A rapid digestive technique to expose networks of vascular elastic fibers for SEM observation

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H2O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.