In Vivo Measurement of Blood‐Brain Barrier Permeability

Kathleen M.K. Boje1

1 University at Buffalo, School of Pharmacy, Buffalo, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.19
DOI:  10.1002/0471142301.ns0719s15
Online Posting Date:  August, 2001
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Abstract

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood ‐ brain barrier. Methods for the measurement of drug influx or efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier‐mediated or saturable transport.

     
 
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Table of Contents

  • Basic Protocol 1: Blood‐Brain Barrier Influx Measurement: In Situ Brain Perfusion Procedure for Rats
  • Alternate Protocol 1: Blood‐Brain Barrier Influx Measurement: Simplified In Situ Brain Perfusion for Rats
  • Basic Protocol 2: Blood‐Brain Barrier Influx Measurement: In Situ Brain Perfusion Procedure for Mice
  • Support Protocol 1: Construction of PE50–Stainless Steel Connectors and Cut 22‐G Hypodermic Needles
  • Support Protocol 2: Construction of In Situ Perfusion Cannulas
  • Support Protocol 3: Experimental Determination of Intravascular Capillary Volume Using Radiolabeled Inulin
  • Support Protocol 4: Experimental Determination of Cerebral Perfusion Fluid Flow Using Radiolabeled Diazepam
  • Support Protocol 5: Use of the In Situ Perfusion Technique for Mechanistic Assessment of Carrier‐Mediated or Saturable Transport
  • Basic Protocol 3: Blood‐Brain Barrier Influx Measurement: Intravenous Administration/Multiple Time Point Procedure for Rats
  • Support Protocol 6: Construction of Jugular Vein Catheters
  • Support Protocol 7: Capillary Depletion Method
  • Basic Protocol 4: Blood‐Brain Barrier Efflux Measurement: The Brain Efflux Index Method
  • Support Protocol 8: Construction of Cerebrospinal Fluid Collection Units
  • Reagents And Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Blood‐Brain Barrier Influx Measurement: In Situ Brain Perfusion Procedure for Rats

  Materials
  • Krebs‐bicarbonate buffer (see recipe), 37°C
  • Perfusate solution: test compound(s) dissolved in Krebs‐bicarbonate buffer, 37°C
  • Laboratory rats, either gender, 200 to 350 g
  • Anesthetic (as specified by the approved animal care protocol; appendix 4B)
  • Saline: 0.9% (w/v) NaCl (sterile)
  • Tissue solubilizer (e.g., Soluene 350; Packard) or 2 N NaOH, for radiolabeled test compounds only
  • Vials (scintillation vials, 7 to 20 ml, with caps, can be used when working with radiolabeled compounds)
  • 1‐, 10‐, 20‐, and 50‐ml syringes
  • 22‐G hypodermic needles with the beveled end cut off (see protocol 4)
  • Warming pads (e.g., Deltaphase Isothermal Pad; Braintree Scientific), preheated to 37°C
  • Syringe infusion pump (e.g., Pump 22; Harvard Apparatus)
  • Animal fur shaver
  • Surgical instruments (e.g., Harvard Apparatus), including:
  •  4.5‐in. (∼11.4‐cm) operating scissors, sharp or blunt
  •  Disposable scalpels
  •  4‐in. microdissecting forceps, full or strongly curved
  •  5.5‐in. Olsen‐Hegar needle holder
  •  3.5‐in. Hartman hemostatic forceps
  •  6‐in. probe with eye (eye probe)
  •  3‐in. Vannas spring scissors, straight
  •  Tissue forceps (straight) or bone rongeurs
  •  Inox no. 7 sharp‐point forceps
  • Suture, USP size 4.0
  • Electrocautery (e.g., Change‐A‐Tip low‐temperature power handle and disposable sterile tips; Aaron Medical Industries), 1100° to 1300°F
  • Microvessel arterial clamp (Kleinert‐Kutz microvessel clip; blades 6 mm × 1 mm, curved; 11 mm in length; Pilling Weck Surgical)
  • Perfusion cannula (see protocol 5)
  • PE50 polyethylene tubing, 0.58 mm (0.023 in.) i.d., 0.965 mm (0.038 in.) o.d. (e.g., Intramedic Clay Adams; Becton Dickinson Labware)
  • 22‐G stainless steel connectors (see protocol 4)
  • Rodent guillotine
  • Filter paper
  • 50°C water bath, optional
  • Additional reagents and equipment for analyzing nonradiolabeled test compounds (see , discussion of sensitive analytical method), optional

Alternate Protocol 1: Blood‐Brain Barrier Influx Measurement: Simplified In Situ Brain Perfusion for Rats

  Materials
  • Krebs‐bicarbonate buffer (see recipe), 37°C
  • Perfusate solution: test compound(s) dissolved in recipeKrebs‐bicarbonate buffer, 37°C
  • Laboratory mice (adult weight, either gender)
  • Anesthetic (as specified by the approved animal care protocol; appendix 4B)
  • Vials (scintillation vials, 7 to 20 ml, with caps, can be used when working with radiolabeled compounds)
  • 1‐ and 10‐ml syringes
  • 22‐G hypodermic needles with the beveled end cut off (see protocol 4)
  • Warming pads (e.g., Deltaphase Isothermal Pad; Braintree Scientific), preheated to 37°C
  • Syringe infusion pump (e.g., Pump 22; Harvard Apparatus)
  • Animal fur shaver
  • Surgical instruments (e.g., Harvard Apparatus; see protocol 1)
  •  4.5‐in. (∼11.4‐cm) operating scissors, sharp or blunt
  •  Disposable scalpels
  •  4‐in. microdissecting forceps, full or strongly curved
  •  5.5‐in. Olsen‐Hegar needle holder
  •  3.5‐in. Hartman hemostatic forceps
  •  Tissue forceps (straight) or bone rongeurs
  •  Inox no. 7 sharp‐point forceps
  • Suture, USP size 4.0
  • Perfusion cannula configuration 1 or 2 (see protocol 5), but made with a 26‐G (not 25‐G) hypodermic needle
  • PE50 polyethylene tubing, 0.58 mm (0.023 in.) i.d., 0.965 mm (0.038 in.) o.d. (Intramedic Clay Adams; Becton Dickinson Labware)
  • 22‐G stainless steel connectors (see protocol 4)
  • Sharp surgical shears or rodent guillotine
  • Additional reagents and equipment for harvesting and analyzing tissue (see protocol 1)

Basic Protocol 2: Blood‐Brain Barrier Influx Measurement: In Situ Brain Perfusion Procedure for Mice

  Materials
  • Metal file
  • 22‐G stainless steel disposable hypodermic needles
  • 5.5‐in. (14‐cm) Olsen‐Hegar needle holder (e.g., Harvard Apparatus)
  • Hartman hemostatic forceps, 3.5 in.

Support Protocol 1: Construction of PE50–Stainless Steel Connectors and Cut 22‐G Hypodermic Needles

  Materials
  • PE50 polyethylene tubing, 0.58 mm (0.023 in.) i.d., 0.965 mm (0.038 in.) o.d. (e.g., Intramedic Clay Adams; Becton Dickinson Labware)
  • Forceps and scissors
  • Boiling water bath

Support Protocol 2: Construction of In Situ Perfusion Cannulas

  • Perfusate solution: 0.5 µCi/ml [3H]inulin (American Radiolabeled Chemicals) or 0.3 µCi/ml [14C]methoxyinulin (American Radiolabeled Chemicals) dissolved in Krebs‐bicarbonate buffer (see recipe), 37°C

Support Protocol 3: Experimental Determination of Intravascular Capillary Volume Using Radiolabeled Inulin

  • Perfusate solution: 0.5 µCi/ml [3H]diazepam (American Radiolabeled Chemicals) or 0.3 µCi/ml [14C]diazepam (American Radiolabeled Chemicals) dissolved in Krebs‐bicarbonate buffer (see recipe), 37°C

Support Protocol 4: Experimental Determination of Cerebral Perfusion Fluid Flow Using Radiolabeled Diazepam

  • Perfusate solution: variable concentrations of a test compound with or without a competitive inhibitor dissolved in Krebs‐bicarbonate buffer (see recipe), 37°C

Support Protocol 5: Use of the In Situ Perfusion Technique for Mechanistic Assessment of Carrier‐Mediated or Saturable Transport

  Materials
  • Laboratory rats, either gender, 200 to 350 g
  • Anesthetic, antibiotic, and other required medications for rodent survival surgery and rapid sacrifice (as specified by the approved animal care protocol; appendix 4B)
  • Betadine (povidone/iodine solution)
  • Saline: 0.9% (w/v) NaCl (sterile), 37° and 4°C
  • 10 U/ml heparinized saline, sterile
  • Test compound(s) dissolved in Krebs‐bicarbonate buffer (see recipe), 37°C
  • Tissue solubilizer (e.g., Soluene 350; Packard) or 2 N NaOH, for radiolabeled test compounds only
  • Hydrogen peroxide, 30% (e.g., Sigma)
  • Warming pads (e.g., Deltaphase Isothermal Pad; Braintree Scientific), preheated to 37°C
  • Animal fur shaver
  • Surgical instruments (e.g., Harvard Apparatus), including:
  •  4.5‐in. (∼11.4‐cm) operating scissors, sharp or blunt
  •  Scalpel
  •  4‐in. (10‐cm) microdissecting forceps, full or strongly curved
  •  3.5‐in. Hartman hemostatic forceps
  •  6‐in. (15‐cm) eye probe
  •  3‐in. (7.5‐cm) Vannas spring scissors, straight
  •  Dumont no. 7 microdissecting forceps
  •  5.5‐in. Olsen‐Hegar needle holder
  •  Inox no. 7 sharp‐point forceps
  • Sterile drapes
  • Sterile sutures with and without needle, USP size 2.0 and 4.0
  • Jugular vein catheter (see protocol 10)
  • 22‐G hypodermic needles with the beveled end cut off (see protocol 4)
  • 1‐, 3‐, and 5‐ml syringes
  • 22‐G stainless steel wire, sterile
  • Vials (scintillation vials, 7 to 20 ml, with caps, can be used when working with radiolabeled compounds)
  • Rodent guillotine
  • PE50 polyethylene tubing, 0.58 mm (0.023 in.) i.d., 0.965 mm (0.038 in.) o.d. (Intramedic Clay Adams; Becton Dickinson Labware)
  • 22‐G stainless steel connectors (see protocol 4)
  • Tissue forceps or bone rongeurs
  • Filter paper
  • 50°C water bath, optional
  • 1.5‐ml microcentrifuge tubes with caps
  • Additional reagents and equipment for analyzing nonradiolabeled test compounds (see , discussion of sensitive analytical method), optional

Basic Protocol 3: Blood‐Brain Barrier Influx Measurement: Intravenous Administration/Multiple Time Point Procedure for Rats

  Materials
  • Chloroform
  • PE50 polyethylene tubing, 0.58 mm (0.023 in.) i.d., 0.965 mm (0.038 in.) o.d. (e.g., Intramedic Clay Adams; Becton Dickinson Labware)
  • Silastic tubing, 0.50 mm (0.020 in.) i.d., 0.965 mm (0.038 in.) o.d. (e.g., Silastic Medical Grade; Dow Corning)
CAUTION: Chloroform is a toxic irritant and mild carcinogen; take suitable precautions and work in a fume hood.

Support Protocol 6: Construction of Jugular Vein Catheters

  • Krebs‐bicarbonate buffer (see recipe), 4°C
  • 26% (w/v) dextran solution (average MW 74,000), 4°C
  • Glass homogenizer with glass pestle, prechilled

Support Protocol 7: Capillary Depletion Method

  Materials
  • Laboratory rats (200 to 350 g, either gender)
  • Anesthetic (as specified by the approved animal care protocol; appendix 4B)
  • 2% (w/v) lidocaine/0.001% (w/v) epinephrine (commercially available as Xylocaine 2% with epinephrine 1:100,000; J.A. Webster Co.)
  • Injectate solution: test and reference compounds dissolved in ECF buffer (see recipe), 37°C
  • Saline: 0.9% (w/v) NaCl (sterile)
  • Tissue solubilizer (e.g., Soluene 350; Packard) or 2 N NaOH
  • Scintillation vials, 7 to 20 ml, with caps
  • Stereotaxic atlas (e.g., Paxinos and Watson, , for rats)
  • 5‐µl syringe (e.g., Hamilton Gas‐Tight removable needle microliter syringe; Hamilton Company), with 28‐G, 2‐in. (5‐cm), point‐style no. 4 (12° bevel) needle, 0.18 mm (0.007 in.) nominal i.d., 0.36 mm (0.014 in.) nominal o.d. (may need to be custom fabricated by Hamilton Company)
  • Rodent stereotaxic frame
  • Animal fur shavers
  • Warming pads (Deltaphase Isothermal Pad; Braintree Scientific), preheated to 37°C
  • Scalpel
  • Drill and small drill bit, e.g., 1/32‐in. (0.8‐mm) bit inserted in a Dremel unit or affixed to a manual handle
  • Surgical shears
  • Cerebrospinal fluid (CSF) collection unit (see protocol 13)
  • Olsen‐Hegar needle holder (e.g., Harvard Apparatus)
  • Rodent guillotine, optional
  • Tissue forceps or bone rongeurs
  • Filter paper
  • Inox no. 7 sharp‐point forceps (e.g., Harvard Apparatus)
  • 50°C water bath, optional
NOTE: Because the amounts of injected test compounds are small, a highly sensitive analytical detection method is required. Radiolabeled test compounds are frequently used. Suggested reference compounds include 3H‐ or 14C‐labeled inulin or carboxyinulin. If the radiolabeled form of a test compound is to be used, an isotopic form different from the inulin reference must be selected.

Basic Protocol 4: Blood‐Brain Barrier Efflux Measurement: The Brain Efflux Index Method

  Materials
  • 5.5‐in. (14‐cm) Olsen‐Hegar needle holder (e.g., Harvard Apparatus)
  • Metal file
  • 22‐ and 25‐G stainless steel disposable hypodermic needles
  • Silastic tubing, 0.50 mm (0.020 in.) i.d., 0.965 mm (0.038 in.) o.d. (e.g., Silastic Medical Grade; Dow Corning)
  • Suture, USP size 4.0
  • 1‐ml syringe
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Figures

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Literature Cited

Literature Cited
   Adkison, K.D. and Shen, D.D. 1996. Uptake of valproic acid into rat brain is mediated by a medium‐chain fatty acid transporter. J. Pharmacol. Exp. Ther. 276:1189‐1200.
   Allen, D.D., Oki, J., and Smith, Q.R. 1997. An update on the in situ rat brain perfusion technique: Simpler, faster, better. Pharm. Res. 14:S337.
   Aoyagi, M., Agranoff, B.W., Washburn, L.C., and Smith, Q.R. 1988. Blood‐brain barrier transport of 1‐aminocyclohexanecarboxylic acid, a non‐metabolizable amino acid for in vivo studies of brain transport. J. Neurochem. 50:1220‐1226.
   Dagenais, C., Rousselle, C., Pollack, G., and Scherrmann, J.M. 2000. Development of an in situ mouse brain perfusion model and its application to mdr1a P‐glycoprotein‐deficient mice. J. Cereb. Blood Flow Metab. 20:381‐386.
   Fukui, S., Schwarcz, R., Rapoport, S.I., Takada, Y., and Smith, Q.R. 1991. Blood‐brain barrier transport of kynurenines:Implications for brain synthesis and metabolism. J. Neurochem. 56:2007‐2017.
   Hosoya, K., Sugawara, M., Asaba, H., and Terasaki, T. 1999. Blood‐brain barrier produces significant efflux of L‐aspartic acid but not D‐aspartic acid:In vivo evidence using the brain efflux index method. J. Neurochem. 73:1206‐1211.
   Kakee, A., Terasaki, T., and Sugiyama, Y. 1996. Brain efflux index as a novel method of analyzing efflux transport at the blood‐brain barrier. J. Pharmacol. Exp. Ther. 277:1550‐1559.
   Kakee, A., Terasaki, T., and Sugiyama, Y. 1997. Selective brain to blood efflux transport of para‐aminohippuric acid across the blood‐brain barrier:In vivo evidence by use of the brain efflux method. J. Pharmacol. Exp. Ther. 283:1018‐1025.
   Kusuhara, H., Suzuki, H., Terasaki, T., Atsuyuki, K., Lemaire, M., and Sugiyama, Y. 1997. P‐glycoprotein mediates the efflux of quinidine across the blood‐brain barrier. J. Pharmacol. Exp. Ther. 283:574‐580.
   Ohno, K., Pettigrew, K.D., and Rapoport, S.I. 1978. Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat. Am. J. Physiol. 235:H299‐H307.
   Pardridge, W.M. 1998. Introduction to the Blood‐Brain Barrier: Methodology, Biology and Pathology. Cambridge University Press, Cambridge.
   Paxinos, G. and Watson, C. 1986. The rat brain in stereotaxic coordinates. Academic Press, San Diego.
   Rapoport, S.I., Fredericks, W.R., Ohno, K., and Pettigrew, K.D. 1980. Quantitative aspects of reversible osmotic opening of the blood‐brain barrier. Am. J. Physiol. 238:R421‐R431.
   Smith, Q.R. 1989. Quantitation of blood‐brain barrier permeability. In Implications of the Blood‐Brain Barrier and Its Manipulation (E.A. Neuwelt, ed.) pp. 85‐118. Plenum, New York.
   Smith, Q.R., Momma, S., Aoyagi, M., and Rapoport, S. 1987. Kinetics of neutral amino acid transport across the blood‐brain barrier. J. Neurochem. 49:1651‐1658.
   Smith, Q.R., Ziylan, Y.Z., Robinson, P.J., and Rapoport, S.I. 1988. Kinetics and distribution volumes for tracers of different sizes in the brain plasma space. Brain Res. 462:1‐9.
   Smith, Q.R., Nagura, H., Takada, Y., and Duncan, M.W. 1992. Facilitated transport of the neurotoxin, β‐N‐methylamino‐L‐alanine, across the blood‐brain barrier. J. Neurochem. 58:1330‐1337.
   Stoll, J., Wadhwani, K.C., and Smith, Q.R. 1993. Identification of the cationic amino acid transporter (system y+) of the rat blood‐brain barrier. J. Neurochem. 60:1956‐1959.
   Takada, Y., Greig, N.H., Vistica, D.T., Rapoport, S.I., and Smith, Q.R. 1991. Affinity of antineoplastic amino acid drugs for the large neutral amino acid transporter of the blood‐brain barrier. Cancer Chemother. Pharmacol. 29:89‐94.
   Takasato, Y., Rapoport, S.I., and Smith, Q.R. 1984. An in situ brain perfusion technique to study cerebrovascular transport in the rat. Am. J. Physiol. 247:H484‐H493.
   Takasawa, K., Terasaki, T., Suzuki, H., and Sugiyama, Y. 1997. In vivo evidence for carrier‐mediated efflux transport of 3′‐azido‐3′‐deoxythymidine and 2′‐3′dideoxyinosine across the blood‐brain barrier via a probenecid‐sensitive transport system. J. Pharmacol. Exp. Ther. 281:369‐375.
   Triguero, D., Buciak, J., and Pardridge, W. 1990. Capillary depletion method for quantification of blood‐brain barrier transport of circulating peptides and plasma proteins. J. Neurochem. 54:1882‐1888.
   Zlokovic, B.V., Begley, D.J., Djuricic, B.M., and Mitrovick, D.M. 1986. Measurement of solute transport across the blood‐brain barrier in the perfused guinea‐pig brain:Method and application to N‐methyl‐α‐aminoisobutyric acid. J. Neurochem. 46:1444‐1451.
Key References
   Boje, K.M.K. 1995. Cerebrovascular permeability changes during experimental meningitis in the rat. J. Pharmacol. Exp. Ther. 274:1199‐1203.
  This paper illustrates the application of the intravenous injection/multiple time point procedure to studies of altered blood‐brain barrier permeability during disease.
   Kakee et al., 1996. See above.
  This seminal paper introduces and validates the brain efflux index method.
   Smith, 1989. See above.
  This comprehensive review provides a theoretical and practical overview of the influx techniques presented in this unit.
   Smith et al., 1987. See above.
  This exemplary paper utilizes the in situ brain perfusion method for mechanistic studies of active transport.
   Zlokovic, B.V. 1995. Cerebrovascular permeability to peptides: Manipulations of transport systems at the blood‐brain barrier. Pharm. Res. 12:1395‐1406.
  This excellent review critically evaluates peptide transport across the blood‐brain barrier.
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