Peripheral Type Benzodiazepine Binding Sites as a Tool for the Detection and Quantification of CNS Injury

Jesús Benavides1, Alain Dubois1, Bernard Scatton1

1 Synthélabo Recherche, Bagneux Cédex
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.16
DOI:  10.1002/0471142301.ns0716s09
Online Posting Date:  May, 2001
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Abstract

The concentration of peripheral type benzodiazepine binding sites (PTBS) in the brain parenchyma is greatly increased following brain lesions, reflecting the glial reaction and/or presence of hematogenous cells. Thus, PTBS density is a sensitive and reliable marker of brain injury in a large number of experimental models (ischemia, trauma, excitotoxic lesions, brain tumors) and equivalent human neuropathological conditions. PTBS density can be measured using specific radioligands and a conventional binding technique, or by quantitative autoradiography in tissue sections.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of PTBS in Brain Homogenates Using a Ligand Binding Technique
  • Basic Protocol 2: PTBS Autoradiography
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of PTBS in Brain Homogenates Using a Ligand Binding Technique

  Materials
  • Experimental animals
  • Tris‐buffered saline (TBS): 120 mM NaCl/50 mM Tris⋅Cl, pH 7.4 (4°C)
  • 1 mCi/ml [3H]PK 11195 (NEN Life Sciences; >50 Ci/mmol)
  • 1 mM stock unlabeled PK 11195 (Research Biochemicals) in ethanol (store up to 1 year at −20°C)
  • Scintillation fluid (e.g., Ultima Gold; Packard Instruments)
  • Polytron homogenizer (Brinkmann)
  • 3‐ml tubes or 96‐well plates capable of holding 250 µl/well
  • Filtration system: depending on number of samples, a manual system (e.g., 12‐well filtration system from Millipore) or semiautomatic system (e.g., Mach III harvester from Tomtec)
  • GF/B glass filters (Whatman)
  • Plastic 5‐ml scintillation vials
  • 40°C drying oven

Basic Protocol 2: PTBS Autoradiography

  Materials
  • Experimental animals
  • Isopentane (Aldrich)
  • 4% (w/v) carboxymethylcellulose (CMC; medium viscosity; Sigma), gel prepared in H 2O (store up to 1 month at 4°C)
  • [3H]PK 11195 (NEN Life Sciences)
  • 1 mM stock of unlabeled PK 11195 (Research Biochemicals) in ethanol (store up to 1 year at −20°C)
  • Tris‐buffered saline (TBS): 120 mM NaCl/50 mM Tris⋅Cl, pH 7.4 (4°C)
  • Encephalotome
  • Metal beaker
  • Self‐sealable plastic bags (Minigrip or equivalent)
  • Cryostat chucks
  • Cryostat microtome (e.g., Leica CM 3000)
  • Ready‐to‐use Superfrost/Plus slides (Menzel/Glazer)
  • Racks for slides
  • 250‐ml plastic tanks
  • Radioactivity microscales (Amersham)
  • Autoradiographic films (e.g., 3H‐Ultrofilm, Amersham)
  • Densitometric system (e.g., BIOCOM)
  • Additional reagents and equipment for cryostat sectioning (unit 1.1) and autoradiography (CPMB APPENDIX and appendix 1A in this manual).
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Figures

Videos

Literature Cited

Literature Cited
   Anholt, R.R.H., De Souza, E.B., Oster‐Granite, M.L., and Snyder, S.H. 1985. Peripheral‐type benzodiazepine receptors: Autoradiographic localization in whole‐body sections of neonatal rats. J. Pharmacol. Exp. Ther. 233:517‐526.
   Benavides, J., Fage, D., Carter, C., and Scatton, B. 1987. Peripheral type benzodiazepine are a sensitive indirect index of neuronal damage. Brain Res. 421:167‐172.
   Benavides, J., Cornu, P., Dennis, T., Dubois, A., Hauw, J.J., MacKenzie, E.T., Sazdovitch, V., and Scatton, B. 1988. Imaging of human brain lesions with an ω3 site radioligand. Ann. Neurol. 24:708‐712.
   Benavides, J., Dubois, A., Dennis, T., Hamel, E., and Scatton, B. 1989. ω3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: An autoradiographic study with the photoaffinity ligand [3H]PK 14105. J. Pharmacol. Exp. Ther. 249:333‐339.
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