Structure and Function of the Choroid Plexus and Other Sites of Cerebrospinal Fluid Formation

Structure and Function of the Choroid Plexus and Other Sites of Cerebrospinal Fluid Formation THOMASH. MILHORAT Departments of Neurosurgery, Children’s Hospital National Medical Center, and George Washington University School of Medicine, Washington, D.C. I. Introduction . . . . . . 11. Choroid Plexus Structure . . . A. Embryology . . . . . B. Gross Anatomy . . . . . C. Microscopic Anatomy . . . D. Ultrastructure . . . . . E. Blood-CSF Bamer . . . . F. Ultracytochemistry . . . . 111. Evidence for Choroid Plexus Secretion. A. Dandy’sThesis . . . . . B. Choroid Plexus Papillomas . . IV. Evidence for Extrachoroidal Secretion . V. Formation of the CSF . . . . A. Chemical Composition . . . B. Brain Extracellular Fluid . . . C. Choroidal Fluid. . . . . . . . D. Leptomeningeal Fluid VI. Summary . . . . . . . References . . . . . .

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I. Introduction

Although the presence of fluid in and around the brain was doubtless noted by the earliest anatomists, the origin of this peculiar watery medium, not to mention its functions, was scarcely questioned until the beginning of the twentieth century. According to Galen, a vaporous substance (the spiritus animalis) and not a fluid, was manufactured within the cerebral ventricles, and to this was ascribed the functions of energy and motion for all parts of the body. So influential was Galenic dogma that it survived for 1000 years, and as late as the sixteenth century, Vesalius (1%3), the first anatomist of the Renaissance, was able to hold this view. Some authorities such as Thomas Willis (1664) came to the conclusion that the choroidal or pineal glands pumped fluid into the cerebral ventricles, but most continued to teach, as von Haller (1760) did, that the cerebral ventricles contained a vapor which condensed after death as water and gravitated to the spaces surrounding the brain and spinal cord. Thus it remained for 225

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Cotugno (1764) to prove beyond question that the cavities of the brain contained a fluid rather than a vapor during life. Following Cotugno’s discovery of the cerebrospinal fluid (or Liquor Cotunnii as he preferred to regard it) almost a century and a half passed before serious attention was again given to the origin and functions of the third great fluid of the body. Not surprisingly, this renewed attention did not originate with anatomists or physiologists, who were more concerned with the localization of function within the brain than with intracranial dynamics, but with clinicians faced with more practical matters such as the treatment of patients with hydrocephalus. It is often remarked that one of the first and most lasting contributions of Harvey Cushing, the father of modern neurosurgery, was his establishment of “The Old Hunterian,” a laboratory for experimental surgery at the Johns Hopkins Hospital. With this modest beginning and without precise goals other than to better understand hydrocephalus, Cushing and his followers-among them S. J. Crowe, James Bordley, Jr., Emil Goetsch, Walter E. Dandy, and Lewis Weed-ventured into the backwaters of cerebrospinal fluid (CSF) physiology. In 1914, Dandy, in conjunction with a pediatrician, Kenneth Blackfan, hit upon an ingenious technique for producing experimental hydrocephalus in dogs. By slipping a pledget of cotton into the aqueduct of Sylvius, these investigators showed that the cerebral ventricles proximal to the block became greatly enlarged and distended with fluid (Dandy and Blackfan, 1914). This finding was soon confirmed by others and could hardly have left doubt that a considerable volume of the CSF was formed within the cerebral ventricles and that the route of circulation was toward the subarachnoid space. During the past decade, the study of the CSF has taken on new dimensions. The development of the electron microscope and the introduction of radiopharmaceuticals into clinical and experimental medicine have greatly increased our knowledge of fundamental brain processes, and we can now discuss in a language unknown to our predecessors the structural basis of the blood-brain barrier, the fine anatomy of the brain interspaces, and the circulatory currents within the CSF cavities. However, if we judge from what is presented in modern classrooms of medicine, students learn little of this, and they are likely to be taught that the CSF is a simple secretion of the choroidal glands having but one purpose in circulating over the brain and spinal cord, namely, to cushion nervous tissue. This view, which denies a truly significant physiological function to the CSF, fails to explain the formation of fluid within the brain interspaces, within the cerebral ventricles following choroid plexectomy, or for that matter within certain pathological cavities lacking a choroid

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plexus. Nor does it explain how the brain, which lacks a lymphatic system of the usual type, removes its products of metabolism. And so, in considering these important questions in the light of modern scientific knowledge, it is to our advantage to take a broad view of the third circulation.

11. Choroid Plexus Structure That a considerable volume of CSF is formed continuously within the cerebral ventricles, and that this fluid flows in bulk toward the subarachnoid space, we may be certain. Yet, more than 3 centuries after William Harvey (1628) transformed medicine with his celebrated studies on the blood, the origin of the CSF remains in doubt. Since the choroid plexuses have been traditionally regarded as the primary source of the CSF, it is appropriate to consider these structures first.

A. EMBRYOLOGY Nature saw fit to provide the nervous system of higher animals with a specialized watery environment and, to contain this medium, she arranged a series of interconnecting fluid-filled chambers which support, surround, and protect the neural elements. In man, the formation of the cerebral ventricles may be said to begin at about 4 weeks, when the primitive neural groove begins to close dorsally. On closure of the anterior neuropore at 5 weeks, the lumen of the newly formed neural tube dilates along two points of constriction, separating the prosencephalon, the mesencephalon, and the rhombencephalon (Kappers et al., 1936). These dilatations then give rise to the bilobed prosencephalic cavity (the lateral ventricles), the mesencephalic cavity (the third ventricle), and the rhombencephalic cavity (the fourth ventricle). It has been emphasized by Kappers (1958) that fluid of some sort is formed within the cerebral ventricles of the human fetus and the fetal pig before the choroid plexus anlage appears. Following closure and segmentation of the neural tube, the choroid plexuses originate in common with the ependymal epithelium from spongioblasts lining the cerebral ventricles (Kappers, 1958; Netsky and Shuangshoti, 1975). In the lateral ventricles, the choroid plexuses arise as a club-shaped primordium from the medial walls of the cerebral hemispheres (Fig. 1).Anteriorly, the choroid plexus of the lateral ventricle develops as an invagination of the multilayered roof plate directly beneath the paraphyseal arch; posteriorly, the plexus extends along a long thin lamina (the so-called area choroidea of His). In the third and fourth ventricles the choroid plexuses arise as simple in-

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FIG. 1. Coronal section of 7-week-old human embryo brain (19 mm in length) showing club-shaped primordium of lateral ventricle choroid plexuses (lower arrow) suspended from developing telencephalon (upper arrow). At this stage, the choroid plexus consists of a vascular mesenchymal stroma covered by a pseudostratified neuroepithelium. From Netsky and Shuangshoti (1975), reproduced by permission of University Press of Virginia.

vaginations of the single-layered roof plate. It is generally agreed that the choroid plexus of the fourth ventricle develops first, followed in turn b y the choroid plexuses of the lateral and third ventricles (Kappers, 1958; Netsky and Shuangshoti, 1975). In man, the choroid plexuses first appear at 6-8 weeks of gestation.

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FIG.2. A 7-week-old human choroid plexus primordium (same brain as in Fig. 1). Note pseudostratified epithelium with brush border and mitotic figures (arrow). Stroma consists of loose mesenchymal tissue, islets of nucleated blood corpuscles, and a few primitive endothelial cells. Hematoxylin and eosin. X485. From Netsky and Shuangshoti (1975),reproduced by permission of University Press of Virginia.

This is the anlage stage. The choroidal epithelium is pseudostratified and surrounds a stroma containing simple mesenchymal tissue (Fig. 2). Thereafter, the epithelial cells rapidly accumulate glycogen (Kappers, 1958), and continuity is established between the choroid plexuses of the lateral and third ventricles. During the anlage stage, the primary role of the mesenchymal stroma is probably hematopoiesis. This function is suggested by the rapid differentiation of stromal cells into angioblasts and hemocytoblasts, so that by the 22-mm stage a vascular endothelium is apparent (Kappers, 1953, 1958). At 8-15 weeks, the choroid plexus epithelium is transformed into a single layer of high cuboidal cells whose cytoplasm is rich in glycogen. At this stage, the choroid plexus occupies most of the lumen of the lateral ventricle and is lobularly shaped. True villi are not yet

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apparent, and mitochondria are seen only infrequently (Kiszely, 1951). The fluid formed within the cerebral ventricles has a high protein content, and its chemical composition is more typical of extracellular fluid than of CSF (Flexner, 1938; Arnhold and Zetterstrom, 1958; Otila, 1948). After the fourth gestational month, the glycogen content of the choroid plexus epithelium is progressively reduced. The cells becomes more cuboidal, true villi and basal infoldings appear, and the mesenchymal stroma is gradually replaced by fibrous connective tissue. In this manner, the size of the plexus decreases, and the ratio of epithelium relative to stroma greatly increases. Although structural evidence of choroid plexus secretion is not evident up to 24 weeks (Kappers, 1958; Kiszely, 1951), it is interesting to point out that congenital hydrocephalus can occur in human fetuses considerably before this time (Milhorat, 1972).

B. GROSSANATOMY When visualized directly at surgery, or viewed through a ventriculoscope, the human choroid plexus appears as a velvety vascular membrane which floats lazily in the CSF. Gross pulsations are not apparent, but the ventricular pulse, reflecting transmitted cardiac and respiratory influences, causes the plexus to bounce to and fro like an anchored boat at sea. When the choroid plexus is excised and fixed in Formalin, it loses its gossamer appearance and becomes rather polypoid and villiform. In man, the telencephalic choroid plexus runs along the floor of the lateral ventricle (Fig. 3) suspended from a vascular invagination of pia mater, the tela choroidea. Beginning at the foramen of Monro, the plexus extends caudally through the body of the ventricle, just medial to the thalamostriate vein. In the trigone area of the ventricle, the plexus enlarges, forming the glomus, and turns anteriorly into the temporal (inferior) horn where it terminates at the tip of that chamber. Choroid plexus tissue is not found in the frontal or occipital horns of man after the fifteenth week of gestation. The blood supply of the telencephalic choroid plexus is primarily derived from the posterior choroidal arteries. These vessels arise from the posterior cerebral artery and distribute as three or four medial branches to the choroid plexus within the body of the lateral ventricle, and four or five lateral branches to the area of the glomus. The anterior choroidal artery, which is a direct branch of the internal carotid artery, enters the choroidal fissure just medial to the uncus of the temporal lobe to supply a variable amount of choroid plexus tissue within the

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FIG.3. Horizontal section of human brain. From Sobotta (1948), reproduced by permission of Urban and Schwarzenberg.

temporal (inferior) horn. Venous drainage of the telencephalic choroid plexus is by route of numerous choroidal veins which empty into the paired internal cerebral veins at the foramina of Monro. These vessels drain to the great vein of Galen (vena cerebralis magna), which in turn drains to the straight sinus, transverse sinuses, and jugular veins. In the third ventricle, the choroid plexus consists of an ependymalined invagination of tela choroidea which hangs down from the roof

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BOUNDARIES OF ClSlERNA MAGNA

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FIG. 4. Drawing of fourth ventricle choroid plexus, human. From Netsky and Shuangshoti (1975), reproduced by pemiission of University Press of Virginia.

of the chamber. The plexus appears as two folds on either side of the midline, which extend from the suprapineal recess to the foramina of Monro. At the foramina1 outlets, the folds fuse with each other and diverge laterally to join the choroid plexuses of the lateral ventricles. The blood supply of the third ventricle choroid plexus is derived from small branches of the superior cerebellar arteries (Larsell, 1953). The choroid plexus of the fourth ventricle, like that of the third ventricle, arises as an invagination of the tela choroidea from the roof of its chamber (Fig. 4).The choroid plexus epithelium overlies this vascular tuft and is continuous with the ependyma lining the walls of the ventricle. The blood supply of the fourth ventricle choroid plexus comes from the posterior inferior cerebellar arteries. In most cases, the choroidal arteries form two vertical strands, on each side of the midline, which join each other just cephalad to the nodule of the vermis and diverge at right angles to enter the foramina of Luschka.

C. MICROSCOPICANATOMY Histologically, the choroid plexus appears as a series of tightly packed villous folds which contain a central core of highly vascularized tissue lined by high cuboidal-low columnar epithelium (Figs. 5 and 6). The nucleus of the choroid plexus epithelial cell is round, centrally located, and associated with one or more nucleoli. On the apical (ventricular) surface of the cell, the plasma membrane is extended as a polypoid or brush border (Kalwaryjski, 1924), which greatly increases the surface area of the epithelium. In many species, cilia are found along the apical border of some cells (Studnicka, 1900),

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FIG.5 . Human choroid plexus consisting of tightly packed villous folds containing a central core of highly vascularized connective tissue. The choroidal villi are lined by high cuboidal-low columnar epithelium. Hematoxylin and eosin. x 160. From Milhorat (IY72), “Hydrocephalus and the Cerebrospinal Fluid.” @ 1972 The Williams i k Wilkins Co., Baltimore, Maryland.

but choroidal cilia become increasingly less frequent in man after infancy (Milhorat, 1972; Netsky and Shuangshoti, 1975). According to Voetmann (1949),the capillaries of the choroid plexus stroma are considerably larger in diameter than capillaries found elsewhere in the body (15 pm as compared to 3 pm). The choroidal arterioles are innervated by unmyelinated fibers, presumably serving a vasomotor function, and a few scattered myelinated nerves which may serve a sensory function (Voetmann, 1949). The nervous supply of the choroid plexus originates from the vagus nerve (Benedikt, 1874), the glossopharyngeal nerve (Stohr, 1922), and the sympathetics of the anterior and posterior choroidal arteries.

D. ULTRASTRUCTURE To date, the fine structure of fetal and adult choroid plexus tissue has been extensively studied in a number of animal species including

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FIG.6. Drawing of choroidal villus. From Millen and Woollam (1962),reproduced by permission of Oxford University Press.

the cat (Maxwell and Pease, 1956), chick (Birge and Doolin, 1965), dog (Shryock and Case, 1956; Wislocki and Ladman, 1958), frog (Maxwell and Pease, 1956; Pontenagel, 1962), pig (Davis et al., 1973), lamprey (Ladman and Roth, 1958), lizard (Murakami, 1961), man (Bargmann and Katritsis, 1966; Dohrmann and Bucy, 1970), monkey (Wislocki and Ladman, 1955), rabbit (Maxwell and Pease, 1956; Millen and Rogers, 1956; Pappas and Tennyson, 1962; Tennyson and Pappas, 1961; Wislocki and Ladman, 1958), rat (Dempsey and Wislocki, 1955; Becker and Sutton, 1963; Cancilla et al., 1966; Maxwell and Pease, 1956; Wislocki and Ladman, 1958), mouse (Dohrmann and Herdson, 1969), opossum (Wislocki and Ladman, 1958), salamander (Carpenter, 1966), toad (Rodriguez, 1967), and woodchuck (Wislocki and Ladman, 1958). With few exceptions, the choroid plexuses of mammals are sufficiently alike so that a species distinction, based on ultrastructural criteria, cannot be made (Davis e t al., 1973). Recent comparative studies of the choroid plexuses from the lateral, third, and fourth ventricles have revealed no significant differences between these structures of different embryological origin (Davis et al., 1973). Transmission electron microscopy reveals choroid plexus tissue to consist of a single layer of epithelial cells in continuity with a subepithelial region containing fibrillar elements (Fig. 7). The stroma con-

FIG.7. Choroid plexus epithelium, immature pig. Note numerous long digitiform microvilli and cilia (solid arrow indicates basal body) extending into the ventricular lumen (L) and a subepithelial region with fibrillar elements (S). The cytoplasm shows numerous mitochondria, an extensive Colgi apparatus (G), lipoid inclusions (open arrow), nuclei, RER, SER, and pinocytotic vesicles. ~ 8 9 2 5 From . Davis et al. (1973), reproduced by permission of Anatomical Record.

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tains numerous capillaries and is rich in alkaline phosphatase (Wislocki and Leduc, 1952). Scattered throughout the stroma are cells of pial origin which are greatly flattened and appear to form a protoplasmic layer between the capillaries and the choroidal epithelium. This protoplasmic “barrier,” however, has been shown by Maxwell and Pease (1956) to be incomplete. The capillaries of the choroid plexus sb-oma are of the fenestrated type (Fig. 8),and their appearance has been compared with the fenestrated capillaries of the kidney glomerulus (Dohrmann, 1970). Whereas there is some question whether these fenestrations are true pores or simply areas of marked cytoplasmic thinning (Millen and Rogers, 1956; Rhodin, 1962), the cells of the choroidal endothelium can be readily distinguished from their counterparts in the brain by the absence of intercellular tight junctions. The fine structure of the choroid plexus epithelial cell is distinctive. The apical surface is extended as numerous digitiform microvilli and, at irregular intervals, tufts of cilia occur with a typical 9 + 2 subfibrillar arrangement (Fig. 7 ) .On the basal surface of the cell, the plasmalemma tends to be extensively infolded (Fig. 9), although this feature is quite variable. The lateral cell membranes of adjoining cells are tortuous, interdigitating, and possess an apical tight junction (Fig. 10). In recent years, scanning electron microscopy (Fig. 11) has provided broad vistas of surface membrane topography, and freezefracture techniques (Figs. 12 and 13) have permitted a detailed examination of tight junctions and the internal aspects of cell membranes. The cytoplasm of the choroid plexus epithelial cell provides few hints concerning its functions. Early electron microscope studies called attention to “secretory granules” or “apical blebs” suggesting apocrine secretion (Millen and Rogers, 1956; Wislocki and Ladman, 1958), but such findings have been subsequently shown to be artifacts (Tennyson and Pappas, 1961). In the immature pig, the cytoplasm of the epithelial cell may be examined to good advantage (Figs. 7 and 14). Smooth endoplasmic reticulum (SER) is present throughout the cell cytoplasm and appears to be a continuous tubular structure. In contrast, the rough endoplasmic reticulum (RER) consists of long cisternae usually concentrated along the apical border. The concentration of these cisternae varies from cell to cell and may reflect differences in cellular activity (Davis et al., 1973). The nucleus of the choroid plexus epithelial cell is usually spherical or lobular in shape (see Fig. 7 ) . The nucleoplasm is delimited by a typical nuclear envelope and often contains dense chromatin matter

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FIG. 8. Choroid plexus capillary, immature pig. Note fenestrations (arrows).

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in the peripheral region. A nucleolus, often containing a pars amorpha, is typically present. The Golgi apparatus is extensive, paranuclear in location, and frequently appears as parallel arrays of smooth-

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FIG.9. Complex interdigitations of basilar region of lateral plasmalemmas of three From Davis et al. (1973), reprochoroid plexus epithelial cells, immature pig. ~20,825. duced by permission of Anatomical Record.

membraned saccules in close proximity to components of the SER. Occasional microtubules, as well as microfilaments, may be evident. Mitochondria and glycogen are randomly distributed throughout the cell cytoplasm (Figs. 7 and 14). The mitochondria range in shape from ovoid to elongated forms and are surrounded by a typical double membrane, the innermost of which is infolded as cristae. The matrix is dense and relatively homogeneous in appearance. Three membrane-bound cytoplasmic inclusions are typically present (Figs. 7 and 14). These include a number of small bristlebordered vesicles (50-70 nm), which appear to originate from pinocytotic pits along the lateral cell membranes and are frequently found in proximity to elements of the SER and the Golgi apparatus. A lesser number of larger vesicles (200-250 nm) are also observed. These vesicles contain dense spherical inclusions or granules and are pre-

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FIG. 10. Apical tight junction, mouse choroid plexus. Intravascularly injected horseradish peroxidase stops at tight junction (arrow). x 100,000. From Brightman and Reese (1969), reproduced by permission of the Journal of Cell Biology.

sumably multivesicular bodies and/or lysosomes. Finally, inclusions thought to contain lipid (200-250 nm) are often present in aggregates or singly in the basal cytoplasm. Because the choroid plexus epithelium has long been regarded as a site of CSF formation, its distinctive morphology has invited comparisons with cells of known secretory or absorptive function. For example, the ultrastructural feature of an apical surface extended as digitiform microvilli is common to cells mainly concerned with absorption; of these, the best known examples are the epithelial cells of the intestinal mucosa and the proximal tubule of the kidney (Pease, 1956; Tennyson and Pappas, 1961). However, the specialization of an infolded basal plasmalemma is associated with cells having either secretory or absorptive functions (Fawcett, 1962), and here we may cite examples such as the epithelium of the avian salt gland, the proximal tu-

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FIG. 11. Scanning electron micrograph of rhesus monkey choroid plexus (lateral ventricle). The apical ends of numerous epithelial cells are seen and possess fine interdigitating microvilli. Note the presence of numerous epiplexus (Kolmer) cells (arrow) Courtesy of Phillip P. Mcwhich are thought to have a phagocytic function. ~1300. Grath.

bule of the kidney, the epithelium of the submaxillary gland, and the epithelium of the ciliary body. Overall, it is apparent that no conclusions can be reached on the basis of such analogies.

E. BLOOD-CSF BARRIER The vertebrate brain, in contrast to other organs, is provided with a specialized circulation which restricts the passage of substances in and out of blood. In most areas of the brain, the cells of the cerebral endothelium are found in close apposition and are joined by pentalaminar tight junctions (zonulae occludentes) which halt the intercellular movement of proteins and other colloidal tracers (Becker et al., 1967, 1968; Bodenheimer and Brightman, 1968; Brightman, 1968; Brightman et aZ., 1970; Milhorat et al., 1973, 1975a; Reese and

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FIG. 12. Freeze-fracture technique showing a tight junction between epithelial cells of the mouse choroid plexus epithelium. Junction consists of six parallel rows of ridges on the inner half of one cell’s membrane and of complementary grooves with attached particles (vertical arrow) on the outer half of the adjoining cell’s membrane. Discontinuities (diagonal arrow) are common in the ridges on the inner half of the membrane. A small cluster of particles (asterisk) might belong to a gap junction. X66,OOO. From Brightman et al. (1975), reproduced by permission of S. Karger.

Brightman, 1968; Reese and Karnovsky, 1967). These junctions form complete circumferential belts and are to be distinguished from plaquelike “gap junctions” found between astrocytes, ependymal cells, and certain neuronal processes (Brightman and Reese, 1967, 1968, 1969). In certain areas of the brain, including the choroid plexus, median eminence, and area postrema, the cells of the cerebral endothelium are not joined by tight junctions and the vessels are “open.” However,

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FIG. 13. Freeze-fracture technique showing epithelium of mouse small intestine. Cells are linked by tight junctions which appear as anatomosing ridges on the inner half of one cell’s membrane and grooves (G) on the outer half of the adjoining cell’s membrane. Only a few discontinuities (arrows) are noted, and the junctions are presumably “tighter” than those of the choroid plexus epithelium. ~41,250.From Brightman et al. (1975),reprinted by permission of S. Karger.

as demonstrated by Reese and Brightman (1968) and Brightman et al. (1970), each of these areas of “functional leakage” is covered by a specialized epithelium, unlike ependyma found elsewhere, which possesses tight junctions between adjoining cells and is capable of halting the intercellular movement of colloidal markers such as ferritin and horseradish peroxidase. When these tracers are injected into the CSF, their distribution forms a “mirror image” of their distribution following intravascular injection. That is, the tracers do not enter the choroid plexus or other structures lined by specialized ependyma, but penetrate the ventricular and pial surfaces of the brain and extend through the extracellular space up to the endothelium of cerebral vessels (Brightman, 1965a,b, 1968). In recent years, studies employing cytochrome c as an electrondense marker have shed new light on the barrier systems of the choroid plexus (Milhorat et al., 1973, 1975a; Davis and Milhorat, 1975). Following intravascular injection, this naturally occurring hemochromogen, which is a considerably smaller protein (MW 13,000; diameter 25-30 A) than ferritin (MW 400,000; diameter 100 A) or horseradish peroxidase (MW 40,000; diameter 50-60 A), is prevented

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FIG. 14. Cytoplasm of two adjoining choroid plexus epithelial cells, immature pig. Note lateral plasmalemmas (lp), numerous mitochondria (m), numerous pinocytotic vesicles (arrows), multivesicular bodies (mvb), Golgi apparatus (G), SER and RER. X 13.000.

from leaving cerebral capillaries (Fig. 15) but passes rapidly out of choroidal capillaries (Fig. 16). Within 2 minutes after intravenous injection, the tracer is evident as an electron-dense reaction product at the following sites within the choroid plexus: within capillary lumina, in the perivascular region surrounding choroidal capillaries, in the extracellular space between epithelial cells (but not beyond the apical tight junction), within invaginations of the basal plasmalemma, and within epithelial cells in pinocytotic pits and vesicles associated with the lateral and basal plasmalemmas (Milhorat et al., 1973).This distribution is associated with an apparent increase in the number of small Golgi-derived vesicles (terminal vesicles) and multivesicular bodies at the lateral extremes of the Golgi cisternae (Davis and Milhorat, 1975). Since it is known that cytochrome c does not enter the CSF up to 9

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FIG.15. Electron micrograph showing the cerebral capillary of a rat 2 minutes after intravascular injection of cytochrome c. Reaction product is confined to the capillary lumen. Uranyl acetate. X4675. From Milhorat et ul. (1975a), reprinted by permission of Journal of Neurosurgery.

hours after intravascular injection (Milhorat et al., 1973), its fate following intravascular injection is of particular interest. Between 10 minutes and 1 hour, the marker is progressively cleared from the tissue interspaces and taken up by intracytoplasmic pinocytotic vesicles, multivesicular bodies, and dense bodies (Fig. 17). Characteristically, pinocytotic vesicles containing reaction product may be seen juxtaposed (suggesting fusion) to multivesicular and dense bodies (Fig. 18), but tracer-laden vesicles do not fuse with the apicaI plasmalemma (Davis and Milhorat, 1975). When the acid phosphatase activity of the choroidal epithelium is examined aAer the intravascular injection of cytochrome c, it is found to be greatly increased and localized at the same intracellular sites as cytochrome c activity (Fig. 19) (Davis and Milhorat, 1975; Milhorat et al., 1975a). This suggests that cytochrome c, and possibly other proteins that penetrate the choroidal stroma, are actively taken up and degraded by the choroidal epithe-

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FIG.16. Electron micrograph showing choroid plexus of a rat 2 minutes after intravascular injection of cytochrome c. Reaction product is apparent in perivascular space (P), extracellular space between epithelial cells, and small intracytoplasmic vesicles . (arrows) adjacent to lateral and basal plasmalemmas. Uranyl acetate. ~ 8 5 0 0 From Milhorat et aZ. (1975a), reprinted by permission ofJournal of Neurosurgery.

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FIG.17. Electron micrograph of epithelial cell of rat choroid plexus 1 hour after intravascular injection of cytochrome c. Reaction product is no longer apparent in extracellular space (arrow) and is largely confined to multivesicular (mvb) and dense (db) bodies. Note that reaction product is not found in relation to the apical plasmalemma. Uranyl acetate. x10,625. From Milhorat et al. (1975a), reprinted by permission of Journal of Neurosurgery.

lium. Since cytochrome c and acid phosphatase activity were not found in relation to the apical plasmalemma, it may be concluded that the pinocytosis of cytochrome c is not a mechanism for transcellular transport, but represents the initial step in lysosomal degradation (heterolysis) of the protein (Davis and Milhorat, 1975; Milhorat et al., 1975a). With the information presented here, it is likely that the following scheme of heterolysis, similar to that established for other tissues (Miller and Palade, 1964; Straus, 1964, 1971; d e Duve and Wattiaux, 1966; Graham and Karnovsky, 1966; Maunsbach, 1966, 1969; Friend and Farquhar, 1967), is responsible for the intracellular absorption and degradation of certain proteins by the choroid plexus: Following filtration by the fenestrated choroidal capillaries, substances are taken up by the epithelial cells via small pinocytotic vesicles which arise in abundance from the lateral and basal plasmalemmas. These vesicles

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then migrate to the Golgi region of the cell. Concurrent with the introduction of exogenous material into the cytoplasm, the production of lysosomal enzymes is triggered, as suggested by the apparent increase in the number of small acid phosphatase-rich vesicles (primary or protolysosomes) and multivesicular bodies (secondary or heterolysosomes) at the lateral extremes of the Golgi cisternae. Multivesicular bodies, which may acquire additional lytic enzymes from terminal vesicles (Friend and Farquhar, 1967), subsequently incorporate exogenous material via fusion with pinocytotic vesicles. The multivesicular bodies then serve as digestive vesicles, progressively condensing their contents and eventually appearing as dense bodies. Although the next stage of degradation is unclear, it is possible that a dense body combines with a multivesicular body or proceeds to degrade its lysosomal enzymes to become a residual body (de Duve and Wattiaux, 1966; Maunsbach, 1966, 1969). Overall, the foregoing heterolytic mechanism may be an important feature of the blood-CSF barrier which prevents the entry of certain substances into the CSF, and subsequently into nervous tissue.

F. ULTRACYTOCHEMISTRY Although histochemical and cytochemical studies of the choroid plexus have attempted to localize enzymes such as carbonic anhydrase (Fisher and Copenhaver, 1959), acid phosphatase (Becker et al., 1960; Becker and Sutton, 1963), alkaline phosphatase (Leduc and Wislocki, 1952), and the important nucleoside phosphatases (Becker et al., 1960; Becker and Sutton, 1963; Cancilla et al., 1966; Torack and Barmett, 1964), these techniques have lacked specificity, and a close correlation between structure and function has not been possible. For interested readers, an excellent review of choroid plexus histochemistry and cytochemistry has been provided by Becker and Sutton (1975). In recent years, the development by Ernst (1972a,b) of an ultracytochemical technique for localizing ouabain-sensitive, potassiumdependent phosphatase activity in secretory epithelia has been an important new advance which has led to certain conclusions concerning the cellular route of active sodium transport in the avian salt gland (Ernst, 1972a,b), the nasal gland of the desert iguana (Ellis and Goertemiller, 1974), the rat cornea (Leuenberger and Novikoff, 1974), and rat renal tubules (Firth, 1974). This method has resolved many of the difficulties inherent in the older Wachstein-Meisel method and its modifications, and is highly specific if appropriate control experiments are performed (Firth, 1974).

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FIG. 18. (A) Ten minutes after intravascularinjection of cytochrome c. The tracer is noted in pinocytotic vesicles (pv) in juxtaposition to multivesicular bodies (mvb,) near the Colgi apparatus (C). ~40,800.(B) Thirty minutes after intravascularinjection of cy-

CHOROID PLEXUS

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FIG. 19. Acid phosphatase activity 10 minutes after intravascular injection of cytochrome c. Reaction product is significantly increased in Golgi cisternae (G), adjacent terminal vesicles, and multivesicular bodies (arrows). x 10,200. From Davis and Milhorat (1975), published by permission of Anatomical Record.

In studies of frog, rabbit, and rat choroid plexus utilizing the Ernst technique, Milhorat et al. (197513) have recently localized Na,KATPase activity along the outer leaflets of the basal and lateral plasmalemmas of choroid plexus epithelial cells, and in the perivascular space apparently bound to collagen fibers (Fig. 20). No reaction product was noted along the apical plasmalemma or along the capillary endothelium, except for occasional pinocytotic pits or vesicles. In other tochrome c. Tracer-ladened pinocytotic vesicle apparently fusing (arrow) with multivesicular body (mvb,) as initial step in degradation of cytochrome c. Note the large number of electron-lucent small vesicles adjacent to the multivesicular body. x41,650. (C) Thirty minutes after intravascular injection of cytochrome c. Tracer-ladened pinocytotic vesicles apparently fusing (arrow) with dense body (d) surrounded by electronlucent small vesicles. x31,450. (D) One hour after intravascular injection of cytochrome c. The tracer is largely confined to early (mvb,) and late ( m v h ) stages of multivesicular bodies and dense bodies (d). ~37,400.From Davis and Milhorat (1975), published by permission of Anatomical Record.

250

THOMAS H. MILHORAT

FIG. 20. Rat choroid plexus epithelium showing Na,K-ATPase activity along the outer leaflets of the lateral and basal plasmalemmas (small arrows). NPPase reaction product is also evident in the perivascular space apparently bound to collagen (lower right-hand corner). Appreciable reaction product was not found along the apical plasmalemma (large arrow) or in endothelial cells of choroidal capillaries (not shown). x 13,090. From Milhorat et al. (1975b), published by permission of Bruin Research.

CHOROID PLEXUS

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experiments, significant reaction product was not observed in the cells of the ventricular ependyma, providing the first evidence of a cytochemical distinction between the choroidal and the ventricular ependyma (T. H . Milhorat, unpublished). These findings were similar and reproducible in the three species studied. Al-though it is tempting to speculate about the functional significance of Na,K-ATPase in choroid plexus tissue, it is appropriate to point out that the localization of sodium pumps along the basolateral membranes is opposite to that predicted by physiological experiments (Wright, 1972), and Quinton et al. (1973), utilizing an autoradiographic technique, have localized a ouabain-sensitive sodium pump to the apical surface of the frog choroid plexus epithelium. Appreciable ouabain binding was not found by the latter investigators along the basolateral membranes. It is of some interest that the localization of Na,K-ATPase along the basolateral membranes of the choroid plexus epithelium is similar to that found in the epithelia of the avian salt gland (Ernst, 1972a,b) and the nasal gland of the desert iguana (Ellis and Goertemiller, 1974). Since both glands produce a sodium-rich effluent, it has been suggested that sodium pumps are oriented to move sodium ions into epithelial cells from basolateral surfaces (Ellis and Goertemiller, 1974). However, in the distal convoluted tubule of the rat, Na,KATPase is also localized along the basolateral membranes (Firth, 1974), and the role of this epithelium in absorbing water rather than secreting sodium is well known. In view of the foregoing, it is apparent that no conclusions can be drawn concerning the functional significance of Na,K-ATPase in choroid plexus epithelium at this time. We may say that the epithelial cell appears ideally suited for the task of transporting fluid and electrolytes, but the direction of such transfers, not to mention the quantitative aspects of secretion, cannot be inferred on a morphological basis. Until more is known about the physiological mechanisms of solvent-solute transport, the significance of Na,K-ATPase activity in secretory epithelia must remain unclear (Milhorat et al., 1975b). 111. Evidence for Choroid Plexus Secretion A. DANDY’S THESIS

Of the various observations advanced as proof that the choroid plexuses secrete the CSF, none has influenced modern thinking more than Dandy’s crucial experiment (1919) concerning the consequences

252

THOMAS H. MILHORAT

of choroid plexectomy. Dandy reported that, if the choroid plexus of one lateral ventricle was removed, and if the foramina of Monro of both lateral ventricles were obstructed, the ventricle containing a choroid plexus would dilate and the ventricle lacking a choroid plexus would collapse. This observation, which was made in a single dog experiment-without a histological study, be it said-led Dandy to conclude that the choroid plexuses were the sole source of the CSF, a view that was accepted without serious criticism for many years. Since the publication of Dandy’s important report, a variety of other data has been adduced to support the view that the choroid plexuses are the primary source of CSF. Cushing (1914,1926a), for example, reported that serous fluid could frequently be seen to collect on the surface of the surgically exposed choroid plexus and, what is more, this exudation could be stopped by placing a silver clip on one of the choroidal arteries. However, as most neurosurgeons are now aware, fluid collects on the exposed surface of any ependyma-lined structure, a point that has been emphasized by Jacobi and Magnus (1925) and others (Milhorat, 1972; Pollay, 1972). Almost certainly, the grossly unphysiological state of the open, evacuated ventricle renders observations such as these invalid (Milhorat, 1972). A similar criticism can be made of experimental studies in which the choroid plexuses are removed from their hydrostatically precise environment and exposed to atmospheric conditions. This applies most especially to two crucial sets of experiments, those by Ames et al. (1964, 1965a,b) and those by Welch (1963),which are regarded as the most convincing arguments in support of Dandy’s thesis. Ames and his co-workers, by directly exposing the choroid plexus of the lateral ventricle of the cat, collected choroidal fluid under pantopaque oil using a micropipet technique. In a series of articles dealing with the microchemical analysis of the fluid so collected, these investigators reported that the electrolyte composition of choroidal fluid was sufficiently different from that of a plasma ultrafiltrate to suggest that it is formed as a characteristic secretion. Although these findings have been widely cited as conclusive evidence of choroid plexus secretion (Cserr, 1971; Davson, 1967; Dohrmann, 1970), it is appropriate to point out that, in addition to the unphysiological conditions imposed by directly exposing the choroid plexus, the technique of collecting choroidal fluid under oil is questionable, since it has been recently shown that pantopaque is a toxic agent which produces acute ventriculitis and inflammatory changes in the choroid plexus following intraventricular administration (Clark et al., 1971). In Welch’s important experiments, the choroid plexus of the lateral

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ventricle of the rabbit was directly exposed and its choroidal vein was cannulated. By determining that the hematocrit of choroid plexus venous blood was 1.15 times that of systemic arterial blood, and by computing this value with the estimated arterial blood flow through the choroid plexus (determined by cinematographically measuring the rate of transit of systemically injected oil particles), Welch came up with a secretion rate of 0.37 pl per minute per gram of choroid plexus tissue, or approximately 8 p1 per minute for the choroid plexuses of all four ventricles (Welch, 1963). Since this value is very close to the total estimated rate of CSF formation in the rabbit (10.1pl per minute) (Bradbury and Davson, 1964), Welch‘s findings have been advanced as evidence that most or all of the CSF is formed by the choroid plexuses (Davson, 1967). However, as Cserr (1971) has emphasized, there are large errors inherent in Welch’s technique for estimating choroidal blood flow, and his assumption that the hematocrits of aortic and choroidal blood are equal is probably unjustified. Welch also arrived at important conclusions concerning the effects of Diamox on choroid plexus secretion. By administering the drug intravenously, or by applying it topically to the choroid plexus, Welch found almost complete inhibition of choroid plexus secretion as determined by the arterial-venous hematocrit technique (Welch, 1963). This finding raises an interesting paradox, since it is known that Diamox transiently reduces the rate of CSF formation by a maximum of 50% (Oppelt et al., 1964; Pollay and Davson, 1963), and it could be just as well argued that Welch’s findings indicate that 50%of the CSF is formed extrachoroidally (Milhorat, 1972). Overall, we can see that great caution must be exercised in assessing the results of experiments on the exposed choroid plexus. B. CHOROIDPLEXUSPAPILLOMAS The proposition that papillary tumors of the choroid plexus may be associated with overproduction of the CSF has been widely held for almost a century. This view is supported by the apparent secretory function of normal choroid plexus tissue, by the frequent association of choroid plexus papillomas and hydrocephalus, and by reports of regression of hydrocephalus following extirpation of these tumors (Kahn and Luros, 1952; Wilkins and Rutledge, 1961; Matson and Crofton, 1960). However, it is important to point out that choroid plexus papillomas may be encountered as incidental findings in patients without hydrocephalus (Milhorat, 1972; Zulch, 1956), and that claims of regression of hydrocephalus following total tumor removal can be countered by claims to the contrary (McDonald, 1969).

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Of the various clinical reports of CSF overproduction in patients with choroid plexus papillomas, one of the most persuasive is the case reported by Ray and Peck (1956). In this account, a 3-month-old infant with progressive hydrocephalus became severely dehydrated and excreted large volumes of urine following a ventriculoureteral shunt. When it became apparent that the patient would require daily infusions of half-normal saline in order to maintain adequate hydration, the ventriculoureteral shunt was transplanted to the peritoneal cavity. This was followed by the development of massive ascites requiring repeated paracenteses. Eventually, it was decided that the only therapeutic measure left was cauterization of the choroid plexuses. Bilateral operations, staged 12 days apart, revealed a large choroid plexus papilloma in the trigone area of each lateral ventricle. Both tumors were removed, but the patient died after the second operation, preventing any conclusions about the effects of surgery. From time to time, attempts have been made to estimate directly the rate of CSF formation in patients with choroid plexus papillomas. In 1908, Vigouroux described a patient with a papillary tumor of the fourth ventricle choroid plexus and CSF rhinorrhea, whose estimated rate of nasal drainage was about 800 ml per day. Johnson (1958) measured ventricular drainage in another patient with a fourth-ventricle papilloma, and estimated a formation rate of 45 ml per hour (1080 ml per day) at a drainage pressure of 450 mm H20. Fairburn (1960),measuring ventricular drainage in an infant with a lateral ventricle papilloma, collected 500, 400, and 950 ml per day on three consecutive days at a drainage pressure of 50 mm HzO. Whereas these data have been cited as evidence of CSF overproduction, it has been recently pointed out that such rates are not appreciably greater than those measured by ventricular perfusion techniques in patients with unobstructed CSF pathways (Eisenberg et al., 1974). To date, the more accurate technique of ventricular perfusion has been applied to the study of CSF formation in only two patients with verified papillomas of the choroid plexus. The first, a 5-month-old infant studied by Eisenberg et al. (1974), was suspected of having a choroid plexus papilloma after two apparently functioning ventricular shunts failed to control hydrocephalus. Cerebral arteriography confirmed the presence of a large tumor within the left lateral ventricle, and a ventriculolumbar perfusion was subsequently performed. Although these investigators reported an excessive rate of CSF formation (1.4 ml per minute), the case is far from convincing, since the perfusion was performed in an unorthodox manner (see Milhorat et al., 1976a) and there was no postoperative study to prove that the rate of CSF formation was actually reduced by removal of the tumor.

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FIG.21. Computerized axial tomogram (EM1 scan) demonstrates lobulated tumor within trigone of left lateral ventricle, generalized enlargement of both lateral ventricles, and normal-appearing cerebral subarachnoid space. The area of decreased density within the midportion of the tumor proved to be a central core of blood vessels and stroma when the tumor was removed and sectioned. From Milhorat et al. (1976a),published by permission of Child's Brain.

Recently, a more definitive study of a patient with a choroid plexus papilloma has been reported by Milhorat et al. (1976a). This case concerns a 2-year-old boy who presented with a large head and a history of recurring episodes of vomiting and opisthotonic posturing. The correct diagnosis, choroid plexus papilloma of the left lateral ventricle, was strongly suggested by the preoperative workup (Fig. 21). Before and after surgery, the rate of CSF formation was determined by a ventriculolumbar perfusion technique that has been successfully employed in the study of CSF formation rates in human subjects with hydrocephalus (Lorenzo et al., 1970), brain tumors (Rubin et al., 1966; Cutler et al., 1968), and unobstructed CSF pathways (Rubin et al., 1966; Cutler et al., 1968). The tumor was totally excised without event

256

THOMAS H. MILHORAT

FIG. 22. Excised tumor. Wet weight 74 gm. Arrow points to vascular pedicle. From Milhorat et al. (1976a), published by permission of Child’s Bruin.

and weighed 74 gm (Fig. 22). Light microscopy confirmed the diagnosis of a choroid plexus papilloma (Fig. 23). As shown in Fig. 24, the preoperative and postoperative perfusions achieved a stable steady state within 2 hours. The calculated rate of CSF formation before surgery was 1.05 f 0.01 S.D. ml per minute (1656 ml per day). Postoperatively, the CSF formation rate was reduced to 0.20 0.01 S.D. ml per minute (288 ml per day). The latter value falls within the range of normal CSF formation rates as determined by ventriculolumbar perfusion (0.15 to 0.57 ml per minute) (Cutler et al., 1968). Based on the wet weight of the tumor (74 gm), a secretion rate of 0.01 ml per minute per gram of tissue was calculated. The finding of a fivefold decrease in the rate of CSF formation following removal of a 74-gm choroid plexus papilloma would appear to provide conclusive evidence of CSF overproduction by this tumor (Milhorat et al., 1976a,b). Less certain are the quantitative aspects of hypersecretion. It is important to emphasize that, although the results

*

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257

FIG.23. Photomicrograph demonstrates typical morphology of choroid plexus papilloma. Hematoxylin and eosin. ~ 5 4 From . Milhorat et al. (1976a), published by permission of Child's Brain.

of ventricular perfusion studies are highly reproducible, the absolute rates of formation calculated by this technique are slightly elevated, owing to small losses of marker into the brain and choroid plexus (Milhorat, 1972). Davson et d.(1962) estimated this error to be about 4%, but Curran et al. (1970) have suggested a correction factor of 20%. Since it has been recently shown that normal choroid plexus tissue is capable of taking up and heterolytically digesting proteins injected into the blood or CSF (Davis and Milhorat, 1975), it is possible that, in this case, significant losses of a l b ~ m i n - ' ~into l I the tumor resulted in a spuriously high estimate of CSF overproduction. Clearly, no conclusions can be reached at this time concerning the mechanisms by which choroid plexus papillomas produce fluid. It is interesting to note that a detailed chemical analysis of ventricular and lumbar CSF in this case revealed no differences from normal CSF (Milhorat et al., 1976a). This suggests that the tumor was equipped with an intact blood-CSF barrier, and that the mechanism by which it formed its secretory product was similar, if not identical, to that in-

258

THOMAS H. MILHORAT

Time in Minutes

n

E60 X

g m 8 .

040

'5 30

3

520 10

0

60

120

180 Time in Minutes

240

300

360

FIG.24. Preoperative(top) and postoperative (bottom) graphs of ventriculolumbar perfusion showing counts of albumin-'"'I in influent (c,) and effluent (co) as a function of time. Perfusion rate: 2.29 cc per minute, From Milhorat et ul. (1976a), published by permission of Child's Bruin.

volved in normal CSF formation. These points were supported b y the following morphological data. A detailed ultrastructural study of the tumor revealed features typical of normal mammalian choroid plexus tissue (Milhorat et al., 1976b) (Figs. 25-28). These included: (1)a single layer of high cuboidal cells contiguous with a subepithelial region containing collagen fibers and

FIG. 25. Epithelium of choroid plexus papilloma demonstrating single layer of high cuboidal cells containing lobulated nuclei. The apical plasmalemma is extended as numerous digitiform microvilli. Occasional cilia are present (arrow). The cells are joined by an apical tight junction (inset). Numerous mitochondria, lysosomes, and granular endoplasmic reticulum are present within the cell cytoplasm. The basal plasmalemma is contiguous with the perivascular space (PV). x 10,200.Inset, x52,700. From Milhorat et al. (1976b), published by permission of Child’s Bruin.

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THOMAS H. MILHORAT

FIG.26. Subepithelial region of choroid plexus papilloma demonstrating irregular basal plasmalemma (large arrows) continguoiis with the perivascular space (PV) containing collagen and a choroidal capillary. The endothelium of the capillary is fenestrated (small arrows). Several red blood cells are present within the capillary lumen. x16,OOO. From Milhorat et al. (1976b), published by permission of Child’s Bruin.

FIG. 27. Cytoplasm of epithelial cell demonstrating the Golgi apparatus ( G ) , numerous mitochondria (M), granular endoplasmic reticulum (ER), irregularly shaped lysosomes (L), and a portion ofa nucleus (N). x 19,550. From Milhoratet al. (1967b), published by permission of Child’s Bruin.

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THOMAS H. MILHORAT

FIG.28. (A) Cytoplasm of epithelial cell demonstrating a large Golgi apparatus (G) in continuity with irregularly shaped lysosomal elements (L). X33,150. (B) Lateral plasmalemmas of adjacent epithelial cells. Note maculae adherens (desmosomes) at irregular intervals (large arrows). Cytoplasm contains mitochondria, multivesicular bodies, microfilaments (small arrows), and a portion of a nucleus (N). X34,850. From Milhorat et al. (1976b), published by permission of Child’s Brain.

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blood vessels; (2) an apical surface extended as numerous digitifonn microvilli with scant but definite cilia; (3) lateral cell membranes which were tortuous, interdigitating, and joined by an apical tight junction; (4) a cytoplasmic organelle profile consisting of numerous mitochondria, large aggregates of glycogen, well-developed granular endoplasmic reticulum, Golgi apparatus, vesicles appearing as primary and secondary lysosomes, and lipid droplets; (5)a round to oval lobulated nucleus with one or two nucleoli; and (6) choroidal capillaries of the fenestrated type. It is of some interest that the finding of large aggregates of cytoplasmic glycogen has been noted in other choroid plexus papillomas arising in early life (Carter et al., 1972) and is a characteristic feature of fetal and immature choroid plexus tissue (Kappers, 1958). In a corollary study, ouabain-sensitive, potassium-dependent phosphatase activity was localized in the tumor by the Ernst method (Milhorat et al., 197613). As shown in Fig. 29, Na,K-ATPase was localized along the basolateral membranes of the tumor epithelium but not along the apical plasmalemma. This localization is similar to that found in normal choroid plexus epithelium (see Fig. 20) and is strong evidence that well-differentiated papillomas are capable of transporting fluid and electrolytes by a mechanism similar to that employed by normal choroid plexus tissue (Milhorat et al., 1976a,b). Finally, a word may be said about the pathogenesis of generalized ventricular enlargement in patients with papillomas of the lateral ventricles. Although this finding has been traditionally ascribed to overproduction of the CSF, Russell (1949), and subsequently van Hoytema (1956), have focused attention on a distal obstruction of the subarachnoid pathways. Supporting this view is the following evidence. (1) Spontaneous subarachnoid hemorrhage is a recognized complication of choroid plexus papillomas (Ernsting, 1955; Russell and Rubinstein, 1959); (2) at the time of clinical presentation, many patients with choroid plexus papillomas have xanthochromic CSF and elevated CSF protein (Matson, 1969; Laurence et al., 1961); (3) pneumoencephalography frequently demonstrates obstruction of the basilar cisternae and subarachnoid space (Kahn and Luros, 1952; McDonald, 1969; Laurence e t al., 1961); and (4)chronic inflammation of the leptomeninges is a common finding at autopsy in patients with choroid plexus papillomas (Russell, 1949; Lawrence e t al., 1961). These observations, taken together with evidence that choroid plexus papillomas may arise in the absence of hydrocephalus (Milhorat, 1972; Zulch, 1956), have suggested that oversecretion of the CSF alone may be insufficient to produce hydrocephalus when

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THOMAS H. MILHORAT

FIG. 29. Left: NPPase reaction product along lateral and basal plasmalemmas of three adjacent epithelial cells (E). No reaction is noted along the apical plasmalemmas (arrows). x 8500. Right: Higher magnification reveals reaction product to be localized to the outer leaflets of the lateral plasmalemmas. The apical surfaces of two adjacent epithelial cells are devoid of reaction product (arrows). x20,OOO. From Milhorat et ul. (1976b), published by permission of Child’s Bruin.

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the mechanisms for CSF drainage and absorption are normal (Milhorat, 1972; Milhorat et d., 1967a).

IV. Evidence for Extrachoroidal Secretion Until recent years, evidence of CSF formation at sites other than the choroid plexuses has been less than convincing. Hassin (1924) considered this question in depth and was the first to repeat Dandy’s important experiment in which a plexectomized ventricular system was secondarily obstructed. Unable to confirm Dandy’s finding that the plexectomized ventricle collapsed, Hassin and his associates (1937) concluded that the brain parenchyma and not the choroid plexus was the main source of the CSF. Among the indirect evidence consistent with this view is the observation that fluid of some type is formed within the neural tube of fetal animals (Weed, 1917) and man (Kappers, 1958) before the choroid plexus anlage appears, and the observation that CSF is formed within the ventricular cavities of some lower vertebrates lacking a choroid plexus (Kappers, 1958; Cserr, 1971). More direct evidence of an extrachoroidal source of CSF has been provided by experiments in which areas of ependyma or pia have been technically isolated from choroid plexus tissue and subsequently perfused according to the inulin dilution technique of Pappenheimer et al. (1962). Pollay and Curl (1967), for example, by perfusing the isolated aqueduct of Sylvius in rabbits, have calculated that approximately 30% of the CSF is secreted by the ventricular ependyma and that this value can be cut in half by the systemic administration of Diamox. Sonnenberg et al. (1967), by perfusing the central canal of the cat spinal cord, have arrived at an even higher estimation of the contribution by the ependyma, and Sat0 and Bering (1967) have reported that at least 40% of the CSF in dogs is formed within the subarachnoid space. Unfortunately, all of these studies involved rather drastic experimental procedures [e.g., removal of the cerebral hemispheres (Pollay and Curl, 1967), transection of the spinal cord (Sonnenberg et al., 1967), injection of kaolin into the subarachnoid space (Sat0 and Bering, 1967)], so that we must reserve judgment concerning the validity of these findings. Of the various tests of Dandy’s thesis that the choroid plexuses are the sole source of the CSF, perhaps none has been as damaging as the failure of choroid plexectomy to cure hydrocephalus. This operation, which was introduced by Dandy in 1918, was for many years the most popular form of treatment for infantile hydrocephalus in the United States. As experience with the procedure increased, however, it be-

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came clear that bilateral extirpation and/or cauterization of the choroid plexuses invariably failed to benefit patients with noncommunicating hydrocephalus (Dandy, 1932), and was only occasionally successful in cases of communicating hydrocephalus, most often when the condition was arrested or nearly so (Matson, 1969; Milhorat, 1971, 1972, 1974). Most perplexing was the observation that, in the majority of patients with chronic hydrocephalus, the cerebral ventricles continued to enlarge at a rate equal to or sometimes greater than that recorded preoperatively (Milhorat, 1974). In the 1950s, because of universally poor results, choroid plexectomy was abandoned by most neurosurgeons as a treatment for hydrocephalus. The failure of choroid plexectomy to cure or at least ameliorate progressive hydrocephalus has important physiological implications and has been a subject of continuing study in our laboratories for a number of years. To examine this question, we first developed a technique for removing the choroid plexuses of rhesus monkeys (Milhorat, 1969). After a suitable period of convalescence, never less than 3 months, the animals were subjected to a variety of studies. In 43 animals undergoing bilateral excision of the lateral ventricle choroid plexuses, for example, the fourth ventricle was obstructed with an inflatable balloon (Fig. 30). The findings were as follows. (1) All animals became hydrocephalic; (2) so-called ventricular collapse did not occur, except in cases in which the surgical technique had been excessive (in these cases, a dense fibroglial scar was found along the course of the stripped choroid plexus and obliteration of part or all of the ventricular cavity was found to result from adhesions binding the floor and roof of the chamber); and (3)the rate and degree of ventricular enlargement were found to be only slightly less marked than that occurring in control animals undergoing similar obstructions. To exclude the possibility that the hydrocephalus observed in the foregoing cases was secondary to fluid secreted by the remaining choroid plexus tissue in the third ventricle, the classic experiment of Dandy, namely, obstruction of the foramen of Monro of one plexectomized lateral ventricle, was performed on 19 animals. In these cases, the ipsilateral lateral ventricle became markedly dilated within 3 weeks (Fig. 31). This indicates that hydrocephalus can occur rapidly and progressively in the plexectomized ventricular system and that the choroid plexus is not essential either as a source of ventricular fluid or as a pulsatile mechanism for expanding the ventricles (Milhorat, 1969). In order to obtain a more quantitative estimate of the rate of CSF formation following choroid plexectomy, ventricular perfusion studies

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FIG.30. Ventricular enlargement occurring 5 days after complete obstruction of the fourth ventricle in a bilaterally choroid plexectomized rhesus monkey. The excised plexuses from the lateral ventricles are shown beneath the specimen. Milhorat (1969), published by permission of Science. Copyright 1969 by the American Association for the Advancement of Science.

employing 14C-labeled inulin were performed on a large series of animals by a lateral ventricle-to-lateral ventricle perfusion technique with the fourth ventricle obstructed (Milhorat et al., 1971). When the rates of CSF formation within the lateral ventricles, third ventricle, and aqueduct of Sylvius were compared in control and bilaterally plexectomized animals, the rates in the latter group (13.3 pl per minute) were found to average about 70% of normal (19.2 pl per minute). When corrections were made for the remaining choroid plexus tissue in the third ventricle, the total rate of extrachoroidal fluid formation in the chambers rostra1 to the fourth ventricle was calculated to be about 60% of normal. I t should be emphasized that this value does not necessarily represent a normal rate of extrachoroidal ventricular fluid formation, any more than the output of urine by a solitary kidney indicates the normal output by one kidney when the other is present and functioning (Milhorat, 1975). However, in these and other studies (Bering, 1966; Milhorat, 1969; Hammock and Milho-

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THOMAS H. MILHORAT

FIG.31. Obstruction of right foramen of Monro (arrow) in a rhesus monkey with a previous right choroid plexectomy. The excised plexus is shown beneath the specimen. Note ipsilateral enlargement of the temporal horn. The hydrocephalus is of 3 weeks’ duration. Milhorat (1969), published by permission of Science. Copyright 1969 by the American Association for the Advancement of Science.

rat, 1973; Milhorat et al., 1976c), choroid plexectomy was not found to alter the chemical composition of the CSF. This indicates, at least, that the CSF formed by the plexectomized ventricular system is not a pathological exudate and that sites other than the choroid plexuses can elaborate a fluid whose composition of water, electrolytes, and protein is the same as that of normal CSF. From a clinical standpoint, the formation of extrachoroidal CSF is of considerable importance, since it provides a logical explanation for the frequent failure of choroid plexectomy as a treatment for hydrocephalus (Milhorat, 1969,1971,1974,1975). Recently, the rate of CSF formation has been measured in a human following choroid plexectomy (Milhorat et al., 1976~).This patient, a 5-year-old child with communicating hydrocephalus, had undergone bilateral excision of the lateral ventricle choroid plexuses during infancy, and at the time of the study (ventriculolumbar perfusion) was suffering from a failed

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ventriculoperitoneal shunt and progressive ventriculomegaly. The calculated rate of CSF formation, 0.355 0.02 S.D. ml per minute (504 ml per day) was within normal limits for patients with unobstructed 1966) and was slightly CSF pathways (Cutler et al., 1968; Rubin et d., higher than the mean rate of 0.30 f 0.02 S.D. ml per minute for patients with hydrocephalus (Lorenzo et d.,1970). The chemical composition of the CSF, except for a slightly elevated total protein, was normal. This suggests that, over prolonged periods of time, compensatory secretion from extrachoroidal sites and/or residual choroid plexus tissue may eventually reestablish normal intracranial dynamics (Milhorat et d,1976~).Taken together, the foregoing data reaffirm the view that choroid plexectomy is an operation of historic interest only and has no place in the current treatment of hydrocephalus (Milhorat, 1972). Finally, although the exact contributions of fluid from choroidal and extrachoroidal sites remain to be determined, data concerning the transport of 24Nafrom the blood to the CSF in control and plexectomized animals provide interesting hints. For example, in both groups of animals, intravenously infused 24Naenters the CSF promptly and reaches appreciable concentrations in the steady state (Fig. 32). In the plexectomized group, however, the isotope enters the CSF less rap-

10

-

c

NDNPLEXECTOMIZEDOROUP

(CSFPmductlon: 10.2pl/mlnfSD2.2 )

8 -

1

4 (

PLEXECTOYIZED ORWP

CSF Pmduction:W.3pl/mhfSD2X I

TIME IN MINUTES

FIG. 32. Concentration curves of %Nain CSF of normal and choroid plexectomized rhesus monkeys during a 3-hour steady-state intravenous infusion of “Na. Milhorat et al. (1971), published by permission of Science. Copyright 1971 by the American Association for the Advancement of Science.

270

THOMAS H. MILHORAT nonplexectomised group

FIG.33. Distribution of UNa in brain and CSF of rhesus monkeys at conclusion of a 3-hour steady-state intravenous infusion of 24Na.The radioactivity is given in counts per minute per milligram. There was no difference in the intracerebral distribution of %Na in normal and choroid plexectomized animals. Milhorat et al. (1971),published by permission of Science. Copyright 1971 by the American Association for the Advancement of Science.

idly and never reaches the same concentrations as the control group. As might be expected, these differences are proportional to the reduced rate of CSF formation (Milhorat et al., 1971). When the intracerebral distribution of intravenously administered 24Nais examined, furthermore, the brain tracer profiles are found to be identical in the control and plexectomized groups, with a markedly greater concentration of 24Nain two areas of the brain (Fig. 33): (1)in the grey matter surrounding the cerebral ventricles, and (2) in the grey matter surrounding the subarachnoid space. In view of the established agreement between the rate of CSF formation and the rate of 24Naentry into the CSF,these data have suggested that a major fraction of the CSF is formed within the cerebral parenchyma (Milhorat et al., 1971; Milhorat, 1972; Hammock and Milhorat, 1973).

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V. Formation of the CSF A. CHEMICALCOMPOSITION

In terms of appearance, specific gravity, and chemical composition, CSF closely resembles an ultrafiltrate of plasma (Table I), and it is mainly for this reason that Mestrezat (1912), and subsequently Foley (1923) and Fremont-Smith (1927), concluded that the CSF is formed by a process of simple filtration from the blood plasma. In 1938, how-

COMPOSITION OF

TABLE I CSF AND BLOOD PLASMA

Measurement

IN

CSF average

MAN"

Blood plasma average ~

Specific gravity Total solids (gml100 ml) Water content (gm/100 ml) Reducing substances (mg/100 ml) (as glucose) Glucose (as glucose) Nonglucose (as glucose) Sodium (meqlliter) Potassium (meqlliter) Calcium (meqlliter) Magnesium (meqlliter) Total base (meq/liter) Chloride (meqlliter) Bicarbonate (meqlliter) Phosphate (mM P/liter) Lactate (meqlliter) Nonprotein nitrogen (mg N/100 ml) Urea Uric acid Amino acids Creatinine Cholesterol (mgl100 ml) Protein (mg/100 ml) Albumin Globulin Fibrinogen Protein (mgl100 ml) Ventricular fluid Cistemal fluid Lumbar fluid ~

'I

Modified from Holmes and Tower. 1955.

1.0075 1.0 99.0 65.0 61.0 4.0 141 3.3 2.5 2.4 155 124 21 0.48 1.7 19 14 0.6 1.6 4 0.14 28 23 5

0

5-15 15-25 15-45

1.025 8.7 91.3 98.0 92.0 6.0 137 4.9 5.0 1.64 162 101 23 1.3 1.7 27 14 1.6 5 6 160 7000 4430 2270 300

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ever, Flexner compared the chemical composition of plasma and CSF and found that CSF contained higher concentrations of magnesium and chloride ions and lower concentrations of glucose, proteins, amino acids, uric acid, calcium, phosphate, and potassium ions. These disparities, and the energy calculated to create them, suggested to Flexner that the CSF could not be formed entirely as an ultrafiltrate of the plasma and that active processes must somehow be involved. In recent years this view has been put on firmer ground by Davson (1967), who has shown that the chemical composition of the CSF is in fact different from a dialyzate of plasma, the latter being defined as the product formed by allowing plasma in a sealed collodion sac to come into equilibrium with a surrounding solution, usually a mixture of sodium chloride and bicarbonate at physiological pH. Since the CSF can be shown to have a characteristic composition that differs quantitatively from a plasma dialyzate, it is correct to define it as a secretion. In describing the CSF as a secretion it is important to emphasize that we are using the modern definition of the term, the classic definition of a secretion being quite different, signifying a synthetic product of a gland as, for example, the secretory juices of the parotid, pancreas, and stomach. Classic secretions are therefore inherently different from the CSF. Their composition bears little resemblance to plasma, and they are formed by the selective uptake of materials from the blood and by the addition of substances specifically elaborated by secretory cells. The modern definition of a secretion is considerably broader. It includes any product of the body whose formation depends on the expenditure of energy-that is, active transport-and it is in this context that we speak of the CSF as a secretion. In discussing the formation of the CSF, it is necessary to discuss not only the formation of fluid within the cerebral ventricles and subarachnoid space, but also within the extracellular compartment of the nervous parenchyma. This is apparent, since it has been recently established that the interspaces of the brain and spinal cord are generally patent and that they communicate freely with the surrounding CSF cavities. Brightman (1965a,b, 1968), for example, has shown that, when proteins such as ferritin or horseradish peroxidase are injected into the CSF, these large markers move with relative ease between ependymal and pial cells to distribute widely throughout the brain extracellular space. Physiological studies have further clarified the continuous relationship of the interstitial and CSF compartments for molecules as large as inulin (Levin and Fenstermacher, 1969; Rall et al., 1962), so that we may conclude that, in specific regions of the CSF system, the chemical compositions of the extracellular fluid and CSF

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are very similar or identical (Cserr, 1971; Davson, 1967; Milhorat, 1975). If CSF is withdrawn from the cerebral ventricles, cisterna magna, and subarachnoid space, significant variations in chemical composition are encountered. The concentration of potassium ion, for example, decreases steadily as fluid passes from the cerebral ventricles to the subarachnoid space (Bito and Davson, 1966). The opposite holds true for the concentrations of urea, albumin, and globulin (Davson, 1969). Recently, Franklin et al. (1975) have demonstrated regional variations in the concentrations of a number of amino acids, including arginine, serine, glycine, lysine, alanine, taurine, and glutamine. On the basis of such findings, it is obvious that sites in addition to the choroid plexuses are involved with the transport of solutes into and out of the CSF (Franklin et al., 1975). Having defined CSF as a secretion, we come to the question of how this medium, which is virtually protein-free, is separated from the parent plasma. A popular hypothesis is that CSF is formed as a specific secretion. Davson (1967, 1969) has postulated that the active transport of certain solutes, for example, sodium ion, is followed by the passive diffusion of water which then produces an approximately isosmolar fluid. This hypothesis assumes that there is a carriermediated transport of solutes across a highly selective membrane, the solutes being attached to hypothetical carriers on the blood side of the system and detached on the CSF side. To the extent that the concentrations of certain constituents of the CSF (e.g., potassium, calcium, and magnesium ions) are relatively independent of their concentrations in plasma, this hypothesis seems quite reasonable. However, we must ask why any organ that possesses a highpressure capillary circulation would elect to form its fluids-and here we include the brain extracellular fluid as well as the fluid within the CSF cavities-by the sole process of active transport. Davson (1967) has argued that the CSF cannot possibly be filtered to any appreciable extent, since it can be shown, based on the assumption that the plasma and brain fluids are separated by a highly selective membrane, that the pressure necessary to maintain the differences in composition between the two media, when added to the colloid osmotic pressure of plasma, is well above the theoretical filtration pressure of cerebral capillaries. The weakness in this line of reasoning, however, is that it assumes that there is a “highly selective membrane” which separates the plasma and brain fluids. As we shall see, this assumption has no basis in fact, a point that can be better appreciated when we consider the following.

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B. BRAIN EXTRACELLULAR FLUID From a morphological standpoint, the interface between the plasma and brain extracellular fluid is formed by a single layer of cells, the capillary endothelium, which is surrounded by a perivascular investment of astrocytes. In most areas of the brain, the cells of the cerebral endothelium are joined by pentalaminar tight junctions (zonulae occludentes) which form complete circumferential belts capable of restricting the intercellular movement of proteins and other colloidal markers having a diameter of 20 A or more (Bodenheimer and Brightman, 1968; Feder et al., 1969; Reese and Karnovsky, 1967). Whereas the cerebral endothelium is unquestionably impermeable to large molecules, it is nonetheless permeable to urea (Pappius et al., 1967), a variety of lipid-soluble substances (Katzman and Pappius, 1973), and a number of small nonelectrolytes (Crone, 1965). Indeed, on the basis of currently available morphological and physiological data, it is estimated that the endothelial tight junctions are perforated by aqueous channels having a diameter of approximately 8 A (Fenstermacher and Johnson, 1966; Brightman and Reese, 1969; Milhorat et al., 1975a). Although it has been suggested that the cerebral endothelium may be directly involved with the active transport of ions and other small molecules between the blood and brain extracellular fluid, evidence for this has been unconvincing or contradictory. For example, it is a clinical fact that when the plasma sodium level falls below 120 meq per liter, human subjects become obtunded and develop signs of a nonspecific metabolic encephalopathy (Plum and Posner, 1966). Since an energy-dependent system should be expected to protect the brain from such a small percentage decrease in serum sodium concentration, it is unlikely that active transport is the principal mechanism by which this ion is separated from the plasma and carried into the brain extracellular fluid. In support of this view, Torack and Barrnett (1964), utilizing the Wachstein-Meisel method for localizing nucleoside phosphatases, reported little or no activity in the endothelial cells of rat cerebral capillaries. However, enzymic activity resulting in the hydrolysis of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, cytidine triphosphate, inosine diphosphate, and guanosine triphosphate was localized to the capillary basement membrane and glial end-foot processes surrounding the capillary endothelium. If such findings are confirmed by the more specific ultracytochemical techniques currently available, the absence of

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phosphatidic activity within endothelial cells will provide a strong case against the active secretion of sodium ion (and consequently water) by the cerebral endothelium. But perhaps the major argument against the cerebral endothelium as a highly selective membrane across which the brain extracellular fluid is actively secreted is the enormous and unnecessary expenditure of energy that this would entail. Recently, Pappenheimer (1970) has questioned the ability of the cerebral endothelium to support metabolically the active transport of all the many substances known to enter the brain, and, based on a detailed study of the active transport of bicarbonate ion by the glia, he has proposed a model for the blood-brain barrier that depends on the passive permeability of the cerebral endothelium and the active transport of substances by the surrounding layer of astrocytes. In this model the cerebral endothelium restricts the diffusion of large molecules but allows the diffusion of small molecules including ions, glucose, and amino acids through the intercellular tight junctions. The metabolic energy for the system is then provided by astrocytes which are capable of transferring substances to and from the capillary basement membrane. It is of interest that this model is not unlike that proposed for the transport of sodium ion by the intestinal epithelium (Katzman and Pappins, 1973) and is consistent with evidence that glial cells contain high concentrations of carbonic anhydrase (Giacobini, 1962) and enzymes capable of hydrolyzing nucleoside phosphates (Torack and Barrnett, 1964). It is appropriate to add that, beyond the capillary-glia complex, and clearly downstream from the blood-brain interface, lie a number of transport systems which further modify the chemical composition of substances entering the brain. These include not only the sodium and potassium pumps associated with neurons (Haniberger et al., 1970; Cummins and Hydbn, 1962) but also the elaborate transport systems possessed by the choroid plexus (see Section V,C). Although there is much to be learned about the active transport of substances by the cellular elements of the brain, it is likely that the brain extracellular fluid is carefully controlled, perhaps with considerable variation in regional concentrations, so that an ideal internal milieu for the nervous system is maintained. Taken together, the foregoing observations suggest that the brain extracellular fluid is not formed as a specific secretion but as an ultrafiltrate of the plasma upon which active processes are secondarily enacted. We can see how such a mechanism, which is basically similar to the mechanism by which the urine is formed, is in the general interest of energy conservation within the brain. To put this view to proof it

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will be necessary to demonstrate, as Starling (1909) did when called upon to prove that the lymph is filtered, that the formation of the brain extracellular fluid is dependent upon the filtration pressure of cerebral capillaries and the osmotic pressure of the plasma. Whereas a variety of clinical and experimental observations suggest that both factors are important (Go and Pratt, 1975; Carey and Vela, 1974), there are as yet no definitive data that resolve these questions. Granted that the formation of the brain extracellular fluid involves the combined processes of ultrafiltration and active transport, it is necessary to consider the fate of this secretory product. Early investigators, including Cushing (1914), Weed (1914), and Flexner (1933) favored the view that there is a steady bulk flow of extracellular fluid from the perineuronal spaces into the perivascular spaces, and subsequently into the CSF. In contrast, a number of later investigators have argued that the CSF cavities do not receive accessions of fluid from the brain interspaces and that the extracellular fluid is neither formed nor circulated in the conventional sense but that it serves primarily as a medium for diffusional exchange between the blood and brain, or between the CSF and brain. This latter point of view has been especially emphasized during the past 2 decades in the work and writings of Davson (1963, 1967, 1969). Renewed interest in the proposal that there is a “lymphaticlike” drainage of brain extracellular fluid into the CSF cavities has come about as a result of many new pieces of information. These include evidence that a substantial fraction of the CSF is formed at extrachoroidal sites (Pollay and Curl, 1967; Sato and Bering, 1967; Sonnenberg et al., 1967; Milhorat, 1969), that the sodium exchange kinetics of the brain parenchyma can be correlated with the rate of CSF formation (Milhorat et al., 1971), that regional variations in the composition of fluid within the cerebral ventricles, cisterna magna, and subarachnoid space cannot be accounted for by the secretory activity of the choroid plexuses (Franklin et al., 1975), and that some and perhaps many substances are cleared from the brain into the CSF by net transport (Cserr, 1971). Unquestionably, the most compelling argument that the brain extracellular fluid serves as a specialized lymph is the fact that the brain, which lacks a lymphatic apparatus of the usual type, has no alternative mechanism for removing its products of metabolism (Cushing, 1926b). As Krogh (1946) and subsequently Cserr (1971) have emphasized, since the exchange of lipid-insoluble substances between the blood and brain extracellular fluid is very slow, and since the exchange of lipid-soluble substances is very rapid, the permeability properties of

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the blood-brain barrier are rather like those of a single cell membrane. This means that, for lipid-insoluble substances entering the brain, there are but two theoretical mechanisms that can effectively account for their removal: (1) facilitated transport back across the blood-brain barrier, and (2) net transport into the CSF. Cserr (1971) has argued convincingly that, if one considers the large number of lipid-insoluble substances known to enter the brain, they far exceed the number of carrier-mediated systems known to exist. Pinocytosis and phagocytosis are also limited in this respect, so that the only system sufficiently nonspecific to transport all the substances that must be removed from the brain is the CSF. It is obvious that conclusive proof of the lymphatic role of the brain extracellular fluid will depend on evidence that substances entering the brain, or substances synthesized by the brain, are cleared by net transport into the CSF. This goal has already been achieved for a number of compounds including the following list of lipid-insoluble substances: inulin (Cserr et al., 1967), mannitol (Cserr et al., 1967), sulfanilic acid (Cserr et al., 1967), urea (Cserr et al., 1970), albumin (Hochwald and Wallenstein, 1967a), globulin (Hochwald and Wallenstein, 1967b), dopamine (Guldberg and Yates, 1968; Portig et d., 1968; Portig and Vogt, 1968), homovanillic acid (Portig e t al., 1968), serotonin (Guldberg and Yates, 1968), norepinephrine (Schanberg et al., 1968), and cyclic nucleoside phosphodiesterase (Hidaka et al., 1975). Recently, Cserr and Ostrach (1974) have reported that, when dextran blue 2000, a molecule so large that it does not move appreciably by diffusion, is injected into the caudate nucleus of rats, the dye is rapidly transported, apparently along extracellular and perivascular channels, to the globus pallidus, internal capsule, stria terminalis, and junction of the lateral and third ventricles. Provided that these findings do not reflect a pathological phenomenon, we may accept them as the first solid evidence of a bulk flow of fluid within the brain interspaces. Overall, the foregoing data would appear to provide strong support for Cushing’s premise (1926a,b) that the brain extracellular fluid drains in bulk to the adjacent CSF cavities as a protein-poor lymph. It is appropriate to add that, although the blood-brain barrier is highly effective in restricting the movement of large or polar substances into the nervous parenchyma, some physiological leakage is inevitable. This of course is greatly compounded when the brain is damaged b y disease, and it is in the removal of substances as varied as proteins, erythrocytes, and bacteria that the lymphatic role of the CSF becomes truly important. For owing to its continuous production, and by virtue

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of its perpetual circulation which irrigates the brain and spinal cord as it flows in bulk toward the arachnoid villi, the CSF serves as an effective, permanent, and circular drain for the tissue it surrounds.

FLUID C. CHOROIDAL Despite the fact that a physiologically acceptable technique for isolating and collecting fluid from the surface of the choroid plexus has yet to be developed, we may assume that the chemical composition of freshly formed choroidal fluid is quite close to that of ventricular fluid as a whole, insofar as the choroid plexuses contribute a significant fraction of the fluid formed within the cerebral ventricles and the chemical composition of ventricular fluid is essentially unchanged following choroid plexectomy (Bering, 1966; Milhorat, 1969; Milhorat et al., 1976~). This conclusion, which is in agreement with the somewhat questionable findings of Ames et al. (1964, 1965a,b), indicates that choroidal fluid, like ventricular fluid, is not a simple ultrafiltrate of plasma and that its formation requires the participation of certain active processes. As mentioned earlier, the capillaries of the choroid plexus stroma are of the fenestrated type and can be readily distinguished on a morphological basis from their counterparts in the brain parenchyma. When an electron microscopic tracer such as ferritin, horseradish peroxidase, or cytochrome c is injected intravascularly, the marker can be shown to pass rapidly out of the choroidal capillaries and to distribute widely in the choroid plexus extracellular space up to, but not beyond, the apical tight junctions joining adjacent epithelial cells. From these observations we can conclude that the fenestrated capillaries of the choroid plexus stroma filter a protein-rich extracellular fluid which is separated from the CSF by a relatively impermeable layer of cells constituting the choroidal epithelium (Milhorat, 1975). Since this epithelium prohibits the movement of large molecules between the choroidal plasma and the CSF, it is the obvious surface across with the choroidal fluid is formed. In recent years, evidence has been accumulating that the choroid plexus epithelium does not restrict the movement of certain very small molecules. Utilizing a newly developed technique for visualizing the in vivo movement of exogenously injected calcium ion (Milhorat et al., 1974, 1975a), for example, it has been shown that, when this divalent cation is injected into blood, it moves rapidly into the CSF via the extracellular route between choroid plexus epithelial cells (Fig. 34). Conversely, when the ion is injected into the CSF, it moves promptly between epithelial cells to enter the choroid plexus

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stroma (Milhorat et ul., 1975a). These findings are consistent with physiological evidence of passive ion movement through the intercellular tight junctions of the choroid plexus epithelium (Wright, 1972) and other low-resistance epithelia including the gallbladder (Barry and Diamond, 1971), the small intestine (Frizzell and Schultz, 1972), and the proximal tubule of the kidney (Fromter et ul., 1971). In spite of a variety of data suggesting that the epithelial cells of the choroid plexus are involved with active transport, it remains to be determined what substances are actually transported, in what direction net transport occurs, and whether or not transport varies according to physiological conditions. Early investigators raised the possibility that the choroid plexuses might serve to remove substances from the CSF. Askanazy (1914) and Hassin (1924) pointed out that the epithelial cells of the choroid plexus accumulate hemosiderin in considerable amounts following intraventricular hemorrhage, and the ability of the choroid plexuses to accumulate substances in vitro has been subsequently demonstrated for a long list of compounds including iodide, thiocyanate, sulfate and thiosulfate, glucose and galactose, xanthine, certain organic acids, certain organic bases, and certain neutral amino acids (Cserr, 1971). Although it is not clear whether such in vitro accumulation represents active transport or simply reflects the processes of tissue binding and /or pinocytosis, direct proof of choroid plexus absorption has been demonstrated for at least one class of compounds-the organic acids-that are actively concentrated by isolated fragments of the choroid plexus (Rall and Sheldon, 1962) and by fetal and newborn choroid plexuses grown in tissue culture (Cameron, 1953; Lumsden, 1958). Recently, Wright (1972) has provided evidence that sodium ion, in addition to passively permeating the choroid plexus epithelium via intercellular tight junctions, is transported from the serosal to the ventricular surface by a ouabainsensitive, electrically silent pump which is unaffected by anoxia or pharmacological agents including Diamox, pitressin, and hydrocortisone. Other experiments by Wright have suggested that potassium ion is actively transported in the opposite direction, and that the rate of transport is dependent on the extracellular concentration of the ion (Wright, 1972). Needless to say, more data will be necessary before such findings can be regarded as conclusive evidence of active transport by the choroid plexus epithelium. On the basis of the foregoing data, it is evident that the choroid plexuses are involved with complex exchanges between the blood and CSF. The exact nature of these exchanges remains unclear, but there is growing evidence that the choroid plexus epithelium serves

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THOMAS H. MILHORAT

FIG.34. Electron micrographs of choroid plexus 1 minute after intravascular injection of calcium ion. (A) Low-power view of pig choroid plexus showing crystals of calcium phosphate in perivascular space (P) surrounding capillaries and in extracellular

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as a fine filter for the choroidal plasma and that it is capable of actively transporting substances in more than one direction. By this means, it is likely that the choroid plexus elaborates a carefully regulated fluid and, at the same time, participates in the homeostasis of the CSF (Milhorat, 1975).

D. LEPTOMENINGEAL FLUID Only scanty information is available concerning the possible formation of fluid by the leptomeninges. Although it is now known that a number of substances are freely exchanged between the blood and CSF across the pia arachnoid (Bowsher, 1960; Levin et al., 1974; Sweet et al., 1951),it has not been established whether or not such exchanges can be equated with net transport. We must keep in mind that the experiments of Sat0 and his associates (1971, 1972; Sato and Bering, 1967) provide a strong case for the extraventricular formation of some CSF and that the pathogenesis of arachnoidal entrapment cysts can hardly be explained by the accumulation of fluid originally formed within the cerebral ventricles. In addition, recent ultrastructural studies have shown that calcium and strontium ions pass rapidly out of arachnoidal arterioles (Fig. 35), so that the sink action of the dural sinuses-that is, the pressure differential between the subarachnoid space and the dural sinuses favoring continuous absorption-implies a net movement of some filtrable substances from the blood to the CSF across the leptomeninges (Milhorat et al., 1975a). VI.

Summary

In this article, the structure and function of the choroid plexuses have been considered in the light of recent advances in CSF physiology. Although many questions remain to be answered, the following conclusions seem justified.

(1) A considerable volume of the CSF is formed continuously within the cerebral ventricles. The choroid plexuses contribute to this formation, but a significant fraction of the CSF is formed extrachoroidally. The exact contributions of fluid from choroidal and extrachoroidal sites remain to be determined. space (arrows) between epithelial cells. Unstained, x4250. (B) High-power view of' rhesus monkey choroid plexus showing crystals of calcium phosphate in extracellular space between epithelial cells (arrow),and free in cerebral ventricles (cv) between microvilli. Unstained. X39,950. From Milhorat et d.(1975a), published by permission of Journal of Neurosurgery.

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FIG.35. Electron micrograph of cerebral cortex of rhesus monkey 1 minute after intravascular injection of calcium ion. Crystals of calcium phosphate are present in a subarachnoid arteriole (upper left) and free i n the subarachnoid space (arrows). Unstained. x3825. From Milhorat et al. (1975a) published by permission ofJournal of Neurosurgery.

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(2) The elaboration of choroidal fluid probably involves the following steps: filtration of the .blood plasma across fenestrated choroidal capillaries, formation of a protein-rich interstitial fluid within the choroidal stroma, and movement of constituents of the interstitial fluid across the choroidal epithelium by the combined processes of ultrafiltration and active transport. Contributing to the choroid plexus blood-CSF barrier are at least three specialized features: a system of circumferential tight functions joining adjacent epithelial cells; a heterolytic system of pinocytotic vesicles and lysosomes within epithelial cells; and a system of epithelial cell enzymes concerned with the active bidirectional transport of substances between the plasma and CSF. It is likely that the choroid plexus epithelium elaborates a carefully regulated fluid and, at the same time, participates in the homeostasis of the CSF. ( 3 )Evidence exists that the extracellular fluid of the brain is continuously formed across the cerebral endothelium, that it drains in bulk to the adjacent CSF cavities, and that it serves as a vehicle for removing intracerebral metabolites. Overall, there is probably a steady, net addition of new fluid at all points along the pathways of CSF circulation until the major sites of absorption (arachnoid villi) are reached. REFERENCES Ames, A., 111, Sakanoue, M., and Endo, S. (1964).J. Neurosurg. 27, 672. Ames, A., 111, Higashi, K., and Nesbett, F. B. (1965a).J. Physiol. (London) 181, 506. Ames, A., 111, Higashi, K., and Nesbett, F. B. (196513).J. Physiol. (London) 181, 516. Arnhold, R. G., and Zetterstrom, R. (1958).Pediatrics 21,279. Askanazy, M. (1914). Verh. Dtsch. Ges. Pathol. 17, 85. Bargmann, W., and Katritsis, E. (1966). 2. Zellforsch. Mikrosk. Anat. 41, 372. Barry, P. H., and Diamond, J. M. (1971).J. Mernbr. Biol. 4,295. Becker, N. H., and Sutton, C. H. (1963).Am. J. Pathol. 43, 1017. Becker, N. H., and Sutton, C. H . (1975).In “The Choroid Plexus in Health and Disease” (M. G. Netsky and S. Shuangshoti, eds.), pp. 113-150. University Press of Virginia, Charlottesville. Becker, N. H., Goldfischer, S., Shin, W. Y., and Novikoff, A. B. (1960).J.Biophys. Biochem. Cytol. 8, 649. Becker, N. H., Novikoff, A. B., and Zimmerman, H. M . (1967).J.Histochem. Cytochem. 15, 160. Becker, N. H., Hirano, A., and Zimmerman, H. M . (1968).J.Neuropathol. E x p . Neurol. 27,439. Benedikt, M. (1874).Arch. Pathol. Physiol. Klin. Med. Anat. 59,395. Bering, E. A., Jr. (1966).In “Workshop in Hydrocephalus” (K. Shulman, ed.), pp. 9-28. Univ. of Pennsylvania Press, Philadelphia. Birge, W. J., and Doolin, P. F. (1975). Int. Neurol. Congr., 8th, p. 122. Bito, L. Z., and Davson, H. (1966). Exp. Neurol. 14,264. Bodenheimer, T. S., and Brightman, M. W. (1968).Am. J. Anat. 122,249.

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