Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives?

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.

Spreading depolarizations trigger caveolin‐1–dependent endothelial transcytosis

“…We next examined cerebral extravasation of Evans blue (EB) dye administered intravascularly immediately after the last CSD. At the selected dose, vast majority of EB binds to serum albumin (~65 kDa), and the complex (~70 kDa) does not cross an intact BBB (Saunders et al, 2015). As with cortical water content, EB extravasation significantly increased within 6 hours after CSDs compared to the contralateral hemisphere, and progressed for at least 24 hours (Figure 1C, left ).…”

Quantitative measurement of lymphatic function in mice by noninvasive near-infrared imaging of a peripheral vein

“…However, during these studies it could not be excluded that the protein-dye complexes directly entered blood capillaries or that a significant amount of the protein-dye complexes disappeared from the circulation over time. In addition, it is unknown how stable these complexes are in the interstitial tissue/serous cavity and the blood; i.e., there may be disassociation of dye occurring over time or some Evans blue may not be binding serum proteins at all (32). Importantly, the approach described here, using 40-kDa PEG-dye conjugates, addresses such concerns.…”

Effects of Exposure to Blast Overpressure on Intracranial Pressure and Blood-Brain Barrier Permeability in a Rat Model

“…Both, changes in ICP and EB leakage were related to the BOP intensity with a more pronounced effect observed in multiple exposures to the higher levels of BOP. While EB is a commonly used marker for the assessment of BBB integrity, its use is limited by some disadvantages such as its non-specific binding to albumin and potential presence of free dye in the brain tissue [33]. Other markers for the determination of BBB permeability, such as radiolabeled compounds, sodium fluorescein (NaFl), and dextrans are less subject to these limitations.…”

“…Other markers for the determination of BBB permeability, such as radiolabeled compounds, sodium fluorescein (NaFl), and dextrans are less subject to these limitations. In particular, biotin and fluorophor labeled dextrans are valuable markers of BBB integrity as they appear to be non-toxic in small quantities, have not been reported to bind with proteins or tissue, and can be visualized under light or electron microscopy [33]. Nonetheless, a study using systemic administration of NaFl, EB, and dextrans to estimate the pore size of BBB opening and the time required for recovery in an in vivo bTBI model, [26] revealed no difference in the time course of extravasation of all three markers in the brain after exposure to blast.…”