Assessment of Ex Vivo Transport Function in Isolated Rodent Brain Capillaries

Gary N.Y. Chan1, Ronald E. Cannon1

1 National Institutes of Health, National Institute of Environmental Health Sciences, Intracellular Regulatory Group, Signal Transduction Laboratory, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.16
DOI:  10.1002/cpph.21
Online Posting Date:  March, 2017
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The blood‐brain barrier plays an important role in neuroprotection; however, it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and biochemical elements of the blood‐brain barrier are essential to regulate the permeability of nutrients, ions, hormones, toxic metabolites, and xenobiotics into and out of the brain. In particular, high expression of ATP‐driven efflux transporters at the blood‐brain barrier is a major obstacle in the delivery of CNS pharmacotherapeutics to the brain. The complete understanding of these elements can offer insights on how to modulate barrier functions for neuroprotection against CNS drug toxicity and to enhance drug delivery to the brain. In the literature, preclinical models of the blood‐brain barrier are widely utilized to predict drug pharmacokinetics and pharmacodynamics properties in the brain. In addition, these models are essential tools to investigate cellular mechanisms and novel interventions that alter barrier function and permeability. This unit presents procedures to isolate fresh and viable rodent brain capillaries for the assessment of ex vivo transport activity at the blood‐brain barrier. © 2017 by John Wiley & Sons, Inc.

Keywords: blood‐brain barrier; brain; capillaries; microvessel; rodent

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Brain Capillaries Using Filters and BSA
  • Alternate Protocol 1: Isolation of Brain Capillaries Using Filters, but Without BSA
  • Basic Protocol 2: Ex Vivo Assessment of Transport Activity in Brain Capillaries
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Brain Capillaries Using Filters and BSA

  • Rodents (see step 1 annotation)
  • CO 2 (for rodent euthanization)
  • Isolation buffer (see recipe)
  • Ice buckets containing wet ice
  • 30% Ficoll PM400 solution (see recipe)
  • Isolation buffer containing 1% BSA (see recipe)
  • Surgical equipment including:
    • Surgical scalpels
    • Scalpel blades
    • Spatula
    • Scissors
  • Littauer bone cutter (Fine Science Tools, cat. no. 16152‐15)
  • 50‐ml Falcon polypropylene conical tubes (Becton, Dickinson and Company)
  • Scale
  • Polystyrene petri dishes (small: 30 × 15 mm & large: 100 × 15 mm; Becton, Dickinson and Company)
  • Forceps (Dumont no. 7: fine and Iris, curved; Fine Science Tools)
  • Stereomicroscope
  • Size C 55‐ml tissue grinding vessel (Thomas Scientific, cat. no. 3431E55) and pestle size C serrated (Thomas Scientific, cat. no. 3431F25) (clearance: 150 to 230 µm)
  • 250‐ and 1000‐ml glass beakers
  • 100‐ml glass bottles
  • 15 ml KONTES dounce tissue grinders and pestle size B (VWR, cat. no. 885300‐0015; clearance: 165 to 889 µm)
  • 50‐ml polycarbonate centrifuge tubes with polyethylene caps (Beckman Coulter, cat. no. 363664)
  • Sorvall RC‐5B centrifuge with a SS‐34 rotor (Beckman Coulter)
  • Long, gel‐loading pipette tips
  • Squirt bottle
  • 7‐ml bulb polyethylene transfer pipette (USA Scientific, cat. no. 1020‐2500)
  • Spectra/Mesh woven nylon filters (sheet cut into desired size) (Spectrum Lab, cat. no. 146486; mesh opening: 300 µm, thickness 200 µm)
  • pluriStrainer (Life Science, cat. no. 43‐50030‐50, mesh opening: 30 µm) and connector rings (pluriSelect Life Science, cat. no. 41‐50000‐03)
  • 25‐ml serological pipettes and a pipette controller (any brand)
  • VWR microscope glass slide, clear, 1‐in. × 3‐in. (any brand)
  • Glass coverslips, #1 thickness, square (any brand)
  • Allegra X‐22R benchtop centrifuge, Beckman Coulter

Alternate Protocol 1: Isolation of Brain Capillaries Using Filters, but Without BSA

  • Freshly isolated brain capillaries suspension in ice‐chilled isolation buffer (from protocol 1)
  • Isolation buffer (see recipe)
    • Experimental treatment solutions: Use DMSO to prepare stock solutions for all chemicals listed below
    • NBD‐CSA, [N‐ϵ(4‐nitrobenzofurazan‐7‐yl)‐D‐Lys8]‐cyclosporine A, custom synthesized by R. Wenger (Schramm, Fricker, Wenger, & Miller, )
    • BODIPY‐Prazosin (Life Technologies)
    • Sulforhodamine 101 free acid (Texas Red; Sigma‐Aldrich)
    • PSC‐833, KO143, and MK571 (Tocris Bioscience)
  • Fluorescence probe solutions (see recipe)
  • Transporter inhibition solutions (see recipe)
  • Chambered no. 1.0 borosilicate cover glass (ThermoFisher Scientific)
  • Inverted confocal laser scanning microscope, with a 40 × 1.2 numeric aperture water‐immersion objective, with functional 488‐nm, and 543‐nm laser and detector lines (Carl Zeiss, LSM 510)
  • ImageJ software (NIH)
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Literature Cited

Literature Cited
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  Hartz, A. M., Bauer, B., Soldner, E. L., Wolf, A., Boy, S., Backhaus, R.,..., Schlachetzki, F. (2012). Amyloid‐beta contributes to blood‐brain barrier leakage in transgenic human amyloid precursor protein mice and in humans with cerebral amyloid angiopathy. Stroke, 43, 514‐523. doi: 10.1161/STROKEAHA.111.627562.
  Hartz, A. M. S., Zhong, Y., Wolf, A., LeVine, H., III, Miller, D., & Bauer, B. (2016). Aβ40 reduces p‐glycoprotein at the blood–brain barrier through the ubiquitin–proteasome pathway. Journal of Neuroscience, 36, 1930‐1941. doi: 10.1523/JNEUROSCI.0350‐15.2016.
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  Miller, D. S. (2010). Regulation of P‐glycoprotein and other ABC drug transporters at the blood‐brain barrier. Trends in Pharmacological Sciences, 31, 246‐254. doi: 10.1016/
  Miller, D. S. (2015). Regulation of ABC transporters blood‐brain barrier: The good, the bad, and the ugly. Advances in Cancer Research, 125, 43‐70. doi: 10.1016/bs.acr.2014.10.002.
  Schramm, U., Fricker, G., Wenger, R., & Miller, D. S. (1995). P‐glycoprotein‐mediated secretion of a fluorescent cyclosporin analogue by teleost renal proximal tubules. American Journal of Physiology ‐ Renal Fluid and Electrolyte Physiology, 268, F46‐F52.
  Song, L., & Pachter, J. S. (2003). Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction‐associated proteins. In Vitro Cellular & Developmental Biology. Animal, 39, 313‐320. doi: 10.1290/1543‐706X(2003)039%3c0313:COMBME%3e2.0.CO;2.
  Wu, Z., Hofman, F. M., & Zlokovic, B. V. (2003). A simple method for isolation and characterization of mouse brain microvascular endothelial cells. Journal of Neuroscience Methods, 130, 53‐63. doi: 10.1016/S0165‐0270(03)00206‐1.
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