31
J. Anat. (1979), 129, 1, pp. 31-44 With 23 figures Printed in Great Britain
A semithin light microscopic, transmission electron microscopic and scanning electron microscopic study of macrophages in the lateral ventricle of mice from embryonic to adult life R. R. STURROCK
Department of Anatomy, University of Dundee, Dundee, Scotland
(Accepted 9 June 1978) INTRODUCTION
The population of intraventricular macrophages is largely made up of cells attached to the choroid plexus (Kolmer cells) and cells attached to the ependyma (supraependymal cells). In prenatal mice there is also a fairly large number of cells apparently lying free within the ventricles, but after birth such cells are few in number (Sturrock, 1978a). A quantitative study of intraventricular macrophages during pre- and postnatal development of the mouse brain indicated that these cells probably originated from within the choroid plexus, as had been suggested as early as 1921 by Kolmer and later by Carpenter, McCarthy & Borison (1970) and by Lewis (1974). Intraventricular macrophages in the mouse are known to make their debut adjacent to the presumptive choroid plexus 11 days after conception. The present study sets out to examine the morphology of these cells using a combination of semithin light microscopic sections, transmission electron microscopy and scanning electron microscopy. Transmission electron microscopy of intraventricular macrophages has been carried out by Tennyson & Pappas (1964), Carpenter et al. (1970) and Bleier (1977), while scanning electron microscopy of intraventricular macrophages has been the subject of reports by Allen (1975) and Bleier (1975). Intraventricular macrophages in the fetal (Chamberlain, 1974) and in the neonatal and adult rat (Walsh, Brawer & Sun Lin, 1978) have been described, but so far no systematic developmental study of these cells using both TEM and SEM has been carried out from fetal to adult life. MATERIALS AND METHODS
Mouse embryos and fetuses were removed from pregnant mothers by Caesarean section under Nembutal anaesthesia at daily intervals from 11 days post-conception to birth. Each embryo or fetus was placed in a holder (made from a razor blade cartridge; Sturrock, 1978b), the heart was exposed and cannulated with a 30 G needle, using micromanipulators. The embryo or fetus was then killed by perfusionfixation with a solution of 2 % glutaraldehyde and 2 % paraformaldehyde in a 0 05 M cacodylate buffer, each 100 ml of which contained 1 2 ml dimethylsulphoxide and 0 3 ml of 10 % CaCl2. Perfusion was carried out for 10-15 minutes, the embryos were decapitated and the heads placed in fresh fixative for 4 hours at 4 'C. The brains were then removed and left in fresh fixative at 4 'C overnight. The following morning each brain was coronally bisected with a razor blade just rostral to thte optic chiasm. 0021-8782/79/2828-6090 $02.00 C 1979 Anat. Soc. G.B. & I.
R. R. STURROCK 32 One half was prepared for transmission electron microscopy and the other half for scanning electron microscopy. Both halves were rinsed for an hour in 0-1 M cacodylate buffer, post-fixed in 1 % osmium tetroxide in a 01 M phosphate buffer and dehydrated in graded alcohols. The halves to be used for transmission electron microscopy were placed in epoxy propane for 15 minutes, then in a 50:50 solution of epoxy propane and Spurr's resin for 30 minutes and flat embedded in Spurr's resin. Semithin (0 5-1 /tm) sections were stained with toluidine blue, then the blocks were trimmed and ultrathin sections, cut on a Reichert ultramicrotome, were stained with uranyl and lead citrate and examined in an AEI 801 electron microscope. The halves to be scanned were changed from absolute alcohol into graded mixtures of absolute alcohol and Freon 113 until they were finally left in 100 % Freon 113. They were then dried in a Sorval critical point drying apparatus using Freon 13, mounted on stubs, coated with gold, and examined in a Cambridge S4 Stereoscan scanning electron microscope. Newborn mice, and mice aged 5, 8, 11, 15 and 90 days post-conception, were anaesthetised with an injection of Nembutal, and killed by perfusion fixation with the same solution of mixed aldehydes. The postnatal brains were prepared in a similar manner to the prenatal brains except that, instead of bisecting the brain, 1-2 mm coronal slides were obtained with a razor blade and each was bisected in the mid-line. One half was used for scanning and the other half was trimmed to include the lateral ventricle and surrounding brain tissue and embedded in Spurr's resin. RESULTS
Semithin light microscopy In semithin sections three main morphological varieties of intraventricular macrophages were observed. Cells of the first type were almost circular in cross section and contained a variable number of vacuoles (Figs. 1, 2, 3, 5) and occasion a Fig. 1. This shows a supraependymal macrophage from a 13 days post-conception mouse. It is roughly circular in cross section and contains a dense body and a variety of cytoplasmic vacuoles (v). Toluidine blue-stained 1 ,um section. x 2400. Fig. 2. This supraependymal macrophage from the brain of a 12 days post-conception mouse brain has a pale irregular nucleus similar to that of the macrophage shown in Fig. 1, but although still roughly circular on cross section the majority of the cytoplasmic volume is made up of large vacuoles (v). This cell may be transitional between the type shown in Fig. 1 and the type shown in Fig. 4. Toluidine blue-stained 1 ,um section. x 2400. Fig. 3. This macrophage from a 13 days post-conception mouse is lying between theependyma on the left and the choroid plexus on the right. It is similar to the macrophage shown in Fig. 1, but contains more dense bodies and a pair of fairly extensive processes (p) which are probably in the form of sheets. Toluidine blue-stained 1 ,gm section. x 2400. Fig. 4. This is an 'irregular' macrophage from a 15 days post-conception mouse. The cytoplasm is scarce in relation to the vacuolar volume, and has a ragged appearance. The nucleus is much darker and resembles that of the mature macrophage (cf. Fig. 6). Toluidine blue-stained 1 ,um section.
x
2400.
Fig. 5. The macrophage shown is of the circular vaiiety, with a fine process (p): it is from the same section as the cell in Fig. 4. Toluidine blue-stained 1 ,um section. x 2400. Fig. 6. This epiplexus cell from a 90 days post-natum mouse is of the 'flattened' variety and contains a few cytoplasmic vacuoles (small arrows) and a dense body (large arrow). Toluidine blue-stained 1 jim section. x 2400. Fig. 7. This shows a fairly typical flattened or mature epiplexus cell spread out over the surface of the choroid plexus. This type of cell has a squamoid appearance and the cytoplasm is thinnest over the nucleus (90 days post-natum). Toluidine blue-stained 1 ,um section. x 2400.
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dense bodies (Figs. 1, 3, 5). This was the form of macrophage most frequently seen at 11 and 12 days post-conception. The nucleus was fairly pale (Figs. 1-3), and usually irregular in outline. Occasional thread-like projections could be seen extending from the surface (Figs. 3, 5). Cells of the second type were also found early in development. Initially these were less common than the circular variety, but by 14 days postconception they were the most common type present. The cytoplasm was very irregular (Fig. 4) and was filled with huge spaces. With increasing age the nuclei of these cells stained more darkly, with thin irregular clumps of chromatin lining the nuclear envelope (Fig. 4). A nucleolus was frequently observed. Cells of the third type were the last to appear, and were uncommon before birth. Cells of this type appeared to be flattened out over the surface to which they were attached (Figs. 6, 7). The cytoplasm stained moderately darkly and often contained small vacuoles (Fig. 6) and dense bodies (Figs. 6, 7). Examples of all three types were found attached to either the choroid plexus or ependyma. With increasing age the simple circular type became less and less common, and in the mature mouse almost all intraventricular macrophages were of the third type, although occasional large vacuolated irregular macrophages were found. Transmission electron microscopy Intraventricular macrophages observed by TEM could be equated fairly readily with one or other of the three types observed in semithin sections. The surface of the first type of cell was relatively smooth (Fig. 8), although a few small pseudopodial projections were evident (Fig. 8). The cytoplasm contained numerous empty vacuoles (Figs. 8, 9), strands of rough endoplasmic reticulum, mitochondria, rosettes of free ribosomes, while some vacuoles contained material which appeared to be undergoing phagocytosis (Figs. 8, 9). A piece of debris in Figure 9 seems to be in the process of being taken up by macrophage pinocytosis. As early as 11 days post-natum irregular macrophages were present (Fig. 10). These cells often contained a centriole, a Golgi apparatus and numerous processes. In older animals the processes became more numerous and much narrower. The appearance of these processes (Fig. 11) makes it likely that they were sheets rather than villous processes. The cytoplasm of irregular macrophages in older animals contained more organelles, particularly rough endoplasmic reticulum, mitochondria, dense bodies and vacuoles of varying sizes (Fig. 11). The flattened macrophages were first observed at 17 days post-conception and had a moderately dense cytoplasm packed with organelles. Pseudopodia were common (Fig. 12). Debris of various kinds was often observed within the vacuoles (Fig. 13).
Scanning electron microscopy Four varieties of intraventricular macrophages were seen in the SEM in contrast to the three varieties seen in semithin sections and in the TEM. Fig. 8. 'Apparently spherical' macrophage with relatively pale cytoplasm containing numerous vacuoles (v) many of which appear empty but some of which contain debris. Two small pseudopodia can be seen. 12 days post-conception. TEM. x 12600. Fig. 9. This is a serial section of the cell shown in Fig. 8. A piece of debris can be seen apparently about to be taken up by pinocytosis (arrow). This cell contains a Golgi apparatus (G), mitochondria, strands of rough endoplasmic reticulum, rosettes of free ribosomes and vacuoles. TEM. x 20000. 3-2
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Intraventricular macrophages 37 In the youngest embryos the most common type was roughly spherical with few blebs, ruffles, or processes (Figs. 14-18). These cells appeared to be attached to the ependyma or choroid plexus by fine processes (Fig. 16). The second type was much less regular in shape with surface ruffles and sheet-like processes (Figs. 18, 19). These cells were most common from 14 days to 18 days post-conception. Cells, possibly transitional between the first and second types, were observed (Fig. 17). The cell shown in Figure 17 appears to be in the process of changing from a smooth to a ruffled cell, and seems to be attached to the choroid plexus by a leash of fine processes spreading out over the surface, although it is possible that these are choroidal microvilli. The third type was commonest at 17 days post-conception (Fig. 20), although one was found as late as 5 days post-natum (Fig. 21). The characteristic feature of these cells was the vast number of blebs almost completely covering their surface (Figs. 20, 21). The fourth type was the last to appear chronologically, and cells of this type were flattened and roughly triangular or circular in outline. Some were smooth-surfaced (Fig. 23), but many, especially in prenatal and newborn animals, had extremely irregular surfaces with numerous ruffles (Fig. 22). These ruffles would appear on section like the processes shown in Figure 11. As well as broad processes, numerous thread-like processes could be seen extending from these cells to the surface of the ependyma (Fig. 22) or choroid plexus. DISCUSSION
A number of authors (Chamberlain, 1974; Allen, 1975; Bleier, 1975) have commented on the wide variety of intraventricular macrophages observed with the scanning electron microscope. The three types identified in the young dog by Allen (1975) are basically similar to the three types identified in the present study. Such differences as have been described are probably associated with a number of factors, including the species (rat: Hosoya & Fujita, 1973; Chamberlain, 1974; mouse: Bleier, 1975; dog: Chamberlain, 1974), the ventricle examined (lateral ventricles: Chamberlain, 1974; third ventricle: Bleier, 1975; fourth ventricle: Allen, 1975), the age of the animal (Walsh et al. 1978), and the technique for preparing material for
scanning. In the present study three main morphological types were apparent in both semithin sections and in the TEM. The changes in the relative frequency of each type at successive ages, the ages at which each type appeared, and the ultrastructural differences between each type, particularly in respect of the variety and number of organelles present in the cytoplasm, support the hypothesis that the three types are really stages in the development of the same cell line. The sequence appears to be that the round, smooth-surfaced macrophages are the most primitive and that these are followed by cells of the irregular large multivacuolated variety (although some of Fig. 10. This macrophage from an 11 days post-conception mouse appears transitional between the type shown in Figs. 8 and 9 and the type shown in Fig. 11. It has a number of fairly broad processes (p). Note the centriole (c). TEM. x 12600. Fig. 11. This 17 days post-conception epiplexus cell has numerous and extensive processes. The distance for which they can be followed suggests they are sheets rather than cylinders. TEM. x 18900.
38
R. R. STURROCK
Intraventricular macrophages
39
these 'vacuoles' may be extracellular caves) and then finally the mature flattened macrophages appear. The presence of spherical intraventricular macrophages in the third ventricle of the neonatal rat and the absence of similar cells in adult rats, has recently been reported by Walsh et al. (1978). It is possible that rather than being simply a developmental stage the irregular macrophage is either an amoeboid macrophage, as these are most common at the stage when intraventricular macrophages appear to be migrating most rapidly from choroid plexus to ependyma (Sturrock, 1978 a), or that irregular macrophages are actively phagocytic, which is a reasonable possibility during a period of rapid brain growth. The three types observed in semithin sections and in the TEM compare fairly well with three of the four types observed in the SEM. The one SEM type which is difficult to fit into the TEM categories is the multi-blebbed cell. This may be a mitotic cell. Seymour & Berry (1975) have demonstrated that surface blebbing is a characteristic feature during the later stages of mitosis in the neural tube. Price (1967) described such blebbing in cultured human epithelial cells, and a similar foamy cytoplasm has been found in mitotic cells in the postnatal nervous system (Sturrock, 1974). The epiplexus cells in Figures 21 and 22 certainly could be interpreted as telophase cells about to undergo separation. Mitotic intraventricular cells have been demonstrated previously (Sturrock, 1978 a) and were most common at 17 days post-conception. One other possibility is that these were cells in the process of entering the ventricles via the choroid plexus or else leaving the ventricles via the ependymal wall. Cells in the process of doing this have a cytoplasm which appears 'foamy' in semithin sections (Sturrock, 1978 a). Multi-blebbed cells were demonstrated by Fig. 12. A 17 days post-conception epiplexus cell with darkly staining cytoplasm containing numerous organelles but relatively few vacuoles. Note the pseudopodia (arrows). TEM. x 8000. Fig. 13. This epiplexus cell from an 18 days post-conception mouse is of the flattened variety but contains more vacuoles than the macrophage in Fig. 12. These vacuoles contain a variety of inclusions. Pseudopodia appear to be absent. TEM. x 12600. Fig. 14. This shows two round relatively smooth epiplexus cells lying on the developing choroid plexus of a 12 days post-conception mouse lateral ventricle. These cells have relatively few processes. SEM. x 2250.
Fig. 15. This shows a relatively smooth epiplexus cell from a 13 days post-conception mouse. A few ruffles and blebs (arrows) can be seen but this cell is fairly similar to those shown in Fig. 14. SEM. x 5000. Fig. 16. Two 'circular' fairly smooth epiplexus cells from a 14 days post-conception mouse. Note the attachment of these cells to the choroid plexus by fine processes (arrows). SEM. x 2250. Fig. 17. This macrophage seems to be transitional between the type shown in Figs. 14-16 and the type seen in Figs. 18 and 19. It appears to be tethered to the choroid plexus by fine processes with a leash of these at the lower pole (arrow). 17 days post-conception. SEM. x 2250. Fig. 18. This irregular macrophage is attached to the surface of the 17 days post-conception choroid plexus. The surface is covered with ruffles. SEM. x 5600.
Fig. 19. 'Irregular' macrophage from a 15 days post-conception mouse. Note the fairly complex sheet-like folds forming extracytoplasmic 'caves' (arrows). SEM. x 6100. Fig. 20. 17 days post-conception. This multi-blebbed macrophage was found in the choroid plexus. It may be a mitotic cell in early telophase. SEM. x 5000. Fig. 21. 5 days post-natum. Multi-blebbed macrophage lying on the ependymal surface. SEM. x 2500.
R. R. STURROCK
40
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Figs. 15-18. For legends see p. 39.
Intraventricular macrophages
Figs. 19-21. For legends see p. 39.
41
42
R. R. STURROCK
Fig. 22. Newborn mouse. This supraependymal macrophage is fairly flattened but is still covered with numerous ruffles. Note the fine processes (arrows) extending from the cytoplasm. This cell is possibly transitional between the type shown in Fig. 15 and that shown in Fig. 23. SEM. x 6100. Fig. 23. Newborn mouse. This shows two flattened epiplexus cells. The surface of these cells is fairly smooth. A common feature of this type of cell is the number of broad processes (arrows) extending all around it. SEM. x 2250.
Intraventricular macrophages
43
Chamberlain (1974) within the ventricles of fetal rats whose mothers had received an injection of 6-aminonicotinamide at 13 days post-conception. These cells were not identical with those shown in Figures 20 and 21, but more closely resembled that shown in Figure 18. One of the Figures in Chamberlain's (1974) paper illustrates a multi-blebbed macrophage apparently in the process of ingesting a red blood cell, but the cells which he described have probably undergone morphological changes as a result of the pathological processes resulting from the injection of an anti-vitamin. On balance it seems likely that the multi-blebbed cells in the present study are mitotic. If the developmental sequence proposed above is correct the postnatal population appears to be relatively quiescent as far as cell turnover is concerned, since almost all the intraventricular macrophages are of the flattened variety. Lewis (1974) suggested that a turnover of intraventricular macrophages takes place in adult rats in conjunction with the production of microglia. In mature animals it is possible that less mature macrophages arising elsewhere undergo a fair degree of differentiation before entering the ventricles. In support of this suggestion, it may be noted that the macrophage observed by Carpenter et al. (1970) migrating through the wall of a choroid plexus capillary in a young cat appears to be a relatively well differentiated cell. SUMMARY
A developmental study of intraventricular macrophages was carried out from 11 days post-conception up to 90 days post-natum, using semithin sections, transmission electron microscopy and scanning electron microscopy. In semithin sections and in the TEM three main morphological types (and some transitional forms) were identified and are described as circular, irregular and flattened, respectively. In the SEM three corresponding types were identified, a relatively smooth spherical type, a highly ruffled type and a fairly smooth flattened type. A fourth, multi-blebbed type, was occasionally seen in late prenatal and early postnatal mice. It seemed most likely that these cells were mitotic. The likely sequence of development is that the spherical or circular type is the most primitive, although capable of phagocytosis; and that this differentiates into the ruffled or irregular variety, which in turn differentiates into the flattened type, which is by far the most common type found after birth. REFERENCES
ALLEN, D. J. (1975). Scanning electron microscopy of epiplexus macrophages (Kolmer cells) in the dog. Journal of Comparative Neurology 161, 197-214. BLEIER, R. (1975). Surface fine structure of supraependymal elements and ependyma of hypothalamic third ventricle of mouse. Journal of Comparative Neurology 161, 555-568. BLEIER, R. (1977). Ultrastructure of supraependymal cells and ependyma of hypothalamic third ventricle of mouse. Journal of Comparative Neurology 174, 359-376. CARPENTER, S. J., MCCARTHY, L. E. & BORISON, H. L. (1970). Electron microscopic study of the epiplexus (Kolmer) cells of the cat choroid plexus. Zeitschrift fur Zellforschung und mikroskopische Anatomie 110, 471-486. CHAMBERLAIN, J. B. (1974). Scanning electron microscopy of epiplexus cells (macrophages) in the fetal rat brain. American Journal of Anatomy 139, 443-447. HOSOYA, Y. & FUJITA, T. (1973). Scanning electron microscope observations of intraventricular macrophages (Kolmer cells) in the rat brain. Archivum histologicum japonicum 35, 133-140. KOLMER, W. (1921). Uber eine eigenartige Beziehung von Wanderzellen zu den Choroidealplexus des Gehirns der Wirbeltiere. Anatomischer Anzeiger 54, 15-19. LEWIS, P. D. (1974). Glial reactions to cerebral injury. Proceedings of the Royal Society of Medicine 67, 130-131.
44 R. R. STURROCK PRICE, Z. H. (1967). The micromorphology of zeiotic blebs in cultured human epithelial (HEp) cells. Experimental Cell Research 48, 82-92. SEYMOUR, R. M. & BERRY, M. (1975). Scanning and transmission electron microscope studies of interkinetic nuclcar migration in the cerebral vesicles of the rat. Journal of Comparative Neurology 160, 105-126. STURROCK, R. R. (1974). Histogenesis of the anteror limb of the anterior commissure of the mouse brain. II. A quantitative study of pre- and postnatal mitosis. Journal of Anatomy 117, 27-35. STURROCK, R. R. (1978a). A developmental study of epiplexus cells and supraependymal cells and their possible relationship to microglia. Neuropathology and Applied Neurobiology 4, 307-322. STURROCK, R. R. (1978b). A simple holder for small embryos during perfusion fixation. Journal of Anatomy 126, 441. TENNYSON, V. M. & PAPPAS, G. D. (1964). Fine structure of the developing telencephalic and myelencephalic choroid plexus in the rabbit. Journal of Comparative Neurology 123, 379-412. WALSH, R. M., BRAWER, J. R. & SUN LIN, P. (1978). Supraependymal cells in the third ventricle of neonatal rat. Anatomical Record 190, 257-270.
J. Anat. (1979), 129, 1, pp. 31-44 With 23 figures Printed in Great Britain
A semithin light microscopic, transmission electron microscopic and scanning electron microscopic study of macrophages in the lateral ventricle of mice from embryonic to adult life R. R. STURROCK
Department of Anatomy, University of Dundee, Dundee, Scotland
(Accepted 9 June 1978) INTRODUCTION
The population of intraventricular macrophages is largely made up of cells attached to the choroid plexus (Kolmer cells) and cells attached to the ependyma (supraependymal cells). In prenatal mice there is also a fairly large number of cells apparently lying free within the ventricles, but after birth such cells are few in number (Sturrock, 1978a). A quantitative study of intraventricular macrophages during pre- and postnatal development of the mouse brain indicated that these cells probably originated from within the choroid plexus, as had been suggested as early as 1921 by Kolmer and later by Carpenter, McCarthy & Borison (1970) and by Lewis (1974). Intraventricular macrophages in the mouse are known to make their debut adjacent to the presumptive choroid plexus 11 days after conception. The present study sets out to examine the morphology of these cells using a combination of semithin light microscopic sections, transmission electron microscopy and scanning electron microscopy. Transmission electron microscopy of intraventricular macrophages has been carried out by Tennyson & Pappas (1964), Carpenter et al. (1970) and Bleier (1977), while scanning electron microscopy of intraventricular macrophages has been the subject of reports by Allen (1975) and Bleier (1975). Intraventricular macrophages in the fetal (Chamberlain, 1974) and in the neonatal and adult rat (Walsh, Brawer & Sun Lin, 1978) have been described, but so far no systematic developmental study of these cells using both TEM and SEM has been carried out from fetal to adult life. MATERIALS AND METHODS
Mouse embryos and fetuses were removed from pregnant mothers by Caesarean section under Nembutal anaesthesia at daily intervals from 11 days post-conception to birth. Each embryo or fetus was placed in a holder (made from a razor blade cartridge; Sturrock, 1978b), the heart was exposed and cannulated with a 30 G needle, using micromanipulators. The embryo or fetus was then killed by perfusionfixation with a solution of 2 % glutaraldehyde and 2 % paraformaldehyde in a 0 05 M cacodylate buffer, each 100 ml of which contained 1 2 ml dimethylsulphoxide and 0 3 ml of 10 % CaCl2. Perfusion was carried out for 10-15 minutes, the embryos were decapitated and the heads placed in fresh fixative for 4 hours at 4 'C. The brains were then removed and left in fresh fixative at 4 'C overnight. The following morning each brain was coronally bisected with a razor blade just rostral to thte optic chiasm. 0021-8782/79/2828-6090 $02.00 C 1979 Anat. Soc. G.B. & I.
R. R. STURROCK 32 One half was prepared for transmission electron microscopy and the other half for scanning electron microscopy. Both halves were rinsed for an hour in 0-1 M cacodylate buffer, post-fixed in 1 % osmium tetroxide in a 01 M phosphate buffer and dehydrated in graded alcohols. The halves to be used for transmission electron microscopy were placed in epoxy propane for 15 minutes, then in a 50:50 solution of epoxy propane and Spurr's resin for 30 minutes and flat embedded in Spurr's resin. Semithin (0 5-1 /tm) sections were stained with toluidine blue, then the blocks were trimmed and ultrathin sections, cut on a Reichert ultramicrotome, were stained with uranyl and lead citrate and examined in an AEI 801 electron microscope. The halves to be scanned were changed from absolute alcohol into graded mixtures of absolute alcohol and Freon 113 until they were finally left in 100 % Freon 113. They were then dried in a Sorval critical point drying apparatus using Freon 13, mounted on stubs, coated with gold, and examined in a Cambridge S4 Stereoscan scanning electron microscope. Newborn mice, and mice aged 5, 8, 11, 15 and 90 days post-conception, were anaesthetised with an injection of Nembutal, and killed by perfusion fixation with the same solution of mixed aldehydes. The postnatal brains were prepared in a similar manner to the prenatal brains except that, instead of bisecting the brain, 1-2 mm coronal slides were obtained with a razor blade and each was bisected in the mid-line. One half was used for scanning and the other half was trimmed to include the lateral ventricle and surrounding brain tissue and embedded in Spurr's resin. RESULTS
Semithin light microscopy In semithin sections three main morphological varieties of intraventricular macrophages were observed. Cells of the first type were almost circular in cross section and contained a variable number of vacuoles (Figs. 1, 2, 3, 5) and occasion a Fig. 1. This shows a supraependymal macrophage from a 13 days post-conception mouse. It is roughly circular in cross section and contains a dense body and a variety of cytoplasmic vacuoles (v). Toluidine blue-stained 1 ,um section. x 2400. Fig. 2. This supraependymal macrophage from the brain of a 12 days post-conception mouse brain has a pale irregular nucleus similar to that of the macrophage shown in Fig. 1, but although still roughly circular on cross section the majority of the cytoplasmic volume is made up of large vacuoles (v). This cell may be transitional between the type shown in Fig. 1 and the type shown in Fig. 4. Toluidine blue-stained 1 ,um section. x 2400. Fig. 3. This macrophage from a 13 days post-conception mouse is lying between theependyma on the left and the choroid plexus on the right. It is similar to the macrophage shown in Fig. 1, but contains more dense bodies and a pair of fairly extensive processes (p) which are probably in the form of sheets. Toluidine blue-stained 1 ,gm section. x 2400. Fig. 4. This is an 'irregular' macrophage from a 15 days post-conception mouse. The cytoplasm is scarce in relation to the vacuolar volume, and has a ragged appearance. The nucleus is much darker and resembles that of the mature macrophage (cf. Fig. 6). Toluidine blue-stained 1 ,um section.
x
2400.
Fig. 5. The macrophage shown is of the circular vaiiety, with a fine process (p): it is from the same section as the cell in Fig. 4. Toluidine blue-stained 1 ,um section. x 2400. Fig. 6. This epiplexus cell from a 90 days post-natum mouse is of the 'flattened' variety and contains a few cytoplasmic vacuoles (small arrows) and a dense body (large arrow). Toluidine blue-stained 1 jim section. x 2400. Fig. 7. This shows a fairly typical flattened or mature epiplexus cell spread out over the surface of the choroid plexus. This type of cell has a squamoid appearance and the cytoplasm is thinnest over the nucleus (90 days post-natum). Toluidine blue-stained 1 ,um section. x 2400.
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35
dense bodies (Figs. 1, 3, 5). This was the form of macrophage most frequently seen at 11 and 12 days post-conception. The nucleus was fairly pale (Figs. 1-3), and usually irregular in outline. Occasional thread-like projections could be seen extending from the surface (Figs. 3, 5). Cells of the second type were also found early in development. Initially these were less common than the circular variety, but by 14 days postconception they were the most common type present. The cytoplasm was very irregular (Fig. 4) and was filled with huge spaces. With increasing age the nuclei of these cells stained more darkly, with thin irregular clumps of chromatin lining the nuclear envelope (Fig. 4). A nucleolus was frequently observed. Cells of the third type were the last to appear, and were uncommon before birth. Cells of this type appeared to be flattened out over the surface to which they were attached (Figs. 6, 7). The cytoplasm stained moderately darkly and often contained small vacuoles (Fig. 6) and dense bodies (Figs. 6, 7). Examples of all three types were found attached to either the choroid plexus or ependyma. With increasing age the simple circular type became less and less common, and in the mature mouse almost all intraventricular macrophages were of the third type, although occasional large vacuolated irregular macrophages were found. Transmission electron microscopy Intraventricular macrophages observed by TEM could be equated fairly readily with one or other of the three types observed in semithin sections. The surface of the first type of cell was relatively smooth (Fig. 8), although a few small pseudopodial projections were evident (Fig. 8). The cytoplasm contained numerous empty vacuoles (Figs. 8, 9), strands of rough endoplasmic reticulum, mitochondria, rosettes of free ribosomes, while some vacuoles contained material which appeared to be undergoing phagocytosis (Figs. 8, 9). A piece of debris in Figure 9 seems to be in the process of being taken up by macrophage pinocytosis. As early as 11 days post-natum irregular macrophages were present (Fig. 10). These cells often contained a centriole, a Golgi apparatus and numerous processes. In older animals the processes became more numerous and much narrower. The appearance of these processes (Fig. 11) makes it likely that they were sheets rather than villous processes. The cytoplasm of irregular macrophages in older animals contained more organelles, particularly rough endoplasmic reticulum, mitochondria, dense bodies and vacuoles of varying sizes (Fig. 11). The flattened macrophages were first observed at 17 days post-conception and had a moderately dense cytoplasm packed with organelles. Pseudopodia were common (Fig. 12). Debris of various kinds was often observed within the vacuoles (Fig. 13).
Scanning electron microscopy Four varieties of intraventricular macrophages were seen in the SEM in contrast to the three varieties seen in semithin sections and in the TEM. Fig. 8. 'Apparently spherical' macrophage with relatively pale cytoplasm containing numerous vacuoles (v) many of which appear empty but some of which contain debris. Two small pseudopodia can be seen. 12 days post-conception. TEM. x 12600. Fig. 9. This is a serial section of the cell shown in Fig. 8. A piece of debris can be seen apparently about to be taken up by pinocytosis (arrow). This cell contains a Golgi apparatus (G), mitochondria, strands of rough endoplasmic reticulum, rosettes of free ribosomes and vacuoles. TEM. x 20000. 3-2
R. R. STURROCK
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Intraventricular macrophages 37 In the youngest embryos the most common type was roughly spherical with few blebs, ruffles, or processes (Figs. 14-18). These cells appeared to be attached to the ependyma or choroid plexus by fine processes (Fig. 16). The second type was much less regular in shape with surface ruffles and sheet-like processes (Figs. 18, 19). These cells were most common from 14 days to 18 days post-conception. Cells, possibly transitional between the first and second types, were observed (Fig. 17). The cell shown in Figure 17 appears to be in the process of changing from a smooth to a ruffled cell, and seems to be attached to the choroid plexus by a leash of fine processes spreading out over the surface, although it is possible that these are choroidal microvilli. The third type was commonest at 17 days post-conception (Fig. 20), although one was found as late as 5 days post-natum (Fig. 21). The characteristic feature of these cells was the vast number of blebs almost completely covering their surface (Figs. 20, 21). The fourth type was the last to appear chronologically, and cells of this type were flattened and roughly triangular or circular in outline. Some were smooth-surfaced (Fig. 23), but many, especially in prenatal and newborn animals, had extremely irregular surfaces with numerous ruffles (Fig. 22). These ruffles would appear on section like the processes shown in Figure 11. As well as broad processes, numerous thread-like processes could be seen extending from these cells to the surface of the ependyma (Fig. 22) or choroid plexus. DISCUSSION
A number of authors (Chamberlain, 1974; Allen, 1975; Bleier, 1975) have commented on the wide variety of intraventricular macrophages observed with the scanning electron microscope. The three types identified in the young dog by Allen (1975) are basically similar to the three types identified in the present study. Such differences as have been described are probably associated with a number of factors, including the species (rat: Hosoya & Fujita, 1973; Chamberlain, 1974; mouse: Bleier, 1975; dog: Chamberlain, 1974), the ventricle examined (lateral ventricles: Chamberlain, 1974; third ventricle: Bleier, 1975; fourth ventricle: Allen, 1975), the age of the animal (Walsh et al. 1978), and the technique for preparing material for
scanning. In the present study three main morphological types were apparent in both semithin sections and in the TEM. The changes in the relative frequency of each type at successive ages, the ages at which each type appeared, and the ultrastructural differences between each type, particularly in respect of the variety and number of organelles present in the cytoplasm, support the hypothesis that the three types are really stages in the development of the same cell line. The sequence appears to be that the round, smooth-surfaced macrophages are the most primitive and that these are followed by cells of the irregular large multivacuolated variety (although some of Fig. 10. This macrophage from an 11 days post-conception mouse appears transitional between the type shown in Figs. 8 and 9 and the type shown in Fig. 11. It has a number of fairly broad processes (p). Note the centriole (c). TEM. x 12600. Fig. 11. This 17 days post-conception epiplexus cell has numerous and extensive processes. The distance for which they can be followed suggests they are sheets rather than cylinders. TEM. x 18900.
38
R. R. STURROCK
Intraventricular macrophages
39
these 'vacuoles' may be extracellular caves) and then finally the mature flattened macrophages appear. The presence of spherical intraventricular macrophages in the third ventricle of the neonatal rat and the absence of similar cells in adult rats, has recently been reported by Walsh et al. (1978). It is possible that rather than being simply a developmental stage the irregular macrophage is either an amoeboid macrophage, as these are most common at the stage when intraventricular macrophages appear to be migrating most rapidly from choroid plexus to ependyma (Sturrock, 1978 a), or that irregular macrophages are actively phagocytic, which is a reasonable possibility during a period of rapid brain growth. The three types observed in semithin sections and in the TEM compare fairly well with three of the four types observed in the SEM. The one SEM type which is difficult to fit into the TEM categories is the multi-blebbed cell. This may be a mitotic cell. Seymour & Berry (1975) have demonstrated that surface blebbing is a characteristic feature during the later stages of mitosis in the neural tube. Price (1967) described such blebbing in cultured human epithelial cells, and a similar foamy cytoplasm has been found in mitotic cells in the postnatal nervous system (Sturrock, 1974). The epiplexus cells in Figures 21 and 22 certainly could be interpreted as telophase cells about to undergo separation. Mitotic intraventricular cells have been demonstrated previously (Sturrock, 1978 a) and were most common at 17 days post-conception. One other possibility is that these were cells in the process of entering the ventricles via the choroid plexus or else leaving the ventricles via the ependymal wall. Cells in the process of doing this have a cytoplasm which appears 'foamy' in semithin sections (Sturrock, 1978 a). Multi-blebbed cells were demonstrated by Fig. 12. A 17 days post-conception epiplexus cell with darkly staining cytoplasm containing numerous organelles but relatively few vacuoles. Note the pseudopodia (arrows). TEM. x 8000. Fig. 13. This epiplexus cell from an 18 days post-conception mouse is of the flattened variety but contains more vacuoles than the macrophage in Fig. 12. These vacuoles contain a variety of inclusions. Pseudopodia appear to be absent. TEM. x 12600. Fig. 14. This shows two round relatively smooth epiplexus cells lying on the developing choroid plexus of a 12 days post-conception mouse lateral ventricle. These cells have relatively few processes. SEM. x 2250.
Fig. 15. This shows a relatively smooth epiplexus cell from a 13 days post-conception mouse. A few ruffles and blebs (arrows) can be seen but this cell is fairly similar to those shown in Fig. 14. SEM. x 5000. Fig. 16. Two 'circular' fairly smooth epiplexus cells from a 14 days post-conception mouse. Note the attachment of these cells to the choroid plexus by fine processes (arrows). SEM. x 2250. Fig. 17. This macrophage seems to be transitional between the type shown in Figs. 14-16 and the type seen in Figs. 18 and 19. It appears to be tethered to the choroid plexus by fine processes with a leash of these at the lower pole (arrow). 17 days post-conception. SEM. x 2250. Fig. 18. This irregular macrophage is attached to the surface of the 17 days post-conception choroid plexus. The surface is covered with ruffles. SEM. x 5600.
Fig. 19. 'Irregular' macrophage from a 15 days post-conception mouse. Note the fairly complex sheet-like folds forming extracytoplasmic 'caves' (arrows). SEM. x 6100. Fig. 20. 17 days post-conception. This multi-blebbed macrophage was found in the choroid plexus. It may be a mitotic cell in early telophase. SEM. x 5000. Fig. 21. 5 days post-natum. Multi-blebbed macrophage lying on the ependymal surface. SEM. x 2500.
R. R. STURROCK
40
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Figs. 15-18. For legends see p. 39.
Intraventricular macrophages
Figs. 19-21. For legends see p. 39.
41
42
R. R. STURROCK
Fig. 22. Newborn mouse. This supraependymal macrophage is fairly flattened but is still covered with numerous ruffles. Note the fine processes (arrows) extending from the cytoplasm. This cell is possibly transitional between the type shown in Fig. 15 and that shown in Fig. 23. SEM. x 6100. Fig. 23. Newborn mouse. This shows two flattened epiplexus cells. The surface of these cells is fairly smooth. A common feature of this type of cell is the number of broad processes (arrows) extending all around it. SEM. x 2250.
Intraventricular macrophages
43
Chamberlain (1974) within the ventricles of fetal rats whose mothers had received an injection of 6-aminonicotinamide at 13 days post-conception. These cells were not identical with those shown in Figures 20 and 21, but more closely resembled that shown in Figure 18. One of the Figures in Chamberlain's (1974) paper illustrates a multi-blebbed macrophage apparently in the process of ingesting a red blood cell, but the cells which he described have probably undergone morphological changes as a result of the pathological processes resulting from the injection of an anti-vitamin. On balance it seems likely that the multi-blebbed cells in the present study are mitotic. If the developmental sequence proposed above is correct the postnatal population appears to be relatively quiescent as far as cell turnover is concerned, since almost all the intraventricular macrophages are of the flattened variety. Lewis (1974) suggested that a turnover of intraventricular macrophages takes place in adult rats in conjunction with the production of microglia. In mature animals it is possible that less mature macrophages arising elsewhere undergo a fair degree of differentiation before entering the ventricles. In support of this suggestion, it may be noted that the macrophage observed by Carpenter et al. (1970) migrating through the wall of a choroid plexus capillary in a young cat appears to be a relatively well differentiated cell. SUMMARY
A developmental study of intraventricular macrophages was carried out from 11 days post-conception up to 90 days post-natum, using semithin sections, transmission electron microscopy and scanning electron microscopy. In semithin sections and in the TEM three main morphological types (and some transitional forms) were identified and are described as circular, irregular and flattened, respectively. In the SEM three corresponding types were identified, a relatively smooth spherical type, a highly ruffled type and a fairly smooth flattened type. A fourth, multi-blebbed type, was occasionally seen in late prenatal and early postnatal mice. It seemed most likely that these cells were mitotic. The likely sequence of development is that the spherical or circular type is the most primitive, although capable of phagocytosis; and that this differentiates into the ruffled or irregular variety, which in turn differentiates into the flattened type, which is by far the most common type found after birth. REFERENCES
ALLEN, D. J. (1975). Scanning electron microscopy of epiplexus macrophages (Kolmer cells) in the dog. Journal of Comparative Neurology 161, 197-214. BLEIER, R. (1975). Surface fine structure of supraependymal elements and ependyma of hypothalamic third ventricle of mouse. Journal of Comparative Neurology 161, 555-568. BLEIER, R. (1977). Ultrastructure of supraependymal cells and ependyma of hypothalamic third ventricle of mouse. Journal of Comparative Neurology 174, 359-376. CARPENTER, S. J., MCCARTHY, L. E. & BORISON, H. L. (1970). Electron microscopic study of the epiplexus (Kolmer) cells of the cat choroid plexus. Zeitschrift fur Zellforschung und mikroskopische Anatomie 110, 471-486. CHAMBERLAIN, J. B. (1974). Scanning electron microscopy of epiplexus cells (macrophages) in the fetal rat brain. American Journal of Anatomy 139, 443-447. HOSOYA, Y. & FUJITA, T. (1973). Scanning electron microscope observations of intraventricular macrophages (Kolmer cells) in the rat brain. Archivum histologicum japonicum 35, 133-140. KOLMER, W. (1921). Uber eine eigenartige Beziehung von Wanderzellen zu den Choroidealplexus des Gehirns der Wirbeltiere. Anatomischer Anzeiger 54, 15-19. LEWIS, P. D. (1974). Glial reactions to cerebral injury. Proceedings of the Royal Society of Medicine 67, 130-131.
44 R. R. STURROCK PRICE, Z. H. (1967). The micromorphology of zeiotic blebs in cultured human epithelial (HEp) cells. Experimental Cell Research 48, 82-92. SEYMOUR, R. M. & BERRY, M. (1975). Scanning and transmission electron microscope studies of interkinetic nuclcar migration in the cerebral vesicles of the rat. Journal of Comparative Neurology 160, 105-126. STURROCK, R. R. (1974). Histogenesis of the anteror limb of the anterior commissure of the mouse brain. II. A quantitative study of pre- and postnatal mitosis. Journal of Anatomy 117, 27-35. STURROCK, R. R. (1978a). A developmental study of epiplexus cells and supraependymal cells and their possible relationship to microglia. Neuropathology and Applied Neurobiology 4, 307-322. STURROCK, R. R. (1978b). A simple holder for small embryos during perfusion fixation. Journal of Anatomy 126, 441. TENNYSON, V. M. & PAPPAS, G. D. (1964). Fine structure of the developing telencephalic and myelencephalic choroid plexus in the rabbit. Journal of Comparative Neurology 123, 379-412. WALSH, R. M., BRAWER, J. R. & SUN LIN, P. (1978). Supraependymal cells in the third ventricle of neonatal rat. Anatomical Record 190, 257-270.
