Brain Research, 134 (1977) 407-415 © Elsevier/North-HollandBiomedicalPress
407
PURIFICATION OF VIABLE CILIATED CUBOIDAL EPENDYMAL CELLS FROM RAT BRAIN
C. MARSTON MANTHORPE, GRAHAM P. WlLKIN and JOHN E. WILSON Department of Biochemistry, Michigan State University, East Lansing, Mich. 48824 (U.S.A.)
(Accepted January 20th, 1977)
SUMMARY Trypsinization of coronal sections of rat brain, followed by incubation with the chelating agent, ethyleneglycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), results in selective release of ciliated cuboidal ependymal cells which may be further purified by centrifugal methods. The isolated cells are motile, viable, and exhibit ultrastructure comparable to that in situ.
INTRODUCTION Ependymal cells line the ventricular space of the central nervous system, representing an interface separating the cerebrospinal fluid (CSF) from the neuroglial parenchyma. Ultrastructural studies of the ventricular surface have shown the presence of at least 4 basic cell types3,6,21,23. The major part of the ventricular surface proper is lined by either the ciliated cuboidal ependymal cells or the generally non-ciliated tanycytes, the relative proportion of these cell types varying in different regions of the ventricles. For example, tanycytes predominate in the ventral aspect of the third ventricle while the ciliated cuboidal cells predominate in the dorsal aspect 9,16,23-27,al. Continuous with the ependymal lining, the choroid plexus extends into the ventricular cavity and is lined with another ependymal cell type, the choroid plexus epitheliumS, s, 21,29,39. Certain very localized areas of the ventricle wall contain supraependymal cells, non-ciliated cells possessing pseudopodia-like extensions, which appear to rest upon the ventricle surface1,6,27. It seems generally accepted that the choroid plexus epithelium is involved in the secretion and perhaps to some extent reabsorption of CSF a,29. The physiological function of the other cell types comprising the ventricular lining is considerably more speculative. Tanycytes have cellular features and spatial relationships to the neuroglial parenchyma suggesting an absorptive or secretory function, and some have speculated that they function in neuroendocrine contro12,16,~8,~9,25,26,31. Supra-
408 ependymal cells phagocytize intraventricularly administered latex beads and it has been proposed that this specific cell type may be a resident phagocytic system in the brain 1. The function of the ciliated cuboidal ependymal cell is also obscure. It has been suggested that the presence of uniformly beating cilia extending into the ventricle might provide localized mixing of the CSF at the ventricle-CNS interface 4,26,29 or perhaps have a role in ciliary pinocytosis2L More recently Bleier et al. 1 have proposed that the cuboidal ependyma might serve a phagocytic function similar to that proposed for supraependymal cells and thereby constitute a first line of defense against pathogenic invasion of the brain. In support of their proposal, these authors cite studies from other laboratories showing that (a) viruses can reach the CSF from the bloodstream via the choroid plexus 15,17 and (b) certain viruses (e.g. mumps) that infiltrate the CSF restrict their infection to the ependyma and do not infect the neuroglial parenchyma35, 37. Since the cuboidal ependymal cell has been shown to possess numerous microvilli-like protrusions on its apical surface in addition to its cilia 3,26, it is also reasonable to postulate that this cell type might play a role in the secretion and/or absorption of CSF constituents. Although morphological studies of the ependyma are relatively abundant, there is virtually nothing known about the biochemical features of these cells. Biochemical studies on neural tissue, including the ependyma, are frequently complicated by the heterogeneity of cell types represented. Thus, although explants of the choroid plexus 14,2°,~8 and ventricle wall 7,H-la,32 have been maintained in culture for extended periods, these specimens inherently contain large numbers of non-ependymal cell types (e.g. neurons, glia, capillary elements, and/or connective tissues). This report describes a procedure which leads to the selective purification of ciliated cuboidal ependymal cells, with good preservation of cellular ultrastructure, ciliary mobility, and cell viability. This procedure should greatly facilitate definitive biochemical studies of this ependymal cell type. MATERIALS AND METHODS Cells were prepared for transmission electron microscopy as previously described a6 and examined using a Phillips 201 electron microscope. Scanning electron microscopy was performed by depositing the cell suspension on a 1.0 # m pore size Nucleopore filter (Nucleopore Corp.), followed by treatment with 2 ~o glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, for 10 min. After washing in buffer, dehydration with ethanol and critical point drying, the specimen was coated with gold and examined with an ISI Super Miniscan scanning electron microscope. The following procedure was routinely employed for the isolation of rat brain ependymal cells. Sixteen 175 g adult albino rats (either sex) were etherized until respiratory arrest and perfused with 0 . 9 ~ (w/v) sodium chloride for 10 min at room temperature through the left ventricle (the descending aorta was ligated and the right atrium cut). The brains were removed intact and both the anterior 5 mm of the cerebrum as well as the cerebellum were removed. The remaining forebrain, which contained most of the ventricular system, was then cut into 6-8 coronal sections, 1-2 mm in
409 thickness. The slices from all 16 brains were combined and placed in a 1 liter Erlenmeyer flask with 160 ml of sterile medium [F-12 culture solution (Grand Island Biological Company) plus 1% bovine serum albumin (BSA) and 25 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.2] containing 0.05% trypsin (EC 3.4.21.4) and incubated for 15 min. This and all subsequent incubations were at 37 °C with reciprocal shaking (80 cycles/min). After this preliminary incubation the supernate was gently decanted through an 80-mesh nylon screen, any particulate material trapped on the nylon filter being returned to the flask with the remaining slices. The trypsinized slices were washed twice by resuspension in 100 ml of trypsin-free medium (37 °C) followed by decanting through nylon mesh, and the filtrates discarded. The washed slices were then incubated for two consecutive 10 min periods, each time with fresh medium without trypsin; following each of these two incubations the slices were washed twice as described above. Finally, the slices were incubated for 5 min with 160 ml medium containing 2.5 m M EGTA. The ciliated ependymal cells were quickly released into the supernate before extensive numbers of non-ciliated cell types became detached. The filtered supernate was combined with that from two subsequent washes of the slices with medium containing EGTA. The combined supernates were centrifuged at 1000 x g for 2 min in conical plastic centrifuge tubes and the pellets resuspended in a total of two ml of medium containing 2.5 m M EGTA. This crude preparation, consisting mainly of ciliated cells and debris, was cooled on ice and mixed with 26 ml of ice cold 25 % BSA in medium plus 2.5 m M E G T A and centrifuged at 7000 x g for 15 min (4 °C) in a Beckman SW25.1 rotor. The supernate, which retained most of the debris, was discarded and the pelleted cells either fixed for electron microscopy or resuspended in 1 ml of cold medium. TABLE I Yield o f ciliated rat brain ependymal cells * Cell category
Crude preparation cells brain
- -
Ciliated beating cells Ciliated non-beating cells* ** Non-ciliated cells§ Total of all cells§§
155 40 88 283
x 10-3**
Final preparation cells brain
% total
- -
55 14 31 100
110 32 45 187
x 10-3**
% total
59 17 24 100
* Cell counts were taken using a standard hemacytometer and a Spencer A/O Phase microscope at450 x. ** Variation in cell yield from one preparation to another was -4- 15 %. *** Loss of ciliary motility may be reversible; Hild11has reported a temporary loss of ciliary motility in lateral ventricle explants. § It is likely that a certain number of cells in this category were indeed ciliated but that the cilia were either lost during the purification procedure or the cilia were oriented so that they were not visible through the light microscope. §§ Greater than 95 % of all the cells, includingthe non-beating ciliated cells, were viable as determined by trypan blue exclusion.
410 RESULTS AND DISCUSSION Phase microscopy showed the preparation to consist of three basic categories of cells: actively beating ciliated cells, non-beating ciliated cells, and non-ciliated cells. The relative proportion of each cell category for both the crude and purified preparation is shown in Table I. The final yield is 142,000 ciliated ependymal cells per rat brain, these being 76 ~ of the total isolated cell population. A greater yield of these ciliated cells can be obtained (up to about 300,000 per brain) by extending the time of incubation with E G T A and/or by increasing the shaking speed, but these changes also increase the relative proportion of the non-ciliated cells to about 50 % of the total yield. Indeed when the procedure is scaled up or down (i.e., using larger or fewer numbers of rats), several variables including incubation time with EGTA, flask size and shape, and shaking speed must be reoptimized to maintain a high ratio of ciliated to non-ciliated cells. As noted m Table I, more than 95 ~ of the isolated cells, both ciliated and nonciliated, were judged to be viable on the basis of their exclusion of trypan blue. Further testament to the preservation of normal function in the ciliated cuboidal ependymal cells was that (a) the metachronal beat seen in explants (we are grateful to Dr. D. E. Scott, Dept. of Anatomy, Univ. of Rochester, for providing motion pictures of ciliary
Fig. 1. Light micrographs taken of thick sections prepared prior to ultrathin sectioning for electron microscopy and stained with methylene blue. This low power field shows the preparation to consist of a population of cells with very little surrounding debris. Notice at higher magnification (inset) the presence of cilia protruding from the cells and also the general lack of debris most of which was eliminated during the BSA centrifugation step.
411
Fig. 2. Transmission electron microscopy of a preparation of ciliated cuboidal ependymal cells. A: low power scan of typical field of cells. B : isolated ependymal cell showing cilia (c) and ribosomal clusters (r). The cilia frequently appear to protrude from an indentation or trough on the apical cell surface; this particular cell has been sectioned through such an identation (cf. the scanning electron micrograph, Fig. 3B). C: cell shows perinuclear filaments (f) and basal bodies (b) terminating in a granular zone (g). D: notice the presence of microvilli-like extensions (m) and rough endoplasmic reticulum (e). m o t i o n in these explants) is also noted with the isolated cells, (b) the preservation o f ciliary motility for up to several days (the longest period examined) after isolation o f the cells attests to the structural integrity o f the ciliary apparatus a s w e l l a s the preservation o f necessary supporting biochemical processes (e.g. energy generation). It is difficult to accurately assess the proportion o f the total cuboidal ependymal cell population that is obtained by this procedure. However, dividing the total ventricular surface area, estimated using a stereotaxic atlas o f the rat brain aa, by the crosssectional area o f one ceil, estimated from phase microscopy o f the isolated ciliated ependymal cells, we calculate that there would be 400,000-700,000 ciliated ependymal cells in the brain region we utilize. This estimate is very likely too high since not all o f the ventricular surface is lined with ciliated cells 25,27. Hence, our isolation o f 140,000-300,000 cells per brain clearly suggests that this m e t h o d yields a substantial p r o p o r t i o n (perhaps ~ 5 0 ~ ) o f the total population o f ciliated ependymal cells in the brain. F r o m light micrographs, we estimate that greater than 95 ~ of the mass o f the
412
Fig. 3. Scanning electron microscopy of ciliated ependymal cell preparation. A: low power field of cells on a 1.0 ¢tm pore size Nucleopore filter. B: as is sometimes evident in phase microscopy these two ceils appear to have remained fused during the isolation procedure or to have reaggregated after separation. The orientation of the cells in such 'dimers' appears comparable to that in situ; thus, if they are the result of reaggregation, it must have occurred with preservation of normal spatial relationships. C: notice the contoured cell surface and the presence of cilia and microvilli-likeextensions which are limited to the apical surface. D : occasionally, the cell surface has a 'spongy' appearance. p r e p a r a t i o n consists o f well defined cells, the other 5 ~,~ being debris (Fig. 1). A t higher magnification (Fig. 1, inset) one can m o r e easily see the cilia p r o t r u d i n g from most o f the cell bodies. T r a n s m i s s i o n electron m i c r o s c o p y (Fig. 2) confirms the m i n o r degree o f acellular c o n t a m i n a t i o n indicated by light m i c r o s c o p y and also confirms that the p r e p a r a t i o n consists mainly o f ultrastructurally intact ciliated c u b o i d a l e p e n d y m a l cells. N o t e in particular the excellent preservation o f characteristic features which have been previously r e p o r t e d for these cells in situ3: cilia, with their basal bodies t e r m i n a t i n g in a g r a n u l a r zone; microvilli-like c y t o p l a s m i c extensions; perinuclear filaments; a n d the p r e p o n d e r a n c e o f r i b o s o m a l clusters as o p p o s e d to an extensive r o u g h e n d o p l a s m i c reticulum. Other m o r e general cellular c o m p o n e n t s such as m i t o c h o n d r i a , Golgi bodies, p l a s m a l e m m a , nuclei, lysosomes, a n d p h a g o s o m e s are also seen to retain an ultrastructure t h a t c o m p a r e s f a v o r a b l y with that seen in situ. Scanning electron microscopy again reflects the purity o f the p r e p a r a t i o n and
413 also provides a perspective not readily appreciated from light microscopy or transmission electron microscopy (Fig. 3). Immediately evident is the retention of microvilli-like extensions protruding mainly from the apical (i.e., ciliated) surface which, in vivo, would be oriented toward the CSF-filled ventricular cavity; some cells show such microvilli-like protrusions over their entire visible surface. Although the cell surfaces generally appear smooth, occasionally one sees cells having a 'spongy' appearance (e.g., Fig. 3D). Generally, the cells are highly contoured as if they have partially retained their in situ shape. Notice also the presence of a few round smooth cells (Fig. 3A), many of which lack visible cilia or microvilli-like structures; these presumably correspond to non-ciliated cells seen in phase microscopy (Table I). The origin of the non-ciliated cells remains uncertain. As noted above (Table 1), some of them may, in fact, be ciliated cells which have lost their cilia during the isolation procedure. Additionally, apparently non-ciliated cells can be seen in certain regions of the ventricles where the ependymal lining consists of more than one cell layer 1°,18,19. These might be released when the surface layer of cells is removed. It is unlikely that the non-ciliated cells originate from the choroid plexus; if our dissociation procedure is applied to choroid plexus explants, very few cells are released into the medium. Furthermore, the non-ciliated cells in our preparation do not exhibit the ultrastructure of choroid plexus epitheliaS,21,a~, 39. An alternative source of these nonciliated cells is, of course, the cut surface of the brain slice (i.e., from the neuroglial parenchyma itself or the surrounding pia). The size and ultrastructure of the nonciliated cells is not characteristic of mature intact neurons or glia; however, a pial origin cannot be excluded on an ultrastructural basis 2s,3°,~4. The physiological role of the cuboidal ependyma in the central nervous system is unknown. We have, for the first time, established a preparative technique for the purification of this cell type from brain, and have characterized this cell preparation by phase, light, transmission electron and scanning electron microscopy. This cell preparation should be useful in determining the biochemical and metabolic properties of the cuboidal ependyma in various regions of the brain and under various experimentally induced conditions. These types of studies should be helpful in elucidating the physiological function of this specific brain cell type. ACKNOWLEDGEMENTS We wish to thank the Center for Electron Optics and the College of Osteopathic Medicine at Michigan State University for permitting us the generous use of their electron microscope facilities, and gratefidly acknowledge the financial support of NIH Grant NS 09910. GPW is pleased to acknowledge the award of a Wellcome Research Travel Grant and CMM is pleased to acknowledge an NIH Postdoctoral Fellowship (1 F32 NS05336-01).
414 REFERENCES 1 Bleier, R., Albrecht, R. and Cruce, J. A. F., Supraependymal cells of the hypothalamic third ventricle: Identification as resident phagocytes of the brain, Science, 189 (1975) 299-301. 2 Brawer, J. R., Lin, P. S. and Sonnenschein, C., Morphological plasticity in the wall of the third ventricle during the estrous cycle in the rat: A scanning electron microscopy study, Anat.Rec., 179 (1974) 481-490. 3 Brightman, M. W. and Palay, S. L., The fine structure of ependyma in the brain of the rat, J. Cell Biol., 19 (1963) 415-439. 4 Brightman, M. W., Reese, T. S. and Feder, N., Assessment with the electronmicroscope of the permeability to peroxidase of cerebral endothelium and epithelium in mice and sharks. In C. Crone and N. A. Lassen (Eds.), Capillary Permeability, Munksgaard, Copenhagen, 1970, pp. 462-476. 5 Clementi, F. and Marini, D., The surface fine structure of the walls of cerebral ventricles and of choroid plexus in cats, Z. ZellJbrsch., 123 (1972) 82-95. 6 Coates, P. W., Supraependymal cells: light and transmission electron microscopy extends scanning electron microscopic demonstration, Brain Research, 57 (1973) 502-507. 7 Dalen, H., Schlapfer, W. T. and Mamoon, A., Cilia on cultured ependymal cells examined by scanning electron microscopy, Exp. Cell Res., 67 (1971) 375 379. 8 Davson, H., Physiology of Cerebrospinal Fluid, Little, Brown, Boston, Mass., 1967, pp. 120-148. 9 Dierickx, K. and De Waele, G., Scanning electron microscopy of the wall of the third ventricle of the brain of Rana temporaria, Cell Tiss. Res., 161 (1975) 343-349. 10 Dodson, R. F. and Chu, L. W.-F., Ultrastructure of the ependymal and subependymal cells in the lateral ventricle of the squirrel monkey, Cytobios, l 0 (1974) 145-156. 11 Hild, W., Ependymal cells in tissue culture, Anat. Rec., 99 (1947) 523 529. 12 Hild, W., Takenaka, T. and Walker, F., Electrophysiological properties of ependymal cells from the mammalian brain in tissue culture, Exp. Neurol., 11 (1965) 493-501. 13 Hogue, M. J., Human fetal ependymal cells in tissue cultures, Anat. Rec., 99 (1947) 523-529. 14 Hogue, M. J., Tissue cultures of the brain: intercellular granules, J. comp. Neurol., 85 (1946) 519-530. 15 Johnson, R. and Mims, C. A., Pathogenesis of viral infections of the nervous system, N. engl. J. Med., 278 (1968) 23-30. 16 Kobayashi, H., Wada, M. and Uemura, H., Uptake of peroxidase from the third ventricle by ependymal cells of the median eminence, Z. Zell]brsch., 127 (1972) 545-551. 17 Lipton, H. and Johnson, R., The pathogenesis of rat virus infections in the newborn hamster, Lab. Invest., 27 (1972) 508-513. 18 Ltifgren, F., The infundibular recess, a component in the hypothalamoadenohypophyseal system, Acta morph, neerl.-Scand., 2 (1959) 220-229. 19 L6fgren, F., New aspects of the hypothalamic control of the adenohypophysis, Acta morph. neerl.-scand., 3 (1959) 55-78. 20 Lumsden, C. E., Observations on the choroid plexus maintained in tissue culture. In G. E. W. Wolstenholme and C. M. O'Connor (Eds.), CIBA, Foundation Symposium on the Cerebrospinal Fluid, Little, Brown, Boston, Mass., 1958, pp. 97-123. 21 Maxwell, D. S. and Pease, D. C., The electron microscopy of the choroid plexus, J. biophys, biochem. CytoL, 2 (1956) 467-474. 22 Mikami, S.-I., A correlative ultrastructural analysis of the ependymal cells of the third ventricle of Japanese quail, Coturnix coturnixjaponica. In K. M. Knigge, D. E. Scott and H. Kobayashi (Eds.), Brain Endocrine Interaction II. The Ventricular System in Neuroendocrine Mechanisms, Karger, Basel, 1975, pp. 80-93. 23 Millhouse, O. E., A Golgi study of third ventricle tanycytes in the adult rodent brain, Z. Zellforseh., 121 (1971) 1-13. 24 Millhouse, O. E., Light and electron microscopic studies of the ventricular wall, Z. ZellJbrsch, 127 (1972) 149-174. 25 Millhouse, O. E., Lining of the third ventricle in the rat. In D. E. Scott, K. M. Knigge and H. Kobayashi (Eds.), Brain Endocrine htteraction II. The Ventricldar System hi Neuroendocrine Mechanisms, Karger, Basel, 1975, pp. 3-18. 26 Mitchell, J. A., Functional anatomy of the hypothalamus: possible role of the ependyma. In E. Hafez and J. Reel (Eds.), Perspectives in Human Reproduction: Hypothalamic Hormones, Ann Arbor Science, Publ., Ann Arbor, Mich., 1975, pp. 1 16.
415 27 Mitchell, J. A., Surface morphology of the ependyma lining of the 3rd ventricle of the guinea pig. In Proceedings of the 6th Annual Meeting of the Michigan Chapter of Society for Neuroscience, Ann Arbor, Mich., 1976. 28 Morse, D. E. and Low, F. N., The fine structure of the pia mater of the rat, Amer. J. Anat., 133 (1972) 349-368. 29 Netsky, M. G. and Shuangshoti, S., Studies on the choroid plexus. In S. Ehrenpreis and O. C. Solnitzky, (Eds.), Neurosciences Research, Vol. 3, Academic Press, New York, 1970, pp. 131-173. 30 Pease, D. C. and Schultz, R. L., Electron microscopy of rat cranial meninges, Amer. J. Anat., 102 (1958) 301-321. 31 Scott, D. E., Dudley, G. K. and Knigge, K. M., The ventricular system in neuroendocrine mechanisms, Cell and Tiss. Res., 154 (1974) 1-16. 32 Singer, I. and Goldman, S. J., Mammalian ependyma: some physiochemical determinants of ciliary activity, Exp. Cell Res., 43 (1966) 367 380. 33 Skinner, J. E., Neuroscience: A Lab Manual, Saunders, Philadelphia, Pa., 1971, pp. 195-237. 34 Waggener, J. D. and Beggs, J., The membranous coverings of neural tissues : an electron microscopy study, J. Neuropath. exp. Neurol., 26 (1967) 412-426. 35 Walker, D. H., Murphy, F. A., Whitfield, S. G. and Bauer, S. P., Lymphocytic choriomenengitis : ultrastructural pathology, Exp. molec. Pathol., 23 (1975) 245-265. 36 Wilkin, G. P., Bal~zs, R., Wilson, J. E., Cohen, J. and Dutton, G. R., Preparation of cell bodies from developing cerebellum: Structural and metabolic integrity of the isolated 'cells', Brain Research, 115 (1976) 181-199. 37 Wolinsky, J. S., Baringer, Jr., Margolis, G. and Kilham, L., Ultrastructure of mumps virus replication in newborn hamster central nervous system, Lab. Invest., 31 (1974) 403-412. 38 Wright, E. M., Mechanisms of ion transport across the choroid plexus, J. Physiol. (Lond.), 226 (1972) 545-571. 39 Yamadori, T., A scanning electron microscopic observation of the choroid plexus in rats, Arch. Histol. jap., 35 (1972) 88-97.
407
PURIFICATION OF VIABLE CILIATED CUBOIDAL EPENDYMAL CELLS FROM RAT BRAIN
C. MARSTON MANTHORPE, GRAHAM P. WlLKIN and JOHN E. WILSON Department of Biochemistry, Michigan State University, East Lansing, Mich. 48824 (U.S.A.)
(Accepted January 20th, 1977)
SUMMARY Trypsinization of coronal sections of rat brain, followed by incubation with the chelating agent, ethyleneglycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), results in selective release of ciliated cuboidal ependymal cells which may be further purified by centrifugal methods. The isolated cells are motile, viable, and exhibit ultrastructure comparable to that in situ.
INTRODUCTION Ependymal cells line the ventricular space of the central nervous system, representing an interface separating the cerebrospinal fluid (CSF) from the neuroglial parenchyma. Ultrastructural studies of the ventricular surface have shown the presence of at least 4 basic cell types3,6,21,23. The major part of the ventricular surface proper is lined by either the ciliated cuboidal ependymal cells or the generally non-ciliated tanycytes, the relative proportion of these cell types varying in different regions of the ventricles. For example, tanycytes predominate in the ventral aspect of the third ventricle while the ciliated cuboidal cells predominate in the dorsal aspect 9,16,23-27,al. Continuous with the ependymal lining, the choroid plexus extends into the ventricular cavity and is lined with another ependymal cell type, the choroid plexus epitheliumS, s, 21,29,39. Certain very localized areas of the ventricle wall contain supraependymal cells, non-ciliated cells possessing pseudopodia-like extensions, which appear to rest upon the ventricle surface1,6,27. It seems generally accepted that the choroid plexus epithelium is involved in the secretion and perhaps to some extent reabsorption of CSF a,29. The physiological function of the other cell types comprising the ventricular lining is considerably more speculative. Tanycytes have cellular features and spatial relationships to the neuroglial parenchyma suggesting an absorptive or secretory function, and some have speculated that they function in neuroendocrine contro12,16,~8,~9,25,26,31. Supra-
408 ependymal cells phagocytize intraventricularly administered latex beads and it has been proposed that this specific cell type may be a resident phagocytic system in the brain 1. The function of the ciliated cuboidal ependymal cell is also obscure. It has been suggested that the presence of uniformly beating cilia extending into the ventricle might provide localized mixing of the CSF at the ventricle-CNS interface 4,26,29 or perhaps have a role in ciliary pinocytosis2L More recently Bleier et al. 1 have proposed that the cuboidal ependyma might serve a phagocytic function similar to that proposed for supraependymal cells and thereby constitute a first line of defense against pathogenic invasion of the brain. In support of their proposal, these authors cite studies from other laboratories showing that (a) viruses can reach the CSF from the bloodstream via the choroid plexus 15,17 and (b) certain viruses (e.g. mumps) that infiltrate the CSF restrict their infection to the ependyma and do not infect the neuroglial parenchyma35, 37. Since the cuboidal ependymal cell has been shown to possess numerous microvilli-like protrusions on its apical surface in addition to its cilia 3,26, it is also reasonable to postulate that this cell type might play a role in the secretion and/or absorption of CSF constituents. Although morphological studies of the ependyma are relatively abundant, there is virtually nothing known about the biochemical features of these cells. Biochemical studies on neural tissue, including the ependyma, are frequently complicated by the heterogeneity of cell types represented. Thus, although explants of the choroid plexus 14,2°,~8 and ventricle wall 7,H-la,32 have been maintained in culture for extended periods, these specimens inherently contain large numbers of non-ependymal cell types (e.g. neurons, glia, capillary elements, and/or connective tissues). This report describes a procedure which leads to the selective purification of ciliated cuboidal ependymal cells, with good preservation of cellular ultrastructure, ciliary mobility, and cell viability. This procedure should greatly facilitate definitive biochemical studies of this ependymal cell type. MATERIALS AND METHODS Cells were prepared for transmission electron microscopy as previously described a6 and examined using a Phillips 201 electron microscope. Scanning electron microscopy was performed by depositing the cell suspension on a 1.0 # m pore size Nucleopore filter (Nucleopore Corp.), followed by treatment with 2 ~o glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, for 10 min. After washing in buffer, dehydration with ethanol and critical point drying, the specimen was coated with gold and examined with an ISI Super Miniscan scanning electron microscope. The following procedure was routinely employed for the isolation of rat brain ependymal cells. Sixteen 175 g adult albino rats (either sex) were etherized until respiratory arrest and perfused with 0 . 9 ~ (w/v) sodium chloride for 10 min at room temperature through the left ventricle (the descending aorta was ligated and the right atrium cut). The brains were removed intact and both the anterior 5 mm of the cerebrum as well as the cerebellum were removed. The remaining forebrain, which contained most of the ventricular system, was then cut into 6-8 coronal sections, 1-2 mm in
409 thickness. The slices from all 16 brains were combined and placed in a 1 liter Erlenmeyer flask with 160 ml of sterile medium [F-12 culture solution (Grand Island Biological Company) plus 1% bovine serum albumin (BSA) and 25 m M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.2] containing 0.05% trypsin (EC 3.4.21.4) and incubated for 15 min. This and all subsequent incubations were at 37 °C with reciprocal shaking (80 cycles/min). After this preliminary incubation the supernate was gently decanted through an 80-mesh nylon screen, any particulate material trapped on the nylon filter being returned to the flask with the remaining slices. The trypsinized slices were washed twice by resuspension in 100 ml of trypsin-free medium (37 °C) followed by decanting through nylon mesh, and the filtrates discarded. The washed slices were then incubated for two consecutive 10 min periods, each time with fresh medium without trypsin; following each of these two incubations the slices were washed twice as described above. Finally, the slices were incubated for 5 min with 160 ml medium containing 2.5 m M EGTA. The ciliated ependymal cells were quickly released into the supernate before extensive numbers of non-ciliated cell types became detached. The filtered supernate was combined with that from two subsequent washes of the slices with medium containing EGTA. The combined supernates were centrifuged at 1000 x g for 2 min in conical plastic centrifuge tubes and the pellets resuspended in a total of two ml of medium containing 2.5 m M EGTA. This crude preparation, consisting mainly of ciliated cells and debris, was cooled on ice and mixed with 26 ml of ice cold 25 % BSA in medium plus 2.5 m M E G T A and centrifuged at 7000 x g for 15 min (4 °C) in a Beckman SW25.1 rotor. The supernate, which retained most of the debris, was discarded and the pelleted cells either fixed for electron microscopy or resuspended in 1 ml of cold medium. TABLE I Yield o f ciliated rat brain ependymal cells * Cell category
Crude preparation cells brain
- -
Ciliated beating cells Ciliated non-beating cells* ** Non-ciliated cells§ Total of all cells§§
155 40 88 283
x 10-3**
Final preparation cells brain
% total
- -
55 14 31 100
110 32 45 187
x 10-3**
% total
59 17 24 100
* Cell counts were taken using a standard hemacytometer and a Spencer A/O Phase microscope at450 x. ** Variation in cell yield from one preparation to another was -4- 15 %. *** Loss of ciliary motility may be reversible; Hild11has reported a temporary loss of ciliary motility in lateral ventricle explants. § It is likely that a certain number of cells in this category were indeed ciliated but that the cilia were either lost during the purification procedure or the cilia were oriented so that they were not visible through the light microscope. §§ Greater than 95 % of all the cells, includingthe non-beating ciliated cells, were viable as determined by trypan blue exclusion.
410 RESULTS AND DISCUSSION Phase microscopy showed the preparation to consist of three basic categories of cells: actively beating ciliated cells, non-beating ciliated cells, and non-ciliated cells. The relative proportion of each cell category for both the crude and purified preparation is shown in Table I. The final yield is 142,000 ciliated ependymal cells per rat brain, these being 76 ~ of the total isolated cell population. A greater yield of these ciliated cells can be obtained (up to about 300,000 per brain) by extending the time of incubation with E G T A and/or by increasing the shaking speed, but these changes also increase the relative proportion of the non-ciliated cells to about 50 % of the total yield. Indeed when the procedure is scaled up or down (i.e., using larger or fewer numbers of rats), several variables including incubation time with EGTA, flask size and shape, and shaking speed must be reoptimized to maintain a high ratio of ciliated to non-ciliated cells. As noted m Table I, more than 95 ~ of the isolated cells, both ciliated and nonciliated, were judged to be viable on the basis of their exclusion of trypan blue. Further testament to the preservation of normal function in the ciliated cuboidal ependymal cells was that (a) the metachronal beat seen in explants (we are grateful to Dr. D. E. Scott, Dept. of Anatomy, Univ. of Rochester, for providing motion pictures of ciliary
Fig. 1. Light micrographs taken of thick sections prepared prior to ultrathin sectioning for electron microscopy and stained with methylene blue. This low power field shows the preparation to consist of a population of cells with very little surrounding debris. Notice at higher magnification (inset) the presence of cilia protruding from the cells and also the general lack of debris most of which was eliminated during the BSA centrifugation step.
411
Fig. 2. Transmission electron microscopy of a preparation of ciliated cuboidal ependymal cells. A: low power scan of typical field of cells. B : isolated ependymal cell showing cilia (c) and ribosomal clusters (r). The cilia frequently appear to protrude from an indentation or trough on the apical cell surface; this particular cell has been sectioned through such an identation (cf. the scanning electron micrograph, Fig. 3B). C: cell shows perinuclear filaments (f) and basal bodies (b) terminating in a granular zone (g). D: notice the presence of microvilli-like extensions (m) and rough endoplasmic reticulum (e). m o t i o n in these explants) is also noted with the isolated cells, (b) the preservation o f ciliary motility for up to several days (the longest period examined) after isolation o f the cells attests to the structural integrity o f the ciliary apparatus a s w e l l a s the preservation o f necessary supporting biochemical processes (e.g. energy generation). It is difficult to accurately assess the proportion o f the total cuboidal ependymal cell population that is obtained by this procedure. However, dividing the total ventricular surface area, estimated using a stereotaxic atlas o f the rat brain aa, by the crosssectional area o f one ceil, estimated from phase microscopy o f the isolated ciliated ependymal cells, we calculate that there would be 400,000-700,000 ciliated ependymal cells in the brain region we utilize. This estimate is very likely too high since not all o f the ventricular surface is lined with ciliated cells 25,27. Hence, our isolation o f 140,000-300,000 cells per brain clearly suggests that this m e t h o d yields a substantial p r o p o r t i o n (perhaps ~ 5 0 ~ ) o f the total population o f ciliated ependymal cells in the brain. F r o m light micrographs, we estimate that greater than 95 ~ of the mass o f the
412
Fig. 3. Scanning electron microscopy of ciliated ependymal cell preparation. A: low power field of cells on a 1.0 ¢tm pore size Nucleopore filter. B: as is sometimes evident in phase microscopy these two ceils appear to have remained fused during the isolation procedure or to have reaggregated after separation. The orientation of the cells in such 'dimers' appears comparable to that in situ; thus, if they are the result of reaggregation, it must have occurred with preservation of normal spatial relationships. C: notice the contoured cell surface and the presence of cilia and microvilli-likeextensions which are limited to the apical surface. D : occasionally, the cell surface has a 'spongy' appearance. p r e p a r a t i o n consists o f well defined cells, the other 5 ~,~ being debris (Fig. 1). A t higher magnification (Fig. 1, inset) one can m o r e easily see the cilia p r o t r u d i n g from most o f the cell bodies. T r a n s m i s s i o n electron m i c r o s c o p y (Fig. 2) confirms the m i n o r degree o f acellular c o n t a m i n a t i o n indicated by light m i c r o s c o p y and also confirms that the p r e p a r a t i o n consists mainly o f ultrastructurally intact ciliated c u b o i d a l e p e n d y m a l cells. N o t e in particular the excellent preservation o f characteristic features which have been previously r e p o r t e d for these cells in situ3: cilia, with their basal bodies t e r m i n a t i n g in a g r a n u l a r zone; microvilli-like c y t o p l a s m i c extensions; perinuclear filaments; a n d the p r e p o n d e r a n c e o f r i b o s o m a l clusters as o p p o s e d to an extensive r o u g h e n d o p l a s m i c reticulum. Other m o r e general cellular c o m p o n e n t s such as m i t o c h o n d r i a , Golgi bodies, p l a s m a l e m m a , nuclei, lysosomes, a n d p h a g o s o m e s are also seen to retain an ultrastructure t h a t c o m p a r e s f a v o r a b l y with that seen in situ. Scanning electron microscopy again reflects the purity o f the p r e p a r a t i o n and
413 also provides a perspective not readily appreciated from light microscopy or transmission electron microscopy (Fig. 3). Immediately evident is the retention of microvilli-like extensions protruding mainly from the apical (i.e., ciliated) surface which, in vivo, would be oriented toward the CSF-filled ventricular cavity; some cells show such microvilli-like protrusions over their entire visible surface. Although the cell surfaces generally appear smooth, occasionally one sees cells having a 'spongy' appearance (e.g., Fig. 3D). Generally, the cells are highly contoured as if they have partially retained their in situ shape. Notice also the presence of a few round smooth cells (Fig. 3A), many of which lack visible cilia or microvilli-like structures; these presumably correspond to non-ciliated cells seen in phase microscopy (Table I). The origin of the non-ciliated cells remains uncertain. As noted above (Table 1), some of them may, in fact, be ciliated cells which have lost their cilia during the isolation procedure. Additionally, apparently non-ciliated cells can be seen in certain regions of the ventricles where the ependymal lining consists of more than one cell layer 1°,18,19. These might be released when the surface layer of cells is removed. It is unlikely that the non-ciliated cells originate from the choroid plexus; if our dissociation procedure is applied to choroid plexus explants, very few cells are released into the medium. Furthermore, the non-ciliated cells in our preparation do not exhibit the ultrastructure of choroid plexus epitheliaS,21,a~, 39. An alternative source of these nonciliated cells is, of course, the cut surface of the brain slice (i.e., from the neuroglial parenchyma itself or the surrounding pia). The size and ultrastructure of the nonciliated cells is not characteristic of mature intact neurons or glia; however, a pial origin cannot be excluded on an ultrastructural basis 2s,3°,~4. The physiological role of the cuboidal ependyma in the central nervous system is unknown. We have, for the first time, established a preparative technique for the purification of this cell type from brain, and have characterized this cell preparation by phase, light, transmission electron and scanning electron microscopy. This cell preparation should be useful in determining the biochemical and metabolic properties of the cuboidal ependyma in various regions of the brain and under various experimentally induced conditions. These types of studies should be helpful in elucidating the physiological function of this specific brain cell type. ACKNOWLEDGEMENTS We wish to thank the Center for Electron Optics and the College of Osteopathic Medicine at Michigan State University for permitting us the generous use of their electron microscope facilities, and gratefidly acknowledge the financial support of NIH Grant NS 09910. GPW is pleased to acknowledge the award of a Wellcome Research Travel Grant and CMM is pleased to acknowledge an NIH Postdoctoral Fellowship (1 F32 NS05336-01).
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