Amino-Cupric-Silver (A-Cu-Ag) Staining to Detect Neuronal Degeneration in the Mouse Brain: The de Olmos Technique

Silver staining procedures have classically been used to study the structure of the nervous system. However, reduced silver staining methods, using substances that reduce silver ions against the natural reducing properties of the tissue, can also successfully reveal degenerative changes in the nervous system. It is not known how silver binds these degenerating elements. During degeneration, silver ions may form complexes with exposed amino acid chains in denatured proteins that are then seen as black-stained elements over an unstained background (of non-degenerating elements). The reduced Amino-Cupric-Silver method developed by de Olmos, where cupric ions are added as an external reducer, provides the greatest contrast between the degenerating and non-degenerating neuronal elements when compared with other reduced silver staining protocols. This method is unique to study degenerative morphological changes and, when combined with other staining procedures, to identify which specific neuronal population is degenerating. After a trauma, neurons undergo several physical changes that can be visualized with different markers, such as FluoroJade, caspases, or Hematoxylin and Eosin stains. Some neurons can overcome this damage and are restored, while others undergo irreversible changes and die. The Amino-Cupric-Silver method can reveal early irreversible neuronal damage before cell death. After these irreversible changes the neurons cannot regenerate, so detecting these degenerative changes will improve the understanding of pathological changes following certain injuries of the nervous system.