1. Methods Mol Biol. 2021;2367:87-103. doi: 10.1007/7651_2020_316.

Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers:

Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish
Peroxidase.

Ahishali B(1), Kaya M(2)(3).

Author information:
(1)Department of Histology and Embryology, Koç University School of Medicine,
Istanbul, Turkey.
(2)Department of Physiology, Koç University School of Medicine, Istanbul,
Turkey. mehmetkaya@ku.edu.tr.
(3)Koç University Research Center for Translational Medicine, Istanbul, Turkey.
mehmetkaya@ku.edu.tr.

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.

DOI: 10.1007/7651_2020_316
PMID: 32785841 [Indexed for MEDLINE]