Acta neuropath. (Berl.) 32, 313 324 (1975) 9 by Springer-Verlag 1975
Human Nervous System Tumors Observations b y High Voltage Electron Microscopy Katherine M. Lyser* Institut d'Embryologie exp6rimentale du Coll~gede France et du C.N.R.S., Nogent-sur-Marne, France (Director: Prof. Etielme Wolff) Received September 12, 1974; Accepted June 13, 1975
Summary. Thick sections (0.5--2 Ex) of biopsies from human nervous system tumors (Sehwannoma, ependymoma, medulloblastoma), fixed in aldehydes followed by osmium, and stained with uranyl acetate and lead, have been studied at 2.5 MV, and compared to thin sections of the same material observed by ordinary low voltage electron microscopy. High voltage electron microscopy permits direct observation of cell fine structure in three dimensions, including the spatial relations of organelles. Details of contours of nuclear envelopes, shapes of mitochondria, fine aspects of the structure of cell surfaces and processes, such as the flat sheet-like and irregular cylindrical processes of Schwannoma cells, the small projections and ridges on their surfaces, and microvilli and cilia of elJendymoma cells, and other features have been observed. These initial observations demonstrate the applicability of high voltage electron microscopy to further study of neural neoplasms. Key words: High Voltage Electron Microscopy -- Nervous System Tumors -- Fine Structure -- Schwannoma. Introduction
High voltage electron microscopy has recently been applied to the study of various biological materials, in order to assess and begin to exploit the advantages of the much greater penetrating power of the electron beam, allowing transmission microscopy of thick specimens, and possible higher resolution, at increased accelerating voltages (tIama and Porter, 1969; Dupouy et al., 1970; Dupouy and Perrier, 1972; Dupouy, 1973; Favard et al., 1971 ; and Favard and Carasso, 1973). So far, the greatest advantage for biologieal material is the direct visualization of three dimensional organization of cells and organelles with electron microscopic resolution in thick sections or in preparations of whole cells (Parsons et al., 1972). As part of a study of human nervous system neoplasms (Lyser, 1974, 1975), several central and peripheral nervous system tumors were observed in the 3 million volt electron microscope (Dupouy, 1973), apparently the first time tot this material. For this initial investigation, they were prepared by adaptations of standard electron microscopic methods, and the observations made by high voltage microscopy are compared directly with ordinary low voltage microscopy of thin sections of the same material, and with previous electron microscopic studies of these types of tumors. * On Sabbatical leave from the Department of Biological Sciences, :Hunter College of the City University of New York, New York 10021, U.S.A.
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The following tumors have been studied: 1. a malignant Schwannoma of the cauda equina in a 14-year old boy, 2. a papillary ependymoma of the floor of the fourth ventricle in an 11-year old boy, 3. a medulloblastoma of the posterior cerebellum in a 2-year old boy. Comparison is also made with a neuroblastoma metastasis of a spinal ganglion described previously (Lyser, 1974). Pieces of tumor obtained during surgery were fixed by immersion in 40/0 paraformaldehyde and 0.5O/o glutaraldehyde, or 3~ glutaraldehyde, in 0.1 M phosphate buffer with 0.001 M CaCI~ (Sabatini et al., 1963; Tilney and Porter, 1965; Vaughn and Peters, 1967) at room temperature. The pieces were postfixed in 1~ in phosphate buffer, dehydrated in ethanol followed by propylene oxide, and embedded in Epon. Tissues were block stained before or during dehydration in aqueous or alcoholic uranyl acetate. Sections were cut on a Reichert ultramicroteme. Thin sections for conventional electron microscopy (Hitachi HS 7) were further stained with lead citrate (Reynolds, 1963). For high voltage microscopy, 0.5--2 ~zsections were picked up on folding grids. In some cases they were stained with lead citrate, followed by a dilute nitric acid rinse to remove precipitate (Favard and Carasso, 1973). The sections were observed in the 3 million volt (3 MV) electron microscope at the Laboratoire d'Optique Electronique du C.N.R.S. at Toulouse, France (see Dupouy et al., 1970; Dupouy and Perrier, 1972; Dupouy, 1973). The microscope was operated at 2.5 1VfVunder bright field conditions, with a 5 tz objective aperture. Micrographs were taken at initial magnifications of • 5000 to • 10000, using the tilting stage to obtain stereo sets
(-lO o, oo, +ioo). For surveys of the tumor tissue at the level of light microscopy, 1--2 Ezsections from the blocks used for electron microscopy, stained with toluidine blue or viewed unstained by phase microscopy, and routine serial sections stained with hematoxylin and eosin with orange G were used.
Results Schwannoma
I n r o u t i n e sections for light microscopy, t h e region which was s t u d i e d was a c o m p a c t S c h w a n n cell t u m o r (Antoni t y p e A, I%ussell a n d R u b i n s t e i n , 1971). T h e r e were some cells w i t h v e r y large, b i z a r r e nuclei, in accordance w i t h t h e histop a t h o l o g i c a l diagnosis of m a l i g n a n c y . The fine s t r u c t u r e as seen b y low v o l t a g e electron m i c r o s c o p y was in general c o m p a r a b l e to t h a t d e s c r i b e d p r e v i o u s l y for S c h w a n n cell t u m o r s , m a i n l y those of acoustic nerve (Wcchsler a n d H o s s m a n n , 1965 ; W a g g e n e r , 1966 ; P o i r i e r a n d Escourolle, 1967 ; Luse, 1968 ; Cravioto, 1969). The p r e s e n t t u m o r was p a r t i c u l a r l y c o m p a c t , w i t h m o s t of the a r e a b e t w e e n t h e cell bodies filled b y cell processes, a n d little e x t r a c e l l u l a r " s p a c e " or s t r u c t u r e d intercellular material. The high v o l t a g e m i e r o g r a p h s showed t h e presence of b o t h small, shallow, a n d larger, v e r y deep, folds in t h e nuclear envelope of the S c h w a n n o m a cells, corr e s p o n d i n g to i r r e g u l a r i t y of profiles in t h i n sections viewed b y s t a n d a r d electron microscopy. T h o u g h t h e sections were o n l y t h i c k enough to include a p o r t i o n of t h e nucleus, the p a r t of t h e surface which was w i t h i n t h e section could be seen in relief, b y shading a n d contours in i n d i v i d u a l m i c r o g r a p h s , a n d in t h r e e dimensions w i t h stereo viewing. The small folds, p a r t i c u l a r l y , could be seen to a d v a n t a g e . Much of t h e n u c l e a r surface in m a n y cells was covered w i t h these small e l e v a t i o n s a n d depressions, r o u n d e d or e l o n g a t e d in v a r i o u s directions (Fig. 1). The contours of cells, a n d p a r t i c u l a r l y , of m a n y of t h e processes, could be seen because of differences in c y t o p l a s m i c d e n s i t y , even t h o u g h t h e c o n t r a s t in general was n o t g r e a t (Fig. 1). T h e i r spacial r e l a t i o n s h i p s could be s t u d i e d b e s t b y stereo-
Fig. 1. Schwannoma, high voltage electron micrograph of several elongated cells (N, nucleus) and intervening cell processes. A t the center is a group of fiat processes perpendicular to the plane of section (P), which can be followed due to the differences in their cytoplasmic densities. Small folds are visible in the nuclear envelopes, especially in the cell a t the right (-->). Section approximately 1.5 ~z. • 11500
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scopic viewing. (The impression of three dimensional relief was much less striking than in material with greater contrast. An a t t e m p t was made, for example, to impregnate nervous system tumors by classical silver carbonate methods. The results were unsatisfactory in t h a t the silver was coarse, and was not deposited in the finer cell processes; however, it could be seen t h a t sections of the same thickness gave a much greater impression of depth.) I n thinner sections the plasma membranes tended to be visible more frequently, and there was less overlapping of processes than in thicker sections. High voltage microscopy emphasized the irregularity of the overall shapes of the cells, as well as demonstrating t h a t frequently the surface of the cell body was not smooth, but had ridges and projections and depressions. There were also short, broad extensions, characteristically with small finger-like projections, which could be seen to advantage in three dimensions b y stereo viewing. The thick sections demonstrated two general forms of cell processes : flattened, somewhat sheet-like processes, and elongated, vaguely cylindrical processes. The flat processes appeared as long, narrow profiles, sometimes only a few A units in width, with low voltage microscopy (Fig. 2). Their sheet-like form was demonstrated best b y high voltage microscopy when the flat surface was more or less parallel to the plane of section--when viewed from the flat surface (Fig. 3). The expanded, flat area appeared as a shadowed area of irregular shape, often with thin projections extending from the edges. The latter could be seen in stereo viewing to be finger-like and to extend up or down within the thickness of the section. Flat processes lying perpendicular or at an angle to the place of section were included as narrow strips, outlines of which could be followed b y contrast in the densities of different processes, and sometimes visible plasma membranes (Fig. 1). The roughly cylindrical shape of other processes was varified by favorable instances where they were viewed approximately perpendicularly to the long axis and could be seen in three dimensions extending through the thickness of' the section. When the long axis was more or less parallel to the plane of section, the variations in diameter and bulges and depressions in the surface were e v i d e n t - - t h e processes had a twisted and very lumpy appearance.
Ependymoma This tumor was very "well differentiated", organized in folded sheets of columnar epithelium, with a small amount of supportive tissue and blood vessels adjacent to the basal aspect of the epithelium. The fine structure of the cells (Figs.4--6) was similar to t h a t of normal cylindric ependymal cells (Tennyson and Pappas, 1968), except for the presence of a basal lamina. I t resembled that of the more differentiated of the cpendymomas previously described (Lure, 1960; Tani and Ametani, 1970; Goebel and Cravioto, 1972), however, the epithelia] arrangement was much more pronounced than usual, and elongated basal processes were not evident (see Russell and Rubinstein, 1971). In the ependymoma, cilia and microvilli particularly could be observed to advantage b y high voltage microscopy, since they were contrasted against the bakgronnd of the cell free space of the lumen. Microvilli were seen best where the apical surface was at an angle to the plane of section. (The contrast between the
Fig. 2. Schwannoma, low voltage electron micrograph of s thin section from the same block as Fig. 1, cut perpendieular to several fiat sheet-like processes of various thickness. • 34500 Fig. 3. Schwannoma, high voltage electron micrograph with a surface view of a flat process (P), which appears as a grey area. Finger-like projections (-->) could be seen to extend up or downward within the section by stereo viewing. Section approximately i.5 ~. • l i 5 0 0
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Fig.4. Ependymal cell tumor, low voltage electron micrograph showing the apical ends of tumor cells. Both longitudinal (right) and tangential (left) views are included in the section, due to the folding of the epithelium. Basal bodies (B) and bases of several cilia are visible in the cell at the left. m Microvilli. • 19200 Fig. 5. Ependymal cell tumor, high voltage electron micrograph. At the apical end of the central cell are several cilia with basal bodies (B). The surface of the nucleus (N) has sharp ridges and folds. Section approximately 2 ~. • 13700
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Fig. 6. Ependymona, high voltage electron micrograph, from a n area with a b u n d a n t apical microvilli (m). N, nucleus. Section approximately 2 ~. • 11300
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lumen and the cells did, however, present difficulties in photography, and the microvilli do not show to advantage in single prints.) High voltage micrographs showed the apical surface of some cells to be covered with a dense m a t of mierovilli, which were about 1 ~ or more in length and had a somewhat flexible a p p e a r a n c e - - t h e y were more or less perpendicular to the cell surface, but individually slightly bent in various directions (Fig. 6). I n most eases, the dense covering of mierovilli was continuous along the apical surface, from one cell to the next. Cilia were usually seen in a cluster toward the center of the apical surface, frequently a half dozen or more visible in one cell, especially in tangential sections for low voltage microscopy and in sections for high voltage microscopy (Figs. 4 and 5). However, in any one section, including sections for high voltage microscopy, there were numerous cells in which no cilia were seen, suggesting variations in the complement of surface organelles, as has been described in normal ependyma (Worthington and Catheart, 1963 ; Bruni et al., 1972). Other organelles within the cells were again discernible in three dimensions. Thick sections viewed at 2.5 MV showed the irregularities in nuclear profiles in these cells to be long ridges and grooves in the nuclear envelope (Fig. 5).
Medulloblastoma The cells in the area observed were polygonal or elongated, with relatively large nuclei and scant cytoplasm. At least some of the ceils had processes, but all had the fine structural features of immature cells, similar to those described in several other medulloblastomas (Voigt, 1968; Kadin et al., 1970; Matakas et al., 1970). The processes were generally cylindrical, but none was found which had numerous microtubules, like those typical of neurites, or as seen in some other medulloblastomas (Eseourolle and Poirier, 1967; Matakas et al., 1970). However, no conclusion about cell types can be drawn, since fine structural morphology, including filaments and mierotubules, can not be used to distinguish between neuronal and glial cells at early stages of development (see Lyser, 1971). Thick sections viewed at 2.5 MV had less contrast than the Schwannoma, due to the lack of variation in general fine structure and overall cytoplasmic density among the medulloblastoma cells and their processes (Fig.7). However, cell boundaries and processes could often be observed, including stereo viewing. Where the plane of section was perpendicular to plasma membranes, they were seen as parallel lines, as in thin sections. Plasma membranes viewed obliquely were not as clear. Where they could be observed, the cell bodies and processes of the medulloblastoma generally had smooth, rounded contours; small projections or folds in the cell surfaces were not seen. The nuclear envelopes, as in the other tumors, could be observed through the entire thickness of the sections. I n this case, there were very moderate folds in the nuclear envelope, usually in the form of rounded shallow depressions and low elevations; no deep indentations were seen. Of the cytoplasmic organelles, mitochondria and polysomes were most easily distinguishable. Many of the mitochondria of the medulloblastoma cells were included within one thick section, and thus could be seen to be more or less spherical in shape.
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Fig. 7. Medulloblastoma, high voltage electron mierograph. Mitochondria (mi) are spherical and overlap within the thickness of the section. Undulations can be seen in the nuclear envelope, especially at the lower edge. (N~ nueleolus.) In some areas (->) adjacent plasma membranes have been cut perpendicular to the plane of section, and appear as parallel lines. Section approximately I lx. X 18 900
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As would be expected, high voltage electron microscopy of tumor tissues entails problems and advantages like those encountered with the various normal tissues which have been studied (Favard et al., 1971). Fine structure can be analyzed in sections up to about 2 ~ in thickness at 2.5 MV, especially with stereoscopic viewing of micrographs, in material in which organe]les are contrasted by osmium post-fixation and block staining with uranyl acetate (lack of penetration prevents satisfactory staining of sections, see Carasso et al., 1973), with or without lead staining of sections. In sections thicker than about 2 ~, the picture is obscured by overlap and superposition of too many organe]les, all stained to a greater or lesser extent. In the 0.5--2 ~ sections, various aspects of fine structure can be compared in different tissues, and high voltage observations related directly to those of thin sections. In the present study, three dimensional observations of the contours of nuclear envelopes indicated some differences in pattern among the tumors observed. In the Schwannoma and ependymal tumor, deep grooves and prominent ridges were seen, along with numerous small depressions and elevations in the case of the Schwannoma, while the nuclei of the neuroblastoma (Lyser, 1974) and the medulloblastoma observed here tended to be more rounded, with smoother and shallower grooves and elevations. Mitochondria of the neuroblastoma tended to be more elongated, those of the medulloblastoma more rounded. Cell surface can also be compared. Even though a whole cell is not included in a section of this thickness, and contrast is sometimes a problem, good samples of the surfaces from favorable areas can be observed in three dimensions, and features such as small projections--with their connections to the rest of the cell--can be seen directly without laborious reconstructions. (For similar observations of other tissues, see t t a m a and Porter, 1969 ; Glauert and Mayo, 1973.) Among the neural tumors investigated, neuroblastoma and medulloblastoma cells appeared to have generally smooth, rounded surfaces and cylindrical or tapering processes, while the Schwannoma was characterized by folds and small projections in the cell surtaces and sheet-like as well as very irregular cylindrical processes. The ependymoma had parallel and interdigitated lateral surfaces, and epithelial specializations characteristic of normal ependyma on the apical surface. Further exploitation of high voltage electron microscopy in the study of neoplasms should make use of complementary techniques to give a composite picture of differences and similarities in fine structure between tumors and corresponding normal tissues, as well as among various tumors. The present observations demonstrate the possibilities of 0.5--2 ~ sections: use of preparation techniques applicable to all sorts of normal and neoplastic tissues, and so forming a link between special methods limited to particular tissues or cells; and the preservation of in vivo relationships among cells, a necessary basis for interpretation of results obtained with technically advantageous methods of culture, cell dissociation, etc. For nervous system tumors, complementary techniques should include high voltage microscopy of impregnated material, to study detailed fine structure of cell surfaces and processes in material with more contrast and in thicker sections, in comparison with high voltage observations of impregnated
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n o r m a l n e u r o n s a n d glial cells, such as C h a n - P a l a y a n d P M a y ' s studies of Golgi m a t e r i a l ( C h a n - P a l a y a n d P a l a y , 1972a a n d b; P a l a y a n d C h a n - P a l a y , 1973). Surface f e a t u r e s o b s e r v e d in t h i c k sections a t 2.5 MV ean be c o m p a r e d to s c a n n i n g e l e c t r o n microscope o b s e r v a t i o n s , such as those of n o r m a l e p e n d y m a l cell surfaces (Bruni et aI., 1972; S c o t t etal., 1972; Allen a n d Low, 1973). I n high v o l t a g e m i c r o g r a p h s , a s m a l l e r a r e a of t h e surface is viewed a t once; however, o t h e r features of fine s t r u c t u r e , i n c l u d i n g organelles w i t h i n the cells, can be seen a t t h e s a m e t i m e as surface f e a t u r e s such as cilia a n d microvilli. I t is h o p e d t h a t these o b s e r v a t i o n s will lead to f u r t h e r i n v e s t i g a t i o n of t h e t h r e e d i m e n s i o n a l a s p e c t s of t h e c o m p l e x fine s t r u c t u r a l o r g a n i z a t i o n o f n e r v o u s s y s t e m t u m o r s now possible b y high v o l t a g e electron microscopy.
Acknowledgements. I am happy to thank Prof. Etienne Wolff for his help during my stay in his laboratory; Dr. O. Schweisguth of the Institut Gustave-Roussy, Villejuif, and Prof. J. F. Hirsch and colleagues in the Service de iNeuro-Chirurgie, Groupe Hospitalier NeckerEnfants Malades, Paris, for making the tumors available; Profs. G. Dupouy and F. Pettier of the Laboratoire d'Optique i~lectronique, Toulouse, for making possible my work there, and the staff of the laboratory for all their help during my visits; and Prof. P. Favard for his kindness in extending the use of facilities in his laboratory at the Centre de t~eeherches d'Ivry du C.N.R.S. References Allen, D. J., Low,/;. iN. : The ependymal surface of the lateral ventricle of the dog as revealed by scanning electron microscopy. Amer. J. Anat. 137, 483--489 (1973) Bruni, J . E . , Montemurro, D.G., Clattenburg, R.E., Singh, R. P. : A scanning electron microscopic study of the ependymal surface of the third ventricle of the rabbit, rat, mouse and human brain. Anat. Rec. 174, 407--420 (1972) Carasso, N., Delaunay, M.-C., Favard, P., Lechaire, J.-P. : Obtention et coloration de coupes epaisses pour la microscopic electronique s haute tension. J. Mier. (Paris) 16, 257--268 (1973) Chan-Paley, V., Palay, S. L. : Nigh voltage electron microscopy of rapid Golgi preparations. Neurons and their processes in the cerebellar cortex of monkey and rat. Z. Anat. EntwickI.Gesch. 137, 125--152 (1972a) Chan-Palay, V., Palay, S. L. : The form of relate astrocytes in the cerebellar cortex of monkey and rat: High voltage electron microscopy of rapid Golgi preparations. Z. Anat. Entwickl.Geseh. 138, 1--19 (1972b) Cravioto, H. : The ultrastructure of acoustic nerve tumors. Acta neuropath. (Berl.) 12, 116 to 140 (1969) Dupouy, G. : Performance and applications of the Toulouse 3 million volt electron microscope. J. Micr. (Lond.) 97, 3--28 (1973) Dupouy, G., Perrier, F.: Microscopie ~lectronique s 3 millions de volts. C. 1%. Acad. Sci. (Paris) 274, 1170--1174 (1972) Dupouy, G., Pettier, F., Durrieu, L. : Microscope 61ectronique trois millions de volts. J. Micr. (Paris) 9, 575--592 (1970) Escourolle, R., Poirier, J. : L'ultrastrueture du m4dulloblastome cgrgbelleux. Ann. Anat. path. 12, 121--136 (1967) Favard, P., Carasso, iN. : The preparation and observation of thick biological sections in the high voltage electron microscope. J. Micr. (Lond.) 97, 59--81 (1973) Favard, P., Ovtraeht, L., Carasso, iN. : Observations de sp6cimens biologiques en microscopic @lectronique &haute tension. I. Coupes 4paisses. J. Mier. (Paris) 12, 301--316 (1971) Glauert, A. M., Mayo, C. R. : The study of the three-dimensional structural relationships in connective tissues by high voltage electron microscopy. J. Micr. (Lond.) 97, 83--94 (1973) Goebel, H. H., Cravioto, H. : Ultrastructure of human and experimental ependymomas. J. iNeuropath, exp. iNeuroI. 31, 54--71 (1972)
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Hama, K., Porter, K. R. : An application of high voltage electron microscopy to the study of biological materials. High voltage electron microscopy. J. Micr. (Paris) 8, 149--158 (1969) Kadin, M. E., Rubinstein, L. J., Nelson, J. S.: Neonatal cerebellar medulloblastoma originating from the fetal external granular layer. J. Neuropath. exp. Neurol. 29, 583--600 (1970) Luse, S. A. : Electronmicroscopic studies of brain tumors. Neurology (Minneap.) 10, 881--905 (1960) Luse, S.: The Schwann cell. In: Pathology of the nervous system, vol. 1, pp. 585--607. J. Minckler (ed.). New York: McGraw Hill Book Co. 1968 Lyser, K. 1~. : Microtubules and filaments in developing axons and optic stalk cells. Tissue & Cell 8, 395--404 (1971) Lyser, K. M. : Low- and high-voltage electron microscopy of a human neuroblastoma in longterm organ culture. Cancer Res. 34, 594--602 (1974) Lyser, K. M. : Organ cultures of human nervous system tumors. I n Vitro (in press 1975) Matakas, F., Cerv6s-Navarro, J., Gullotta, F. : The ultrastructure of medulloblastomas. Acta neuropath. (Berl.) 16, 271--284 (1970) Palay, S. L., Chan-Palay, V. : High voltage electron microscopy of the central nervous system in Golgi preparations. J. Micr. (Lond.) 97, 41--47 (1973) Parsons, D. F., Matricardi, V. 1%, Subjeck, J., Uydess, I., Wray, G. : High-voltage electron microscopy of wet whole cancer and normal cells. Visualization of cytoplasmic structures and surface projections. Biochim. biophys. Acta (Amst.) 290, 110--124) (1972) Poirier, J., Escourolle, R.: Ultrastrncture des neurinomes de l'acoustique. Z. mikr.-anat. Forsch. 76, 509--525 (1967) ]~eynolds, E. 9. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17,208--212 (1963) Russell, D. S., Rubinstein, L. J. : Pathology of tumours of the nervous system, 3rd Ed. London: Edw. Arnold Ltd. 1971 Sabatini, D. D., Bensch, K., Barrnett, R. J. : Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19--58 (1963) Scott, D. E., Paull, W . K . , Dudley, G . K . : A comparative scanning electron microscopic analysis of the human cerebral ventricular system. 1. The third ventricle. Z. Zellforsch. 132, 203--215 (1972) Tani, E., Ametani, T. : Polygonal crystalline structures in human ependymoma cells. Acta neuropath. (Berl.) 15, 359--362 (1970) Tennyson, V. M., Pappas, G. D.: Ependyma. In: Pathology of the nervous system, vol. 1, pp. 518--531. J. Minckler (ed.). New York: McGraw Hill Book Co. 1968 Tilney, L. G., Porter, K. R. : Studies on microtubules in Helioza. I. The fine structure of Actino~phaerium nucleo/ilum (Barrett), with particular reference to the axial rod structure. Protoplasma (Wien) 60, 317--344 (1965) Vaughn, J. E., Peters, A. : Electron microscopy of the early postnatal development of fibrous astrocytes. Amer. J. Anat. 121, 131--152 (1967) Voigt, W.-H. : Elektronenmikroskopisehe Beebachtungen an menschlichen Medulloblastomen. Dtsch. Z. Nervenheilk. 192, 290--309 (1968) Waggener, J. D. : Ultrastrueture of benign peripheral nerve sheath tumors. Cancer (Philad.) 19, 699--709 (1966) Wechsler, W., Hossmann, K.-A. : Zur Feinstruktur menschlicher Acustieusneurinome. Beitr. path. Anat. 132, 319--343 (1965) Worthington, W. C., Cathcart, R. S., I I I : Ependymal cilia: Distribution and activity in the adult human brain. Science 189, 221--222 (1963) Katherine M. Lyser, Ph.D. Hunter College Box 1030 695 Park Avenue New York, N. Y. 10021 U.S.A.
Human Nervous System Tumors Observations b y High Voltage Electron Microscopy Katherine M. Lyser* Institut d'Embryologie exp6rimentale du Coll~gede France et du C.N.R.S., Nogent-sur-Marne, France (Director: Prof. Etielme Wolff) Received September 12, 1974; Accepted June 13, 1975
Summary. Thick sections (0.5--2 Ex) of biopsies from human nervous system tumors (Sehwannoma, ependymoma, medulloblastoma), fixed in aldehydes followed by osmium, and stained with uranyl acetate and lead, have been studied at 2.5 MV, and compared to thin sections of the same material observed by ordinary low voltage electron microscopy. High voltage electron microscopy permits direct observation of cell fine structure in three dimensions, including the spatial relations of organelles. Details of contours of nuclear envelopes, shapes of mitochondria, fine aspects of the structure of cell surfaces and processes, such as the flat sheet-like and irregular cylindrical processes of Schwannoma cells, the small projections and ridges on their surfaces, and microvilli and cilia of elJendymoma cells, and other features have been observed. These initial observations demonstrate the applicability of high voltage electron microscopy to further study of neural neoplasms. Key words: High Voltage Electron Microscopy -- Nervous System Tumors -- Fine Structure -- Schwannoma. Introduction
High voltage electron microscopy has recently been applied to the study of various biological materials, in order to assess and begin to exploit the advantages of the much greater penetrating power of the electron beam, allowing transmission microscopy of thick specimens, and possible higher resolution, at increased accelerating voltages (tIama and Porter, 1969; Dupouy et al., 1970; Dupouy and Perrier, 1972; Dupouy, 1973; Favard et al., 1971 ; and Favard and Carasso, 1973). So far, the greatest advantage for biologieal material is the direct visualization of three dimensional organization of cells and organelles with electron microscopic resolution in thick sections or in preparations of whole cells (Parsons et al., 1972). As part of a study of human nervous system neoplasms (Lyser, 1974, 1975), several central and peripheral nervous system tumors were observed in the 3 million volt electron microscope (Dupouy, 1973), apparently the first time tot this material. For this initial investigation, they were prepared by adaptations of standard electron microscopic methods, and the observations made by high voltage microscopy are compared directly with ordinary low voltage microscopy of thin sections of the same material, and with previous electron microscopic studies of these types of tumors. * On Sabbatical leave from the Department of Biological Sciences, :Hunter College of the City University of New York, New York 10021, U.S.A.
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The following tumors have been studied: 1. a malignant Schwannoma of the cauda equina in a 14-year old boy, 2. a papillary ependymoma of the floor of the fourth ventricle in an 11-year old boy, 3. a medulloblastoma of the posterior cerebellum in a 2-year old boy. Comparison is also made with a neuroblastoma metastasis of a spinal ganglion described previously (Lyser, 1974). Pieces of tumor obtained during surgery were fixed by immersion in 40/0 paraformaldehyde and 0.5O/o glutaraldehyde, or 3~ glutaraldehyde, in 0.1 M phosphate buffer with 0.001 M CaCI~ (Sabatini et al., 1963; Tilney and Porter, 1965; Vaughn and Peters, 1967) at room temperature. The pieces were postfixed in 1~ in phosphate buffer, dehydrated in ethanol followed by propylene oxide, and embedded in Epon. Tissues were block stained before or during dehydration in aqueous or alcoholic uranyl acetate. Sections were cut on a Reichert ultramicroteme. Thin sections for conventional electron microscopy (Hitachi HS 7) were further stained with lead citrate (Reynolds, 1963). For high voltage microscopy, 0.5--2 ~zsections were picked up on folding grids. In some cases they were stained with lead citrate, followed by a dilute nitric acid rinse to remove precipitate (Favard and Carasso, 1973). The sections were observed in the 3 million volt (3 MV) electron microscope at the Laboratoire d'Optique Electronique du C.N.R.S. at Toulouse, France (see Dupouy et al., 1970; Dupouy and Perrier, 1972; Dupouy, 1973). The microscope was operated at 2.5 1VfVunder bright field conditions, with a 5 tz objective aperture. Micrographs were taken at initial magnifications of • 5000 to • 10000, using the tilting stage to obtain stereo sets
(-lO o, oo, +ioo). For surveys of the tumor tissue at the level of light microscopy, 1--2 Ezsections from the blocks used for electron microscopy, stained with toluidine blue or viewed unstained by phase microscopy, and routine serial sections stained with hematoxylin and eosin with orange G were used.
Results Schwannoma
I n r o u t i n e sections for light microscopy, t h e region which was s t u d i e d was a c o m p a c t S c h w a n n cell t u m o r (Antoni t y p e A, I%ussell a n d R u b i n s t e i n , 1971). T h e r e were some cells w i t h v e r y large, b i z a r r e nuclei, in accordance w i t h t h e histop a t h o l o g i c a l diagnosis of m a l i g n a n c y . The fine s t r u c t u r e as seen b y low v o l t a g e electron m i c r o s c o p y was in general c o m p a r a b l e to t h a t d e s c r i b e d p r e v i o u s l y for S c h w a n n cell t u m o r s , m a i n l y those of acoustic nerve (Wcchsler a n d H o s s m a n n , 1965 ; W a g g e n e r , 1966 ; P o i r i e r a n d Escourolle, 1967 ; Luse, 1968 ; Cravioto, 1969). The p r e s e n t t u m o r was p a r t i c u l a r l y c o m p a c t , w i t h m o s t of the a r e a b e t w e e n t h e cell bodies filled b y cell processes, a n d little e x t r a c e l l u l a r " s p a c e " or s t r u c t u r e d intercellular material. The high v o l t a g e m i e r o g r a p h s showed t h e presence of b o t h small, shallow, a n d larger, v e r y deep, folds in t h e nuclear envelope of the S c h w a n n o m a cells, corr e s p o n d i n g to i r r e g u l a r i t y of profiles in t h i n sections viewed b y s t a n d a r d electron microscopy. T h o u g h t h e sections were o n l y t h i c k enough to include a p o r t i o n of t h e nucleus, the p a r t of t h e surface which was w i t h i n t h e section could be seen in relief, b y shading a n d contours in i n d i v i d u a l m i c r o g r a p h s , a n d in t h r e e dimensions w i t h stereo viewing. The small folds, p a r t i c u l a r l y , could be seen to a d v a n t a g e . Much of t h e n u c l e a r surface in m a n y cells was covered w i t h these small e l e v a t i o n s a n d depressions, r o u n d e d or e l o n g a t e d in v a r i o u s directions (Fig. 1). The contours of cells, a n d p a r t i c u l a r l y , of m a n y of t h e processes, could be seen because of differences in c y t o p l a s m i c d e n s i t y , even t h o u g h t h e c o n t r a s t in general was n o t g r e a t (Fig. 1). T h e i r spacial r e l a t i o n s h i p s could be s t u d i e d b e s t b y stereo-
Fig. 1. Schwannoma, high voltage electron micrograph of several elongated cells (N, nucleus) and intervening cell processes. A t the center is a group of fiat processes perpendicular to the plane of section (P), which can be followed due to the differences in their cytoplasmic densities. Small folds are visible in the nuclear envelopes, especially in the cell a t the right (-->). Section approximately 1.5 ~z. • 11500
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scopic viewing. (The impression of three dimensional relief was much less striking than in material with greater contrast. An a t t e m p t was made, for example, to impregnate nervous system tumors by classical silver carbonate methods. The results were unsatisfactory in t h a t the silver was coarse, and was not deposited in the finer cell processes; however, it could be seen t h a t sections of the same thickness gave a much greater impression of depth.) I n thinner sections the plasma membranes tended to be visible more frequently, and there was less overlapping of processes than in thicker sections. High voltage microscopy emphasized the irregularity of the overall shapes of the cells, as well as demonstrating t h a t frequently the surface of the cell body was not smooth, but had ridges and projections and depressions. There were also short, broad extensions, characteristically with small finger-like projections, which could be seen to advantage in three dimensions b y stereo viewing. The thick sections demonstrated two general forms of cell processes : flattened, somewhat sheet-like processes, and elongated, vaguely cylindrical processes. The flat processes appeared as long, narrow profiles, sometimes only a few A units in width, with low voltage microscopy (Fig. 2). Their sheet-like form was demonstrated best b y high voltage microscopy when the flat surface was more or less parallel to the plane of section--when viewed from the flat surface (Fig. 3). The expanded, flat area appeared as a shadowed area of irregular shape, often with thin projections extending from the edges. The latter could be seen in stereo viewing to be finger-like and to extend up or down within the thickness of the section. Flat processes lying perpendicular or at an angle to the place of section were included as narrow strips, outlines of which could be followed b y contrast in the densities of different processes, and sometimes visible plasma membranes (Fig. 1). The roughly cylindrical shape of other processes was varified by favorable instances where they were viewed approximately perpendicularly to the long axis and could be seen in three dimensions extending through the thickness of' the section. When the long axis was more or less parallel to the plane of section, the variations in diameter and bulges and depressions in the surface were e v i d e n t - - t h e processes had a twisted and very lumpy appearance.
Ependymoma This tumor was very "well differentiated", organized in folded sheets of columnar epithelium, with a small amount of supportive tissue and blood vessels adjacent to the basal aspect of the epithelium. The fine structure of the cells (Figs.4--6) was similar to t h a t of normal cylindric ependymal cells (Tennyson and Pappas, 1968), except for the presence of a basal lamina. I t resembled that of the more differentiated of the cpendymomas previously described (Lure, 1960; Tani and Ametani, 1970; Goebel and Cravioto, 1972), however, the epithelia] arrangement was much more pronounced than usual, and elongated basal processes were not evident (see Russell and Rubinstein, 1971). In the ependymoma, cilia and microvilli particularly could be observed to advantage b y high voltage microscopy, since they were contrasted against the bakgronnd of the cell free space of the lumen. Microvilli were seen best where the apical surface was at an angle to the plane of section. (The contrast between the
Fig. 2. Schwannoma, low voltage electron micrograph of s thin section from the same block as Fig. 1, cut perpendieular to several fiat sheet-like processes of various thickness. • 34500 Fig. 3. Schwannoma, high voltage electron micrograph with a surface view of a flat process (P), which appears as a grey area. Finger-like projections (-->) could be seen to extend up or downward within the section by stereo viewing. Section approximately i.5 ~. • l i 5 0 0
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Fig.4. Ependymal cell tumor, low voltage electron micrograph showing the apical ends of tumor cells. Both longitudinal (right) and tangential (left) views are included in the section, due to the folding of the epithelium. Basal bodies (B) and bases of several cilia are visible in the cell at the left. m Microvilli. • 19200 Fig. 5. Ependymal cell tumor, high voltage electron micrograph. At the apical end of the central cell are several cilia with basal bodies (B). The surface of the nucleus (N) has sharp ridges and folds. Section approximately 2 ~. • 13700
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Fig. 6. Ependymona, high voltage electron micrograph, from a n area with a b u n d a n t apical microvilli (m). N, nucleus. Section approximately 2 ~. • 11300
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lumen and the cells did, however, present difficulties in photography, and the microvilli do not show to advantage in single prints.) High voltage micrographs showed the apical surface of some cells to be covered with a dense m a t of mierovilli, which were about 1 ~ or more in length and had a somewhat flexible a p p e a r a n c e - - t h e y were more or less perpendicular to the cell surface, but individually slightly bent in various directions (Fig. 6). I n most eases, the dense covering of mierovilli was continuous along the apical surface, from one cell to the next. Cilia were usually seen in a cluster toward the center of the apical surface, frequently a half dozen or more visible in one cell, especially in tangential sections for low voltage microscopy and in sections for high voltage microscopy (Figs. 4 and 5). However, in any one section, including sections for high voltage microscopy, there were numerous cells in which no cilia were seen, suggesting variations in the complement of surface organelles, as has been described in normal ependyma (Worthington and Catheart, 1963 ; Bruni et al., 1972). Other organelles within the cells were again discernible in three dimensions. Thick sections viewed at 2.5 MV showed the irregularities in nuclear profiles in these cells to be long ridges and grooves in the nuclear envelope (Fig. 5).
Medulloblastoma The cells in the area observed were polygonal or elongated, with relatively large nuclei and scant cytoplasm. At least some of the ceils had processes, but all had the fine structural features of immature cells, similar to those described in several other medulloblastomas (Voigt, 1968; Kadin et al., 1970; Matakas et al., 1970). The processes were generally cylindrical, but none was found which had numerous microtubules, like those typical of neurites, or as seen in some other medulloblastomas (Eseourolle and Poirier, 1967; Matakas et al., 1970). However, no conclusion about cell types can be drawn, since fine structural morphology, including filaments and mierotubules, can not be used to distinguish between neuronal and glial cells at early stages of development (see Lyser, 1971). Thick sections viewed at 2.5 MV had less contrast than the Schwannoma, due to the lack of variation in general fine structure and overall cytoplasmic density among the medulloblastoma cells and their processes (Fig.7). However, cell boundaries and processes could often be observed, including stereo viewing. Where the plane of section was perpendicular to plasma membranes, they were seen as parallel lines, as in thin sections. Plasma membranes viewed obliquely were not as clear. Where they could be observed, the cell bodies and processes of the medulloblastoma generally had smooth, rounded contours; small projections or folds in the cell surfaces were not seen. The nuclear envelopes, as in the other tumors, could be observed through the entire thickness of the sections. I n this case, there were very moderate folds in the nuclear envelope, usually in the form of rounded shallow depressions and low elevations; no deep indentations were seen. Of the cytoplasmic organelles, mitochondria and polysomes were most easily distinguishable. Many of the mitochondria of the medulloblastoma cells were included within one thick section, and thus could be seen to be more or less spherical in shape.
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Fig. 7. Medulloblastoma, high voltage electron mierograph. Mitochondria (mi) are spherical and overlap within the thickness of the section. Undulations can be seen in the nuclear envelope, especially at the lower edge. (N~ nueleolus.) In some areas (->) adjacent plasma membranes have been cut perpendicular to the plane of section, and appear as parallel lines. Section approximately I lx. X 18 900
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K.M. Lyser Discussion
As would be expected, high voltage electron microscopy of tumor tissues entails problems and advantages like those encountered with the various normal tissues which have been studied (Favard et al., 1971). Fine structure can be analyzed in sections up to about 2 ~ in thickness at 2.5 MV, especially with stereoscopic viewing of micrographs, in material in which organe]les are contrasted by osmium post-fixation and block staining with uranyl acetate (lack of penetration prevents satisfactory staining of sections, see Carasso et al., 1973), with or without lead staining of sections. In sections thicker than about 2 ~, the picture is obscured by overlap and superposition of too many organe]les, all stained to a greater or lesser extent. In the 0.5--2 ~ sections, various aspects of fine structure can be compared in different tissues, and high voltage observations related directly to those of thin sections. In the present study, three dimensional observations of the contours of nuclear envelopes indicated some differences in pattern among the tumors observed. In the Schwannoma and ependymal tumor, deep grooves and prominent ridges were seen, along with numerous small depressions and elevations in the case of the Schwannoma, while the nuclei of the neuroblastoma (Lyser, 1974) and the medulloblastoma observed here tended to be more rounded, with smoother and shallower grooves and elevations. Mitochondria of the neuroblastoma tended to be more elongated, those of the medulloblastoma more rounded. Cell surface can also be compared. Even though a whole cell is not included in a section of this thickness, and contrast is sometimes a problem, good samples of the surfaces from favorable areas can be observed in three dimensions, and features such as small projections--with their connections to the rest of the cell--can be seen directly without laborious reconstructions. (For similar observations of other tissues, see t t a m a and Porter, 1969 ; Glauert and Mayo, 1973.) Among the neural tumors investigated, neuroblastoma and medulloblastoma cells appeared to have generally smooth, rounded surfaces and cylindrical or tapering processes, while the Schwannoma was characterized by folds and small projections in the cell surtaces and sheet-like as well as very irregular cylindrical processes. The ependymoma had parallel and interdigitated lateral surfaces, and epithelial specializations characteristic of normal ependyma on the apical surface. Further exploitation of high voltage electron microscopy in the study of neoplasms should make use of complementary techniques to give a composite picture of differences and similarities in fine structure between tumors and corresponding normal tissues, as well as among various tumors. The present observations demonstrate the possibilities of 0.5--2 ~ sections: use of preparation techniques applicable to all sorts of normal and neoplastic tissues, and so forming a link between special methods limited to particular tissues or cells; and the preservation of in vivo relationships among cells, a necessary basis for interpretation of results obtained with technically advantageous methods of culture, cell dissociation, etc. For nervous system tumors, complementary techniques should include high voltage microscopy of impregnated material, to study detailed fine structure of cell surfaces and processes in material with more contrast and in thicker sections, in comparison with high voltage observations of impregnated
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n o r m a l n e u r o n s a n d glial cells, such as C h a n - P a l a y a n d P M a y ' s studies of Golgi m a t e r i a l ( C h a n - P a l a y a n d P a l a y , 1972a a n d b; P a l a y a n d C h a n - P a l a y , 1973). Surface f e a t u r e s o b s e r v e d in t h i c k sections a t 2.5 MV ean be c o m p a r e d to s c a n n i n g e l e c t r o n microscope o b s e r v a t i o n s , such as those of n o r m a l e p e n d y m a l cell surfaces (Bruni et aI., 1972; S c o t t etal., 1972; Allen a n d Low, 1973). I n high v o l t a g e m i c r o g r a p h s , a s m a l l e r a r e a of t h e surface is viewed a t once; however, o t h e r features of fine s t r u c t u r e , i n c l u d i n g organelles w i t h i n the cells, can be seen a t t h e s a m e t i m e as surface f e a t u r e s such as cilia a n d microvilli. I t is h o p e d t h a t these o b s e r v a t i o n s will lead to f u r t h e r i n v e s t i g a t i o n of t h e t h r e e d i m e n s i o n a l a s p e c t s of t h e c o m p l e x fine s t r u c t u r a l o r g a n i z a t i o n o f n e r v o u s s y s t e m t u m o r s now possible b y high v o l t a g e electron microscopy.
Acknowledgements. I am happy to thank Prof. Etienne Wolff for his help during my stay in his laboratory; Dr. O. Schweisguth of the Institut Gustave-Roussy, Villejuif, and Prof. J. F. Hirsch and colleagues in the Service de iNeuro-Chirurgie, Groupe Hospitalier NeckerEnfants Malades, Paris, for making the tumors available; Profs. G. Dupouy and F. Pettier of the Laboratoire d'Optique i~lectronique, Toulouse, for making possible my work there, and the staff of the laboratory for all their help during my visits; and Prof. P. Favard for his kindness in extending the use of facilities in his laboratory at the Centre de t~eeherches d'Ivry du C.N.R.S. References Allen, D. J., Low,/;. iN. : The ependymal surface of the lateral ventricle of the dog as revealed by scanning electron microscopy. Amer. J. Anat. 137, 483--489 (1973) Bruni, J . E . , Montemurro, D.G., Clattenburg, R.E., Singh, R. P. : A scanning electron microscopic study of the ependymal surface of the third ventricle of the rabbit, rat, mouse and human brain. Anat. Rec. 174, 407--420 (1972) Carasso, N., Delaunay, M.-C., Favard, P., Lechaire, J.-P. : Obtention et coloration de coupes epaisses pour la microscopic electronique s haute tension. J. Mier. (Paris) 16, 257--268 (1973) Chan-Paley, V., Palay, S. L. : Nigh voltage electron microscopy of rapid Golgi preparations. Neurons and their processes in the cerebellar cortex of monkey and rat. Z. Anat. EntwickI.Gesch. 137, 125--152 (1972a) Chan-Palay, V., Palay, S. L. : The form of relate astrocytes in the cerebellar cortex of monkey and rat: High voltage electron microscopy of rapid Golgi preparations. Z. Anat. Entwickl.Geseh. 138, 1--19 (1972b) Cravioto, H. : The ultrastructure of acoustic nerve tumors. Acta neuropath. (Berl.) 12, 116 to 140 (1969) Dupouy, G. : Performance and applications of the Toulouse 3 million volt electron microscope. J. Micr. (Lond.) 97, 3--28 (1973) Dupouy, G., Perrier, F.: Microscopie ~lectronique s 3 millions de volts. C. 1%. Acad. Sci. (Paris) 274, 1170--1174 (1972) Dupouy, G., Pettier, F., Durrieu, L. : Microscope 61ectronique trois millions de volts. J. Micr. (Paris) 9, 575--592 (1970) Escourolle, R., Poirier, J. : L'ultrastrueture du m4dulloblastome cgrgbelleux. Ann. Anat. path. 12, 121--136 (1967) Favard, P., Carasso, iN. : The preparation and observation of thick biological sections in the high voltage electron microscope. J. Micr. (Lond.) 97, 59--81 (1973) Favard, P., Ovtraeht, L., Carasso, iN. : Observations de sp6cimens biologiques en microscopic @lectronique &haute tension. I. Coupes 4paisses. J. Mier. (Paris) 12, 301--316 (1971) Glauert, A. M., Mayo, C. R. : The study of the three-dimensional structural relationships in connective tissues by high voltage electron microscopy. J. Micr. (Lond.) 97, 83--94 (1973) Goebel, H. H., Cravioto, H. : Ultrastructure of human and experimental ependymomas. J. iNeuropath, exp. iNeuroI. 31, 54--71 (1972)
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Hama, K., Porter, K. R. : An application of high voltage electron microscopy to the study of biological materials. High voltage electron microscopy. J. Micr. (Paris) 8, 149--158 (1969) Kadin, M. E., Rubinstein, L. J., Nelson, J. S.: Neonatal cerebellar medulloblastoma originating from the fetal external granular layer. J. Neuropath. exp. Neurol. 29, 583--600 (1970) Luse, S. A. : Electronmicroscopic studies of brain tumors. Neurology (Minneap.) 10, 881--905 (1960) Luse, S.: The Schwann cell. In: Pathology of the nervous system, vol. 1, pp. 585--607. J. Minckler (ed.). New York: McGraw Hill Book Co. 1968 Lyser, K. 1~. : Microtubules and filaments in developing axons and optic stalk cells. Tissue & Cell 8, 395--404 (1971) Lyser, K. M. : Low- and high-voltage electron microscopy of a human neuroblastoma in longterm organ culture. Cancer Res. 34, 594--602 (1974) Lyser, K. M. : Organ cultures of human nervous system tumors. I n Vitro (in press 1975) Matakas, F., Cerv6s-Navarro, J., Gullotta, F. : The ultrastructure of medulloblastomas. Acta neuropath. (Berl.) 16, 271--284 (1970) Palay, S. L., Chan-Palay, V. : High voltage electron microscopy of the central nervous system in Golgi preparations. J. Micr. (Lond.) 97, 41--47 (1973) Parsons, D. F., Matricardi, V. 1%, Subjeck, J., Uydess, I., Wray, G. : High-voltage electron microscopy of wet whole cancer and normal cells. Visualization of cytoplasmic structures and surface projections. Biochim. biophys. Acta (Amst.) 290, 110--124) (1972) Poirier, J., Escourolle, R.: Ultrastrncture des neurinomes de l'acoustique. Z. mikr.-anat. Forsch. 76, 509--525 (1967) ]~eynolds, E. 9. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17,208--212 (1963) Russell, D. S., Rubinstein, L. J. : Pathology of tumours of the nervous system, 3rd Ed. London: Edw. Arnold Ltd. 1971 Sabatini, D. D., Bensch, K., Barrnett, R. J. : Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19--58 (1963) Scott, D. E., Paull, W . K . , Dudley, G . K . : A comparative scanning electron microscopic analysis of the human cerebral ventricular system. 1. The third ventricle. Z. Zellforsch. 132, 203--215 (1972) Tani, E., Ametani, T. : Polygonal crystalline structures in human ependymoma cells. Acta neuropath. (Berl.) 15, 359--362 (1970) Tennyson, V. M., Pappas, G. D.: Ependyma. In: Pathology of the nervous system, vol. 1, pp. 518--531. J. Minckler (ed.). New York: McGraw Hill Book Co. 1968 Tilney, L. G., Porter, K. R. : Studies on microtubules in Helioza. I. The fine structure of Actino~phaerium nucleo/ilum (Barrett), with particular reference to the axial rod structure. Protoplasma (Wien) 60, 317--344 (1965) Vaughn, J. E., Peters, A. : Electron microscopy of the early postnatal development of fibrous astrocytes. Amer. J. Anat. 121, 131--152 (1967) Voigt, W.-H. : Elektronenmikroskopisehe Beebachtungen an menschlichen Medulloblastomen. Dtsch. Z. Nervenheilk. 192, 290--309 (1968) Waggener, J. D. : Ultrastrueture of benign peripheral nerve sheath tumors. Cancer (Philad.) 19, 699--709 (1966) Wechsler, W., Hossmann, K.-A. : Zur Feinstruktur menschlicher Acustieusneurinome. Beitr. path. Anat. 132, 319--343 (1965) Worthington, W. C., Cathcart, R. S., I I I : Ependymal cilia: Distribution and activity in the adult human brain. Science 189, 221--222 (1963) Katherine M. Lyser, Ph.D. Hunter College Box 1030 695 Park Avenue New York, N. Y. 10021 U.S.A.
