Table of Contents
The central nervous system (CNS) is composed of various types of cells including neurons, glial cells (astrocytes, oligodendrocytes, ependyma and choroid plexus epithelium), blood vessel elements and microglia. Leptomeninges (pia mater and arachnoid mater) surround the CNS, and the pachymeninges (dura mater) form an outer coating that separates the brain from ‘the skull and the spinal cord from the spine.
The neuropil is composed of the complex and intricate network of neuronal cell processes. This intermingling and interconnection of myelinated and unmyelinated axons and dendrites may appear random, but these processes form the neuronal circuitry of a particular area. Cell processes of astrocytes, oligodendrocytes and microglial cells further add to the variety of structures in the neuropil (Fig. 1.3).
Cell processes and perikarya, bounded by typical unit membranes, are separated by an extra-cellular space with an average width of 15-20 run. Electron-dense markers, electrical impedance and the distribution of radiotracers have conclusively demonstrated the existence of this extracellular space, which constitutes 18~25o() of the total gre~’ and white matter.29
Myelin in the central nervous system is produced by oligodendrocytes. The myclin sheath is formed by the spiral wrapping of the cytoplasmic processes of the myelinating cells around axons, and the subsequent extrusion of the cytoplasm leads to a compact, tightly spiralled, multi-layered envelope. Electron microscopy reveals the characteristic structure of the myelin sheath: major dense lines alternate with thinner intraperiod lines to form the repeating units. The major dense line results from the fusion of the thicker, inner leaflet of the oligodendrocytic plasma membrane, whereas the intraperiod line is formed by the apposition of the thinner, outer leaflet of this membrane. Unlike Schwann cells (the myelinating cells of the peripheral nervous system which are concerned with the myelination of a single axon), each oligodendrocyte may provide myelin for many axons. From this, it follows that the motion by which the sheath is produced by the myelinating cell may be different in the central and peripheral nervous systems. Whilst Schwann cells can, in principle, lay down myelin by rotating around a single axon, the oligodendrocyte, being in connection with many axons, cannot perform a revolving motion. Consequently, ensheathment takes place by the progressive lengthening of the oligodendrocytic process which encircles the axon completely and an internal mesaxon is formed by apposition of the free edges of the myclinating process. The outermost lamella of the sheath encloses the outer tongue and this, together with the internal mesaxon, represents the tenuous connections which remain between the fully formed myclin and the myelinating cell.
Nodes of Ranvier occur regularly along the course of central myelinated fibres; the segment between two nodes is referred to as internodal myelin, and the region where the lamellae terminate is the paranode. The thickness of the myelin sheath is related to axonal diameter: larger axons are surrounded by thicker myelin sheaths. Evidence from experimental animals has convincingly shown that the formation of myelin is preceded by the proliferation of glial precursors which develop into young oligodendrocytes. The processes of these active oligodendrocytes are frequently wrapped around axons, but this direct connection is difficult to demonstrate in later life.’ Biologically, the formation of myelin is a two-step process: first, the oligodendrocyte relates to the axon in response to an axonal myelinogenic stimulus and, second, it produces myelin, the volume of which is determined by the internodal axonal surface area. 30
Mvelination in man progresses slowly and generally proceeds centripetally: it commences peripherally and whilst many axons, particularly in the spinal cord) are myelinated at birth, the more central fibres in the frontal and parietal lobes remain unmyelinated well into postnatal life. Completion of myelin formation is achieved largely during the first two years after birth.31 Immunohistochemistry for myelin basic protein confirms earlier findings of the chronological sequence of myelination: phylogenetically older regions acquire myelin first. The sequential nature of myelination reflects physiological demand: the time and rapidity of myelination are related to the relative significance of a fibre system at various periods of cerebral development.
Biochemically, myelin is composed of alternating layers of proteins and lipids. Of the myelin proteins, a proteolipid protein constitutes approximately 50 mu, basic protein (the antigenic agent capable of inducing experimental allergic encephalomyclitis) 30%, and an acidic proteolipid protein 200/0 .3′ Myelin-associated glycoprotein is an acidic, concanavalin A binding minor component which is also present in the cytoplasm of oligodendrocytes before and during myelination. The various lipids to be found in the myelin include cholesterol, phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, sphingomyelin and glycolipids. Galactocerebroside is the main glycolipid component and the cell membrane of the myelinating cell contains a high percentage of this compound. This suggests that galactocerebroside may play an important role in the successive layering of cell membranes, a process unique to myelination.
Nerve cells can be identified in most cases in sections stained with haematoxylin and eosin, but various ‘special’ methods are used in order to demonstrate particular organelles, cell processes or myelin sheaths. Cresyl violet (Nissl’s stain) displays the coarse granular Nissi substance composed of rough endoplasmic reticulum and consequently is ideal for detecting chromatolysis in which Nissi substance is lost.
This technique, combined with Luxol fast blue for the staining of myelin, is one of the most valuable methods for revealing topographical details in sections of the brain and spinal cord.
Since neurofilaments are argyrophilic, there are various silver impregnation techniques for the demonstration of nerve. The Golgi method has, however, remained superior in revealing neuronal configuration in its entirety with all the cell processes. Immunohistochemistry is extensively used in neurocytology, and the various antigens to be found in neurons and other cells can now be demonstrated by both immunofluorescence and immunoperoxidase techniques.
Monoclonal and polyclonal antibodies against these antigens have proved to be valuable in diagnostic work. Monoclonal antibodies against the protein components of neurofilaments are also valuable in the study of abnormalities affecting these organelles9~56 and in the diagnosis of neuronal neop1asms. Of the three forms of enolase to be found in the central nervous system, the isozyme occurs in nerve cells (neuron-specific enolase) and thus provides an immunological marker for neurons.58 However, enolase is widely distributed in extraneural tissues and is not a neuron-specific antigen.
Cerebral capillaries are fundamentally similar to those of other tissues, but there are important differences. There is a paucity of cytoplasmic vesicles in the endothelial cells, and the tight junctions between the endothelial cells differ from those of other tissue capillaries. In addition, astrocytic foot processes surround each capillary. The lumen is lined by endothelial cells which display oval or elongated nuclei located in the thickened part of the capillary wall. The cytoplasm contains the usual set of organelles, of which mitochondria are the most abundant. Complex membrane-bound structures, the Weibel-Palade bodies, are difficult to find. Cytoplasmic vesicles are scarce and fewer than in most other capillaries. The structure of the interendothelial connections is variable, but in general, the adjacent cell membranes are parallel, and, towards the luminal end, the outer leaflets fuse to form tight junctions (zonulac occludentes).
The endothelial cytoplasm is richly endowed with enzymes, including adenosine triphosphatase, nicotinamide adenine dinucleotide, monoamine oxidase, acid and alkaline phosphatases, various dehydrogenases, DOPA decarboxylase and glutamyl transpeptidase. The wealth of these enzyme systems reflects the unique role played by the cerebral endothelium in the blood-brain barrier. Moreover, differences in the intensity of various hydrolytic enzymes at the luminal and abluminal cell membrane strongly indicate the polarity of endothelial function in the control of the blood-brain interface. Outside the endothelium lies a continuous basal lamina or basement membrane approximately 40-50 nm thick and composed of an admixture of substances, including type IV collagen, heparan sulphate proteoglycan, laminin and entactin. Astrocytic foot processes abut onto capillaries, forming a complete envelope in most cases :
occasionally other cells may have direct contact with the basal lamina. Pericytes are completely surrounded by a duplication of the basement membrane, and are frequently seen extending their processes around the capillary; their cytoplasm contains many lysosome-like bodies. The origin and function of pericytes remain to be established, although the view that they could give rise to microglial cells has gained some support.
Arterioles and small arteries differ from capillaries not only in their larger size but also by the presence of smooth muscle cells in their walls. Outside the endothelium, one or two layers of smooth muscle cells are transversely orientated and sandwiched between thick basal laminae. Venules resemble large capillaries and the transition between the two types of vessel is difficult to identify
Histological demonstration of blood vessels Van Gieson’s technique alone or combined with an elastin stain gives good results in demonstrating connective tissue components of vessel walls. The overall pattern of vascularisation is well demonstrated by reticulin stains. Of the enzymes, the allaline phosphatase reaction reliably identifies endothelial cells both by light and electron microscopy. The immunocytochemical demonstration of factor VII I-related antigen is now routinely used to define endothelial cells.
The concept of a blood-brain barrier was first based upon the observation that intravenously injected vital dyes, like Evans (azovan) blue and trypan blue, entered and stained various organs, but not the brain.1~ Later, ultrastructural studies with electron-dense tracers, such as horseradish peroxidase or lanthanum, demonstrated that tracers do not penetrate the interendothelial cell junctions in the brain, neither are they carried across the endothelial cell by vesicular transport.141 The morphological basis for the blood-brain barrier appears to reside in two features of endothelial cells: the presence of tight junctions and the paucity of cytoplasmic vesicles. The oversimplified single-membrane model, however, is no longer accepted; intercellular tight junctions, intracellular enzyme systems and the two endothelial cell membranes all contribute to the barrier effect. It has been demonstrated in vitro that astrocytic foot processes, unique to cerebral capillaries, could contribute to the barrier. Astrocytes are essential for the expression of the endothelial enzymes which play a role in transport mechanisms142 and for the induction of barrier properties in vitro.
The blood-brain barrier does not pertain to all parts of the mammalian brain: a few, relatively small and usually periventricular structures are freely permeable to vital dyes and electron-dense tracers. These structures include the area postrema, median eminence, subcornmissural organ, pineal gland, subfornical organ, supraoptic crest and neurohypophysis. The blood vessels in these areas have ultrastructural, enzymatic and permeability features which are different from those in other areas of the brain.
It has been realised that the blood-brain barrier is more a regulatory interface between the blood flow and the cerebral parenchyma than a simple rigid physical barrier. The passage of a particular substance across the blood-brain barrier may depend upon various factors, including its lipid solubility, electrical charge, molecular size, dissociation constant, an affinity for a carrier molecule and the nature of the substance in relation to the capacity of the blood-brain barrier for active transport.
The function of the blood-brain barrier is threefold. First, it prevents or hinders the entry of most water-soluble substances into the brain; the permeation rates are usually determined by lipid solubility. Secondly, the blood-brain barrier promotes the transport of certain materials, including some hexoses and several amino acids, by stereo-specific carrier transport systems which are present in the cerebral endothelium. The transport of various materials across the blood-brain barrier has been recently reviewed. Thirdly, the blood-brain barrier plays an important r6le in the volume regulation of the central nervous system. This is achieved by two mechanisms that limit the bulk flow of water across the blood-brain barrier: these are the low hydraulic conductivity of capillaries and the high osmotic activity of the major solutes.
The choroid plexus is composed of the vascular fold of the pia mater and an epithelial layer derived from the ependymal lining. There are four choroid plexuses, one in the medial wall of each lateral ventricle and one each in the roofs of the third and fourth ventricles. They have clearly defined attachments to the ventricular wall and their free edges are invaginated into the ventricles. The surface area of the choroid plexus is greatly increased by the many fronds, which in turn consist of tiny villous processes. The arteries supplying the choroid plexus branch out into capillaries, one for each villus, which then join to form a vein.
The epithelium consists of a single layer of cuboidal cells mounted on a basement membrane; in a few areas, however, pseudostratification or even true stratification may occur. Choroid plexus epithelium can be identified immunocytochemically by the presence of carbonic anhydrase 11(c) in the cytoplasm; this enzyme probably plays a role in the production of cerebrospinal fluid (CSF). The round or oval nucleus of the cell is usually centrally located in the cytoplasm, which has the usual complement of organelles. Mitochondria are particularly numerous and are mainly in the apical portion of the cell: they provide the energy necessary for the active transport carried out during the production of the CSF. Smooth and coated vesicles of various sizes are seen throughout the cytoplasm and these take part in the transport of materials. The apical surface of the epithelial cell is greatly increased in area by masses of microvilli and occasional cilia which tend to be grouped together. The lateral plasma membranes are bound together by complex connections: by tight junctions (zonulac occludentes) at the apical end, by zonulac adhaerentes, and by intricate infoldings at the basal end. Occasional phagocytes, the epiplexus or Kolmer cells, are seen on the surface of the choroid plexus epithelium; they may play a role in keeping this surface free of debris.
The fibrovascular core of the choroid plexus supporting the epithelium contains arachnoid cells; whorls of collagen fibres in the fibrous stroma become calcified with advancing age.1~0 Blood vessels of various sizes include small arteries, arterioles, capillaries and venous sinuses. Capillaries in the villi are fenestrated and their endothelial lining is very thin.
The main function of the choroid plexus is the production of the CSF, although a proportion of the CSF, estimated to be 10-20%, is derived from extrachoroidal sources. In man, approximately 500-700 ml is produced every day; of this only 140 ml can be accommodated at any one time:
25-30 ml in the ventricles and the rest in the subarachnoid space. Although it has been disputed whether the CSF is the result of passive dialysis or active secretion, evidence now favours the latter mechanism. Factors that are involved in the formation of CSF include pressure, serum osmolality, temperature, age, innervation of the plexus and prostaglandins. The enzyme systems of the choroid plexus and various theories of CSF production have been recently reviewed.
The choroid plexus may also take part in the absorption of materials as demonstrated in experimental animals but this function has not been unequivocally confirmed. An estimated l0% of the CSF may be absorbed by the choroid plexus.
The meninges covering the central nervous system are composed of three layers: the dura mater, the arachnoid mater and the pia mater. The arachnoid and pia jointly form the leptomeninges.
The dura mater (pachymeninx) is a tough, dense membrane that surrounds the brain. It has two extensions: the faix, between the two cerebral hemispheres, and the tentorium cerebelli, separating the contents of the posterior fossa from the rest of the brain. The cranial dura is closely attached to the skull; its two layers, the periosteal and meningeal dura, are fused and separate only to form the venous sinuses. In the spinal canal, however, the dura is separated from the vertebral periosteum by the epidural space, which contains fibro-fatty tissue and an epidural venous plexus.
The dura is formed by dense, interlacing bundles of collagen in which flattened fibroblasts are embedded. The central part contains more cells and occasional blood vessels. Its outer surface is covered by thin, overlapping cell processes and its inner border also has a covering of flattened cells. The subdural space is artefactual since the dura and arachnoid are closely apposed in life with no appreciable gap between them.
The arachnoid mater has a variable thickness, in places being formed by several cell layers. Its outer, dural aspect is smoother than the inner, pial -aspect from which trabeculae emerge to bridge the subarachnoid space. The arachnoid cells are joined together by specialised contacts, including tight junctions, which ensure an effective physiological barrier impermeable to CSF.
The cells of the pia mater are similar to those of the arachnoid, but the pia itself is thinner than the arachnoid. Pial cells form a complete layer joined by desmosomes and gap junctions.156 The subpial space separates the pia from the glia limitans of the underlying neural tissue and the pia mater separates the subarachnoid space from the perivascular (Virchow-Robin) spaces of the brains.
Arachnoid villi are diverticula of the arachnoid mater and the subarachnoid space which extend into veins and venous sinuses of the dura. Arachnoid granulations are larger than villi and are visible to the naked eye, whereas villi are microscopical structures. Each villus or granulation is coated on its venous aspect by endothelial cells and is bathed by venous blood. As the villus or granulation penetrates the dura, it forms a narrow neck which then expands to form a central core composed of channels and collagenous trabeculae. Towards the apex of the granulations, there is a cap of arachnoid cells with wide channels running through to the coating endothelium. These structures are a major pathway for the drainage of cerebrospinal fluid, which percolates through the cores of the villi or granulations and is transported across the endothelium into the blood.
The subependymal plate has long been recognised as a layer of primitive cells beneath the ependymal lining of the lateral ventricles in the adult human ~ It is the remnant of the embryonal matrix (the subventricular zone). Studies of the subependymal plate in various animal species have revealed that in the fetus it gives rise to both neurons and glia, whilst after birth, it is a source of glial cells only. The cells of the subependymal plate display ultrastructural features common to primitive cells: high nuclear-cytoplasmic ratio, the dominance of free ribosomes over membrane-bound ribosomes and a general scarcity of organelles. Mitotic activity persists into later adult life in various species, including primates. Unfortunately, information on the human subependymal extrapolation may be misleading.
The human subependymal plate is limited, and extrapolation from experimenta] animals to man may be misleading.
In addition to the subependymal plate, there are other secondary germinal sites in the mammalian central nervous system, including the dentate gyrus of the hippocampus, the olfactory bulb and the external granular layer of the cerebellum. This latter zone, which has been more comprehensively studied than the other two, is formed in fetal life and postnatally continues to produce the neurons of the internal granular layer. The proliferative activity of these secondary germinal zones and the hormonal, nutritional and pharmacological factors which influence cellular turnover have been reviewed.
The presence of the subependymal plate with potential mitotic activity in the adult human brain raises the question of the replacement of glial cells and of their proliferative activity in the normal brain. The view that cells of the adult central nervous system do not divide cannot be maintained any longer as there is convincing evidence that astrocytes and cells of the subependymal plate maintain mitotic activity throughout adult life. Although oligodendrocytes undergo mitosis in pathologica] conditions, their ability to divide in the normal brain has not been unequivocally demonstrated. Similarly, microglial cells do not appear to be mitotically active in the normal, adult central nervous system. Neurons, ependymal cells, choroid plexus epithelium and pericytes do not divide after they have become differentiated, whilst endothelial cells continue to undergo mitosis during adult life. The low turnover of cells in the adult central nervous system, coupled with the difficulty of positively identifying dividing cells and the occasional cell which is not fully differentiated, makes a precise assessment of the mitotic activity of a particular cell type difficult. Moreover, recent tissue culture studies of the developing rat optic nerve have revealed that glial precursors, depending on the composition of the culture medium, can differentiate into either astrocyte or oligodendrocyte even without the influence of other brain cells.170 If these cells persist into adult life they may retain their differentiation potential and mitotic activity.