A quantitative computer-assisted morphometric analysis of stimulation-induced injury to myelinated fibers in a...

We describe a computer-assisted morphometric procedure for quantifying acute axonal injury induced in peripheral nerves by prolonged electrical stimulation. The procedure is a two-phase process, with the image analysis implemented via a commercial image analysis program, followed by an automated editing of the morphometric parameters of each object identified by the image analysis software. Both phases are implemented on IBM-compatible personal computers. The custom software counts the number of fibers undergoing early axonal degeneration, using a two-category classification scheme based on the range of myelin cross-sectional area and axonal cross-sectional areas of normal (unstimulated) nerves. When the damaged fibers are counted using this procedure, the correlation between the normalized amplitude of the electric stimulus and the number of degenerating fibers is the same as when the analysis is performed by an experienced histopathologist (R = 0.87) and carries the advantage of being entirely objective. The correlation was higher with a two-category classification (damage/no damage) than when the severity of the damage to each axon was weighted according to the amount of axonal shrinkage. We determined that axons 3.5-9 microm in diameter are the most vulnerable to injury from the electrical stimulation. This has certain implications regarding the mechanism underlying this type of injury.