Fluorescein Isothiocyanate (FITC)‐Dextran Extravasation as a Measure of Blood‐Brain Barrier Permeability

Reka Natarajan1, Nicole Northrop1, Bryan Yamamoto1

1 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.58
DOI:  10.1002/cpns.25
Online Posting Date:  April, 2017
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Abstract

The blood‐brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC‐labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels. © 2017 by John Wiley & Sons, Inc.

Keywords: blood‐brain barrier; FITC‐dextran extravasation; brain capillaries

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of FITC‐Dextran Extravasation from the BBB
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assessment of FITC‐Dextran Extravasation from the BBB

  Materials
  • FITC‐dextran solution (see recipe)
  • 250 to 300 g Sprague‐Dawley rats (Harlan, RRID:RGD_10395233)
  • Ketamine
  • Xylazine (Butler Schein, cat. no. 33197)
  • Heparin
  • 4% paraformaldehyde (PFA) solution (see recipe)
  • 10% and 20% Glycerol solution (see recipe)
  • 2‐methylbutane (Fisher Scientific, cat. no. AC12647‐0010)
  • Dry ice
  • 95% and 70% ethanol
  • 0.1 N Hydrochloric acid
  • Distilled water
  • Gelatin subbing solution (see recipe)
  • TissueTek (Electron Microscopy Sciences, cat. no. 62550‐12)
  • Millipore water
  • DRAQ5 (Cell Signaling, cat.no. 4054S)
  • Fluoromount‐G (Electron Microscopy Sciences, cat. no. 17984‐25)
  • Perfusion pump (Fisher scientific, cat. no. 13‐876‐2)
  • 50‐ml graduated cylinder (Fisher scientific, cat no. 03‐007‐45)
  • Aluminum foil
  • 16‐G, 1.5‐in. needle (Fisher scientific, cat. no. 14‐821‐14H)
  • 26‐G needle (Fisher Scientific, cat. no. 14‐826‐10)
  • 1‐ml syringe (Fisher Scientific, cat. no. 14‐823‐434)
  • Hemostat (Fisher Scientific, cat. no. 16‐100‐118)
  • Blunt scissors (Fisher Scientific, cat. no. 08‐950)
  • Micro dissection scissors (Fisher Scientific, cat. no. 08‐935)
  • Guillotine (Harvard Instruments, cat. no. 55‐0020)
  • Bone rongeurs (Fisher Scientific, cat. no. NC0688492)
  • Spatula (Fisher Scientific, cat. no. S50823)
  • Small sample jars (Weaton, cat.no. W216971)
  • Glass beaker (Fisher Scientific, cat. no 02‐540K, 02‐540T))
  • Ice bucket (Fisher Scientific, cat. no. 07‐210‐129)
  • Tweezers
  • Glass slides (Worldwide Medical, cat. no. 48011066)
  • Slide rack (EMS, cat.no. 71394‐01)
  • Deep tray for submerging slide rack (12"L X 6"W X 2"D)
  • Oven (Norco Model 410)
  • Slide box (Fisher Scientific, cat.no. 03‐446)
  • Cryostat (Fisher Scientific, Cryostar NX50)
  • Pedestals (Fisher Scientific, cat. no. 715870‐CN)
  • Soft bristled paint brush
  • Kimwipes
  • Coverslip (Fisher scientific, cat.no. 12‐545‐100)
  • Microfiber cloth
  • Confocal microscope with 488‐nm, 633‐nm excitation laser
  • Image J software (http://rsb.info.nih.gov/ij/index.html, RRID:SCR_003070)
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Figures

Videos

Literature Cited

Literature Cited
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