Annals of the Royal College of Surgeons of England
(I979)
vol 6i
The significance of the evolution of the cerebrospinal fluid system Gordon Brocklehurst Mchir FRCS Consultant Surgeon, Neurosurgical Service, Hull Royal Infirmary, Hull, E Yorks
Summary A study of the comparative morphology of the cerebrospinal fluid (CSF) system has been made in amphioxus, lamprey, dogfish, goldfish, lungfish, frog, salamander, turtle, pigeon, and mouse. Using mainly intracardiac fixation and a careful histological technique, serial sections have been obtained of the brain in situ surrounded by its various membranes and the skull. The ventricular system, the roof of the hindbrain, the meninges and subarachnoid space, the ependyma with its various derivatives, including the choroid plexuses and paraphysis, and the relationship between the various CSF compartments and the cerebrovascular system have all been compared in these animals. The hypothesis has been derived that the CSF system is primarily developed to maintain the chemical environment necessary to the function of the cells of the central nervous system, including the neuroendocrine pathways.
Introduction In I859, when the first edition of Darwin's The Origin of Species was published, comparative morphology was already established. It had been used as a method of study in the i 8th century by John Hunter and also by Richard Owen, his assistant, who developed the search for structural homologues in the animal kingdom. The effect of Darwin's concept of species originating by means of natural selection was to add a considerable impetus to studies which sought to elucidate the evolution of structures by their comparative morphology. For obvious reasons the central nervous system as a whole figured prominently in this type of comparative study, but the morphology of the cerebrospinal fluid (CSF) system within and around the neuraxis was relatively neglected. The existence of a clear fluid within and around the brain was first demonstrated by Cotugno in the i8th century', and the studies of Francois Arris and Gale Lecture delivered on gth March 1978
Magendie from I825 to i8422'3 demonstrated the continuity of the intracranial CSF with the extracranial and spinal CSF in humans through the midline, forainen in the roof of the fourth ventricle which is now commonly called by his name. He considered that the external fluid was formed by the surface of the brain and that the intraventricular fluid was formed by the frond-like structures within the ventricles known as the choroid plexuses; he thought there was a free exchange between the two fluids by an ebb and flow through his foramen. In i 854 Luschka4 gave an account of openings in the lateral recesses of the fourth ventricle in humans, and the extensive study by Key and Retzius5 in I875 finally defined all of the main structures of the CSF system, including the pia, arachnoid, and dura mater. In i866 Wilder', who was primarily concerned with the comparative morphology of the brain as a whole, confirmed the lateral and midline foramina in the roof of the fourth ventricle in man and reported that the cat lacked a midline foramen (often termed the metapore). From the same laboratory in I893 Gage7 compared the structural features of the brain in three vertebrate species and clearly described a metapore in one of them, the amphibian newt known as Diamectylus viridescens. It was the roof of the hindbrain which was extensively studied by Blake8 in I900 using a technique of formalin fixation and serial section, and he reported the metapore as present in man and three species of Old World monkey but absent from all the other species that he studied, including birds and amphibians. Sporadic observations upon particular morphological aspects of the CSF system in vertebrate animals appeared throughout the first three decades of the 20th century, but during this period the observations of Dandy and Blackfan9 on experimental hydrocephalus in dogs and those of Weed"0 on the CSF system
350
Gordon Brocklehurst
in pig embryos dominated the subject, particularly when the authority of Cushing'1 was added to the concept that CSF was produced within the ventricular system by the choroid plexuses, flowed through the fourth ventricle and subarachnoid space, and was absorbed by the venous sinuses. The incompatibility of this type of CSF circulation with the morphology of the CSF system in other vertebrates was apparently bypassed. More important, the classic concept of CSF circulation has never explained its function. The study presented here was a deliberate return to an examination of the comparative morphology of the CSF system with the objective of defining the structure of its main components in a number of vertebrate species by the careful application of well-established methods of serial section histology in order to look for an evolutionary pattern which might indicate the function of the system. Materials and methods The animals were obtained live and the choice (see table) was determined by size, availability, and the value of a particular species as a representative of a vertebrate class. They were anaesthetised with MS222 (tricaine methane-
sulphonate, Sandoz) by immersion or injection, and a cardiac perfusion of Ringer's solution appropriate for the particular species was performed, followed by perfusion with a gluteraldehyde-formaldehyde fixative (Karnovsky'2). After fixation and decapitation excessive tissue surrounding the cranial vault was removed and the preparation decalcified in RDO (du Pane Kinetic Laboratories Inc.). Dehydration was obtained by serial passage through ethanol solutions and the preparations were cleared in terpineol or cedar wood oil and embedded in paraffin wax or Paraplast. Serial sections were cut at 6 ,um (or 8 ,um for the larger animals), laid on slides, and then stained with haematoxylin and eosin, van Gieson's connective tissue stain, and either Masson or Mallory trichrome stain for successive slides. The arrangement of the sections and the numbering of each slide enabled a serial study to be undertaken of the entire head of the animal. LIMITATIONS OF TECHNIQUE
The time-consuming nature of this type of serial section technique and the great number of sections produced from each specimen limit the number of examples of each species which can conveniently be examined. When only one
Rhombencephalic roof Foramina 1 2 3 4 5 6 7 Other Intact ,_T,Median Lateral Amphioxus Lamprey Dogfish Goldfish Lungfish Frog Salamander Turtle Pigeon Mouse
Cfhorold plexuses A-ii- ---!A
Meninges
- .
Ventricular system
Pia Arachnoid Dura
1 2 34 5
+_
+
_+
_
_
___+
+ ++ ++ + ++
+
++ ++ + ++
+
~+
-~~~
+ + + + + + + + + + +
_+
+__
+ + + + + + +.
+
+ + + + + + + + + +
+__
+
++++
-
+
+
+
+
_+
+
+
+++
_+
+
+
++++_
_+
+
+
++++_
+
+
+
++++_
++~
_
Ventricular system: Nos 1, 2, 3, and 4 refer to the ventricles as numbered in the human, No 5 is the mesencephalic venitricle, and Nos 6 and 7 are the optic ventricles. The heading 'Other' includes the various recesses and cul-de-sacs of the ventricular system seen in the elasmobranch and teleost fishes. of the Choroid plexuses: Nos 1, 2, 3, and 4 refer to the choroid plexuses of the components ventricular system in humans and No 5 refers to the choroid plexus of the mesencephalon.
The significance of the evolution of the cerebrospinal fluid system or two examples have been sectioned the possibility of structural differences within the species is not eliminated. Furthermore, the choice of a particular representative of any class does not ensure that the morphology of all members of that class will be identical. Finally, the process of fixation and preparation for histological study of the brain within the surrounding skull causes relatively more shrinkage of some structures than others, which may artificially enhance spaces and also disrupt delicate membranes. By the application of strict criteria and careful serial section study disruption artefacts can be distinguished from anatomical deficiencies in a membrane consisting of one or two layers of cells. The serious technical difficulties in the study of fluid-filled cavities separated by relatively fine membranes may have been one reason for the apparent neglect of the comparative morphology of the CSF system in the past. Presentation of results In the descriptions of the individual species the zoological terms dorsal, ventral, rostral, and caudal are used for uniformity of orientation and, for simplicity, the terms forebrain, midbrain, and hindbrain are used to describe the main regions of the brain. The ventricles are termed lateral (that is, first and second), third, and fourth as in the human, with the addition of specifically named mesencephalic, optic, and cerebellar ventricles as they are found in some species. Attention has been paid to the following structures: (a) ventricular system; (b) roof of the hindbrain, (c) meninges and subarachnoid spape; (d) ependyma and its various specialised areas; (e) choroid plexuses and paraphysis; and (f relationship between the CSF compartments and the cerebrovascular system. An overall comparison of these structures has been made, and some of these comparative results are presented in the table. VENTRICULAR SYSTEM
351
This region appears to be equivalent to the hindbrain. The lamprey has relatively small lateral and third ventricles, without choroid plexuses, and capacious midbrain and hindbrain ventricles, both of which have large dorsal recesses bearing well-vascularised, extensive choroid plexuses. In the dogfish and goldfish the ventricular systems are very elaborate, extending between and around the various lobes of the brain and forming many double channels and cul-de-sacs. The lungfish has a relatively simple arrangement of the four main ventricles, as have also the frog and turtle, but in all of these the mesencephalic ventricle extends to a varying degree into the optic lobes (Fig. i). In the fishes, reptiles, and amphibians studied there is a ventrocaudal extension of the third ventricle forming a large infundibular recess which usually overlies the adenohypophysis at the level of the midbrain (Fig. i). This infundibular recess is lined by thin ependyma and, in the case of the dogfish, divides into median and lateral lobules surrounded by a
meningeal meshwork containing a large number of blood vessels and known as the saccus vasculosus. The vascularity around the infundibulum is less well developed in the other ver-
... 0., .,,,s+..,,..............................
FIG. I Transverse section through midbrain of frog. The midbrain ventricle with the lateral extensions forming the optic ventricles are shown and the pia can just be seen. The arachnoid is well defined, as is the subarachnoid space, and it is clear that ventrally the infundibular recess from the third ventricle, and the adenohypophysis, lie outside the arachnoid. The dorsolateral structure in the subdural space is part of the endolymphatic system. The dura can just be seen as a thin layer loosely attached to the skull. Magnification X I5. Masson's trichrome
The primitive brain of the amphioxus contains a bilobar dilatation lined by columnar ependyma which is clearly a simple ventricular system. A few segments caudal to this the epithelial-lined central canal has a flattened dorsal area bearing many pigmented cells, and adjacent to this is a layer of large neurones. stain.
352
Gordon Brocklehurst MENINGES
The amphioxus neuraxis is surrounded by a thick layer of fibrous tissue which invests but does not penetrate the neural tissue, and this is a relatively acellular structure. In all of the other specimens studied the neuraxis is invested with a layer of fibrous tissue which penetrates the glia of the brain and spinal cord to a variable degree and accompanies the blood vessels; this has been termed the pia mater. In the lamprey the plugs of pial tissue entering the brain are quite short, but in the goldfish, in contrast, there is a thick fibrous layer surrounding the brain and very thick fibrous septa FIG. 2 Transverse section of caudal end of medulla penetrate the ventral aspect of the brain and of salamander (compare with Fig. 3). The hindbrain carry blood vessels, almost as if this were the roof (posterior tela) consists of a thin, intact ependy- hilum of the organ. Surrounding the pia in mal membrane separating the ventricle from the sur- the lamprey is a special loose meshwork of rounding well-de-fined subarachnoid space. The meningeal tissue the cells of which contain arachnoid can clearly be seen extending laterally to accompany the cranial nerves. There is a subdural glycogen, and the whole structure varies in space outside the arachnoid. Magnification X i8. size with the nutrition of the animal (Fig. 4). Masson's trichrome stain. Between this meningeal meshwork and the surtebrates studied. In the goldfish and pigeon the rounding dura is the fluid-filled subdural space. wall of the infundibulum contains glial tissue The areolar meningeal tissue outside the pia and is much thicker and more akin to the well- in the dogfish, goldfish, and lungfish (Fig. 5) known appearance of the infundibular recess is much looser, and in life a considerable amount of subdural fluid is present in the in mammals. elasmobranchs. HINDBRAIN ROOF The amphibians, reptiles, and birds show a In the lamprey, dogfish, goldfish, and lungfish the roof of the hindbrain consists of a dorsal sac bearing the choroid plexus rostrally and a simple layer of ependyma caudal to this which is delicate but intact; this latter area is known as the posterior tela. In the salamander and other urodele amphibians studied the posterior tela is also a thin, intact layer of ependyma surmounted by pial tissue (Fig. 2). But in the frog (Rana temporaria) and all of the other anuran amphibians studied, excepting xenopus, the posterior tela is deficient caudally (Fig. 3) and many sections show an appearance of fenestration. The turtle has a hindbrain posterior tela consisting of an intact, large, thinwalled dorsal pouch, and the pigeon also has a large ependymal dorsal pouch in place of the posterior tela. The mouse has no midline FIG. 3 Transverse section through caudal part of foramen or metapore but has lateral foramina hindbrain of frog. The hindbrain (posterior tela) is in the roof of the hindbrain. Thus the only deficient at this level and there is continuity between animals in the series studied with a free com- the narrow ventricle and the subarachnoid space. On rhombic lip there is a blood vessel closely attached munication between the ventricular system and the to the pia, and the endolymphatic system intervenes surrounding external CSF compartment are between the arachnoid and the dorsal dura. Magnifithe anuran amphibians and the mouse. cation Xi8. Masson's trichrome stain.
The significance of the evolution of the cerebrospinal fluid system
353
(Fig. 5). In the turtle and pigeon the hindbrain plexus is subarachnoid in position.
choroid
EPENDYMA
Throughout the study the versatility of the ependyma is striking, from the well-defined apparent secretory epithelium of the choroid plexuses, containing a core of connective tissue and blood vessels, and the similar secretory epithelium of the paraphysis, everted against the dorsal skull roof and adjacent venous sinuses, to the simple flat epithelium lining the FIG. 4 Transverse section of cervical spinal cord of lamprey. There is a very small central canal, the pia has separated from the glia, and the meningeal areolar tissue has contracted away from the surrounding dura. Magnification XI8. Masson's trichro-me stain.
clearly defined layer of arachnoid around the neuraxis and, in most places, quite free from it, leaving a very clear subarachnoid space (Figs I, 2, 3, 5, and 6). The arachnoid is very distinctive; it stains only slightly for collagen and under high-power magnification consists of a thick membrane with a number of layers of flattened cells continuous with each other. In all of the animals studied the paraphysis lies outside the arachnoid, as does also the infundibular recess and its accompanying blood vessels (Fig. i). In the frog and the salamander the choroid plexus of the hindbrain is also outside the arachnoid and adjacent to the endosteal dura and the endolymphatic sacs
infundibulum and the dorsal sacs of the third and fourth ventricles. In many areas the ependyma is ciliated and in other areas it is a tall, pigmented epithelium overlying brainstem nuclei in the floor of the fourth ventricle or the subcommissural organ in the rostral end of the midbrain. At the lateral edges of the ventricular system and over the rhombic lips in the fourth ventricle the ependyma is arranged in a number of layers of flattened cells. CHOROID PLEXUSES
All of the vertebrates studied except the amphioxus have choroid plexuses and in some the lateral ventricle choroid plexuses are either not present (frog) or small compared with the third ventricle choroid plexuses. The latter (and the midbrain choroid plexuses of the lamprey) and the hindbrain choroid plexuses of all the animals studied except the birds and mammals are suspended from an elaborate dorsal sac in which the base of the choroid plexus is adjacent to the roof of the skull and the vili hang down within the ventricle (Figs 5 and 7). In the birds Transverse section through rostral part of hindbrain of frog. The folded choroid plexus lies dorsal to the wide fourth ventricle, and the arachnoid can be seen as a layer attached to the FIG. 5
interval of ependyma between the rhombic lip and the choroid plexus; thus the choroid plexus has its base in an extra-arachnoid * " position adjacent to the venous sinuses, which have shrunk away the dura of the dorsum of 4 \*Ae ^wgfrom the skull. Some of the sinusoidal vessels are part of the endolymphatic system. The ventral extension of the subarachnoid space to the perilymph of the vestibular apparatus is apparent on both sides. Magnification X 8. Masfon's trichrome stain.
354
Gordon Brocklehurst
6 Transverse section of cervical spinal cord of turtle. The spinal cord and surrounding pial layer of blood vessels lie within a subarachnoid space surrounded by a well-defined arachnoid. There is a small subdural space which may be partly artefactual ce Van Gieson's Xof from shrinkage. Magnification connective tissue stain. FIG.
and mammals, where the ventricular system is relatively much smaller and situated deep within the elaborately developed cerebral hemispheres, the choroid plexuses maintain a large surface area by the complexity of their folds, but there is no juxtaposition of any of the choroid plexuses with the veins of the roof of the skull. Similarly the cerebellar hemispheres are well developed in these species and the choroid plexus of the hindbrain is deeply buried, although there are lateral extensions through the lateral foramina into the subarachnoid space.
dividual species made by other workers, but this appears to be the first comprehensive study of such a wide range of vertebrates. The ventricular system in mammals has been studied by injection techniques and its configuration related to the development of the cerebral hemispheres and their contained ganglia"3. Our study has also confirmed a recent demonstration of the phylogeny of the ventricular system by a radiological technique"4. The elaborate ventricular system found in fishes has been described by other workers, and the remarkable ventricular system of myxine, a very primitive hagfish, has been demonstrated to have no choroid plexuses and a number of unconnected ventricular cavities'5. Many modern studies have confirmed the appearance of the arachnoid in individual vertebrate species, and the ultrastructure of the arachnoid has been well described in the amphibians'6. The detailed light microscope appearance of the hindbrain roof in the frog, amphibians in general, and the pigeon in particular, have already been described from this laboratory"-9. The microscopic and ultramicroscopic structure of the ependyma and choroid plexuses have been studied in various animals in recent years and the secretory and
RELATIONSHIP TO CEREBROVASCULAR SYSTEM
In the brains of all the animals studied except the amphioxus blood vessels can be traced into the cerebral substance, and in many these are clearly lined by tissue stained for collagen and presumably derived from the surface pia. However, these studies do not define in detail the structural relationships between the cerebral circulation and the surrounding glial tissue. In none of the species examined here are arachnoid granulations present. Blood vessels within the choroid plexus are prominent, as is the close relationship of the choroid plexuses and paraphysis and infundibular recess to the FIG. 7 Transverse section through forebrain of lungvenous plexuses around the brain. fish. The bulky choroid plexuses are present in the shallow lateral ventricles, and the third ventricle Discussion choroid plexus is suspended from a dorsal sac. The dura has shrunk away slightly from the skull, and FINDINGS between the dura and pia there is a very loose meninSome of the findings in this study confirm ob- geal meshwork with blood vessels. Magnification servations on CSF system morphology in in- XI14.5. Masson's trichrome stain.
The significance of the evolution of the cerebrospinal fluid system absorptive activities of this remarkable epithelium elucidated20'21. In summary, the ventricular system of the vertebrate brain is lined by an epithelium which could fulfil secretory, absorptive, or transmitting functions, and the areas of plentiful cilia suggest that there is local movement of the contained CSF. In many vertebrates the fluid within this ventricular system is only separated from adjacent venous sinuses by the simple ependyma of, for instance, the infundibular recess or the ependyma of the paraphysis or choroid plexus epithelium. The elaborate brains of the fishes studied appear to impinge upon the ventricular system and reduce it to complicated recesses, double channels, and cul-de-sacs. In the goldfish the ventricular system is actually everted over the surface of the forebrain to form a double epithelial layer reaching almost round to the ventral side. In the amphibians, reptiles, birds, and mammals the external surface of the neuraxis is covered by CSF contained within the arachnoid, but in the first two of these the choroid plexuses and infundibulum extend outside the arachnoid to retain their proximity to the cranial venous system. In the birds and mammals, with the choroid plexus placed in the ventricles deep within the hemispheres, it is the subarachnoid space which extends the external CSF to lie adjacent to the venous sinuses of the skull. However, only in mammals and the anuran amphibians is there apparently free communication through the roof of the hindbrain between the external CSF compartment and the ventricular system.
355
arachnoid granulations identified. With the exception of the mammals and anuran amphibians, the structural separation of the ventricular system from the external CSF, or the absence of an external CSF altogether, stuggests that bulk-flow out of the ventricular system is not likely to occur. It must be remembered that the classic patterns of bulk-flow have been derived from studies in mammals, and the flow patterns in these other vertebrates with closed ventricular systems have still to be elucidated. Similarly, studies of CSF composition, production, and absorption are based upon mammalian work, and information upon these aspects of CSF in other vertebrates is at present scanty22. The pathology of hydrocephalus also has primarily been studied in mammals and humans, in which blockage of the bulk-flow pathway produces acute followed by chronic hydrocephalus, and the latter form is now known to exist with relatively normal intracranial pressure. In such a closed ventricular system the pattern of CSF production and absorption must be similar to the normal pattern for those vertebrates with naturally closed ventricular systems. In this respect it is of interest that the composition of the CSF within the obstructed ventricular system in humans with hydrocephalus is maintained at normal levels so far as electrolytes, glucose, and protein are concerned. The possibility exists, however, that there may be abnormalities in the composition of minute quantities of neurotransmitter substances which normally appear only transiently in the CSF.
There are many gaps to be filled by further studies of the CSF system and, while it may The findings suggest a hypothesis that the CSF, be justifiable to assume that whatever function the composition of which is controlled by the it has is fulfilled most adequately in the highly multiple activities of the ependymal epithelium, efficient brain of Homo sapiens, some of its is necessary to the functioning of the neurones, very basic features may still be better defined glia, or both and that the proximity of the by the study of less intelligent but biologically fluid to the venous system enables a slow highly successful vertebrates. drainage of fluid to occur. In many vertebrates this is likely to take place across the ependymal CONCLUSION epithelium or choroid plexuses directly from Finally, on the basis of these studies of the the ventricular system, but in those with an comparative morphology of the CSF system, external compartment of subarachnoid fluid it is submitted that the function is primarily sites of potential drainage to the venous system that of maintaining a suitable environment are clearly present, although in none of the for the neurones and glia of the central neranimals in this study were structures such as vous system which are known to be normally HYPOTHESIS
356
Gordon lBrocklehurst
separated from the systemic circulation by a blood-brain barrier. This internal environment consists of the ventricular system as a reservoir within which CSF composition is strictly controlled by the multiple activities of its ependymal lining, varying from secretory or absorptive choroid plexus epithelium to thin transmitting epithelium, columnar ciliated epithelium, and special epithelium overlying subependymal nuclei. In addition to maintaining and redistributing electrolytes it is quite possible that the local currents move neurotransmitters from one part of the ventricular system to another and participate in neuroendocrine transmission or interneural transmission. The ependyma also permits exchange of the reservoir of ventricular fluid with the interstitial fluid of the adjacent brain, and a slow escape of fluid occurs from the reservoir to the venous system directly or via the subarachnoid space, thus enabling the CSF to perform a slow drainage or sink function for the removal of unwanted or excreted substances. This study was performed with the aid of MRC Grant 74oo65 in the Bio-Medical Research Unit of the University of Hull. It contains material which is being submitted for an MD thesis to the University of Cambridge. The serial section histology was principally performed by Gillian S Dolman and the serial sections independently reviewed by my research assistant, Hazel C Jones. The photomicrographs were taken by Mr Alan Marshall of the Photographic Department, Brynmor Jones Library, University of Hull.
References Cotugno, Domenico (1 764) De Ischiade Nervosa Commentarius. Naples. 2 Magendie, F (1825) Journal de physiologie experimentale, 5, 27. I
3 Magendie, F (I842) Recherches physiologiques et cliniques sur le liquide cephalo-rachidien ou cerebro-spinal. Paris, Libraire Medicale de Mequignon-Marris fils. 4 Luschka, H (I854) Archiv fiur physiologische Heilkunde, I3, I. 5 Key, A, and Retzius, G (I875) Studien in der Anatomie des Nervensystem und des Bindgewebes. Stockholm. 6 Wilder, B G (i886) Journal of Nervous and Mental Disease, 13, 206. 7 Gage, S P (i893) The Brain of Diemyctylus viridescens from Larval to Adult Life and Comparisons with the Brain of Amia and Petromyzon. Ithaca, Wilder Quarter-Century Book. 8 Blake, J A (I900) Journal of Comparative Neurology, IO, 79. 9 Dandy, W E, and Blackfan, K D (I914) Journal of the American Medical Association, 8, 406. Io Weed, L H (1917) Contributions to Embryology, 5, 3. ii Cushing, H (I926) Studies in Intracranial Physiology and Surgery. London, Oxford University Press. I 2 Karnowsky, M J (I965) Journal of Cellular Biology, 27, i37A. I3 McFarland, W L, Morgane, P J, and Jacobs, M S (I969) Journal of Comparative Neurology, I35, 275. 14 Kier, E L (I977) in Radiology of the Skull and Brain-Anatomy and Physiology, ed. Newton, T H, and Potts, D G. St Louis, Mosby. I5 Murray, H, Jones, H, Cserr, H, and Rall, D P (I975) Brain Research, 99, 17. i6 Carpenter, S J (I966) Journal of Comparative Neurology, I27, 4I3. I7 Brocklehurst, G (1976) Acta neurochirurgica, 35, 205.
i8 Jones, H C, Dolman, G S, and Brocklehurst, G
(1978) Journal of Zoology, I85, 34I.
I9 Jones, H C, and Dolman, G S (I979) Journal of Anatomy, 128, 13. 20 Netsky, M G, and Shuangshoti, S (I97s5) The Choroid Plexus in Health and Disease. Bristol, Wright. 2I Cserr, H (I967) Federation Proceedings, 26, 1024. 22 Plum, F, and Siesjo, B K (I975) Anesthesiology, 42, 708.
(I979)
vol 6i
The significance of the evolution of the cerebrospinal fluid system Gordon Brocklehurst Mchir FRCS Consultant Surgeon, Neurosurgical Service, Hull Royal Infirmary, Hull, E Yorks
Summary A study of the comparative morphology of the cerebrospinal fluid (CSF) system has been made in amphioxus, lamprey, dogfish, goldfish, lungfish, frog, salamander, turtle, pigeon, and mouse. Using mainly intracardiac fixation and a careful histological technique, serial sections have been obtained of the brain in situ surrounded by its various membranes and the skull. The ventricular system, the roof of the hindbrain, the meninges and subarachnoid space, the ependyma with its various derivatives, including the choroid plexuses and paraphysis, and the relationship between the various CSF compartments and the cerebrovascular system have all been compared in these animals. The hypothesis has been derived that the CSF system is primarily developed to maintain the chemical environment necessary to the function of the cells of the central nervous system, including the neuroendocrine pathways.
Introduction In I859, when the first edition of Darwin's The Origin of Species was published, comparative morphology was already established. It had been used as a method of study in the i 8th century by John Hunter and also by Richard Owen, his assistant, who developed the search for structural homologues in the animal kingdom. The effect of Darwin's concept of species originating by means of natural selection was to add a considerable impetus to studies which sought to elucidate the evolution of structures by their comparative morphology. For obvious reasons the central nervous system as a whole figured prominently in this type of comparative study, but the morphology of the cerebrospinal fluid (CSF) system within and around the neuraxis was relatively neglected. The existence of a clear fluid within and around the brain was first demonstrated by Cotugno in the i8th century', and the studies of Francois Arris and Gale Lecture delivered on gth March 1978
Magendie from I825 to i8422'3 demonstrated the continuity of the intracranial CSF with the extracranial and spinal CSF in humans through the midline, forainen in the roof of the fourth ventricle which is now commonly called by his name. He considered that the external fluid was formed by the surface of the brain and that the intraventricular fluid was formed by the frond-like structures within the ventricles known as the choroid plexuses; he thought there was a free exchange between the two fluids by an ebb and flow through his foramen. In i 854 Luschka4 gave an account of openings in the lateral recesses of the fourth ventricle in humans, and the extensive study by Key and Retzius5 in I875 finally defined all of the main structures of the CSF system, including the pia, arachnoid, and dura mater. In i866 Wilder', who was primarily concerned with the comparative morphology of the brain as a whole, confirmed the lateral and midline foramina in the roof of the fourth ventricle in man and reported that the cat lacked a midline foramen (often termed the metapore). From the same laboratory in I893 Gage7 compared the structural features of the brain in three vertebrate species and clearly described a metapore in one of them, the amphibian newt known as Diamectylus viridescens. It was the roof of the hindbrain which was extensively studied by Blake8 in I900 using a technique of formalin fixation and serial section, and he reported the metapore as present in man and three species of Old World monkey but absent from all the other species that he studied, including birds and amphibians. Sporadic observations upon particular morphological aspects of the CSF system in vertebrate animals appeared throughout the first three decades of the 20th century, but during this period the observations of Dandy and Blackfan9 on experimental hydrocephalus in dogs and those of Weed"0 on the CSF system
350
Gordon Brocklehurst
in pig embryos dominated the subject, particularly when the authority of Cushing'1 was added to the concept that CSF was produced within the ventricular system by the choroid plexuses, flowed through the fourth ventricle and subarachnoid space, and was absorbed by the venous sinuses. The incompatibility of this type of CSF circulation with the morphology of the CSF system in other vertebrates was apparently bypassed. More important, the classic concept of CSF circulation has never explained its function. The study presented here was a deliberate return to an examination of the comparative morphology of the CSF system with the objective of defining the structure of its main components in a number of vertebrate species by the careful application of well-established methods of serial section histology in order to look for an evolutionary pattern which might indicate the function of the system. Materials and methods The animals were obtained live and the choice (see table) was determined by size, availability, and the value of a particular species as a representative of a vertebrate class. They were anaesthetised with MS222 (tricaine methane-
sulphonate, Sandoz) by immersion or injection, and a cardiac perfusion of Ringer's solution appropriate for the particular species was performed, followed by perfusion with a gluteraldehyde-formaldehyde fixative (Karnovsky'2). After fixation and decapitation excessive tissue surrounding the cranial vault was removed and the preparation decalcified in RDO (du Pane Kinetic Laboratories Inc.). Dehydration was obtained by serial passage through ethanol solutions and the preparations were cleared in terpineol or cedar wood oil and embedded in paraffin wax or Paraplast. Serial sections were cut at 6 ,um (or 8 ,um for the larger animals), laid on slides, and then stained with haematoxylin and eosin, van Gieson's connective tissue stain, and either Masson or Mallory trichrome stain for successive slides. The arrangement of the sections and the numbering of each slide enabled a serial study to be undertaken of the entire head of the animal. LIMITATIONS OF TECHNIQUE
The time-consuming nature of this type of serial section technique and the great number of sections produced from each specimen limit the number of examples of each species which can conveniently be examined. When only one
Rhombencephalic roof Foramina 1 2 3 4 5 6 7 Other Intact ,_T,Median Lateral Amphioxus Lamprey Dogfish Goldfish Lungfish Frog Salamander Turtle Pigeon Mouse
Cfhorold plexuses A-ii- ---!A
Meninges
- .
Ventricular system
Pia Arachnoid Dura
1 2 34 5
+_
+
_+
_
_
___+
+ ++ ++ + ++
+
++ ++ + ++
+
~+
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Ventricular system: Nos 1, 2, 3, and 4 refer to the ventricles as numbered in the human, No 5 is the mesencephalic venitricle, and Nos 6 and 7 are the optic ventricles. The heading 'Other' includes the various recesses and cul-de-sacs of the ventricular system seen in the elasmobranch and teleost fishes. of the Choroid plexuses: Nos 1, 2, 3, and 4 refer to the choroid plexuses of the components ventricular system in humans and No 5 refers to the choroid plexus of the mesencephalon.
The significance of the evolution of the cerebrospinal fluid system or two examples have been sectioned the possibility of structural differences within the species is not eliminated. Furthermore, the choice of a particular representative of any class does not ensure that the morphology of all members of that class will be identical. Finally, the process of fixation and preparation for histological study of the brain within the surrounding skull causes relatively more shrinkage of some structures than others, which may artificially enhance spaces and also disrupt delicate membranes. By the application of strict criteria and careful serial section study disruption artefacts can be distinguished from anatomical deficiencies in a membrane consisting of one or two layers of cells. The serious technical difficulties in the study of fluid-filled cavities separated by relatively fine membranes may have been one reason for the apparent neglect of the comparative morphology of the CSF system in the past. Presentation of results In the descriptions of the individual species the zoological terms dorsal, ventral, rostral, and caudal are used for uniformity of orientation and, for simplicity, the terms forebrain, midbrain, and hindbrain are used to describe the main regions of the brain. The ventricles are termed lateral (that is, first and second), third, and fourth as in the human, with the addition of specifically named mesencephalic, optic, and cerebellar ventricles as they are found in some species. Attention has been paid to the following structures: (a) ventricular system; (b) roof of the hindbrain, (c) meninges and subarachnoid spape; (d) ependyma and its various specialised areas; (e) choroid plexuses and paraphysis; and (f relationship between the CSF compartments and the cerebrovascular system. An overall comparison of these structures has been made, and some of these comparative results are presented in the table. VENTRICULAR SYSTEM
351
This region appears to be equivalent to the hindbrain. The lamprey has relatively small lateral and third ventricles, without choroid plexuses, and capacious midbrain and hindbrain ventricles, both of which have large dorsal recesses bearing well-vascularised, extensive choroid plexuses. In the dogfish and goldfish the ventricular systems are very elaborate, extending between and around the various lobes of the brain and forming many double channels and cul-de-sacs. The lungfish has a relatively simple arrangement of the four main ventricles, as have also the frog and turtle, but in all of these the mesencephalic ventricle extends to a varying degree into the optic lobes (Fig. i). In the fishes, reptiles, and amphibians studied there is a ventrocaudal extension of the third ventricle forming a large infundibular recess which usually overlies the adenohypophysis at the level of the midbrain (Fig. i). This infundibular recess is lined by thin ependyma and, in the case of the dogfish, divides into median and lateral lobules surrounded by a
meningeal meshwork containing a large number of blood vessels and known as the saccus vasculosus. The vascularity around the infundibulum is less well developed in the other ver-
... 0., .,,,s+..,,..............................
FIG. I Transverse section through midbrain of frog. The midbrain ventricle with the lateral extensions forming the optic ventricles are shown and the pia can just be seen. The arachnoid is well defined, as is the subarachnoid space, and it is clear that ventrally the infundibular recess from the third ventricle, and the adenohypophysis, lie outside the arachnoid. The dorsolateral structure in the subdural space is part of the endolymphatic system. The dura can just be seen as a thin layer loosely attached to the skull. Magnification X I5. Masson's trichrome
The primitive brain of the amphioxus contains a bilobar dilatation lined by columnar ependyma which is clearly a simple ventricular system. A few segments caudal to this the epithelial-lined central canal has a flattened dorsal area bearing many pigmented cells, and adjacent to this is a layer of large neurones. stain.
352
Gordon Brocklehurst MENINGES
The amphioxus neuraxis is surrounded by a thick layer of fibrous tissue which invests but does not penetrate the neural tissue, and this is a relatively acellular structure. In all of the other specimens studied the neuraxis is invested with a layer of fibrous tissue which penetrates the glia of the brain and spinal cord to a variable degree and accompanies the blood vessels; this has been termed the pia mater. In the lamprey the plugs of pial tissue entering the brain are quite short, but in the goldfish, in contrast, there is a thick fibrous layer surrounding the brain and very thick fibrous septa FIG. 2 Transverse section of caudal end of medulla penetrate the ventral aspect of the brain and of salamander (compare with Fig. 3). The hindbrain carry blood vessels, almost as if this were the roof (posterior tela) consists of a thin, intact ependy- hilum of the organ. Surrounding the pia in mal membrane separating the ventricle from the sur- the lamprey is a special loose meshwork of rounding well-de-fined subarachnoid space. The meningeal tissue the cells of which contain arachnoid can clearly be seen extending laterally to accompany the cranial nerves. There is a subdural glycogen, and the whole structure varies in space outside the arachnoid. Magnification X i8. size with the nutrition of the animal (Fig. 4). Masson's trichrome stain. Between this meningeal meshwork and the surtebrates studied. In the goldfish and pigeon the rounding dura is the fluid-filled subdural space. wall of the infundibulum contains glial tissue The areolar meningeal tissue outside the pia and is much thicker and more akin to the well- in the dogfish, goldfish, and lungfish (Fig. 5) known appearance of the infundibular recess is much looser, and in life a considerable amount of subdural fluid is present in the in mammals. elasmobranchs. HINDBRAIN ROOF The amphibians, reptiles, and birds show a In the lamprey, dogfish, goldfish, and lungfish the roof of the hindbrain consists of a dorsal sac bearing the choroid plexus rostrally and a simple layer of ependyma caudal to this which is delicate but intact; this latter area is known as the posterior tela. In the salamander and other urodele amphibians studied the posterior tela is also a thin, intact layer of ependyma surmounted by pial tissue (Fig. 2). But in the frog (Rana temporaria) and all of the other anuran amphibians studied, excepting xenopus, the posterior tela is deficient caudally (Fig. 3) and many sections show an appearance of fenestration. The turtle has a hindbrain posterior tela consisting of an intact, large, thinwalled dorsal pouch, and the pigeon also has a large ependymal dorsal pouch in place of the posterior tela. The mouse has no midline FIG. 3 Transverse section through caudal part of foramen or metapore but has lateral foramina hindbrain of frog. The hindbrain (posterior tela) is in the roof of the hindbrain. Thus the only deficient at this level and there is continuity between animals in the series studied with a free com- the narrow ventricle and the subarachnoid space. On rhombic lip there is a blood vessel closely attached munication between the ventricular system and the to the pia, and the endolymphatic system intervenes surrounding external CSF compartment are between the arachnoid and the dorsal dura. Magnifithe anuran amphibians and the mouse. cation Xi8. Masson's trichrome stain.
The significance of the evolution of the cerebrospinal fluid system
353
(Fig. 5). In the turtle and pigeon the hindbrain plexus is subarachnoid in position.
choroid
EPENDYMA
Throughout the study the versatility of the ependyma is striking, from the well-defined apparent secretory epithelium of the choroid plexuses, containing a core of connective tissue and blood vessels, and the similar secretory epithelium of the paraphysis, everted against the dorsal skull roof and adjacent venous sinuses, to the simple flat epithelium lining the FIG. 4 Transverse section of cervical spinal cord of lamprey. There is a very small central canal, the pia has separated from the glia, and the meningeal areolar tissue has contracted away from the surrounding dura. Magnification XI8. Masson's trichro-me stain.
clearly defined layer of arachnoid around the neuraxis and, in most places, quite free from it, leaving a very clear subarachnoid space (Figs I, 2, 3, 5, and 6). The arachnoid is very distinctive; it stains only slightly for collagen and under high-power magnification consists of a thick membrane with a number of layers of flattened cells continuous with each other. In all of the animals studied the paraphysis lies outside the arachnoid, as does also the infundibular recess and its accompanying blood vessels (Fig. i). In the frog and the salamander the choroid plexus of the hindbrain is also outside the arachnoid and adjacent to the endosteal dura and the endolymphatic sacs
infundibulum and the dorsal sacs of the third and fourth ventricles. In many areas the ependyma is ciliated and in other areas it is a tall, pigmented epithelium overlying brainstem nuclei in the floor of the fourth ventricle or the subcommissural organ in the rostral end of the midbrain. At the lateral edges of the ventricular system and over the rhombic lips in the fourth ventricle the ependyma is arranged in a number of layers of flattened cells. CHOROID PLEXUSES
All of the vertebrates studied except the amphioxus have choroid plexuses and in some the lateral ventricle choroid plexuses are either not present (frog) or small compared with the third ventricle choroid plexuses. The latter (and the midbrain choroid plexuses of the lamprey) and the hindbrain choroid plexuses of all the animals studied except the birds and mammals are suspended from an elaborate dorsal sac in which the base of the choroid plexus is adjacent to the roof of the skull and the vili hang down within the ventricle (Figs 5 and 7). In the birds Transverse section through rostral part of hindbrain of frog. The folded choroid plexus lies dorsal to the wide fourth ventricle, and the arachnoid can be seen as a layer attached to the FIG. 5
interval of ependyma between the rhombic lip and the choroid plexus; thus the choroid plexus has its base in an extra-arachnoid * " position adjacent to the venous sinuses, which have shrunk away the dura of the dorsum of 4 \*Ae ^wgfrom the skull. Some of the sinusoidal vessels are part of the endolymphatic system. The ventral extension of the subarachnoid space to the perilymph of the vestibular apparatus is apparent on both sides. Magnification X 8. Masfon's trichrome stain.
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Gordon Brocklehurst
6 Transverse section of cervical spinal cord of turtle. The spinal cord and surrounding pial layer of blood vessels lie within a subarachnoid space surrounded by a well-defined arachnoid. There is a small subdural space which may be partly artefactual ce Van Gieson's Xof from shrinkage. Magnification connective tissue stain. FIG.
and mammals, where the ventricular system is relatively much smaller and situated deep within the elaborately developed cerebral hemispheres, the choroid plexuses maintain a large surface area by the complexity of their folds, but there is no juxtaposition of any of the choroid plexuses with the veins of the roof of the skull. Similarly the cerebellar hemispheres are well developed in these species and the choroid plexus of the hindbrain is deeply buried, although there are lateral extensions through the lateral foramina into the subarachnoid space.
dividual species made by other workers, but this appears to be the first comprehensive study of such a wide range of vertebrates. The ventricular system in mammals has been studied by injection techniques and its configuration related to the development of the cerebral hemispheres and their contained ganglia"3. Our study has also confirmed a recent demonstration of the phylogeny of the ventricular system by a radiological technique"4. The elaborate ventricular system found in fishes has been described by other workers, and the remarkable ventricular system of myxine, a very primitive hagfish, has been demonstrated to have no choroid plexuses and a number of unconnected ventricular cavities'5. Many modern studies have confirmed the appearance of the arachnoid in individual vertebrate species, and the ultrastructure of the arachnoid has been well described in the amphibians'6. The detailed light microscope appearance of the hindbrain roof in the frog, amphibians in general, and the pigeon in particular, have already been described from this laboratory"-9. The microscopic and ultramicroscopic structure of the ependyma and choroid plexuses have been studied in various animals in recent years and the secretory and
RELATIONSHIP TO CEREBROVASCULAR SYSTEM
In the brains of all the animals studied except the amphioxus blood vessels can be traced into the cerebral substance, and in many these are clearly lined by tissue stained for collagen and presumably derived from the surface pia. However, these studies do not define in detail the structural relationships between the cerebral circulation and the surrounding glial tissue. In none of the species examined here are arachnoid granulations present. Blood vessels within the choroid plexus are prominent, as is the close relationship of the choroid plexuses and paraphysis and infundibular recess to the FIG. 7 Transverse section through forebrain of lungvenous plexuses around the brain. fish. The bulky choroid plexuses are present in the shallow lateral ventricles, and the third ventricle Discussion choroid plexus is suspended from a dorsal sac. The dura has shrunk away slightly from the skull, and FINDINGS between the dura and pia there is a very loose meninSome of the findings in this study confirm ob- geal meshwork with blood vessels. Magnification servations on CSF system morphology in in- XI14.5. Masson's trichrome stain.
The significance of the evolution of the cerebrospinal fluid system absorptive activities of this remarkable epithelium elucidated20'21. In summary, the ventricular system of the vertebrate brain is lined by an epithelium which could fulfil secretory, absorptive, or transmitting functions, and the areas of plentiful cilia suggest that there is local movement of the contained CSF. In many vertebrates the fluid within this ventricular system is only separated from adjacent venous sinuses by the simple ependyma of, for instance, the infundibular recess or the ependyma of the paraphysis or choroid plexus epithelium. The elaborate brains of the fishes studied appear to impinge upon the ventricular system and reduce it to complicated recesses, double channels, and cul-de-sacs. In the goldfish the ventricular system is actually everted over the surface of the forebrain to form a double epithelial layer reaching almost round to the ventral side. In the amphibians, reptiles, birds, and mammals the external surface of the neuraxis is covered by CSF contained within the arachnoid, but in the first two of these the choroid plexuses and infundibulum extend outside the arachnoid to retain their proximity to the cranial venous system. In the birds and mammals, with the choroid plexus placed in the ventricles deep within the hemispheres, it is the subarachnoid space which extends the external CSF to lie adjacent to the venous sinuses of the skull. However, only in mammals and the anuran amphibians is there apparently free communication through the roof of the hindbrain between the external CSF compartment and the ventricular system.
355
arachnoid granulations identified. With the exception of the mammals and anuran amphibians, the structural separation of the ventricular system from the external CSF, or the absence of an external CSF altogether, stuggests that bulk-flow out of the ventricular system is not likely to occur. It must be remembered that the classic patterns of bulk-flow have been derived from studies in mammals, and the flow patterns in these other vertebrates with closed ventricular systems have still to be elucidated. Similarly, studies of CSF composition, production, and absorption are based upon mammalian work, and information upon these aspects of CSF in other vertebrates is at present scanty22. The pathology of hydrocephalus also has primarily been studied in mammals and humans, in which blockage of the bulk-flow pathway produces acute followed by chronic hydrocephalus, and the latter form is now known to exist with relatively normal intracranial pressure. In such a closed ventricular system the pattern of CSF production and absorption must be similar to the normal pattern for those vertebrates with naturally closed ventricular systems. In this respect it is of interest that the composition of the CSF within the obstructed ventricular system in humans with hydrocephalus is maintained at normal levels so far as electrolytes, glucose, and protein are concerned. The possibility exists, however, that there may be abnormalities in the composition of minute quantities of neurotransmitter substances which normally appear only transiently in the CSF.
There are many gaps to be filled by further studies of the CSF system and, while it may The findings suggest a hypothesis that the CSF, be justifiable to assume that whatever function the composition of which is controlled by the it has is fulfilled most adequately in the highly multiple activities of the ependymal epithelium, efficient brain of Homo sapiens, some of its is necessary to the functioning of the neurones, very basic features may still be better defined glia, or both and that the proximity of the by the study of less intelligent but biologically fluid to the venous system enables a slow highly successful vertebrates. drainage of fluid to occur. In many vertebrates this is likely to take place across the ependymal CONCLUSION epithelium or choroid plexuses directly from Finally, on the basis of these studies of the the ventricular system, but in those with an comparative morphology of the CSF system, external compartment of subarachnoid fluid it is submitted that the function is primarily sites of potential drainage to the venous system that of maintaining a suitable environment are clearly present, although in none of the for the neurones and glia of the central neranimals in this study were structures such as vous system which are known to be normally HYPOTHESIS
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Gordon lBrocklehurst
separated from the systemic circulation by a blood-brain barrier. This internal environment consists of the ventricular system as a reservoir within which CSF composition is strictly controlled by the multiple activities of its ependymal lining, varying from secretory or absorptive choroid plexus epithelium to thin transmitting epithelium, columnar ciliated epithelium, and special epithelium overlying subependymal nuclei. In addition to maintaining and redistributing electrolytes it is quite possible that the local currents move neurotransmitters from one part of the ventricular system to another and participate in neuroendocrine transmission or interneural transmission. The ependyma also permits exchange of the reservoir of ventricular fluid with the interstitial fluid of the adjacent brain, and a slow escape of fluid occurs from the reservoir to the venous system directly or via the subarachnoid space, thus enabling the CSF to perform a slow drainage or sink function for the removal of unwanted or excreted substances. This study was performed with the aid of MRC Grant 74oo65 in the Bio-Medical Research Unit of the University of Hull. It contains material which is being submitted for an MD thesis to the University of Cambridge. The serial section histology was principally performed by Gillian S Dolman and the serial sections independently reviewed by my research assistant, Hazel C Jones. The photomicrographs were taken by Mr Alan Marshall of the Photographic Department, Brynmor Jones Library, University of Hull.
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I9 Jones, H C, and Dolman, G S (I979) Journal of Anatomy, 128, 13. 20 Netsky, M G, and Shuangshoti, S (I97s5) The Choroid Plexus in Health and Disease. Bristol, Wright. 2I Cserr, H (I967) Federation Proceedings, 26, 1024. 22 Plum, F, and Siesjo, B K (I975) Anesthesiology, 42, 708.
