Scanning electron microscopy of the ventricular system in normal and hydrocephalic rabbits Preliminary report and atlas ROBERT B. PagE, M . D .
M.S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania v' The author used the scanning electron microscope to study the ependyma in six control rabbits and six rabbits made hydrocephalic by infusion of silicone oil into the cisterna magna. The ependymal lining of the third ventricle, head of the caudate nucleus, superior angle of the caudate, and atrium of the lateral ventricle was examined. In the hydrocephalic animals, clusters of cilia emanating from the ependyma over periventricular white matter become separated; the author believes this is secondary to ingrowth of new ependymal cell processes covered with microvilli. The addition of these cells to the ependymal surface permits ventricular dilatation without ependymal disruption and provides more surface containing microvilli, presumably capable of increased transventricular fluid transfer. No such changes occur over gray matter masses since their surfaces are not deformed by moderate ventricular dilatation. The morphological alterations in the ependyma that occur in moderate hydrocephalus do not appear to be simply manifestations of ependymal destruction but rather suggest a modification in its function from that of a surface capable of propelling cerebrospinal fluid to one capable of increased transfer of transventricular fluid. As hydrocephalus progresses, compensation may fail because of the relative decrease in microvilli so that the cell surface provides a less efficient mechanism for absorption. Krv WORDS 9 hydrocephalus 9 ependymal cilia and microvUli scanning electron microscopy 9 cerebral ventricles
E
NLARGEMENT of the ventricular system is the sine qua non of hydrocephalus. Several studies of the ependyma and subependymal white matter have been reported in n o r m a l and hydrocephalic animals utilizing light microscopy (LM) and transmission electron 646
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m i c r o s c o p y ( T E M ) . 6'7'9'1''24'25'31's~ These methods have demonstrated expansion of the extracellular space of the subependymal white matter in hydrocephalus, presumably the result of increased transependymal fluid transport? 6,2~ In the event of obstruction to cerebrospinal fluid (CSF) circulation, such a
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Scanning electron microscopy in hydrocephalus mechanism would provide an alternative means of egress of CSF from the ventricular system. However, previous limitations in the available techniques have allowed little knowledge of the fate of ependymal cells in hydrocephalus. Light microscopy has only permitted evaluation of relatively gross alterations in far advanced hydrocephalus,14,25,3x while TEM has been limited to study of ultrathin sections made from minute samples of the ventricular surface. Scanning electron microscopy (SEM) permits examination of a relatively large surface of the ventricle at low magnification. Ventricular topography may be assessed and areas of interest selected for more detailed study. At higher magnification the cellular surface, cilia, and microvilli may be examined. Scanning electron microscopic studies of the ventricular surface have been reported in several mammalian species 4'15,2~ but to date there have been no comparable published studies of hydrocephalic animals. The following SEM study was undertaken to characterize the ventricular system of an experimental animal under normal conditions and under conditions of moderate hydrocephalus, and to assess the following questions: Do structural alterations occur at the surface of the ependymal cell? Are these changes compatible with the increase in transventricular fluid transport postulated to occur in hydrocephalus, or are they simply manifestations of destruction of ependymal cells? Materials and Methods
Twelve adult Dutch Belted rabbits of both sexes were used; six were made hydrocephalic by infusion of silicone into the cisterna magna. 33 With the animals under halothane anesthesia, the foramen magnum was exposed and a No. 60 polyethylene catheter was placed in the cisterna magna. A mixture of equal parts of Dow Corning 200 and 360 medical fluids was infused into the cisterna magna with a Harvard pump at a rate of 0.0136 cc/min to a total of 0.6 cc.* The *Dow Corning 200 and 360 Medical fluids made by Dow Corning Corporation, Medical Products Division, Midland, Michigan 48640. Harvard pump made by Harvard Apparatus, Inc., Millis, Massachusetts.
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Fic. 1. Comparison of ventricular size in control (left) and hydrocephalic (right) rabbits. viscosity of No. 200 fluid is 100,000 centistokes (cs); viscosity of No. 360 fluid is 12,500 cs. Following the infusion the wound was closed in lay(-,: and the animals were returned to their cages. Three animals were sacrificed at 4 weeks after infusion and three at 16 weeks. The control series consisted of two animals that underwent infusion of saline, rather than silicone, into the cisterna magna. In two additional animals the cisterna magna was exposed under halothane anesthesia. The dura was not opened and the animals were maintained under anesthesia for 1 hour prior to closure of the wound. These four animals were sacrificed 16 weeks after surgery. Finally, two unoperated animals were also examined. Specimens were prepared in the following manner: After nembutal anesthesia, intracardiac perfusion with Karno~,sky's solutionla was performed. The brain was immediately removed from the skull and postfixed by immersion in Karno~sky's solution overnight at 4~ Coronal sections were then taken at the level of the anterior commissure to document ventricular size (Fig. l). The brain was dissected under fixative utilizing a Zeiss OPMI 6 operating microscope.t Three samples of ependyma were obtained from each animal: 1) a 0.5 • 0.5-cm segment consisting of the caudate head and adjacent corpus callosum; 2) a triangular segment (0.5 cm base, 0.5 cm height) of the roof of the lateral ventricle at the junction of the body and the temporal horn; and 3) a 1 X 1-cm block of the epentZeiss OPMI 6 operating microscope made by Carl Zeiss, Inc., 444 Fifth Avenue, New York, New York 10018. 647
R . B. P a g e dyma lining the third ventricle limited anteriorly by the lamina terminalis, superiorly by the midportion of the massa intermedia, and posteriorly by a line drawn from the superior colliculus to the mammillary bodies. Samples were also taken at corresponding sites from the opposite caudate nucleus, atrium of lateral ventricle, and third ventricle, and prepared for TEM. For orientation, the TEM observations in control animals are briefly noted in this report. Full descriptions of TEM changes will be the subject of a separate report. After dissection, the specimens were washed with cold 0.1 M cacodylate buffer for 24 hours. Postfixation with collidine-buffered 1.33% osmium tetroxide for 2 hours and subsequent dehydration through serial alcohols and freon followed. The specimens were dried in a critical point dryer, mounted on stubs, coated with gold palladium, and were examined in an AMR scanning electron microscope.* Results
The transition zone lies at the level of the optic chiasm and mammillary bodies (Fig. 4); cilia are present here but clusters become progressively sparse as the infundibulum is approached (Fig. 6). Microvilli are present in abundance on the cell surface between widely spread clusters of cilia (Fig. 3). The ependyma of the infundibulum is not ciliated (Fig. 7 upper). Here, the outlines of cells create the impression of a "cobblestone street." The cell surfaces are covered with microvilli and small, round excrescences (apical blebs) which appear to be budding from the surface of the cells. Hydrocephalic Animals (6). Despite enlargement of the third ventricle the surface morphology of the ependyma is not significantly altered in the six specimens studied. The surface anatomy of the infundibulum is unchanged; both apical blebs and microvilli are still apparent (Fig. 7). The morphology and distribution of cilia and microvilli in the transition zone (Fig. 8) and in the dorsal third ventricle are also unchanged (Fig. 9).
A guide to the cross-sectional anatomy of the ventricular system as seen in TEM is Caudate Nucleus provided in Fig. 2, which shows a third ventriControl Animals (4). Ependyma over the cle. Here the ependymal cells are cuboidal caudate head overlies gray matter as does the and their nuclei are prominent; between these ependyma over the dorsal third ventricle. The cells are the tapering cell processes of ependyma over the corpus callosum covers tanycyte ependymal cells. The ventricular white matter. The superior angle represents surface is characterized by the presence of the junction of gray and white matter at the microvilli and cilia. A typical view taken un- junction of the caudate nucleus and corpus der the SEM shows the elements seen in callosum (Fig. 10). At high power (Fig. 11) characteristic variations over the various ven- the cilia once again can be seen to arise in tricular surfaces differentiated in the clusters. Microvilli are present but not in as remainder of the atlas (Fig. 4). great abundance as over the dorsal third ventricle (compare Fig. 11 with Fig. 5). In addiThird Ventricle tion, neuron-like processes are frequently Control Animals (6). The surface of the found on the surface. The pattern of cilia over third ventricle may be divided into three the head of the caudate is similar to that seen characteristic zones, dorsal, transitional, and over the dorsal third ventricle (compare Fig. infundibular (Fig. 4). In the dorsal zone 12 upper with Fig. 9 upper). At the junction of the caudate nucleus and (beneath the massa intermedia) the ependyma is heavily ciliated (Fig. 5). The cilia arise from corpus callosum (superior angle of the the surface of the ventricle in closely-packed caudate), the pattern of cilia and microvilli clusters, with microvilli covering the cell sur- differs from that seen over the adjacent caudate. The clusters of cilia are not as face between these clusters. densely packed. Between clusters of cilia, *AMR scanning electron microscope made by microvilli are present on the cell surface but Advanced Metals Research Corporation, 160 not in the profusion noted over the caudate Middlesex Turnpike, Bedford, Massachusetts (Fig. 13 upper). In addition, neuron-like 01730, processes are abundant. 648
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Ft~. 2. Transmission electron micrograph showing cross section of third ventricular ependyma. Black arrow points to a typical cilium. Large arrowhead designates microvilli. "T" designates tanycyte ependyma. X 1150.
FrG. 3. SEM appearance of third ventricular ependyma. White arrow designates a sphere capping a cilium. White arrowhead points to rod-like microviIli. X 8000.
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R. B. Page Hydrocephalic Animals (3). The appearance of the surface of the caudate nucleus is not changed in the presence of hydrocephalus with respect either to the pattern and distribution of cilia and microvilli, or to the presence of neuron-like processes (Fig. 12). In the superior angle, at the junction of caudate nucleus and corpus callosum, the pattern of microvilli is altered; here microvilli are not randomly distributed over the surface as in control specimens, but appear to be piled up in rows that stream radially from clusters of cilia (Fig. 13). Lateral Ventricles In control animals the ventricular cavity is small. Synechiae are present between the hippocampus and the roof of the lateral ventricle. With hydrocephalus the ventricular
cavity enlarges. The roof over the atrium of the lateral ventricle expands and becomes dome-shaped. When the specimen is inverted for examination under the scanning microscope the atrium of the ventricle resembles a bowl (Fig. 14). Control Animals (6). While the ependyma over the caudate nucleus overlies gray matter, the ependyma of the " a t r i u m " of the lateral ventricle overlies white matter. The surface of the lateral ventricle in this area has a characteristic pattern (Fig. 15 upper), with clusters of cilia more widely separated in this area than over the caudate head (Fig. 12 upper). Occasional areas lack cilia and have only microvilli on the cell surface. Microvilli do not cover the entire surface between clusters of cilia. Microvilli are abundant between filaments of cilia as they emerge
FIG. 4. Low-power SEM of third ventricle. • 20. MI = massa intermedia; AC --- anterior commissure; MB = mammillary body; OC = optic chiasm; DZ = dorsal zone; TZ = transition zone; IZ = infundibular zone. 650
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Fic. 5. High-power SEM of dorsal third ventricular ependyma. White arrow designates cilia. White arrowhead designates microvilli. X 4000.
F~. 6. Low-power SEM of transition zone (TZ). Note progressive separation of ciliary clusters as infundibulum is approached (left to right). X 180.
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FI~. 7. Upper: High-power SEM of normal infundibulum. Note absence of cilia. White arrowhead points to microvilli. Black arrow points to an apical bleb. E designates the surface of one ependymal cell. Lower: Infundibulum from a hydrocephalic rabbit. White arrowhead designates microvilli; white arrow designates an apical bleb. • 4000.
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Fro. 8. Upper." SEM of normal transition zone. Note clusters of cilia which are separated from one another. Lower." Transition zone of hydrocephalic rabbit. • 1800.
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FI~. 9. SEM of dorsal third ventricle.
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Upper."Normal rabbit.
Lower."Hydrocephalic rabbit. • 1800.
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FIG. 10. Low-power SEM ofcaudate specimen. CN = caudate nucleus; SA = superior angle. X 20.
FIG. 11. High-power SEM of ependyma over caudate nucleus. White arrow designates neuron-like process on ventricular surface. • 8000.
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FIG. 12. SEM of head of caudate nucleus. Upper."Normal rabbit. Note abundance of cilia. Hydrocephalic rabbit, x 1800.
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Lower."
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FIG. 13. SEM of ependyma at superior angle. Upper."Control rabbit. Note distance between ciliary clusters and compare with Fig. 12 upper. White arrow points to ciliary cluster. White arrowhead points to neuron-like process lying within ventricle. Lower: Hydrocephalic rabbit. White arrowhead points to an aggregation of microvilli. Hollow arrow designates debris frequently noted in hydrocephalus. X 1800.
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R. B. Page from the cell surface (Fig. 16), but between clusters of cilia lie areas with no microvilli that appear as shallow depressions. Although microvilli are abundant about the brim of these depressions, the depths are smooth and devoid of surface projections (Fig. 16). Hydrocephalic Animals (4). With moderate hydrocephalus, changes of structure occur on the surface of the atrium of the ventricle; as noted, the atrial specimen resembles a bowl. In each specimen examined, the most marked changes are seen in the depths of the bowl; as the examination progresses from the center of the bowl to the periphery, the changes became less marked. Figure 17 is a medium power view of a normal lateral ventricular surface which may serve as a reference. In moderate hydrocephalus a separation of clusters of cilia occurs (Fig. 18) in the periphery of the bowl; this separation becomes more marked as the center of the bowl is approached (Fig. 19). In more severe hydrocephalus the clusters of cilia are widely separated (Fig. 20); in addition, new cells resembling macrophages become apparent at the center of the bowl (Fig. 21).
Higher power (Fig. 15 lower) reveals that each cluster of cilia, with its associated bare area, is separated from its neighbor. The area between these ciliary clusters is occupied by a cellular surface characterized by the presence of microvilli. (Compare Fig. 15 upper with Fig. 15 lower.) Discussion The degree of hydrocephalus produced by the infusion of silicone was moderate. Wisniewski, et al., 83 noted that severe hydrocephalus was difficult to produce in rabbits because the suprapineal recess frequently ruptured and the degree of hydrocephalus became stabilized. In this investigation, the rabbit was chosen as the experimental animal in order to study the effects of chronic, compensated hydrocephalus. The morphology of the ependyma of the adult rabbit has been characterized by Tennyson and Pappas? ~ who described the aqueductal ependyma as columnar or cuboidal cells whose surfaces are covered with microvilli from which the cilia emerge. This picture might be assumed to be representative of the entire ventricular system; however, ex-
FIG. 14. Low-power SEM of atrium of lateral ventricle of a hydrocephalic rabbit. • 20. 658
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FI6. 15. SEM of atrium of lateral ventricle. Upper." Control rabbit. White arrow points to cilia, white arrowhead points to microvilli, open white arrows point to areas devoid of cilia and microvilli. Lower: Hydrocephalic rabbit. Large arrowhead points to a cluster of cilia, small arrowhead points to microvilli. Note increased distance between ciliary clusters compared with upper. • 1800.
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FIG. 16. High-power SEM of lateral ventricle of a normal rabbit. Large arrow points to a cluster of cilia, large arrowhead to microvilli around emerging filaments of cilia, hollow arrow to a shallow pit devoid of microvilli, and small arrow leads to microvilli around the brim. • 8000.
amination of the surface of the ventricular system with the SEM has demonstrated that such an assumption is untenable. In the present study, each of the regions investigated presented a unique picture with respect to the population and distribution of cilia and microvilli. In the third ventricle, three distinct regions were characterized; these findings are in agreement with previous reports by others. 4,15,~7 Similarly, in the lateral ventricle the head of the caudate nucleus, the superior angle, and the atrium each presented a characteristic distribution of cilia, microvilli, and a presence or absence of neuronal processes within the ventricle?8 Over the gray matter masses examined, namely those in the dorsal third ventricle and the head of the caudate nucleus, the ependyma was heavily ciliated and the clusters of 20 to 25 filaments were closely packed together. On the other hand, over the regions of white matter studied, namely, the superior angle and the atrium of the lateral ventricle, the ependyma was not as heavily ciliated; here 660
each cluster of cilia contained the same number of filaments as over gray matter, but the distance between clusters was greater. In the presence of hydrocephalus the ependyma over gray matter masses was unchanged while that over periventricular white matter was altered. This topographic distribution conforms to that described by Lawson and Raimondi xe and Raimondi, et al. 24 in a light and transmission electron microscopic study of the ependyma of hydrocephalic HY3 mice. There were three types or stages of alteration of the ependymal surface over periventricular white matter in the presence of hydrocephalus. In the first, the clusters of cilia became more separated from one another and, as a result, the population density of cilia was decreased. In the second, broad areas covered with microvilli appeared between clusters of cilia and their associated bare areas. In the third, a new cellular element, presumably a macrophage, appeared on the surface of the ventricle. Two questions J. Neurosurg. / Volume 42 / June, 1975
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Fi6. 17. Medium-power SEM of atrium of lateral ventricle of a normal rabbit. X 300.
FIG. 18. SEM of atrium of lateral ventricle of hydrocephalic rabbit at periphery of bowl. Note separation of clusters of cilia. X 300.
FIG. 19. SEM of atrium of lateral ventricle of hydrocephalic rabbit toward center of bowl. Increased separation of ciliary clusters is noted. Same specimen as Fig. 18. X 300.
FIG. 20. SEM of atrium of lateral ventricle at center of bowl. Ciliary clusters are widely separated and scant. X 300.
FIG. 21. SEM of lateral ventricle at center of bowl. Surface is characterized by presence of large cells (white arrowhead) resembling macrophages. Only a few clusters of cilia may be discerned. x 300.
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R. B. Page can be posed: By what mechanism are such changes brought about, and do these changes have any functional significance? Although the ependyma over gray matter masses and periventricular white matter are presumably subjected to the same ventricular pressure, changes resulting from hydrocephalus are limited to the periventricular white matter; this region presumably undergoes a significant increase in surface area associated with the ventricular distention that occurs in hydrocephalus. This expanded ventricular surface is continuous and not disrupted by moderate ventricular dilatation. Thus the surface covered by microvilli increases as the ciliary clusters become separated. It is postulated that with distention of the periventricular white matter, new cellular processes emanating from germinal cells of the subependymal plate 8'6'~7,~s'~9 may insinuate themselves between preexisting apical processes to gain the ventricular surface. These apical processes are devoid of cilia but rich in microvilli. This response of course would not be expected over gray matter masses since their surfaces would not be stretched by hydrocephalus. The apical processes of ependymal cells form the interface between the fluid-filled ventricular cavities and the surrounding brain. With certain exceptions (see Results) the surface of ependymal cells is characterized by the presence of cilia and microvilli. The coordinated beating of cilia on the surface of the ependyma is considered an important factor in the propulsion of spinal fluid; T M the ependyma may therefore be viewed as an organ system whose function is to assist the cerebrospinal circulation in this fashion. Conversely, the presence of microvilli on an epithelial surface connotes a high degree of resorptive and/or secretory activity? Thus, the ependyma may also be viewed as an organ system whose function is the regulation of fluid transport between the ventricular cavity and the adjacent brain. Increased transependymal transfer of fluid has been postulated to play an important compensatory role in hydrocephalus? '1~ An increased permeability of the ventricular lining to vital dye has been noted in hydrocephalic dogs, s~ while TEM studies have demonstrated an increase in the extracellular space of periventricular white 662
matter suggesting increased transventricular fluid transport, x6,19'21 The present study demonstrates that in hydrocephalus commensurate changes occur at the ventricular surface. Initially there is a relative decrease in the population density of cilia capable of propelling fluid. Since no vacant pits appear in the cell surface from which cilia might have once emerged, the ciliary clusters do not seem to have been destroyed or lost from the cell surface. Rather, these clusters have been separated by new areas covered with microvilli and presumably capable of absorbing fluid. In the presence of hydrocephalus, the surface of the atrium of the lateral ventricle begins to resemble that of the normal transition zone of the third ventricle, an area held to be specifically engaged in fluid transport between the third ventricle and the hypothalamus2 '12'13'2~'~8 The presence of microvilli at the cell surface suggests transport of fluid across cell membranes, while the increase in surface area of the hydrocephalic ventricle accomplished by adding surface units rich in microvilli would provide a mechanism for increased transventricular fluid transfer as well as a means of increasing the surface area of the ventricle. These changes may provide a compensatory mechanism by which the ventricular system responds to hydrocephalus. An ingrowth of new cellular elements (presumably macrophages) and a decrease in the number of both cilia and microvilli accompany further hydrocephalic progression. Areas such as those found deep in the bowl of the atrium appear no longer capable of significant fluid transport. However, it should be noted that at this stage there still are areas replete with microvilli at the periphery of the bowl of the atrium. With more extensive hydrocephalus the compensatory mechanisms may fail as the ependymal cells lose their microvilli at the surface and become incapable of regulating fluid transport between the ventricle and surrounding brain.
Summary An atlas of the scanning electron microscopic appearance of the ependymal surface in the ventricles of normal and hydrocephalic rabbits has been presented and related to the topography of this region. J. Neurosurg. / Volume 42 / June, 1975
Scanning electron microscopy in hydrocephalus Changes associated with moderate hydrocephalus appear to be limited to the ependyma over periventricular white matter, and do not appear to be simply manifestations of destruction of the ependyma but rather manifestations of its plasticity. The sequential modifications identified suggest an initial functional alteration from a surface capable of propelling fluid by ciliary motion to a surface capable of increased transventricular fluid transfer. As the hydrocephalic process progresses, these compensatory mechanisms may fail as the surface loses microvilli and so becomes a less efficient mechanism for absorption. References
l. Adamo N J: Ultrastructural features of the lateral preoptic area, median eminence and arcuate nucleus of the rat. Z Zellforseh Mikrosk Anat 127:483-491, 1972 2. Bering EA, Sato O: Hydrocephalus: changes in formation and absorption of cerebrospinal fluid within the cerebral ventricles. J Neurosurg 20:1050-1063, 1963 3. Blakemore WF, Jolly RD: The subependymal plate and associated ependyma in the dog. An ultrastructural study. J Neurocytol 1:69-84, 1972 4. Bruni JE, Montemurro DG, Clattenburg RE, et al: A scanning electron microscopic study of the ependymal surface of the third ventricle of the rabbit, rat, mouse and human brain. Anat Ree 174:407-420, 1972 5. Cathcart RS 3rd, Worthington WC Jr: Currents and clearing action produced in the rat cerebral ventricle by ependymal cilia. (Abstract) Anat Ree 148:269, 1964 6. Clark RG, Milhorat T: Experimental hydrocephalus. Part 3: Light microscopic findings in acute and subacute obstructive hydrocephalus in the monkey. J Neurosurg 32:400-413, 1970 7. Duckett S: The morphology of Telluriuminduced hydrocephalus. Exp Neurol 31 : 1-16, I971 8. Fawcett D: Surface specializations of absorbing cells. J Histochem Cytochem 13:75-91, 1965 9. Hochwald GM, Sahar A, Sadik AR, et al: Cerebrospinal fluid production and histological observations in animals with experimental hydrocephalus. Exp Neurol 25:190-199, 1969 10. James AE Jr, Strecker EP, Sperber E, et al: An alternative pathway of cerebrospinal fluid absorption in communicating hydrocephalus:
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transependymal movement. Radiology 111: 143-146, 1974 11. Karnovsky M J: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. (Abstract) J Cell Biol 27:137A, 1965 12. Knowles F: Ependyma of the third ventricle in relation to pituitary function. Prog Brain Res 38:255-270, 1972 13. Knowles F, Anand Kumar TC: Structural changes, related to reproduction, in the hypothalamus and in the pars tuberalis of the Rhesus monkey. Part 1. The hypothalamus. Part 2. The pars tuberalis. Philos Trans R Soc Lond (Bioi Sci) 256:357-375, 1969 14. Koyama T, Handa J, Handa H: Experimental hydrocephalus. Post natal observations of hydrocephalus induced by ethylnitrosourea in the SD-JCL rat. Z Neurol 202:21-36, 1972 15. Kozlowski GP, Scott DE, Dudley GK: Scanning electron microscopy of the third ventricle of the sheep. Z Zellforsch Mikrosk Anat 136:169-176, 1973 16. Lawson RF, Raimondi A J: Hydrocephalus-3, a murine mutant: 1. Alterations in fine structure of the choroid plexus and ependyma. Surg Neurol 1:115-128, 1973 17. Lewis PD: The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia. Brain 91:721-736, 1968 18. Lewis PD: A quantitative study of cell proliferation in the subependymal layer of the adult rat brain. Exp Nearol 20:203-207, 1968 19. McLone DG, Bondareff W, Raimondi A J: Brain edema in the hydrocephalic HY-3 mouse: submicroscopic morphology. J Neuropathol Exp Neurol 30:627-637, 1971 20. Milhorat TH, Clark RG: Some observations on the circulation of phenosulfonphthalein in cerebrospinal fluid: normal flow and the flow in hydrocephalus. J Neurosarg 32:522-528, 1970 21. Milhorat TH, Clark RG, Hammock MK, et al: Structural, ultrastructural and permeability changes in the ependymal and surrounding brain favoring equilibration in progressive hydrocephalus. Arch Neurol 22: 397-407, t970 22. Nakai Y: Fine structure and its functional properties of the ependymal cell in the frog median eminence. Z Zellforsch Mikros Anat 122:15-25, 1971 23. Noack W, Dumitrescu L, Schweichel JU: Scanning and electron microscopical investigations of the surface structures of the lateral ventricles in the cat. Brain Res 46:121-129, 1972 24. Raimondi A J, Bailey OT, McLone DG, et al: The pathophysiology and morphology of
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25.
26.
27.
28.
29.
30.
murine hydrocephalus in HY-3 and CH mutants. Surg Neurol 1:50-55, 1973 Russell DS: Observations on the Pathology of Hydrocephalus. London, Her Majesty's Stationary Office (Spec Rep Ser, Med Res Council, No 265), 1949 Sahar A, Hochwald GM, Ransohoff J: Alternate pathway for cerebrospinal fluid absorption in animals with experimental obstructive hydrocephalus. Exp Neurol 25:200-206, 1969 Scott DE, Kozlowski GP, Dudley GK: A comparative ultrastructural analysis of the third cerebral ventricle of the North American mink (Mustela vison). Anat Rec 175:155-168, 1973 Sharp P J: Tanycyte and vascular patterns in the basal hypothalamus of Coturnix quail with reference to their possible neuroendocrine significance. Z Zellforsch Mikrosk Anat 127:552-569, 1972 Smart I: The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-H3 injection. J Comp Neurol 116:325-347, 1961 Tennyson VM, Pappas GD: An electron microscopic study of ependymal cells of the
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31.
32.
33.
34.
fetal, early postnatal and adult rabbit. Z Zellfnrsch Mikros Anat 56:595-618, 1962 Weller RO, Shulman K: Infantile hydrocephalus: clinical, histological and ultrastructural study of brain damage. J Neurosnrg 36:255-265, 1972 Weller RO, Wisniewski H: Histological and ultrastructural changes with experimental hydrocephalus in adult rabbits. Brain 92:819-828, 1969 Wisniewski H, Weller RO, Terry RD: Experimental hydrocephalus produced by the subarachnoid infusion of silicone oil. J Neurosurg 31:10-14, 1969 Worthington WC Jr, Cathcart RS 3rd: Ependymal cilia: distribution and activity in the adult human brain. Science 139:221-222, 1963
This work was presented at the annual meeting of the American Association of Neurological Surgeons, in St. Louis, Missouri, April, 1974. Address reprint requests to: Robert B. Page, M.D., M.S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania.
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M.S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania v' The author used the scanning electron microscope to study the ependyma in six control rabbits and six rabbits made hydrocephalic by infusion of silicone oil into the cisterna magna. The ependymal lining of the third ventricle, head of the caudate nucleus, superior angle of the caudate, and atrium of the lateral ventricle was examined. In the hydrocephalic animals, clusters of cilia emanating from the ependyma over periventricular white matter become separated; the author believes this is secondary to ingrowth of new ependymal cell processes covered with microvilli. The addition of these cells to the ependymal surface permits ventricular dilatation without ependymal disruption and provides more surface containing microvilli, presumably capable of increased transventricular fluid transfer. No such changes occur over gray matter masses since their surfaces are not deformed by moderate ventricular dilatation. The morphological alterations in the ependyma that occur in moderate hydrocephalus do not appear to be simply manifestations of ependymal destruction but rather suggest a modification in its function from that of a surface capable of propelling cerebrospinal fluid to one capable of increased transfer of transventricular fluid. As hydrocephalus progresses, compensation may fail because of the relative decrease in microvilli so that the cell surface provides a less efficient mechanism for absorption. Krv WORDS 9 hydrocephalus 9 ependymal cilia and microvUli scanning electron microscopy 9 cerebral ventricles
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NLARGEMENT of the ventricular system is the sine qua non of hydrocephalus. Several studies of the ependyma and subependymal white matter have been reported in n o r m a l and hydrocephalic animals utilizing light microscopy (LM) and transmission electron 646
9
m i c r o s c o p y ( T E M ) . 6'7'9'1''24'25'31's~ These methods have demonstrated expansion of the extracellular space of the subependymal white matter in hydrocephalus, presumably the result of increased transependymal fluid transport? 6,2~ In the event of obstruction to cerebrospinal fluid (CSF) circulation, such a
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Scanning electron microscopy in hydrocephalus mechanism would provide an alternative means of egress of CSF from the ventricular system. However, previous limitations in the available techniques have allowed little knowledge of the fate of ependymal cells in hydrocephalus. Light microscopy has only permitted evaluation of relatively gross alterations in far advanced hydrocephalus,14,25,3x while TEM has been limited to study of ultrathin sections made from minute samples of the ventricular surface. Scanning electron microscopy (SEM) permits examination of a relatively large surface of the ventricle at low magnification. Ventricular topography may be assessed and areas of interest selected for more detailed study. At higher magnification the cellular surface, cilia, and microvilli may be examined. Scanning electron microscopic studies of the ventricular surface have been reported in several mammalian species 4'15,2~ but to date there have been no comparable published studies of hydrocephalic animals. The following SEM study was undertaken to characterize the ventricular system of an experimental animal under normal conditions and under conditions of moderate hydrocephalus, and to assess the following questions: Do structural alterations occur at the surface of the ependymal cell? Are these changes compatible with the increase in transventricular fluid transport postulated to occur in hydrocephalus, or are they simply manifestations of destruction of ependymal cells? Materials and Methods
Twelve adult Dutch Belted rabbits of both sexes were used; six were made hydrocephalic by infusion of silicone into the cisterna magna. 33 With the animals under halothane anesthesia, the foramen magnum was exposed and a No. 60 polyethylene catheter was placed in the cisterna magna. A mixture of equal parts of Dow Corning 200 and 360 medical fluids was infused into the cisterna magna with a Harvard pump at a rate of 0.0136 cc/min to a total of 0.6 cc.* The *Dow Corning 200 and 360 Medical fluids made by Dow Corning Corporation, Medical Products Division, Midland, Michigan 48640. Harvard pump made by Harvard Apparatus, Inc., Millis, Massachusetts.
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Fic. 1. Comparison of ventricular size in control (left) and hydrocephalic (right) rabbits. viscosity of No. 200 fluid is 100,000 centistokes (cs); viscosity of No. 360 fluid is 12,500 cs. Following the infusion the wound was closed in lay(-,: and the animals were returned to their cages. Three animals were sacrificed at 4 weeks after infusion and three at 16 weeks. The control series consisted of two animals that underwent infusion of saline, rather than silicone, into the cisterna magna. In two additional animals the cisterna magna was exposed under halothane anesthesia. The dura was not opened and the animals were maintained under anesthesia for 1 hour prior to closure of the wound. These four animals were sacrificed 16 weeks after surgery. Finally, two unoperated animals were also examined. Specimens were prepared in the following manner: After nembutal anesthesia, intracardiac perfusion with Karno~,sky's solutionla was performed. The brain was immediately removed from the skull and postfixed by immersion in Karno~sky's solution overnight at 4~ Coronal sections were then taken at the level of the anterior commissure to document ventricular size (Fig. l). The brain was dissected under fixative utilizing a Zeiss OPMI 6 operating microscope.t Three samples of ependyma were obtained from each animal: 1) a 0.5 • 0.5-cm segment consisting of the caudate head and adjacent corpus callosum; 2) a triangular segment (0.5 cm base, 0.5 cm height) of the roof of the lateral ventricle at the junction of the body and the temporal horn; and 3) a 1 X 1-cm block of the epentZeiss OPMI 6 operating microscope made by Carl Zeiss, Inc., 444 Fifth Avenue, New York, New York 10018. 647
R . B. P a g e dyma lining the third ventricle limited anteriorly by the lamina terminalis, superiorly by the midportion of the massa intermedia, and posteriorly by a line drawn from the superior colliculus to the mammillary bodies. Samples were also taken at corresponding sites from the opposite caudate nucleus, atrium of lateral ventricle, and third ventricle, and prepared for TEM. For orientation, the TEM observations in control animals are briefly noted in this report. Full descriptions of TEM changes will be the subject of a separate report. After dissection, the specimens were washed with cold 0.1 M cacodylate buffer for 24 hours. Postfixation with collidine-buffered 1.33% osmium tetroxide for 2 hours and subsequent dehydration through serial alcohols and freon followed. The specimens were dried in a critical point dryer, mounted on stubs, coated with gold palladium, and were examined in an AMR scanning electron microscope.* Results
The transition zone lies at the level of the optic chiasm and mammillary bodies (Fig. 4); cilia are present here but clusters become progressively sparse as the infundibulum is approached (Fig. 6). Microvilli are present in abundance on the cell surface between widely spread clusters of cilia (Fig. 3). The ependyma of the infundibulum is not ciliated (Fig. 7 upper). Here, the outlines of cells create the impression of a "cobblestone street." The cell surfaces are covered with microvilli and small, round excrescences (apical blebs) which appear to be budding from the surface of the cells. Hydrocephalic Animals (6). Despite enlargement of the third ventricle the surface morphology of the ependyma is not significantly altered in the six specimens studied. The surface anatomy of the infundibulum is unchanged; both apical blebs and microvilli are still apparent (Fig. 7). The morphology and distribution of cilia and microvilli in the transition zone (Fig. 8) and in the dorsal third ventricle are also unchanged (Fig. 9).
A guide to the cross-sectional anatomy of the ventricular system as seen in TEM is Caudate Nucleus provided in Fig. 2, which shows a third ventriControl Animals (4). Ependyma over the cle. Here the ependymal cells are cuboidal caudate head overlies gray matter as does the and their nuclei are prominent; between these ependyma over the dorsal third ventricle. The cells are the tapering cell processes of ependyma over the corpus callosum covers tanycyte ependymal cells. The ventricular white matter. The superior angle represents surface is characterized by the presence of the junction of gray and white matter at the microvilli and cilia. A typical view taken un- junction of the caudate nucleus and corpus der the SEM shows the elements seen in callosum (Fig. 10). At high power (Fig. 11) characteristic variations over the various ven- the cilia once again can be seen to arise in tricular surfaces differentiated in the clusters. Microvilli are present but not in as remainder of the atlas (Fig. 4). great abundance as over the dorsal third ventricle (compare Fig. 11 with Fig. 5). In addiThird Ventricle tion, neuron-like processes are frequently Control Animals (6). The surface of the found on the surface. The pattern of cilia over third ventricle may be divided into three the head of the caudate is similar to that seen characteristic zones, dorsal, transitional, and over the dorsal third ventricle (compare Fig. infundibular (Fig. 4). In the dorsal zone 12 upper with Fig. 9 upper). At the junction of the caudate nucleus and (beneath the massa intermedia) the ependyma is heavily ciliated (Fig. 5). The cilia arise from corpus callosum (superior angle of the the surface of the ventricle in closely-packed caudate), the pattern of cilia and microvilli clusters, with microvilli covering the cell sur- differs from that seen over the adjacent caudate. The clusters of cilia are not as face between these clusters. densely packed. Between clusters of cilia, *AMR scanning electron microscope made by microvilli are present on the cell surface but Advanced Metals Research Corporation, 160 not in the profusion noted over the caudate Middlesex Turnpike, Bedford, Massachusetts (Fig. 13 upper). In addition, neuron-like 01730, processes are abundant. 648
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Ft~. 2. Transmission electron micrograph showing cross section of third ventricular ependyma. Black arrow points to a typical cilium. Large arrowhead designates microvilli. "T" designates tanycyte ependyma. X 1150.
FrG. 3. SEM appearance of third ventricular ependyma. White arrow designates a sphere capping a cilium. White arrowhead points to rod-like microviIli. X 8000.
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R. B. Page Hydrocephalic Animals (3). The appearance of the surface of the caudate nucleus is not changed in the presence of hydrocephalus with respect either to the pattern and distribution of cilia and microvilli, or to the presence of neuron-like processes (Fig. 12). In the superior angle, at the junction of caudate nucleus and corpus callosum, the pattern of microvilli is altered; here microvilli are not randomly distributed over the surface as in control specimens, but appear to be piled up in rows that stream radially from clusters of cilia (Fig. 13). Lateral Ventricles In control animals the ventricular cavity is small. Synechiae are present between the hippocampus and the roof of the lateral ventricle. With hydrocephalus the ventricular
cavity enlarges. The roof over the atrium of the lateral ventricle expands and becomes dome-shaped. When the specimen is inverted for examination under the scanning microscope the atrium of the ventricle resembles a bowl (Fig. 14). Control Animals (6). While the ependyma over the caudate nucleus overlies gray matter, the ependyma of the " a t r i u m " of the lateral ventricle overlies white matter. The surface of the lateral ventricle in this area has a characteristic pattern (Fig. 15 upper), with clusters of cilia more widely separated in this area than over the caudate head (Fig. 12 upper). Occasional areas lack cilia and have only microvilli on the cell surface. Microvilli do not cover the entire surface between clusters of cilia. Microvilli are abundant between filaments of cilia as they emerge
FIG. 4. Low-power SEM of third ventricle. • 20. MI = massa intermedia; AC --- anterior commissure; MB = mammillary body; OC = optic chiasm; DZ = dorsal zone; TZ = transition zone; IZ = infundibular zone. 650
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Fic. 5. High-power SEM of dorsal third ventricular ependyma. White arrow designates cilia. White arrowhead designates microvilli. X 4000.
F~. 6. Low-power SEM of transition zone (TZ). Note progressive separation of ciliary clusters as infundibulum is approached (left to right). X 180.
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FI~. 7. Upper: High-power SEM of normal infundibulum. Note absence of cilia. White arrowhead points to microvilli. Black arrow points to an apical bleb. E designates the surface of one ependymal cell. Lower: Infundibulum from a hydrocephalic rabbit. White arrowhead designates microvilli; white arrow designates an apical bleb. • 4000.
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Fro. 8. Upper." SEM of normal transition zone. Note clusters of cilia which are separated from one another. Lower." Transition zone of hydrocephalic rabbit. • 1800.
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FI~. 9. SEM of dorsal third ventricle.
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Upper."Normal rabbit.
Lower."Hydrocephalic rabbit. • 1800.
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FIG. 10. Low-power SEM ofcaudate specimen. CN = caudate nucleus; SA = superior angle. X 20.
FIG. 11. High-power SEM of ependyma over caudate nucleus. White arrow designates neuron-like process on ventricular surface. • 8000.
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FIG. 12. SEM of head of caudate nucleus. Upper."Normal rabbit. Note abundance of cilia. Hydrocephalic rabbit, x 1800.
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Lower."
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FIG. 13. SEM of ependyma at superior angle. Upper."Control rabbit. Note distance between ciliary clusters and compare with Fig. 12 upper. White arrow points to ciliary cluster. White arrowhead points to neuron-like process lying within ventricle. Lower: Hydrocephalic rabbit. White arrowhead points to an aggregation of microvilli. Hollow arrow designates debris frequently noted in hydrocephalus. X 1800.
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R. B. Page from the cell surface (Fig. 16), but between clusters of cilia lie areas with no microvilli that appear as shallow depressions. Although microvilli are abundant about the brim of these depressions, the depths are smooth and devoid of surface projections (Fig. 16). Hydrocephalic Animals (4). With moderate hydrocephalus, changes of structure occur on the surface of the atrium of the ventricle; as noted, the atrial specimen resembles a bowl. In each specimen examined, the most marked changes are seen in the depths of the bowl; as the examination progresses from the center of the bowl to the periphery, the changes became less marked. Figure 17 is a medium power view of a normal lateral ventricular surface which may serve as a reference. In moderate hydrocephalus a separation of clusters of cilia occurs (Fig. 18) in the periphery of the bowl; this separation becomes more marked as the center of the bowl is approached (Fig. 19). In more severe hydrocephalus the clusters of cilia are widely separated (Fig. 20); in addition, new cells resembling macrophages become apparent at the center of the bowl (Fig. 21).
Higher power (Fig. 15 lower) reveals that each cluster of cilia, with its associated bare area, is separated from its neighbor. The area between these ciliary clusters is occupied by a cellular surface characterized by the presence of microvilli. (Compare Fig. 15 upper with Fig. 15 lower.) Discussion The degree of hydrocephalus produced by the infusion of silicone was moderate. Wisniewski, et al., 83 noted that severe hydrocephalus was difficult to produce in rabbits because the suprapineal recess frequently ruptured and the degree of hydrocephalus became stabilized. In this investigation, the rabbit was chosen as the experimental animal in order to study the effects of chronic, compensated hydrocephalus. The morphology of the ependyma of the adult rabbit has been characterized by Tennyson and Pappas? ~ who described the aqueductal ependyma as columnar or cuboidal cells whose surfaces are covered with microvilli from which the cilia emerge. This picture might be assumed to be representative of the entire ventricular system; however, ex-
FIG. 14. Low-power SEM of atrium of lateral ventricle of a hydrocephalic rabbit. • 20. 658
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FI6. 15. SEM of atrium of lateral ventricle. Upper." Control rabbit. White arrow points to cilia, white arrowhead points to microvilli, open white arrows point to areas devoid of cilia and microvilli. Lower: Hydrocephalic rabbit. Large arrowhead points to a cluster of cilia, small arrowhead points to microvilli. Note increased distance between ciliary clusters compared with upper. • 1800.
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FIG. 16. High-power SEM of lateral ventricle of a normal rabbit. Large arrow points to a cluster of cilia, large arrowhead to microvilli around emerging filaments of cilia, hollow arrow to a shallow pit devoid of microvilli, and small arrow leads to microvilli around the brim. • 8000.
amination of the surface of the ventricular system with the SEM has demonstrated that such an assumption is untenable. In the present study, each of the regions investigated presented a unique picture with respect to the population and distribution of cilia and microvilli. In the third ventricle, three distinct regions were characterized; these findings are in agreement with previous reports by others. 4,15,~7 Similarly, in the lateral ventricle the head of the caudate nucleus, the superior angle, and the atrium each presented a characteristic distribution of cilia, microvilli, and a presence or absence of neuronal processes within the ventricle?8 Over the gray matter masses examined, namely those in the dorsal third ventricle and the head of the caudate nucleus, the ependyma was heavily ciliated and the clusters of 20 to 25 filaments were closely packed together. On the other hand, over the regions of white matter studied, namely, the superior angle and the atrium of the lateral ventricle, the ependyma was not as heavily ciliated; here 660
each cluster of cilia contained the same number of filaments as over gray matter, but the distance between clusters was greater. In the presence of hydrocephalus the ependyma over gray matter masses was unchanged while that over periventricular white matter was altered. This topographic distribution conforms to that described by Lawson and Raimondi xe and Raimondi, et al. 24 in a light and transmission electron microscopic study of the ependyma of hydrocephalic HY3 mice. There were three types or stages of alteration of the ependymal surface over periventricular white matter in the presence of hydrocephalus. In the first, the clusters of cilia became more separated from one another and, as a result, the population density of cilia was decreased. In the second, broad areas covered with microvilli appeared between clusters of cilia and their associated bare areas. In the third, a new cellular element, presumably a macrophage, appeared on the surface of the ventricle. Two questions J. Neurosurg. / Volume 42 / June, 1975
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Fi6. 17. Medium-power SEM of atrium of lateral ventricle of a normal rabbit. X 300.
FIG. 18. SEM of atrium of lateral ventricle of hydrocephalic rabbit at periphery of bowl. Note separation of clusters of cilia. X 300.
FIG. 19. SEM of atrium of lateral ventricle of hydrocephalic rabbit toward center of bowl. Increased separation of ciliary clusters is noted. Same specimen as Fig. 18. X 300.
FIG. 20. SEM of atrium of lateral ventricle at center of bowl. Ciliary clusters are widely separated and scant. X 300.
FIG. 21. SEM of lateral ventricle at center of bowl. Surface is characterized by presence of large cells (white arrowhead) resembling macrophages. Only a few clusters of cilia may be discerned. x 300.
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R. B. Page can be posed: By what mechanism are such changes brought about, and do these changes have any functional significance? Although the ependyma over gray matter masses and periventricular white matter are presumably subjected to the same ventricular pressure, changes resulting from hydrocephalus are limited to the periventricular white matter; this region presumably undergoes a significant increase in surface area associated with the ventricular distention that occurs in hydrocephalus. This expanded ventricular surface is continuous and not disrupted by moderate ventricular dilatation. Thus the surface covered by microvilli increases as the ciliary clusters become separated. It is postulated that with distention of the periventricular white matter, new cellular processes emanating from germinal cells of the subependymal plate 8'6'~7,~s'~9 may insinuate themselves between preexisting apical processes to gain the ventricular surface. These apical processes are devoid of cilia but rich in microvilli. This response of course would not be expected over gray matter masses since their surfaces would not be stretched by hydrocephalus. The apical processes of ependymal cells form the interface between the fluid-filled ventricular cavities and the surrounding brain. With certain exceptions (see Results) the surface of ependymal cells is characterized by the presence of cilia and microvilli. The coordinated beating of cilia on the surface of the ependyma is considered an important factor in the propulsion of spinal fluid; T M the ependyma may therefore be viewed as an organ system whose function is to assist the cerebrospinal circulation in this fashion. Conversely, the presence of microvilli on an epithelial surface connotes a high degree of resorptive and/or secretory activity? Thus, the ependyma may also be viewed as an organ system whose function is the regulation of fluid transport between the ventricular cavity and the adjacent brain. Increased transependymal transfer of fluid has been postulated to play an important compensatory role in hydrocephalus? '1~ An increased permeability of the ventricular lining to vital dye has been noted in hydrocephalic dogs, s~ while TEM studies have demonstrated an increase in the extracellular space of periventricular white 662
matter suggesting increased transventricular fluid transport, x6,19'21 The present study demonstrates that in hydrocephalus commensurate changes occur at the ventricular surface. Initially there is a relative decrease in the population density of cilia capable of propelling fluid. Since no vacant pits appear in the cell surface from which cilia might have once emerged, the ciliary clusters do not seem to have been destroyed or lost from the cell surface. Rather, these clusters have been separated by new areas covered with microvilli and presumably capable of absorbing fluid. In the presence of hydrocephalus, the surface of the atrium of the lateral ventricle begins to resemble that of the normal transition zone of the third ventricle, an area held to be specifically engaged in fluid transport between the third ventricle and the hypothalamus2 '12'13'2~'~8 The presence of microvilli at the cell surface suggests transport of fluid across cell membranes, while the increase in surface area of the hydrocephalic ventricle accomplished by adding surface units rich in microvilli would provide a mechanism for increased transventricular fluid transfer as well as a means of increasing the surface area of the ventricle. These changes may provide a compensatory mechanism by which the ventricular system responds to hydrocephalus. An ingrowth of new cellular elements (presumably macrophages) and a decrease in the number of both cilia and microvilli accompany further hydrocephalic progression. Areas such as those found deep in the bowl of the atrium appear no longer capable of significant fluid transport. However, it should be noted that at this stage there still are areas replete with microvilli at the periphery of the bowl of the atrium. With more extensive hydrocephalus the compensatory mechanisms may fail as the ependymal cells lose their microvilli at the surface and become incapable of regulating fluid transport between the ventricle and surrounding brain.
Summary An atlas of the scanning electron microscopic appearance of the ependymal surface in the ventricles of normal and hydrocephalic rabbits has been presented and related to the topography of this region. J. Neurosurg. / Volume 42 / June, 1975
Scanning electron microscopy in hydrocephalus Changes associated with moderate hydrocephalus appear to be limited to the ependyma over periventricular white matter, and do not appear to be simply manifestations of destruction of the ependyma but rather manifestations of its plasticity. The sequential modifications identified suggest an initial functional alteration from a surface capable of propelling fluid by ciliary motion to a surface capable of increased transventricular fluid transfer. As the hydrocephalic process progresses, these compensatory mechanisms may fail as the surface loses microvilli and so becomes a less efficient mechanism for absorption. References
l. Adamo N J: Ultrastructural features of the lateral preoptic area, median eminence and arcuate nucleus of the rat. Z Zellforseh Mikrosk Anat 127:483-491, 1972 2. Bering EA, Sato O: Hydrocephalus: changes in formation and absorption of cerebrospinal fluid within the cerebral ventricles. J Neurosurg 20:1050-1063, 1963 3. Blakemore WF, Jolly RD: The subependymal plate and associated ependyma in the dog. An ultrastructural study. J Neurocytol 1:69-84, 1972 4. Bruni JE, Montemurro DG, Clattenburg RE, et al: A scanning electron microscopic study of the ependymal surface of the third ventricle of the rabbit, rat, mouse and human brain. Anat Ree 174:407-420, 1972 5. Cathcart RS 3rd, Worthington WC Jr: Currents and clearing action produced in the rat cerebral ventricle by ependymal cilia. (Abstract) Anat Ree 148:269, 1964 6. Clark RG, Milhorat T: Experimental hydrocephalus. Part 3: Light microscopic findings in acute and subacute obstructive hydrocephalus in the monkey. J Neurosurg 32:400-413, 1970 7. Duckett S: The morphology of Telluriuminduced hydrocephalus. Exp Neurol 31 : 1-16, I971 8. Fawcett D: Surface specializations of absorbing cells. J Histochem Cytochem 13:75-91, 1965 9. Hochwald GM, Sahar A, Sadik AR, et al: Cerebrospinal fluid production and histological observations in animals with experimental hydrocephalus. Exp Neurol 25:190-199, 1969 10. James AE Jr, Strecker EP, Sperber E, et al: An alternative pathway of cerebrospinal fluid absorption in communicating hydrocephalus:
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transependymal movement. Radiology 111: 143-146, 1974 11. Karnovsky M J: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. (Abstract) J Cell Biol 27:137A, 1965 12. Knowles F: Ependyma of the third ventricle in relation to pituitary function. Prog Brain Res 38:255-270, 1972 13. Knowles F, Anand Kumar TC: Structural changes, related to reproduction, in the hypothalamus and in the pars tuberalis of the Rhesus monkey. Part 1. The hypothalamus. Part 2. The pars tuberalis. Philos Trans R Soc Lond (Bioi Sci) 256:357-375, 1969 14. Koyama T, Handa J, Handa H: Experimental hydrocephalus. Post natal observations of hydrocephalus induced by ethylnitrosourea in the SD-JCL rat. Z Neurol 202:21-36, 1972 15. Kozlowski GP, Scott DE, Dudley GK: Scanning electron microscopy of the third ventricle of the sheep. Z Zellforsch Mikrosk Anat 136:169-176, 1973 16. Lawson RF, Raimondi A J: Hydrocephalus-3, a murine mutant: 1. Alterations in fine structure of the choroid plexus and ependyma. Surg Neurol 1:115-128, 1973 17. Lewis PD: The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia. Brain 91:721-736, 1968 18. Lewis PD: A quantitative study of cell proliferation in the subependymal layer of the adult rat brain. Exp Nearol 20:203-207, 1968 19. McLone DG, Bondareff W, Raimondi A J: Brain edema in the hydrocephalic HY-3 mouse: submicroscopic morphology. J Neuropathol Exp Neurol 30:627-637, 1971 20. Milhorat TH, Clark RG: Some observations on the circulation of phenosulfonphthalein in cerebrospinal fluid: normal flow and the flow in hydrocephalus. J Neurosarg 32:522-528, 1970 21. Milhorat TH, Clark RG, Hammock MK, et al: Structural, ultrastructural and permeability changes in the ependymal and surrounding brain favoring equilibration in progressive hydrocephalus. Arch Neurol 22: 397-407, t970 22. Nakai Y: Fine structure and its functional properties of the ependymal cell in the frog median eminence. Z Zellforsch Mikros Anat 122:15-25, 1971 23. Noack W, Dumitrescu L, Schweichel JU: Scanning and electron microscopical investigations of the surface structures of the lateral ventricles in the cat. Brain Res 46:121-129, 1972 24. Raimondi A J, Bailey OT, McLone DG, et al: The pathophysiology and morphology of
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25.
26.
27.
28.
29.
30.
murine hydrocephalus in HY-3 and CH mutants. Surg Neurol 1:50-55, 1973 Russell DS: Observations on the Pathology of Hydrocephalus. London, Her Majesty's Stationary Office (Spec Rep Ser, Med Res Council, No 265), 1949 Sahar A, Hochwald GM, Ransohoff J: Alternate pathway for cerebrospinal fluid absorption in animals with experimental obstructive hydrocephalus. Exp Neurol 25:200-206, 1969 Scott DE, Kozlowski GP, Dudley GK: A comparative ultrastructural analysis of the third cerebral ventricle of the North American mink (Mustela vison). Anat Rec 175:155-168, 1973 Sharp P J: Tanycyte and vascular patterns in the basal hypothalamus of Coturnix quail with reference to their possible neuroendocrine significance. Z Zellforsch Mikrosk Anat 127:552-569, 1972 Smart I: The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-H3 injection. J Comp Neurol 116:325-347, 1961 Tennyson VM, Pappas GD: An electron microscopic study of ependymal cells of the
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31.
32.
33.
34.
fetal, early postnatal and adult rabbit. Z Zellfnrsch Mikros Anat 56:595-618, 1962 Weller RO, Shulman K: Infantile hydrocephalus: clinical, histological and ultrastructural study of brain damage. J Neurosnrg 36:255-265, 1972 Weller RO, Wisniewski H: Histological and ultrastructural changes with experimental hydrocephalus in adult rabbits. Brain 92:819-828, 1969 Wisniewski H, Weller RO, Terry RD: Experimental hydrocephalus produced by the subarachnoid infusion of silicone oil. J Neurosurg 31:10-14, 1969 Worthington WC Jr, Cathcart RS 3rd: Ependymal cilia: distribution and activity in the adult human brain. Science 139:221-222, 1963
This work was presented at the annual meeting of the American Association of Neurological Surgeons, in St. Louis, Missouri, April, 1974. Address reprint requests to: Robert B. Page, M.D., M.S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania.
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