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Toxicologic Pathology
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Application of Silver Degeneration Stains for Neurotoxicity Testing

Robert C. Switzer

NeuroScience Associates, Knoxville, Tennessee 37922

Silver staining procedures have been used in numerous ways to render a variety of physical and biological features visible. In biological tissue, histologic protocols use silver to visualize diverse structures or features, such as reticulin, melanin, fungi, chromosome bands, nucleolar organizing regions, and different features in the nervous system. A comparison of the specific steps in these protocols indicates that the silver is "directed" to stain any given feature by the type of fixation, the pretreatment ("mordanting"), the composition of the silver-containing solution(s), and the form of development (reduction). Since the mechanisms of staining have not been understood historically (nor are they now), each method was developed by trial and error. Keystone methods such as those of Bodian and Bielschowsky exploit the nervous system's affinity for silver (argyrophilia). The beginning of a new era in brain research came with the recognition that distinct silver-impregnated morphologic changes occurring in damaged axons could be used for tracing axon pathways in experimental animals with specifically placed lesions. Improvements in staining methods used to selectively impregnate the disintegrating axons but to leave normal axons unstained were achieved by Nauta and Gygax (early workers with these procedures) and spawned a host of method variations known as the "Nauta" methods. Of these, the Fink-Heimer and de Olmos cupric-silver methods were able to unambiguously demonstrate disintegrating synaptic terminals, thereby allowing complete tracing of axon pathways. The late 1970s and 1980s witnessed innovative applications of these techniques. The silver methods once used to trace axon pathways became indicators of the extreme endpoint of neurotoxicity: disintegrative degeneration of neurons induced by neurotoxic chemicals that were administered systemically. The hallmark of neurotoxic substances is the selectivity with which each destroys specific populations or subpopulations of neurons. The high contrast and sensitivity of the silver degeneration stains greatly facilitate the screening process to detect these affected populations, especially when there is no basis for knowing where in the brain to look for damage. More recently, in addition to expanded use in screening for neurotoxic effects, the silver degeneration stains are being used to chart the neuron populations undergoing programmed cell death in the developing brain. Other newly developed silver methods have been refined to show nondisintegrative degeneration, such as the plaques and tangles of Alzheimer's disease.

Key Words: Amino cupric silver • apoptosis • brain • electron microscopy • fragmentation of proteins • MultiBrain Technology • neural tracing • neurohistology • neurotoxins • risk assessment

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Toxicologic Pathology, Vol. 28, No. 1, 70-83 (2000)
DOI: 10.1177/019262330002800109
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