Morphological Measurement of Neurotoxic Injury in the Peripheral Nervous System: Preparation of Material for Light and Transmission Electron Microscopic Evaluation

S.K. Hancock1, J. Hinckley1, M. Ehrich1, B.S. Jortner1

1 Virginia Polytechnic Institute and State University, Blacksburg, Virginia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.12
DOI:  10.1002/0471140856.tx1212s22
Online Posting Date:  January, 2005
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Abstract

An important method of assessing experimental neurotoxic injury is the pathologic examination of the nervous system. Methods for fixation, sampling, and preparation of peripheral nervous system tissues for critical pathological neurotoxicology studies are presented. Fixation of tissue is carried out using either perfusion‐fixation of laboratory animals or immersion‐fixation of dissected nerve segments. Dissection of the peripheral nervous system (from perfusion‐fixed animals) is done to allow for multilevel sampling. Focus is on use of epoxy resin embedding tissue sections for optimal light microscopic resolution. Protocols for processing, sectioning, and staining for light and transmission electron microscopy are provided. A protocol for teasing and microscopic study of individual myelinated fibers is provided.

Keywords: neurotoxicity; peripheral nerve; neuropathology; tissue preparation

     
 
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Table of Contents

  • Basic Protocol 1: Perfusion Fixation in Rats
  • Alternate Protocol 1: Immersion Fixation of Peripheral Nerve
  • Basic Protocol 2: Dissection of Peripheral Nervous Tissue Samples from Perfusion–Fixed Rats
  • Basic Protocol 3: Embedding Tissues in Epoxy Resin
  • Basic Protocol 4: Sectioning Epoxy‐Embedded Tissue
  • Basic Protocol 5: Staining Epoxy Sections for Light Microscopy
  • Alternate Protocol 2: Staining Sections for Electron Microscopy
  • Basic Protocol 6: Peripheral Nerve Fiber Teasing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Perfusion Fixation in Rats

  Materials
  • Fixative for perfusion (see recipe)
  • Heparinized saline washout solution (see recipe)
  • 65 mg/ml pentobarbital sodium anesthesia solution
  • Rat, control or treated
  • Peristaltic pump with variable speed control (e.g., Masterflex pump; Cole‐Parmer)
  • Pump tubing (e.g., Masterflex C‐flex tubing, 3.1‐mm i.d.) and connectors
  • Graduated carboys with spigots
  • 3‐way plastic Luer stopcock (Cole‐Parmer)
  • 3‐in. 16‐G straight stainless‐steel feeding needles (Popper)
  • Dissecting board
  • Heavy push pins or plastic‐coated wire
  • Shears
  • Forceps
  • Hemostats
  • Microdissecting scissors
  • Carcass storage bags

Alternate Protocol 1: Immersion Fixation of Peripheral Nerve

  • Fixative: 10% neutral buffered formalin, 3% glutaraldehyde, or mixture of paraformaldehyde and glutaraldehyde (see reciperecipes)
  • Fine, non‐toothed forceps
  • Scalpel handles and blades
  • Index card
  • Specimen vials and labels
  • Dental wax pads

Basic Protocol 2: Dissection of Peripheral Nervous Tissue Samples from Perfusion–Fixed Rats

  Materials
  • Fixative (see recipe)
  • Perfused animal (see protocol 1)
  • Specimen vials
  • Indelible marker
  • Chemical fume hood or downdraft table
  • Forceps
  • Scalpel handles and blades (no. 11 or 15)
  • Bone‐cutting forceps
  • Rongeurs
  • Dental wax pad

Basic Protocol 3: Embedding Tissues in Epoxy Resin

  Materials
  • Vials containing trimmed tissues in fixative (see protocol 3)
  • 0.1 M sodium phosphate buffer, pH 7.4 (see recipe for 0.2 M sodium phosphate stock)
  • 2% (w/v) osmium tetroxide solution in 0.1 M sodium phosphate buffer, pH 7.4 (see recipe)
  • 15%, 30%, 50%, 70%, 95%, and 100% (v/v) ethanol
  • Propylene oxide
  • Epoxy resin embedding medium (e.g., Poly/Bed 812; Polysciences)
  • Chemical fume hood
  • Glass or plastic disposable transfer pipets
  • Disposable plastic beakers
  • Wooden applicator sticks with beveled ends
  • Plastic dishes (e.g., mincing dishes or weigh boats)
  • Flat embedding molds
  • 60°C oven
  • Cardboard boxes (e.g., pill boxes)
NOTE: For all processing steps, use a transfer pipet to remove the liquid in the vial from the previous processing step and replace it with liquid from the next processing step. Recap vials at each step. Perform all steps at room temperature unless otherwise noted.

Basic Protocol 4: Sectioning Epoxy‐Embedded Tissue

  Materials
  • Epoxy resin tissue blocks (see protocol 4)
  • 3:1 (v/v) Permount/xylene mounting medium
  • Ultramicrotome or Butler Block Trimmer (Electron Microscopy Sciences)
  • Dissecting microscope
  • Single‐edge razor blades
  • Glass knives
  • Frosted precleaned glass microscope slides
  • Fine forceps
  • 70° to 90°C hot plate
  • Coverslips
  • Chemical fume hood
  • Dissection needle
  • Bibulous paper
  • Microscope slide boxes
  • Diamond knife
  • Copper grids
  • Petri dishes, glass
  • Filter paper
  • Grid storage boxes
  • Additional reagents and equipment for staining sections with toluidine blue and safranin (see protocol 6) and staining thin sections (see protocol 7)

Basic Protocol 5: Staining Epoxy Sections for Light Microscopy

  Materials
  • Slides with heat‐fixed epoxy tissue sections (see protocol 5)
  • 1% (w/v) toluidine blue in 1% (w/v) sodium borate (see recipe)
  • 0.5% (w.v) safranin in 0.5% (w/v) sodium borate (see recipe)
  • 12‐ml syringes and syringe filters (0.2‐µm)
  • 70° to 90°C hot plate
  • Slide holder for drying slides vertically
  • Coverslips
  • Cardboard slide trays
  • Microscope slide boxes

Alternate Protocol 2: Staining Sections for Electron Microscopy

  Materials
  • Uranyl acetate (see recipe)
  • Reynold's lead citrate (see recipe)
  • NaOH
  • Thin sections collected on grids (see protocol 5, steps to )
  • Parafilm
  • Petri dishes, glass
  • Glass, disposable transfer pipets
  • Anti‐capillary forceps
  • Small beakers
  • Filter paper
  • Grid storage box
CAUTION: Uranyl acetate is radioactive. Wear latex gloves when handling uranyl acetate and lead citrate. Dispose of labware contaminated with uranyl acetate according to institutional radioactive waste policies.

Basic Protocol 6: Peripheral Nerve Fiber Teasing

  Materials
  • Fixed peripheral nerve (see protocol 2 or protocol 3)
  • Fixative (see recipe)
  • 0.05 M sodium phosphate buffer (see recipe for 0.2 M sodium phosphate buffer stock)
  • 2% (w/v) osmium tetroxide in 0.1 M sodium phosphate buffer (see recipe)
  • 30%, 50%, 70%, 95%, and 100% ethanol
  • Cedarwood oil
  • 3:1 (v/v) permount/xylene mounting medium
  • Razor blades and blade holders
  • Scalpel handles and blades
  • Dental wax pads
  • Labeled specimen vials
  • Glass or plastic disposable transfer pipets
  • Fine forceps
  • Glass microscope slides
  • Watchmaker's forceps
  • Butterfly needles inserted into wooden applicator sticks
  • 50°C oven
  • Glass coverslips
  • Cardboard slide tray
  • Slide boxes
NOTE: For all processing steps, use a transfer pipet to remove the liquid in the vial from the previous processing step and replace it with liquid from the next processing step. Recap vials at each step. Perform all steps at room temperature unless otherwise noted.
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Figures

Videos

Literature Cited

Literature Cited
   Dyck, P.J., Giannini, C., and Lais, A. 1993. Pathologic alterations of nerves. In Peripheral Neuropathy, 3rd ed. (P.J. Dyck, P.K. Thomas, J.W. Griffin, P.A. Low, and J.F. Podulso, eds.) pp. 514‐595. W.B. Saunders, Philadelphia.
   Fix, A.S. and Garman, R.H. 2000. Practical aspects of neuropathology: A technical guide for working with the nervous system. Toxicol. Pathol. 28:122‐131.
   Hayat, M.A. 1970. Principles and Techniques of Electron Microscopy. Van Nostrand Reinhold Co., New York.
   Jortner, B.S. 2000. Mechanisms of toxic injury in the peripheral nervous system: Neuropathologic considerations. Toxicol. Pathol. 28:54‐69.
   King, R. 1999. Atlas of Peripheral Nerve Pathology. Arnold, London.
   Krinke, G.J., Vidotto, N., and Weber, E. 2000. Teased‐fiber technique for peripheral myelinated nerves: Methodology and interpretation. Toxicol. Pathol. 28:113‐121.
   Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208‐212.
   Schaumburg, H.H., Berger, A.R., and Thomas, P.K. 1992. Disorders of Peripheral Nerves. 2nd ed. F.A. Davis Co., Philadelphia.
   Spencer, P.S., Bischoff, F.M., and Schaumburg, H.H. 1980. Neuropathological methods for detection of neurotoxic disease. In Experimental and Clinical Neurotoxicology (P.S. Spencer and H.H. Schaumburg, eds.) pp. 743‐757. Williams and Wilkins, Baltimore.
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