Journal of Neurochemistry 34(6):1614-1621, June. Raven Press, New York 0 1980 International Society for Neurochemistry

0022-3042!80/0601- 1614/$02.00/0

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

D. Stephen Snyder, Cedric S. Raine, Muhammad Farooq, and William T. Norton

The Suul R. Korey Deportment oj Neurology, Department of Pathology (Neiiroputhology), Department of Neiiroscience, ond the Robe F. Kennedy Center for Reseurck in Mental Retardation and Hiimun De Lrelopment,

Albert Einstein College oj' Medicine, Bronx, New York 10461, U.S .A.

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from lo-, 30-, and 60-day-old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated, and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase-bright rounded cells having diameters after fixa- tion of 7-12 pm. The contamination was primarily by red blood cells and phase-dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pgicell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals. Key words: Neuroglia-Cell isolation-Ultrastructure.

Although several methods have been published for the bulk isolation of oligodendroglia from CNS white matter (Fewster et al., 1967; 1973; Poduslo and Norton, 1972; Iqbal et al., 1977), these methods are either not applicable or are inconvenient for the isolation of oligodendrocytes from the brains of small laboratory animals. To permit biochemical studies of oligodendroglia during development and myelination, it would be highly desirable to have an isolation procedure suitable for the brains of smaller laboratory animals, such as the rat.

The isolation procedure previously developed in this laboratory (Poduslo and Norton, 1972), one which has been widely adopted by others (Cohen and Bernsohn, 1973; 1978; Benjamins et al., 1974; Deshmukh et al., 1974; Trapp et al., 1975; Banik and Smith, 1976; Pleasure et al., 1977; 19791, fur- nishes highly enriched preparations of oligoden- droglia, which are both structurally intact (Raine et

al., 1971; 1978; Trapp, 1975) and metabolically ac- tive (Cohen and Bernsohn, 1973; 1978; Benjamins et al., 1974; Deshmukh et al., 1974; Banik and Smith, 1976; Poduslo and McKhann, 1977; Poduslo et al., 1978; Pleasure et al., 1977; 1979). The method, however, suffers from two main disadvantages, which limit its usefulness for biochemical studies of cells from small animals. It requires dissected white matter as starting material and uses a non- physiologic medium throughout. The first limitation arises from the fact that it is nearly identical to one developed for the isolation of neurons and astro- cytes (Norton and Poduslo, 1970). Thus, when a p p l i e d t o who le r a t b ra in t h e r e s u l t a n t oligodendrocyte-enriched fraction is heavily con- taminated with neurons. Despite this drawback, the procedure has been successfully applied to white matter of rat brain after dissection (Deshmukh et al., 1974; Banik and Smith, 1976). In young devel-

Received August 29, 1979; accepted November 28, 1979. Address reprint requests to D. Stephen Snyder, The Saul R.

Korey Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, U.S.A.

Abbreviations used: BSA, Bovine serum albumin; BSS, Bal- anced salt solution; HEPES, N-2-Hydroxyethylpiperazine-N'- 2-ethanesulfonic acid.

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BULK ISOLATION OF OLIGODENDROGLIA 1615

oping animals, however, such an approach would be tedious, if not impossible. Even in the adult rat brain, the amount of tissue needed for reasonable numbers of cells is impractical to obtain with hand dissection.

The second limitation, the use of a hypertonic, low-salt medium of pH 6.0 (Poduslo and Norton, 1972), is a potential, rather than proven, disadvan- tage. While cells isolated in this medium are metabolically active, as noted above, and can be transferred to tissue culture media for in vitro studies (Poduslo et al., 1978; Pleasure et al., 1979), such cells from bovine brain do not survive more than 2-3 days in culture.

We have recently reported (Snyder et al., 1978) that oligodendroglia can be obtained from several species with little or no loss in yield and purity, using balanced salt solutions at pH 7.4. This tech- nique was adaptable for use with the brains of small laboratory animals because such media caused neurons and astrocytes to lyse. In this report, the preliminary results of which have been presented elsewhere (Snyder et al., 1979), we describe the ap- plication of these principles to the isolation of oligodendrocytes from undissected rat forebrain and present some biochemical data on the isolates.

MATERIALS AND METHODS

Materials and Reagents

Trypsin (type 111, 2 x crystallized, from bovine pan- creas), HEPES (free acid), deoxyribonuclease (DN-100, from bovine pancreas), and bovine serum albumin (BSA) (Cohn fraction V) were obtained from Sigma Chemical Co., St. Louis, Missouri. All lot numbers of these re- agents used worked equally well, and we did not verify the enzyme activities independently. Hank’s balanced salt solution (BSS) and calf serum were supplied by Grand Island Biological Co., Grand Island, New York. Sucrose (ACS) was obtained from Fisher Scientific Co., Springfield, New Jersey. Nitex screens were obtained from TETKO Inc., Elmsford, New York. The stainless steel was obtained from Greiner Scientific, New York, New York.

Unstained cells were counted in a hemocytometer using a phase microscope. The method used for electron microscope analysis of the cells has been described pre- viously (Raine et al., 1971). Protein was measured by the method of Lowry et al. (1951). Nucleic acids were ex- tracted from whole fractions of unwashed cells using con- ditions recommended by Munro and Fleck (1966). RNA was measured by absorbance a t 260 nm using t (P) of 9380. DNA was determined by the method of Burton (1956) with calf thymus DNA as the standard.

The isolation medium was Hank’s BSS, to which was added HEPES (25 mM) and, in most experiments, BSA (176, wiv). The p H of the final solution was adjusted to 7.2 with NaOH. The trypsinization medium contained 0.1% trypsin, 10 p g h l DNase, and 25 mM-HEPES in Hank’s BSS, p H 7.2. The BSA was omitted from this solution to avoid competition for trypsin activity.

Procedure

Incubation F o r the p r e s e n t s t u d y lo- , 30-, a n d 60-day-old

Sprague-Dawley rats were used. The rats were decapi- tated, the brains removed and trimmed of olfactory bulbs, cerebellum, and brain stem. The remaining brain tissue was collected in ice-cold isolation medium and weighed. The brains were finely minced with a tissue-slicing blade on a cold glass plate, and the minced brain was trans- ferred to a 50-ml Erlenmeyer flask. Usually, 10 g of tissue were processed at a time, through the density-gradient step. During incubation of the first batch of tissue, a further 10 g of brain from an additional group of animals could be collected and processed sequentially. Thus, 20 g of tissue could be processed in a staggered fashion during a 6-7-h period. The trypsinization solution (3 mlig) was added to the flask, and incubation was continued for 30 min at 37°C in an oscillating water bath. After incubation, the suspension was chilled for 5 min on ice, and ice-cold undiluted calf serum ( 1 mlig) was added. All subsequent steps were carried out a t 0-4°C. The minced tissue was apportioned among 30-ml round-bottom centrifuge tubes in such a way that each tube contained the equivalent of up to five brains. The tubes were centrifuged for 5 min at 190 g in a Sorvall RC-5 centrifuge equipped with an HS-4 rotor. The overlying fluid plus the superficial layer of red blood cells on the pellet were removed by aspiration through a Pasteur pipet. The pellet was resuspended in twice its volume with isolation medium and centrifuged for 5 min a t 190 g. T h e overlying medium and the peripheral ring of red blood cells were aspirated as before. This step was repeated three times more.

Disaggregation A 12 x 12 cm piece of nylon screen cloth with 145-pm

openings (100 mesh) was washed in distilled water and then in medium and fitted to a two-piece polypropylene Buchner funnel (9 cm in diameter) from which the porous bed had been removed. The funnel was placed in a filtra- tion apparatus and the tissue pellet was picked up with a spatula and applied to the sieve surface. The tissue was kept wet with generous amounts of isolation medium (to a maximum value of 10 ml/g original tissue) and forced through the screen with very light pressure applied with the bottom of a 10-ml glass beaker. No vacuum was used at this stage, and the screen was not washed since most of the capillaries are retained on its surface. The coarse sus- pension was poured slowly three times through a 200- mesh stainless steel screen (74-pm openings). No pres- sure was applied to the screen surface in these steps. The wetted screen was held in place with just enough vacuum t o maintain its downward concavity over the funnel opening.

Density gradient centrifugation The cell suspension was adjusted with medium to 10

ml/g original tissue, and an equal volume of 70% (w/v) sucrose in medium (density 1.24) was added. A 1s-ml aliquot of this suspension was layered over a discontinu- ous sucrose gradient, having at the bottom 12 ml of 53% (wlv) sucrose (density 1.17) and an intermediate layer of 4 ml of 45% (wiv) sucrose (density l . l S ) , both prepared in isolation medium. Densities were measured at 25°C by weighing the solutions in a 10-ml volumetric flask. When

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I616 D . S . SNYDER ET AL.

working with 30- and 60-day-old animals, but not with younger animals, the interface between the 45% and the 53% sucrose layers was blurred before layering the tissue suspension. This incompletely continuous zone could be made either by briefly stirring the boundary with a Pas- teur pipet or by preforming the interface 2 h prior to cen- trifugation. When processing large amounts of tissue, the following volumes have been used: 100 ml of the cell sus- pension in 35% sucrose in medium, 10 ml of 45% sucrose, and 35 ml of 53% sucrose. The gradients were centrifuged at 3065 g for 15 min in a swinging bucket rotor. The entire volume beneath the red-cell layer (which is below the interface of 45-53% sucrose solutions) and the pellet was collected and diluted fivefold with isolation medium con- taining 10 pg/ml of DNase. This amounted to 8- 10 ml in the small gradient and 25-30 ml in the larger gradient. The suspension was filtered through a nylon screen with 25-pm openings (460 mesh) to trap capillary fragments, and a n aliquot was taken for cell-count determination. Cell preparations were taken directly a t this stage for pro- cessing for electron microscope examination, or were stored frozen for subsequent RNA and DNA analysis.

When preparations were to be analyzed for total pro- tein, BSA was omitted from the procedure and the cells were washed in Dulbecco’s phosphate-buffered saline.

RESULTS

Phase microscope examination showed that 90% of the preparations consisted of small, 6-12 pm phase-bright rounded cells (Figs. 1 and 2). The contamination consisted of 3 -6% red blood cells, 4-5% phase-dark cells, and nuclei, < O . 1% capillary fragments and <0.1% ependyma, often with the cilia still active. The grossly visible 45/53% sucrose zone which was discarded was a mixture of 60-70% red blood cells, 20-30% oligodendroglia, and 5-10% ependyma. Examination of 1-pm thick to- luidine blue-stained plastic sections of the cell prep- arations (Fig. 3) substantiated the phase microscope assessment that the isolates were nearly homogene- ous preparations of oligodendroglia. Ultrastructural examination showed that the majority of cells were well preserved and had, for the most part, intact plasma membranes, attached to which myelin loops were occasionally seen. Little cytoplasmic vesicu- lation was observed. The nucleus of each cell was bounded by an intact nuclear membrane and con- tained clumped heterochromatin, unlike neuronal and astrocytic nuclei in which the nucleochromatin is more diffuse. The amount of cytoplasm was vari- able and relatively dense but not extensive. Within the cytoplasm, well-preserved organelles were evi- dent. In particular, ribosomes were abundant and microtubules were present (Figs. 4 and 5). No 10-nm filaments or glycogen granules have been ob- served. Mitochondria were only slightly swollen and possessed well-preserved cristae.

The values determined for viability, purity, pro- tein, and nucleic acids are included in Table 1 . By trypan blue exclusion, 80-90% of the cells were

deemed viable. The number of phase-bright cells was estimated to be >90%. The phase-bright ap- pearance of freshly isolated cells has, in our laboratory and elsewhere (Poduslo and McKhann, 1977), been taken as an index of cellular integrity. The cell yield per brain was similar for all age groups studied, but when expressed on a weight basis, the yield was higher for the 10-day-old ani- mals than for the older groups. None of the cell parameters measured varied significantly with age in the time period studied, although a possible trend toward lesser amounts of RNA per cell was seen with increasing age. The ratio of RNA to DNA was less than 0.5 at each age.

DISCUSSION

The method described here permits the isolation of oligodendrocytes in bulk amounts from undis- sected rat forebrain, an achievement not possible with previous protocols. This method eliminates the necessity for dissected white matter as a starting material and involves a physiologic medium throughout. We believe that such media should facilitate cell survival in cultures, since we specu- late that exposure to the non-physiologic medium used previously (Poduslo and Norton, 1972; Poduslo et al., 1978) might cause irreversible dam- age to the cells. The success of the present method is due, apparently, to the almost complete lysis of neurons and astroglia in balanced salt solutions while the oligodendroglia remain largely intact. This phenomenon can be seen ultrastructurally in sec- tions of tissue pieces examined during the trypsini- zation process (Snyder, Raine, and Norton, in preparation), and has been verified by our failure to observe any intact neurons and astrocytes in the cell suspensions, either before or after gradient sep- aration. The reasons for the differential stability of brain cells in these media are not known. The method is applicable to whole tissue from rats, at least in the age range of 10 to 60 days, and appears to be generally applicable to other species (Farooq, Cammer, Snyder, Raine, and Norton, in prepara- t i o n ) . Recently, two abstracts have appeared (Szuchet et al., 1978; Hodson et al., 1979) which support our findings (Snyder et al., 1978; 1979) that oligodendroglia can be isolated in isosmotic salt solutions. Szuchet et al. (1978) were able to isolate oligodendroglia from lamb white matter using Hank’s BSS solutions at pH 6.0. Hodson et al. (1979) have modified slightly the method in our original communication (Snyder et al., 1978) for isolation of rat oligodendroglia. Chao and Rumsby (1977) had claimed that neurons and astroglia were lysed and that oligodendroglia could be isolated selectively from whole rat brain by raising the pH of the hexose-phosphate medium of Norton and Poduslo (1970) to pH 7.4. To date, we have not been

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BULK ISOLATION OF OLIGODENDROGLIA 1617

FIG. 1. Oligodendroglial cell suspension from the forebrain of a 30-day-old rat. Phase contrast light micrograph of the suspen- sion after glutaraldehyde fixation. Scale bar = 20 pm. FIG. 2. Higher magnification from the same preparation as Fig. 1. Scale bar = 10 pm. FIG. 3. Light micrograph of a toluidine blue-stained, 1-pm-thick plastic section of an oligodendrocyte pellet from the forebrain of a 30-day-old rat. Scale bar = 10 Fm.

able to duplicate this work, but instead have found that neurons remain intact in this medium, indepen- dent of pH in the range 6.0-7.5.

The percentage of phase-bright cells compares favorably with the measure of viability by trypan blue and eosin exclusion. Thus, the percentage of viable cells obtained by this isolation method is comparable to the value reported (Trapp et al., 1975) for cells isolated by the method of Poduslo and Norton (1972).

Preforming or blurring the 45/53% sucrose inter-

face substitutes a continuous change of density at this step for a sharp discontinuity, which facilitates the separation of red blood cells from oligodendro- cytes, cells that appear to differ only slightly in den- sity. Red blood cells band at approx. 47-48% su- crose in medium. In the 10-day animals, the 45/53% sucrose interface is maintained as a sharp step, since oligodendroglia from the younger animals penetrate into the 53% sucrose at 3065 x g more readily than do cells from older animals.

The omission of BSA from all media, when pro-

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1618 D. S. SNYDER ET AL.

FIG. 4. Intact oligodendroglia from the forebrain of a 60-day-old rat. Note the clumped heterochromatin, prominent rough endoplasmic reticulum, and well-preserved plasmalemma. Scale bar = 1 pm. FIG. 5. Detail of an oligodendrocyte from a 60-day-old rat preparation showing well-preserved organelles. A mitochondrion is seen to the left and a single microtubule (arrow) is seen in the center. Note intact plasmalemma. Nucleus is below. Scale bar = 0.2 wm.

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BULK ISOLATION OF OLIGODENDROGLIA 1619

TABLE 1. Some charucteristics of rut oligodendroglia compared with those obtained for bovine cells

Rat

Age (days) 10 30 60 Bovine"

% Purity > 90 >90 >90 90-95

% Phase-bright 90 90 90 70-90' % Viable (dyes) 80 - 90 80 - 90 80-90 906

Forebrain weight, g 0.74 f .06 1.17 f .02 1.27 ? .02 - Cell yieldbrain 5.3 f 2.1 4.1 f 1.3 4.9 ? .9 -

( X 10-8)

( x 10-6) Cell yieldg 7.1 ? 3.2 3.5 ? 1.2 3.9 ? .7 15,lO-13'

Protein, pg/cell 60 56 57 30-39'

RNA, pg/cell 2.9 ? .4 2.7 ? 1.2 2.5 ? .5 2.0 DNA, pg/cell 7.2 2 .4 6.2 ? 1.5 7.9 f 1 . 1 5.2

RNAIDNA 0.40 0.42 0.31 0.36 ProteiniDNA 8.4 9.1 1.2 6.7 Protein/RNA 21.0 21.1 23.3 17.5

Values given for brain weight, cell yield, DNA, and RNA are the means ? S.D. for four preparations in each age group. Each preparation consists of from 30 to 60 x lo6 cells. Protein was determined from a single preparation in each age group, which consisted of 1.5-3.0 x lo6 cells.

'' Unless otherwise noted, the numbers for bovine oligodendrocytes are from Podu- slo and Norton (1972) and Poduslo and McKhann (1977).

Value from Trapp et a]. (1975). Values from Raine et al. (1978).

tein analyses were performed, had no effect on the numbers of viable cells or fraction purity; but we have the subjective impression that the appearance of the cells is improved by the presence of BSA. It is probable that modifications in the procedure, such as omission of BSA, replacement of trypsin with acetylated trypsin (Guarnieri et al., 1976), and substitution of specific trypsin inhibitors for serum, could make the interpretation of future studies of cell composition and surface antigens less ambigu- ous. However, systematic examination of these variables has not yet been carried out.

The yield of oligodendrocytes per brain varied little from 10 to 60 days of age despite the consider- able differences in brain weight. It has been re- ported for rat (Schonbach, 1968; Kozik, 1976; Skoff et al., 1976~; Tennekoon et al., 1977; Imamoto et al., 1978; Jacobson, 1978) that the majority of oligodendroglial precursors have ceased division and begun to myelinate cerebral axons by postnatal days 10- 15. Thus, it is possible that the number of oligodendrocytes per brain does not change ap- preciably from 10 to 60 days. This has been shown convincingly by Skoff et al. (1976b) for rat optic nerve where the final cell division occurs at about the 10th postnatal day. The decline of cell yield, on a weight basis, may primarily reflect cellular hypertrophy.

The ultrastructural appearance of these rat cells conforms to the characteristics associated with both isolated (Raine et al., 1971; 1978) and in situ oligodendroglia (Kruger and Maxwell, 1966; Bunge,

1968; Mori and Leblond, 1970; Raine, 1976; Skoff et al., 1976u,b). These include a well-defined, densely staining small soma, a nucleus containing abundant heterochromatin, large numbers of free ribosomes, and cytoplasmic microtubules. The cells lack glyco- gen and glial filaments, features associated with mature astrocytes. Immature astrocytes contain few filamentous bundles, but do resemble mature astrocytes in that the nucleus and cytoplasm are electron-lucent, and the chromatin is evenly dis- persed (Ling et al., 1973; Raine, 1976) and would therefore be easily distinguished if they were pres- ent in our preparations.

The protein content of 56-60 pg per cell is rea- sonably consistent with a spherical cell having a diameter of 10- 1 1 pm, a protein content of 10% of the wet weight, and a water content of 78%. The amount of protein is considerably less than the 195 pg and 235 pg reported for rat astrocytes and neurons, respectively (Farooq et al., 1977; Farooq and Norton, 1978), which further indicates that as- troglia and neurons do not contribute appreciably to the final oligodendroglial pellet. The values deter- mined for total protein are from 50 to 90% greater in rat than those previously reported for bovine oligodendroglia (Table 1) and may reflect the devel- opmental stage of the animal, cell count error, dif- ference in cell size, or the extent of preservation.

The amount of DNA determined for these oligodendrocytes is in agreement with the values for rat neurons and astroglia (Farooq et al., 1977; Farooq and Norton, 1978), but it is greater than the

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1620 D . S. SNYDER ET AL.

6.4 pg/cell reported for rat diploid cells (Thompson, 1953; Santen and Agranoff, 1962). The RNA con- tent of rat oligodendroglia is much less than either the 8-29 pg/cell or 21 -26 pg/cell found for astroglia and neurons, respectively (Farooq et al., 1977; Farooq and Norton, 1978).

The cell content of nucleic acid was greater in rat oligodendrocytes than in bovine oligodendroglia. The low RNA/DNA ratio of 0.31-0.42 is in good agreement with the value for bovine oligodendroglia of 0.36 (Poduslo and Norton, 1972) and quite differ- ent from the values for rat neurons and astrocytes, respectively, of 2.6-3.0 and 1.1-2.6 (Farooq et al., 1977; Farooq and Norton, 1978). This characteristic ratio is an additional criterion for the identity and purity of the preparation.

We feel that the ability to obtain preparations of viable cells, sampled at selected stages of develop- ment and differentiation in vivo, should prove valu- able for a wide range of biological and biochemical studies of oligodendroglial function and myelina- tion.

ACKNOWLEDGMENT

This work is in partial fulfillment of the require- ments for the Ph.D. degree to D.S.S. in the Sue Golding Graduate Division of the Albert Einstein College of Medicine, Bronx, New York, and was supported in part by USPHS grants NS 08952, NS 02476, and NS 03356, and National Multiple Sclerosis Research Grants RG 1001-B-2 and

We thank Everett Swanson, Miriam Pakingan, and Howard Finch for expert technical assistance and Mary Palumbo and Marion Levine for sec- retarial help.

1089-B-5.

REFERENCES

Banik N. L. and Smith M. E. (1976) In vitro protein synthesis by oligodendroglial cells. Neiirosci. Lett. 2, 235-238.

Benjamins J . A., Guarnieri M., Miller K., Sonneborn M., and McKhann G . M. (1974) Sulphatide synthesis in isolated oligodendroglial and neuronal cells. J . Neurochem. 23, 751-757.

Bunge R. P. (1968) Glial cells and the central myelin sheath. Physiol. R ~ v . 48, 197-251.

Chao S. W. and Rumsby M. G. (1977) Preparation of astrocytes, neurones and oligodendrocytes from the same rat brain. Bruin Res. 124, 347-351.

Cohen S. R. and Bernsohn 3. (1973) Incorporation of l-I4C labelled fatty acids into isolated neuronal soma, astroglia and oligodendroglia from calf brain. Brain Res. 60,521-525.

Cohen S. R. and Bernsohn J . (1978) The in vivo incorporation of linolenic acid into neuronal and glial cells and myelin. J . Neurochem. 30, 661-669.

Deshmukh D. S. , Flynn T. J., and Pieringer R. A. (1974) The biosynthesis and concentration of galactosyl diglyceride in glial and neuronal enriched fractions of actively myelinating rat brain. J . Neiirochem. 22, 479-485.

Farooq M. and Norton W. T. (1978) A modified procedure for

isolation of astrocyte- and neuron-enriched fractions from rat brain. J . Neiirochem. 31, 887-894.

Farooq M., Ferszt R., Moore C. L., and Norton W. T. (1977) The isolation of cerebral neurons with partial retention of processes. Brain Res. 124, 69-81.

Fewster M. E., Scheibel A. B., and Mead J . F. (1967) The prepa- ration of isolated glial cells from rat and bovine white mat- ter. Brain Res. 6, 401 -408.

Fewster M. E., Blackstone S. C. , and Ihrig T. J. (1973) The pro- portion and characterization of isolated oligodendroglia from bovine white matter. Brain Res. 63, 263-271.

Guarnieri M., Cohen S. R., and Ginns E. (1976) Cells isolated form trypsin-treated brain contain trypsin. J. Neiirochem.

Hodson A. K., Quinn B., Kim S. U., and Pleasure D. (1979) Improved method of oligodendrocyte preparation from rat brain. Trans. A m . Soc. Neurochem. 10, 158.

Imarnoto K . , Paterson J. A. , and Leblond C. P. (1978) Radioautoradiographic investigation of gliogenesis in the corpus callosum of young rats. J . Comp. NFLiroI. 180,

Iqbal K., Grundke-Iqbal I., and Wisniewski H. M. (1977) Oligodendroglia from human autopsied brain: Bulk isolation and some chemical properties. J . Neiirochem. 28,707-716.

Jacobson M. (1978) Developmental Neiirobiology, pp. 166- 180. Plenum Press, New York.

Kozik M. B. (1976) The electron-microscopic picture of post- natal development of oligodendroglia. Folio Hisfochem. Cytochem. (Krakow) 14, 99-106.

Kruger L. and Maxwell D. S. (1966) Electron microscopy of oligodendrocytes in normal rat cerebrum. A m . J . Anat. 118, 411-436.

Ling E. A., Paterson J. A., Privat A., Mori S. , and Leblond C. P. (1973) Investigation of glial cells in semithin sections. J . Comp. Neiirol. 149, 43-72.

Lowry 0. H., Rosebrough N. S. , Farr A. L., and Randall R. J. (195 1) Protein measurement with the Folin phenol reagent. J . B i d . Chem. 193, 265-275.

Mori S. and Leblond C. P. (1970) Electron microscopic identifi- cation of three classes of oligodendrocytes and a preliminary study of their proliferative activity in the corpus callosum of young rats. J . Comp. Neiirol. 139, 1-30.

Munro H. N. and Fleck A. (1966) The determination of nucleic acids. Methods Biochem. Anal. 14, 113-176.

Norton W. T. and Poduslo S. E. (1970) Neuronal soma and whole neuroglia of rat brain: A new isolation technique. Sci- ence 167, 1144- 1146.

Pleasure D., Abramsky O., Silberberg D., Quinn B., Parris J . , and Saida T. (1977) Lipid synthesis by an oligodendroglial fraction in suspension culture. Brain Res. 134, 377-382.

Pleasure D., Lichtman C., Eastman S. , Lieb M., Abramsky O., and Silberberg D. (1979) Acetoacetate and D-(-)-beta- hydroxybutyrate as precursors for sterol synthesis by calf oligodendrocytes in suspension culture; extramitochondrial pathway for acetoacetate metabolism. J . Nearrochem. 32,

Poduslo S. E. and McKhann G. M. (1977) Synthesis of cere- brosides by intact oligodendroglia maintained in culture. Neurosci. Lett. 5, 159- 163.

Poduslo S. E. and Norton W. T. (1972) Isolation and some chemical properties of oligodendroglia from calf brain. J . Neirrochem. 19, 727-736.

Poduslo S. E., Miller K., and McKhann G . M. (1978) Metabolic properties of maintained oligodendroglia purified from brain. J . Biol. Chem. 253, 1592- 1597.

Raine C . S . (1976) Neuroce l lu l a r a n a t o m y , in Bas ic Necrrochemistry (Siege1 G . J., Albers R. W., Katzman R., and Agranoff B. W., eds), pp. 5-33. Little, Brown and Co., Boston.

Raine C. S. , Poduslo S. E., and Norton W. T. (1971) The ul- trastructure of purified preparations of neurons and glial cells. Brain Res. 27, 11-24.

Raine C. S., Traugott U . , Iqbal K., Snyder D. S., Cohen S. R.,

26, 41-44.

115- 138.

1447-1450.

J . Neurochem., Vol. 34, No. 6 , 1980

BULK ISOLATION OF OLIGODENDROGLIA 1621

Farooq M., and Norton W. T. (1978) Encephalitogenic properties of purified preparations of bovine oligodendro- cytes tested in guinea pigs. Brain Res . 142, 85-96.

Santen R. J. and Agranoff B. W. (1962) Studies on the estimation of deoxyribonucleic acid and ribonucleic acid in rat brain. Biochim. Biophys. Acta 72, 251-262.

Schonbach J., Hu K. H., and Friede R. L. (1968) Cellular and chemical changes during myelination. Histologic, au- toradiographic, histochemical and biochemical data on myelination in the pyramidal tract and corpus callosum of rat. J . Comp. Neurol. 134, 21-38.

Skoff R. P., Price D. L., and Stocks A. (197Q) Electron micro- scopic autoradiographic studies of gliogenesis in rat optic nerve. Cell proliferation. J . Cump. Neiirol. 169, 291-312.

Skoff R. P., Price D. L., and Stocks A. (19765) Electron micro- scopic autoradiographic studies of gliogenesis in rat optic nerve. Time of origin. J . Comp. Neiirol. 169, 313-334.

Snyder D. S. , Raine C. S. , Farooq M., and Norton W. T. (1978)

Exploration of new methods for bulk isolation of oligoden- droglia. Tmns. Am. SOC. Neurochem. 9, 64.

Snyder D. S. , Norton W. T., and Raine C. S. (1979) A method for the isolation of oligodendroglia from rat whole brain. J . Neuropathol. Exp. Neurol. 38, 342.

Szuchet S., Arnason B. G . W., and Polak P. E. (1978) A new method for oligodendrocyte isolation. Biophys. J. 21, 51a.

Tennekoon G . I . , Cohen S. R., Price D. L. , and McKhann G. M. (1977) Myelinogenesis in optic nerve. J . Cell B i d . 72,

Thompson R. Y . , Heagy F. C., Hutchison W. C. , and Davidson J . N . (1953) The deoxyribonucleic acid content of the rat cell nucleus and its use in expressing the results of tissue analysis. with particular reference to the composition of liver tissue. Biochem. J . 53, 460-474.

Trapp B. D., Dwyer B., and Bernsohn J. (1975) Light and elec- tron microscopic examination of isolated neurons, astro- cytes and oligodendrocytes. Neurobiology 5, 235-248.

604- 6 16.

J . Neurochem., Vul. 34, N u . 6 , 1980