On Thu, 22 Jun 2000, Rebecca S Smith wrote:

> Through my searching I found the "Marchi" stain for the 
> demonstration of Degenerated Myelin.  It takes 14 days.  
> She would like to  know if there is
> #1 - A newer, better technique?
> #2 - A stain that will get done sometime in this month?
> #3 - A microwave procedure to speed this (Marchi) stain up?
> #4 - Any news on demonstrating this type of syndrome?

Marchi's method (developed and described about 1890) has yielded
much information about human neuroanatomy, comparable to that
obtained from staining degenerated tracts in lab animals, prior
to the development of anterograde and retrograde tracing experiments.
The Swank & Davenport variant, described in the 1930s, seems to be
the best. I've done it, and it worked. Adams' OTAN method (which
includes a histochemically reasoned counterstain for normal myelin)
is a more recent development, reviewed in all histochemistry
textbooks published since 1965. OTAN can be done on frozen 
sections, but is less sensitive than a Marchi method done on
blocks of tissue.
 
Marchi & variants are the only available neuroanatomical tracing
techniques based on staining of degeneration that are applicable to 
the human CNS, and they have the added advantage that the 
histochemistry has been well understood for nearly 40 years (see 
Bayliss High, O. 1984. Lipid Histochemistry. Oxford Univ Press. 
This was Handbook No 6 in the Royal Microscopical Society's
splendid series. It may be out of print.)  There is a method
based on staining with oil red O (degenerating myelin) and luxol
fast blue (normal myelin), but I don't think it's seen a lot of
use. It's described in Culling; I've not seen the original papers
or tried the technique. Like the Marchi method, it picks out the
hydrophobic cholesterol esters in degenerating myelin. (Normal
myelin is rich in hydrophilic lipids that are tightly bound to
protein. That's why it can be stained in paraffin sections. The
hydrophobic lipids of degenerating myelin can be stained only in
frozen sections.)

This is a field in which technical developments occur quite slowly.
Old observations on the detection of cholesterol by polarizing
microscopy have been exploited in the detection of degenerated
tracts in frozen sections of human CNS. The published photos look
impressive. I have tried the method (on sections of human spinal
cord known to contain degenerating corticospinal fibres) but was
unable to see the finely granular birefringence due to degenerating
myelin because of an abundance of coarsely granular birefringent
material throughout the white matter. The specimens I had were
not specifically collected, fixed and fairly promptly sectioned
for the purpose, and I wasn't surprised at my failure to get this
method to work. For all techniques for degenerating axons the
specimens must be specially prepared.
    
If you want to use polarizing microscopy to look at degenerating
myelinated fibre tracts you should consult the following papers.

 Doinikow, B. 1913. Zur Histopathologie der Neuritis mit
   besonderer Berucksichtigung der Regenerationsvorgange. Deutsch.
   Z. Nervenheilk. 46: 20-42.

 Miklossy, J. and Van der Loos, H. 1987. Cholesterol ester
   crystals in polarized light show pathways in the human brain.
   Brain Res. 426: 277-380.
 
 Miklossy, J. and Van der Loos, H. 1991. The long-distance
   effects of brain lesions: visualization of myelinated pathways
   in the human brain using polarizing and fluorescence
   microscopy. J. Neuropathol. Exp. Neurol. 50: 1-15.
   
The last of these is the most informative (and has nice coloured photos),
but the first serves to remind us all that polarizing microscopy,
degenerating myelin and cholesterol have been connected in the minds
of scientists for quite a long time. 

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1