THE ISOLATION AND CHARACTERIZATION OF RAT AND BOVINE OLIGODENDROGLIA

Abstract

This thesis describes the isolation from rat brain and the characterization of oligodendroglia, the myelin-forming cells of the CNS. The method that has been developed has permitted the isolation of oligodendrocytes in bulk amounts, an achievement not possible with previous procedures, and has allowed the detailed analysis of oligodendroglial perikarya during development.;Preliminary experiments to develop an isolation method applicable to rat brain were undertaken with bovine white matter. Some of the variables examined in these experiments were: media tonicity, ionic composition and pH, trypsin concentration and incubation time, the effect of DNase, the means of tissue dissection, density gradient composition and centrifugation time. Cell fractions obtained were assessed by light and electron microscopy, yield and viability. From such experiments, optimal conditions were established for the isolation of oligodendrocytes from bovine white matter. With this technique, tissue was incubated 20-30 minutes with 0.1% trypsin in either Hank's balanced salt solution or Dulbecco's modified Eagle medium supplemented with 25 mM HEPES buffer (pH 7.2-7.3) and 10 (mu)g/ml of DNase. The tissue was washed by centrifugation and disaggregated by passage through nylon and steel screens and the cells were purified by centrifugation through a two-step discontinuous sucrose gradient.;When similar conditions were applied to rat whole cerebrum, well preserved oligodendrocytes were obtained, whereas astrocytes and neurons could not be recovered intact from the gradient. Electron microscope analysis of incubated rat tissue showed that the astrocytes and neurons were lysed during the thirty minute incubation. This selective preservation of cells has made possible the first practical isolation of oligodendrocytes in bulk from the brains of laboratory animals. The method has been applied to brains from 10-, 21-, 30-, and 60-day-old rats. Finely minced rat cerebrums were incubated for 30 minutes at 37(DEGREES)C in a 0.1% trypsin solution in Hank's balanced salt solution containing 25 mM HEPES pH 7.2, and 10 (mu)g/ml DNase. In most experiments bovine serum albumin (0.75-1.0%) was also present, but it is not essential.;Oligodendroglia can be enriched to greater than 90% and were 80-90% viable by dye exclusion. They were obtained in a yield of 3.5-5.0 x 10('6) cells/g brain, depending on age. The contaminants were 3-6% red blood cells, 4-5% small phase-dark cells and nuclei, < 0.1% capillary fragments and < 0.1% ependyma. Ultrastructural examination showed that the majority of cells were well preserved and conformed to the description of isolated and in situ oligodendroglia. The cell diameter was 7-12 (mu)m, the nucleus contained clumped heterochromatin in the cytoplasm, ribosomes were abundant and microtubules were present. No intermediate filaments or glycogen granules were observed. Freshly isolated rat oligodendroglia were readily stained with antibodies to galactocerebroside. After establishing this method, the content of protein, DNA, RNA, lipid (cholesterol, total phospholipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, galactolipid, and sulfatide), carbonic anhydrase, 5'-nucleotidase, and 2', 3'-cyclic nucleotide 3'-phosphohydrolase was measured in oligodendrocytes obtained from rats 10-60 days of age. The oligodendrocytes from 30-day-old rat forebrain have been successfully maintained in vitro. At six days in vitro the cultures were composed mainly of galactocerebroside-positive cells when examined by immunofluorescence using a rabbit antiserum to that lipid hapten.;These studies defined the means for isolation of oligodendrocytes from rat brain and many of the features of these cells in the adult and developing rat. The availability of this method now makes it possible to address many important biological questions concerning oligodendrocytes and myelination.