Morphometric Analysis in Neurodegenerative Disorders

Dejan Milatovic1, Thomas J. Montine2, Snjezana Zaja‐Milatovic1, Jennifer L. Madison1, Aaron B. Bowman1, Michael Aschner1

1 Vanderbilt University School of Medicine, Nashville, Tennessee, 2 University of Washington School of Medicine, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.16
DOI:  10.1002/0471140856.tx1216s46
Online Posting Date:  November, 2010
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Abstract

Golgi impregnation has been commonly used for neuronal morphometry in a number of neurological diseases and brain‐disorder studies. Introduced more than a century ago, it is still the standard and state‐of‐the‐art technique for visualization of neuronal architecture. We successfully applied the Golgi method to mouse, rat, monkey, and human brain tissues for studying both the normal and abnormal morphology of neurons. We were able to discover subtle morphological alterations in neuronal dendrites and dendritic spines in different brain areas. Although Golgi preparations can be examined by electronic microscopy, we used light microscopy and reconstruction using Neurolucida software to quantitatively explore the relationship between total dendritic length and spine density in different types of neurons. This unit summarizes the methodology used to quantify neuronal abnormalities and discusses the utility of these techniques in different models of neurodegeneration. Curr. Protoc. Toxicol. 46:12.16.1‐12.16.14. © 2010 by John Wiley & Sons, Inc.

Keywords: Golgi impregnation; Neurolucida; dendrites; dendritic spine; neuronal morphometry

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Experimental animal, e.g., mouse, rat, monkey
  • FD Rapid Golgi Stain Kit (FD NeuroTechnologies, cat. no. PK‐401)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylenes
  • Paraffin
  • 1% (w/v) bovine serum albumin (Sigma A3912) in PBS
  • Tissue‐Tek O.C.T. Compound (Sakura Finetek, cat. no. 4583)
  • 0.1% cresyl violet
  • Histo‐Clear (National Diagnostics; optional)
  • Cytoseal XYL mounting solution (Thermo Fisher Scientific)
  • Dissecting equipment
  • Razor blade (e.g., surgical carbon steel #12, single edge, VWR 55411‐055)
  • 15‐ml conical plastic tubes
  • Histology cassettes
  • Automated histology processor, e.g., Thermo Scientific Shandon Excelsior
  • Embedding molds
  • Automated rotary microtome (Leica RM 2235) or manual rotary microtome (Leica RM 2125)
  • Superfrost Plus microscope slide (25 × 75 × 1–mm; e.g., VWR, cat. no. 48311‐703)
  • Histology oven
  • Cryostat and chuck
  • Gelatin‐coated microscope slides (see recipe)
  • Slide box
  • Plastic staining dishes and rack (e.g., Sakura Finetek 4451)
  • Coverslips (24 × 50–mm, no. 1 1/ 2; e.g., VWR, cat. no. 48393‐241)
  • Light microscope, e.g., Olympus BX62 with motorized stage
  • Neurolucida and NeuroExplorer software (MBF Bioscience, http://www.mbfbioscience.com)
  • Additional reagents and equipment for anesthesia (Donovan and Brown, ) and euthanasia (Donovan and Brown, ) of animals
NOTE: Keep containers closed tightly at all times. Protect tissues from light during and after exposure to Solutions A and B. Perform all procedures at room temperature, unless specified.
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Figures

Videos

Literature Cited

Literature Cited
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