerging from a venule, whose lumen has been “optically” cut open to reveal the inner vessel wall. Endothelial cells are highlighted with CD31 (Red). (b,c) To gauge endothelial heterogeneity with respect to CLN-5, spinal cord microvessels (capillaries and venules) obtained in confocal z-stacks from the dorsolateral white matter (between T10 and L3 vertebrae) were imaged and categorized into appropriate segments based on their average diameter. (d) Schematic indicating this method effectively allows the surface area of the microvascular endothelium to be spread out in 3D space (x, y, z axes) for quantification of CLN-5 density per unit area, while excluding the luminal volume. (e) To quantify CLN-5 staining associated with a microvessel in 3D, an individual contour for each confocal z-slice was created by cursoring out the vessel of interest, and the individual z-slice contours then merged into a 3D contour surface. This contour surface was utilized to isolate the microvessel of interest from the rest of the dataset (masking), and its area was used as an estimate of the “microvascular surface area” defined by the endothelial layer. An isosurface for the CLN-5 channel was then created from the selected vessel for statistical analysis. Scale bar = 50µm.
Fig. 2
Fig. 2. Heterogeneity in CLN-5 density distribution among different-sized microvessels in naïve spinal cord
Isosurface rendering of the CLN-5 channel was performed in confocal z-stacks of different-sized spinal cord microvessels in tissue sections from naïve mice: (a,b) larger venules; (c,d) smaller venules; (e,f) capillaries. Top row, shows CLN-5 (Green) and nuclei/DRAQ5 (Blue). Bottom row, shows CLN-5 only, to emphasize the disparity in junctional CLN-5 immunostaining between the smaller and larger microvessels. (g,h) 3D contour-based quantification of CLN-5 density (intensity per unit surface area of the endothelium) within naïve spinal cord microvessels. Junctional CLN-5 density was greatest in the capillaries and smaller venules, and least in the larger venules. A total of 5 microvessels were analyzed in each group sampled from 3 mice. *p < 0.001. Scale bar = 15µm.
Fig. 3
Fig. 3. CLN-5 density in spinal cord microvessels during EAE
Isosurface rendering of the CLN-5 channel was performed in confocal z-stacks of spinal cord microvessels at d24 EAE. Top row, shows CLN-5 (Green) and nuclei/DRAQ5 (Blue) to highlight the close association of altered CLN-5 with dense perivascular cellularity. Bottom row, shows staining of only CLN-5 to emphasize significant TJ protein disruption. Inflamed venules demonstrated heterogeneity in CLN-5 loss: (a,b) severely inflamed venules displayed diffuse and extensive disruption of CLN-5; (c,d) moderately inflamed venules showed small punctate regions of CLN-5 loss; and (e,f) capillaries adjacent to severely inflamed venules appeared refractory to CLN-5 loss. 3D quantification of intercellular CLN-5 staining showed a significant reduction in intensity of CLN-5 staining per unit area of the endothelium in the severely inflamed venules compared to the capillaries (g,h). The boundaries of inflamed venules are marked with dashed white lines. A total of 6 microvessels were analyzed in each group sampled from 3 mice. *p < 0.0001. Scale bar = 20µm.
Fig. 4
Fig. 4. CLN-5 density in spinal cord microvessels from naïve vs. EAE mice
Isosurface rendering of z-stacked images of spinal cord sections from naïve mice and mice at d24 EAE. Top row, shows staining of CLN-5 (Green) and DRAQ5 (Blue) to highlight the close association of altered CLN-5 with dense perivascular cellularity (reflective of leukocyte infiltrates) during EAE. Bottom row, shows staining of only CLN-5 to emphasize significant TJ protein disruption that accompanies disease. (a,b) Venules from naïve mice; (c,d) Venules from d24 EAE mice; (e,f) Capillaries from naïve mice; and (g,h) Capillaries from d24 EAE mice. (i,j) 3D quantification of CLN-5 microvascular staining showed a significant reduction in CLN-5 density in inflamed venules compared to the naïve venules and naïve capillaries. The boundary of inflamed venule is marked with dashed white line. A total of 6 microvessels were analyzed in each group sampled from 3 mice. *p < 0.0001. Scale bar = 15µm.
Fig. 5
Fig. 5. Heterogeneity in CLN-5 density in a contiguous venule/capillary pair during EAE
(a) Isosurface rendered 3D reconstruction of a contiguous venule and capillary in spinal cord section from d24 EAE mouse, highlighting basement membrane/Lam1 (Red), CLN-5 (Green), and nuclei/DRAQ-5 (Blue). (b) Isosurface rendered CLN-5 channel only, with boundary of the inflamed venule marked with dashed white line. The venule shows severe loss and fragmentation of junctional CLN-5, while the attached capillary displays intact junctional CLN-5 staining. The insets reveal cross sections through the inflamed venule, optically cut using clipping plane module in Imaris®, demonstrating association of venular CLN-5 loss with seminal signs of inflammation: (top) separation of endothelial and astrocyte basement membranes (BM); and (bottom) increased perivascular cellularity. (c) Schematic representation showing, qualitatively, heterogeneity in CLN-5 density distribution in a contiguous venule/capillary pair at d24 EAE.
Fig. 6
Fig. 6. Endogenous serum IgG leakage from spinal cord microvessels during EAE
(a,c,e) Shows volume rendered images of confocal z-stacks from microvascular segments obtained from naïve mice, and mice at d6 and d24 EAE. (b,d,f) Shows the corresponding isosurface rendered images for purpose of enhanced spatial perspective. Staining of IgG (Green) and basement membrane/LAM 1 (Red) highlights vascular permeability around venules and capillaries. (a,b) Microvessels from naïve mice reveal no visible IgG immunostaining associated with venules or capillaries. (c,d) Microvessels at d6 EAE – prior to evidence of clinical disease – display focal IgG immunoreactivity around both venules and capillaries. (e,f) Microvessels at d24 EAE show pronounced and diffuse IgG immunoreactivity – reflecting endogenous serum protein extravasation – which obscured boundaries between the microvessel segments. Increased perivascular cellularity, indicative of leukocyte infiltration (inset), is highlighted by DRAQ5 staining (Blue). Scale bar = 20µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abbott NJ, et al. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7:41–53. - PubMed
    1. Alvarez JI, Teale JM. Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature. Brain Res. 2007;1169:98–111. - PMC - PubMed
    1. Argaw AT, et al. VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. Proc Natl Acad Sci U S A. 2009;106:1977–1982. - PMC - PubMed
    1. Bake S, et al. Reproductive age-related changes in the blood-brain barrier: Expression of IgG and tight junction proteins. Microvasc. Res. 2009;78:413–424. - PMC - PubMed
    1. Batoulis H, et al. Experimental autoimmune encephalomyelitis--achievements and prospective advances. APMIS. 2011;119:819–830. - PubMed

Publication types

MeSH terms