X-Message-Number: 27994
From: "Basie" <>
Subject: Brain Preservation 
Date: Thu, 25 May 2006 21:43:01 -0400

Cragg, "Brain Preservation of the Extracellular Space During Fixation of the 
Brain for . . . Tissue and Cell," 12:63-72, 1980.

The shrinkage and anatomical distortion routinely seen after perfusion 
fixation of soft tissue are not inevitable. They can be avoided. The 
required procedure is easy, eliminates more red blood cells from the tissue, 
and gives more vivid staining and generally better histological results, 
even for routine cryostat sectioning. This procedure is Electron Microscopy 
and should generally be used for all histology where perfusion is necessary.

After the usual isotonic saline or phosphate buffer wash through the 
vascular system, the tissue has not shrunk. However, when the fixative 
arrives, it attacks extracellular and outer membrane proteins. The sodium 
pumps are deactivated, resulting in an inrush of sodium and water to keep 
the osmolarity balanced. This causes the cells to swell against each other, 
eliminating extracellular space. The membrane proteins are crosslinked to 
neighboring cells. Later, when water leaves the cell after the inside of the 
cell is fixed, everything collapses. The entire soft organ shrinks as much 
as 30 percent and is distorted in shape. Additionally, some cell membranes 
are torn and some cell contents are lost. Membrane damage is further 
worsened by freezing artifact or paraffin processing.

Cragg reasoned that it would be necessary to remove sodium and other ions 
from the extracellular fluid before fixative arrives to prevent shrinkage. 
Common sucrose is not ionic and can be dissolved in water to make an 
isotonic solution. However, it does not cross the blood brain barrier and, 
hence, can replace blood, but not the extracellular fluid in the brain. To 
accomplish this, the blood brain barrier must be broken by pressure. After 
the sucrose wash starts, pressure can be pumped up to 300 mm Hg by using a 
perfusion apparatus, such as the Perfusion One indicated below. This breaks 
the blood brain barrier and results in a change of the extracellular fluid 
to sucrose, washing out the ionic fluid. After the usual wash time, the 
fluid is switched to fixative. As a result, cells are fixed at normal size, 
and there is no inrush of fluid. Tissue can be seen to have extracellular 
space under the microscope, and membranes are not abutting and fixed to each 
other. Cytosol is better retained. The entire organ does not shrink or 
distort, and sections resemble unfixed tissue used in the brain atlases by 
Paxinos and his coauthors.

Why would researchers perfuse the old fashioned way? Apparatus to implement 
the Cragg perfusion protocol is available from the Vibratome Company. To 
find out more about the New Technology: Click Perfusion One


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