Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
, 9, 385
eCollection

Markers for Blood-Brain Barrier Integrity: How Appropriate Is Evans Blue in the Twenty-First Century and What Are the Alternatives?

Affiliations
Review

Markers for Blood-Brain Barrier Integrity: How Appropriate Is Evans Blue in the Twenty-First Century and What Are the Alternatives?

Norman R Saunders et al. Front Neurosci.

Abstract

In recent years there has been a resurgence of interest in brain barriers and various roles their intrinsic mechanisms may play in neurological disorders. Such studies require suitable models and markers to demonstrate integrity and functional changes at the interfaces between blood, brain, and cerebrospinal fluid. Studies of brain barrier mechanisms and measurements of plasma volume using dyes have a long-standing history, dating back to the late nineteenth-century. Their use in blood-brain barrier studies continues in spite of their known serious limitations in in vivo applications. These were well known when first introduced, but seem to have been forgotten since. Understanding these limitations is important because Evans blue is still the most commonly used marker of brain barrier integrity and those using it seem oblivious to problems arising from its in vivo application. The introduction of HRP in the mid twentieth-century was an important advance because its reaction product can be visualized at the electron microscopical level, but it also has limitations. Advantages and disadvantages of these markers will be discussed together with a critical evaluation of alternative approaches. There is no single marker suitable for all purposes. A combination of different sized, visualizable dextrans and radiolabeled molecules currently seems to be the most appropriate approach for qualitative and quantitative assessment of barrier integrity.

Keywords: blood-brain barrier; embryo; fetus; newborn; permeability; tight junctions.

Figures

Figure 1
Figure 1
Evans blue. As used at University College London, Department of Physiology circa 1960 for in vivo plasma volume estimation.
Figure 2
Figure 2
Papers in PubMed using different blood-brain barrier markers since 1953. One curiosity is that radiolabeled albumin was used more than a decade (Ashkenazy and Crawley, 1953) before the first use of Evans blue (Rössner and Temple, 1966) but has only been infrequently used since then compared to Evans blue.
Figure 3
Figure 3
Numbers of papers by year listed in PubMed for “blood-brain barrier Evans blue.” Note the very steep increase in the past 10 years; note also that the value for 2015 is for only 9 months of the year. Thus, the use of Evans blue is still clearly increasing substantially.
Figure 4
Figure 4
Evans blue binding to 1, α1-lipoprotein; 2, albumin; 4, hemopexin; 5, prealbumin; 6, α1X and 8, transferrin. Identified by anodal shifts. From Figure 3 in Emmett et al. (1985).
Figure 5
Figure 5
Electron micrographs of the localization of biotin ethylenediamine (BED) in blood vessels deep inside the cortex of a 2-month-old opossum 10 min after an intravenous injection. Similar staining is found after an intravenous injection of biotin-dextran (BDA3000). (A) Low-power micrograph showing two paired vessels with abundant reaction product within lumen. No reaction product is visible in the surrounding tissue. Pairs of arteries and veins are characteristic of the vascular pattern in marsupial brains (Wislocki and Campbell, 1937). (B) High-power micrograph of an interendothelial cleft showing that the tight junctions in the young adult restrict the passage of BED through the cleft (arrowhead). Scale bar = 4 μm in (A); 300 nm in (B). From Ek et al. (2006).
Figure 6
Figure 6
Cellular localization of dextran probes in postnatal lateral ventricular choroid plexus of the marsupial South American opossum (Monodelphis domestica). (A) Forty-five minutes after intraperitoneal injection with BDA-3 kDa, the probe can be seen in individual epithelial cells of the choroid plexus (filled arrow), as well as in the blood vessel lumen (arrowhead) and precipitated in the CSF (unfilled arrow). (B) Ten minutes after intraventricular injection with BDA-3 kDa–Fluorescein, more epithelial cells take up the probe (filled arrows) following CSF injection compared with intraperitoneal injection (A). Penetration of the fluorescent probe between epithelial cells is stopped by the presence of tight junctions (examples highlighted by arrowheads). Scale: 50 μm. From Liddelow et al. (2009).

Similar articles

See all similar articles

Cited by 51 PubMed Central articles

See all "Cited by" articles

References

    1. Abbott N. J., Patabendige A. A. A., Dolman D. E. M., Yusof S. R., Begley D. J. (2010). Structure and function of the blood-brain barrier. Neurobiol. Dis. 37, 13–25. 10.1016/j.nbd.2009.07.030 - DOI - PubMed
    1. Abraham C. S., Deli M. A., Joo F., Megyeri P., Torpier G. (1996). Intracarotid tumor necrosis factor-alpha administration increases the blood-brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labelling studies and confocal laser scanning analysis. Neurosci. Lett. 208, 85–88. 10.1016/0304-3940(96)12546-5 - DOI - PubMed
    1. Alle T. H., Ochoa M., Jr., Roth R. F., Gregersen M. I. (1953). Spectral absorption of T-1824 in plasma of various species and recovery of the dye by extraction. Am. J. Physiol. 175, 243–246. - PubMed
    1. Allen T. H., Orahovats P. D. (1950). Combination of toluidine dye isomers with plasma albumin. Am. J. Physiol. 161, 473–482. - PubMed
    1. Anderson J. M. (2001). Molecular structure of tight junctions and their role in epithelial transport. News Physiol. Sci. 16, 126–130. - PubMed

LinkOut - more resources

Feedback