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Assessing Blood–Brain Barrier Function Using In Vitro Assays

  • Joseph Bressler
  • Katherine Clark
  • Cliona O’Driscoll
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1066)

Abstract

The impermeability of the blood–brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by their polarity, or oil/water partition coefficient, many lipophilic chemicals are not permeable because of multidrug transporters at the luminal and abluminal membranes. In contrast, many nutrients, which are usually polar, cross the BBB more readily than predicted by their oil/water partition coefficients due to the expression of specific nutrient transporters. In vitro models are being developed because rodent models are of low input and relatively expensive. Isolated brain microvessels and cell culture models each offers certain advantages and disadvantages. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity, whereas cell culture models allow the investigator to measure directional transport and can be genetically manipulated. In this chapter, we describe how to isolate large batches of brain microvessels from freshly slaughtered cows. The different steps in the isolation procedure include density gradient centrifugations and filtering. Purity is determined microscopically and by marker enzymes. Permeability is assessed by measuring the uptake of fluorescein-labeled dextran in an assay that has been optimized to have a large dynamic range and low inter-day variability. We also describe how to evaluate transendothelial cell electrical resistance and paracellular transport in cell culture models.

Key words

Blood–brain barrier Microvessels Tight junctions Transporters Electrical resistance 

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Joseph Bressler
    • 1
    • 2
  • Katherine Clark
    • 2
  • Cliona O’Driscoll
    • 3
    • 2
  1. 1.Hugo Moser Laboratory at the Kennedy KriegerKennedy Krieger InstituteBaltimoreUSA
  2. 2.Department of Environmental Health Sciences, Center In Alternatives In Animal Testing, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  3. 3.Hugo Moser LaboratoryKennedy Krieger InstituteBaltimoreUSA

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