Premium
OLIGODENDROGLIA FROM HUMAN AUTOPSIED BRAIN: BULK ISOLATION AND SOME CHEMICAL PROPERTIES 1
Author(s) -
Iqbal Khalid,
GrundkeIqbal Inge,
Wisniewski Henryk M.
Publication year - 1977
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1977.tb10617.x
Subject(s) - myelin , centrifugation , ficoll , differential centrifugation , biochemistry , biology , ultrastructure , density gradient , microbiology and biotechnology , rna , white matter , tonicity , cell , chemistry , in vitro , anatomy , peripheral blood mononuclear cell , central nervous system , medicine , physics , radiology , quantum mechanics , neuroscience , gene , magnetic resonance imaging
— Oligodendroglia were isolated from fresh and frozen human autopsied brains by a modification of our technique for isolation of neuronal perikarya and astroglia (Iqbal & Tellez‐Nagel, 1972). Cerebral white matter was minced in a hypertonic hexose‐Ficoll buffered medium, passed through successive screens of decreasing pore size, and the cells were then separated on a discontinuous sucrose density gradient using low speed centrifugation. Cells could also be isolated by substituting sucrose for hexoses in the cell isolation medium. About 95% of the isolated cells had morphology characteristic of oligodendroglia. The usual contaminants in this fraction were capillary fragments, red blood cells, and a few astrocytes. Free myelin was only sparsely seen. About 5–10% of the isolated oligodendroglia had one or more loops of loose membranes extending from the cell plasma membrane. These membraneous loops resembled myelin. The ultrastructural preservation of the cells was poor. The average yield per gram of wet tissue was 52 million cells amounting to 2.4 mg protein, 283 μ g DNA, and 94 μ g RNA. The protein, DNA, and RNA contents per average cell were 47, 5.3, and 1.8 pg respectively. About 50% of the tissue DNA was recovered as isolated cells, suggesting a larger proportion of oligodendroglia to astroglia, the other principal cell type in human white matter. Comparison of the total protein profiles of the isolated oligodendroglia with those of the purified human myelin on sodium dodecyl sulfate‐polyacrylamide gels revealed the presence of all human myelin constituent proteins in the isolated cells except the intermediate (or DM‐20) myelin proteins. Densitometry of the gels stained with Fast Green showed that the ratio of protein bands corresponding to myelin encephalitogenic basic protein (BP) to myelin proteolipid protein (PLP) in the isolated cells was three‐fold as high as in purified human myelin. The oligodendroglial BP which constituted about 14% of the total cell protein contents, also coelectrophoresed with the myelin BP on pH 4.3 polyacrylamide gels. Furthermore, the two dimensional peptide maps of the tryptic digest of the cell BP labelled with 125 I were also identical to the similarly treated myelin BP. The data suggest that the BP can be used as a chemical marker for oligodendroglia.