分離ヒト脳微小血管

Abstract

We evaluated activities of 5'-nucleotidase (5'-N), 3'-5'-phosphodiesterase (3'-5'-PDE), AchE, and ChAT on intraparenchymal blood vessels of a human brain. Brain vessels were isolated using the method which we developed. Fragments of human brain weighed 2 or 3g were minced and passed through 250μm nylon mesh. The material on the mesh was resuspended in buffer, after mild homogenization, passed through 73μm mesh. The mesh was washed, then the fluid was passed through 150μm mesh. Two vessel fractions were separated on the mesh and in the filtrate. The fraction on the mesh consisted primarily of larger vessels greater than arterioles, and that in the filtrate consisted predominantly of microvessels, which were purified by passing through a column of glass beads and centrifuged on sucrose density gradient. We evaluated the degree of contamination of isolated vessels using cerebroside as an indicator. The cerebroside level in these fractions was less than 5 percent of that in the brain homogenate.The activity of γGTP was significantly higher than that in the brain homogenate. These results indicate that enzyme activities are preserved well during the isolation procedure. 5'-N level was significantly lower in these fractions. 3'-5'-PDE level was significantly higher in the large vessel than in the small vessel fraction. Changes of these enzyme activities in the vessels may affect micro-circulation and blood brain barrier function via change in the concentration of adenosine and cyclic AMP. ChAT activity was very low in vessel fractions. AchE activity was significantly higher in both vessel fractions than in the brain homogenate, and in small vessel fraction than in the large vessel fraction. The discrepancy of these two enzyme activities in isolated vessels will provide a new tool and scope in studies on brain microvessels.