Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish Peroxidase

The blood-brain barrier (BBB) constituted by endothelial cells of brain microvessels is a dynamic interface, which controls and regulates the transport of various substances including peptides, proteins, ions, vitamins, hormones, and immune cells from the circulation into the brain parenchyma. Certain diseases/disorders such as Alzheimer’s disease, sepsis, and hypertension can lead to varying degrees of BBB disruption. Moreover, impairment of BBB integrity has been implicated in the pathogenesis of various neurodegenerative diseases like epilepsy. In attempts to explore the wide spectrum of pathophysiologic mechanisms of these diseases/disorders, a variety of experimental insults targeted to the BBB integrity in vitro in cell culture models and in vivo in laboratory animals have been shown to alter BBB permeability causing enhanced transport of certain tracers such as sodium fluorescein, cadaverine-Alexa fluor, horseradish peroxidase, FITC-dextran, albumin-Alexa fluor conjugates, and Evans blue dye across the barrier. The permeability changes in barrier-type endothelial cells can be assessed by intravascular infusion of exogenous tracers and subsequent detection of the extravasated tracer in the brain tissue, which enable functional and structural analysis of BBB integrity. In this chapter, we aimed to highlight the current knowledge on the use of four most commonly performed tracers, namely, Evans blue, sodium fluorescein, albumin-Alexa fluor conjugates, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, confocal laser scanning microscopy, and electron microscopy are also discussed. Tracing studies at the morphological level are mainly aimed at the identification of the tracers both in the barrier-related cells and brain parenchyma. In addition, BBB permeability to the tracers can be quantified using spectrophotometric and spectrophotofluorometric assays and image analysis by confocal laser scanning microscopy and electron microscopy. The results of our studies conducted under various experimental settings using the mentioned tracers indicate that barrier-type endothelial cells in brain microvessels orchestrate the paracellular and/or transcellular trafficking of substances across BBB. These efforts may not only contribute to designing approaches for the management of diseases/disorders associated with BBB breakdown but may also provide new insights for developing novel brain drug delivery strategies.