Cells and tissues must undergo extensive processing before they can be seen and analyzed under a microscope. The most common method for preparing animal tissue for microscopy is known as perfusion. With perfusion, a researcher uses an animal's circulatory system to deliver a fixative to tissue cells and thereby remove troublesome red blood cells. Fixatives are agents that lock tissue in a stable state, preventing naturally occurring autolysis and deterioration. The faster a fixative penetrates a tissue, the more closely the preserved tissue resembles the living state. Red blood cells create background labeling in many important histological reactions; their removal improves the contrast and visibility of cells of interest. Because standard instrumentation and protocols for sacrifice perfusion have not been commercialized, laboratories must assemble their own apparatus. This has led to wide variations in apparatus and protocols, which are frequently not described in the methods section of published papers. Although perfusion quality can substantially impact tissue analysis, perfusion techniques have received remarkably little attention. A drawback in the most common perfusion procedures is that they produce shrinkage --about 20% shrinkage for brain tissue -- and thereby alter the gross anatomical and microscopic morphology. The living brain is 20% extracellular space. This space is absent from tissue sections of brain perfused and fixed using common protocols, resulting in sections that are substantially smaller than comparable sections from fresh, unfixed tissue. The inward collapse distorts shape and position of many brain structures. Moreover, neuronal elements may erroneously appear in apposition on an electron microscopic level, due to loss of extracellular space. Shrinkage, however, is not an inevitable result of fixation. An improved perfusion fixation procedure has been published requiring more sophisticated apparatus. This procedure does not cause whole organ shrinkage, does not result in loss of extracellular space, and is more effective in removing red blood cells. This procedure yields consistently reproducible, high tissue quality. The instrumentation it requires can be designed to facilitate replication of perfusion parameters. The proposed research is to develop and test a new commercial perfusion apparatus that will improve the efficiency and reproducibility of histology on animal tissues used in biomedical research, especially neuroscience.
