severe with increasing malignancy. Vas- of ... - Europe PMC

Br. J. exp. Path. (1976) 57, 419

THE FINE STRUCTURE OF BLOOD VESSELS IN ETHYLNITROSOUREAINDUCED TUMOURS OF THE RAT NERVOUS SYSTEM. WITH SPECIAL REFERENCE TO THE BREAKDOWN OF THE BLOOD-BRAIN BARRIER D. J. COX, G. J. PILKINGTON AND P. L. LANTOS From the Department of Neurological Studies and the Bland-Sutton Institute of Pathology, Middlesex Hospital Medical School, London W1P 8AA Received for publication March 3, 1976

Summary.-The fine structure of capillaries in and around ethylnitrosourea-induced tumours, gliomas and schwannomas, was examined in rats. A great variation was observed in the severity of changes: the degree of abnormality depended on the histological type and size of the tumour and on the site of the capillaries within the neoplasm. Endothelial cells, basement membranes and pericytes all demonstrated changes in their fine structure. The most striking alterations occurred in the endothelial cells: luminal cell membranes, tight junctions and pinocytotic activity were all modified. The widened extracellular spaces, particularly around capillaries, were frequently seen to contain proteinaceous material and haematogenous cells. Invasion of these spaces by neoplastic cells, however, rarely occurred. Formation of new capillaries was indicated by the mitotic activity of endothelial cells. These changes in the blood vessels of cerebral tumours have an important role in the breakdown of the blood-brain barrier. IT IS well known that blood vessels in the cerebral tumours of man show structural changes which become more severe with increasing malignancy. Vascular endothelial proliferation can occur in any malignant glioma and this phenomenon is regarded as the earliest evidence of dedifferentiation, being present in 95% of glioblastomas (Russell and Rubinstein, 1971). Although some changes, particularly the pronounced proliferation of endothelial cells, are quite obvious by light microscopy, only electron microscope investigations provide detailed and precise information. All the constituents of normal blood vessels endothelial cells, basement membranes, pericytes and astrocytic foot processes-are altered. The endothelium shows attenuation or hyperplasia, irregularity of the luminal surface, widened intercellular junctions and increased pinocytotic activity. Tortuosity, thickening, disruption and reduplication are the features of basement membranes. There is a loss of astrocytic investiture and

widening of the extracellular space (Luse, 1960; Raimondi, 1966; Long, 1970; Hirano and Matsui, 1975). Some of the changes, particularly those occurring in the endothelium, are thought to indicate an increased transport of material between the circulation and the tumour and to be responsible for the breakdown of the blood-brain barrier. The consequent cerebral oedema is of vital importance to the prognosis of the patient. In order to facilitate the further study of the vascular changes in cerebral tumours, an ideal method of inducing experimental neural neoplasms was needed. This was provided by Druckrey, Ivankovic and Preussmann (1966) who introduced Nethyl-N-nitrosourea (ENU), a simple nitrosamide, into experimental neuropathology. A single dose of this carcinogen induces a high incidence of neoplasms consistently and selectively in the nervous system of foetal and neonatal rats. The morphology of ENU-induced neural neoplasms has been studied

420

D. J. COX, G. J. PILKINGTON AND P. L. LANTOS

extensively (Kleihues, Lantos and Magee, 1976), but very little is known about the fine structure of their vasculature. The purpose of this communication is to describe the fine structural changes of the blood vessels in ENU-induced neural tumours and to relate these morphological alterations to their functional consequences. MATERIALS AND METHODS

Neoplasms of the nervous system were induced transplacentally by a single i.v. injection of 30 mg of ENU/kg of body weight into pregnant BD-IX rats on the 15th day of gestation. The ENU was dissolved in a 3mM citrate buffer, containing 0.9% (w/v) NaCl at pH 6-0. The offspring were killed by whole body perfusion via the ascending aorta when they developed signs of the tumours. For fixation a mixture of glutaraldehyde and formaldehyde, one-half strength Karnovsky fixative at pH 7 4, was used (Karnovsky, 1965): the rats were perfused for 30 min at roomn temperature. The brain and spinal cord, having been left in situ, were placed in the same fixative for a further 4 h. The nervous system was then dissected out and examined for tumours. These were cut into 1-mm3 blocks and washed in 0-2 M sodium cacodylate-sucrose solution for 12 h at pH 7 4. Post-fixation in 1% osmium tetroxide in phosphate buffer was followed by a further wash in buffer before the usual dehydration in ascending strengths of ethanol and subsequent embedding in either Spurr or Epon resin. Thin sections were stained with uranyl acetate and lead citrate and examined in either an AEI 801 or an Hitachi HU12A electron microscope.

RESULTS

Histologically the tumours induced by ENU are various types of glioma (oligodendrocytomas, astrocytomas, mixed oligodendro-astrocytomas, ependymomas, anaplastic gliomas and periventricular pleomorphic gliomas) and schwannomas of the spinal roots and trigeminal nerves. The morphology, including the ultrastructure, of these tumours has been described previously (Kleihues, Lantos and Magee, 1976); here their vasculature will be described in detail.

Gliomas Changes in capillary ultrastructure range from normal to extensively altered. Larger capillaries appear flattened or elliptical in cross-section and they are lined by attenuated endothelium. Smaller vessels, in contrast, retain their circular outline and show less tendency to develop attenuated endothelial cells. It is indbed these small capillaries in which the endothelial cells develop a large, irregular nucleus and bulky cytoplasm; the enlarged cell consequently protrudes into the lumen (Fig. 1) which may therefore become partially occluded. It must, however, be remembered that fixation by perfusion can modify both the outline of the capillaries and the shape of the endothelial cells. The endothelial nuclei vary from small and oval to large and irregular. The chromatin pattern is also variable and hyperchromatic nuclei are frequently seen (Fig. 1). The amount of cytoplasm ranges from sparse to abundant: it shows free ribosomes, occasional short cisternae of the rough-surfaced endoplasmic reticulum, few mitochondria and occasional Golgi complexes. Extremely attenuated endothelial cells containing few organelles are also seen. The most significant feature of the cytoplasm is the large number of smooth and coated vesicles. These originate by budding from invaginations of the luminal cell membrane (Fig. 2). The luminal surface of the endothelial cell is increased by numerous cytoplasmic processes and invaginations. Most tight junctions (zonulae occludentes) between endothelial cells appear fundamentally normal displaying the usual pentalaminar structure, although some show dilatation both at their luminal and abluminal aspects. In attenuated cells these junctions vary from long, straight, fused structures to short, open ones (Fig. 3a). Convoluted junctions in which fused portions alternate with dilated, open segments along their course are also seen (Fig. 3b). Basement membranes vary in number, thickness, course and density. They

FINE STRUCTURE OF BLOOD VESSELS IN ENU-INDUCED TUMOURS

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FIG. 1.-Enlarged endothelial cell shows irregular, hyperchromatic nucleus and numerous pinocytotic vesicles (arrows) beneath the luminal membrane. The basement membrane (BM) is thickened and contains areas of varying electron-density. x 15,880. FIG. 2. The cytoplasm of an endothelial cell (E) and a pericyte (P) shows many vesicles, some of which are in the process of formation. L = lumen; BM = basement membrane. x 48,000.

occasionally form multiple, thick layers around the endothelium and enclose pericytes (Fig. 4a); in other cases they are represented by a single, frayed band. Their outer aspects are frequently indistinct and it is difficult to distinguish

them from the abutting tissue. The usual homogeneity of their substance is lost in many cases; rarefied areas and occasional vacuoles give them a mottled appearance. Collagen fibres seem to be closely applied to the outer aspects of some basement

422


FIG. 3a. A short junction between 2 endothelial cells appears to be patent.

x 176,000.

FIG. 3b.-A long, convoluted junction shows irregular dilatations (arrows) along its course.

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membranes (Fig. 4b). Complete inter- cytes may also be separated by astrocytic ruptions occur only in more abnormal processes from their points of attachment blood vessels. to the basement membrane and these Pericytes are present in many cases: detached cells then lie in the widened they range from attenuated cells with few extracellular space or enter the tumour. organelles to large cells with increased Pinocytotic activity is noticeable in periorganelle content (Fig. 5). Frequently cytes (Fig. 2). The capillaries are usually denuded of they are sandwiched between 2 layers of basement membrane (Fig. 2). Peri- astrocytic foot processes; occasionally,


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FIG. 4a. Layers of basement membranes (arrows) show uneven density and tortuous course. The cytoplasm of a pericyte (P) and a large astrocytic foot process (A) are also present. x 13,360. FIG. 4b. Indistinct basement membrane (arrows) is applied to the attenuated endothelial cell (E). There are numerous collagen fibres (Co) in the widened extracellular space. x 18,720.

however, these are seen winding in bizarre fashion around capillaries (Fig. 6). They contain numerous filaments, mitochondria and glycogen. Neoplastic glial cells sometimes abut on the basement membrane and in some cases they are found inside the membrane encroaching upon endothelial cells (Fig. 7). 28

The extracellular space is greatly increased in gliomas, being particularly widened around capillaries (Fig. 4b). Detached pericytes, degenerated astrocytic processes, neoplastic cells-singly or in groups-and haematogenous cells may all be present (Fig. 8). These cellular elements are often embedded in a floc-

424

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COX, G. J. PILKINGTON AND P. L. LANTOS

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Fia. 5. Pericytes (P) surrounded by basement membranes (arrows) are present adjacent to capillary lumina (L). x 12,600. FiG. 6. Numerous layers of astrocytic processes surround a capillary. x 2,400.

culent material whose appearance is proteinaceous. Endothelial cells are occasionally seen to undergo mitosis-as are pericytes-but marked proliferation of these cells has not been observed and in none of these cases was the diagnosis of gliosarcoma justified. Endothelial cells retain the integrity of

their special cell junctions during division. Centrioles, surrounded by an osmiophilic halo, are sometimes seen; they are suggestive of incipient cell division. The extent of abnormality of blood vessels is seen to vary according to 3 factors: the size of the tumours, the site of the vessel within the tumours and the


FIG. 7.-Invasion of the pericapillary space by undifferentiated neoplastic cells. Intact basement membrane (arrows) separates the tumour cells from the surrounding brain tissue. x 2,950. FIG. 8.-Extravasated erythrocytes (E) are seen in the widened extracellular space around a capillary (C). x 3,200. FIG. 9.-Fenestration (arrow) in the endothelial lining of a capillary of a schwannoma. Flocculent material is present in the extracellular space (ECS). x 13,350.

425

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FIG. 10.-Two endothelial cells undergoing mitosis and bearing slender processes (arrows) at their luminal surface in the blood vessel of a schwannoma. The widened extracellular space (ECS) contains flocculent material. x 4,660. FIG. 11I.-A part of a large cyst (Cy) lined by neoplastic Schwann cells the luminal surface of which is increased by numerous slender processes (arrows). x 1,360.


The histological type of the glioma. degree of abnormality is directly related to the size of the neoplasm: the ultrastructural changes are more severe in larger tumours. The location of the capillary also determines the extent of damage: blood vessels at the periphery of the tumours are less affected. The histological type also influences capillary changes: anaplastic gliomas and periventricular pleomorphic gliomas are the tumours with the most striking capillary abnormalities.

Schwannomas Blood vessels in schwannomas show similar changes to those found in gliomas. Endothelial cells are particularly attenuated with occasional fenestrations (Fig. 9) and open cell junctions. Mitotic activity of endothelial cells is moderate (Fig. 10). The widened extracellular space around capillaries frequently contains many collagen fibres laid down by fibroblasts which are present in large numbers. A characteristic feature of schwannomas is the presence of cysts of varying Multiple, size, shape and number. extensive and irregular cysts occur in larger tumours particularly in degenerating regions. They contain protein-like material and blood cells, mainly erythrocytes. The cysts are lined not by endothelium but by a single row of neoplastic Schwann cells. These cells assume a cuboidal or slightly flattened shape and may display a polarity with cell organelles grouped towards the luminal surface and the nucleus occupying the opposite end of the cell. The lateral surfaces of the cells are closely apposed but gaps may also occur. The luminal surfaces are increased by numerous slender processes while the abluminal surface has a smooth contour (Fig. 11). DISCUSSION

Electron microscope investigation of blood vessels of ENU-induced gliomas and

427

schwannomas reveals that all vascular constituents show changes of varying severity. Similar alterations have been recorded in other experimental neural tumours induced by viruses and chemicals. In intracerebral tumours induced by Rous sarcoma virus in dogs, Vick and Bigner (1972) found 2 types of distinctive endothelial damage. These were fenestrations, covered by a single leaflet of the unit membrane, and small, complete gaps in the endothelial wall. The fenestrations were fundamentally identical to those found normally in the choroid plexus, ciliary body of the eye, intestinal villi and endocrine organs. The small gaps resembled the discontinuities of the normal vasculature of liver, spleen and bone marrow. Unlike normal cerebral capillaries, these fenestrated and discontinuous blood vessels were permeable to cationic dyes: a 1% solution of Evans blue injected i.p. 1 h before killing the animals stained the tumour parenchyma. The authors therefore surmised that the altered endothelium in virally-induced brain tumours was responsible for the breakdown of the blood-brain barrier. They could not, however, exclude the possibility that other, morphologically undetectable, phenomena may also play a role in the increased vascular permeability. Somewhat different observations were made by Haguenau et al. (1971), who found long and wavy tight junctions between the endothelial cells of dog gliomas induced by Rous sarcoma virus. Some endothelial cells were extremely hypertrophied with hyperchromatic and irregular nuclei, but the thickening of the basement membrane and the marked hyperplasia of pericytes were the most striking findings. Hirano, Hasson and Zimmerman (1972) described some fine structural features of blood vessels and cysts found in ENU-induced tumours of the trigeminal nerves and spinal roots of rats. The vascular lumina were lined by a single layer of attenuated endothelial cells which

428


showed occasional fenestrations. The cysts were lined by neoplastic cells: this lining, although continuous in most areas, was interrupted by substantial gaps. Thus, it appears that virally- and chemically-induced tumours of the nervous system have an abnormal vasculature. These abnormalities are similar to those described in the present paper. Hypertrophy, increased pinocytotic activity and abnormal intercellular junctions characterized many endothelial cells. Basement membranes displayed changes in their number, thickness, course and density. Pericytes, occasionally hypertrophied, were seen to become detached from the basement membrane and to enter the parenchyma. Capillaries often lost their astrocytic foot processes, but occasionally were surrounded by many layers of these processes. Evidence obtained from both human and experimental material suggests that some of these alterations are responsible for the breakdown of the blood-brain barrier and for the resulting cerebral oedema (Brightman et al., 1970; Long, 1970). Since cerebral oedema in man carries an ominous prognosis for the patient, the breakdown of the blood-brain barrier merits further consideration. It has long been known that certain dyes, including trypan blue and Evans blue, stain most organs but fail to penetrate the brain parenchyma. This phenomenon has given rise to the concept of the blood-brain barrier: the brain is protected from many substances which can easily reach other organs. In the study of the blood-brain barrier various techniques have been used to localize the morphological structure which is responsible for this effect. In earlier experiments the acidic dyes, trypan blue and Evans blue, were most frequently used: they have a strong affinity for serum proteins and the distribution of these dyes in thick frozen sections therefore provides information about the vascular permeability to proteins. The introduction of fluorescent conjugates has permitted a more precise definition of the blood-brain barrier

(Klatzo, Miquel and Otenasek, 1962), but the complex morphology of blood vessels was only fully revealed by the electron microscope with the study of the transport of electron-opaque materials from blood vessels. Horseradish peroxidase, which after a reaction with diaminobenzidine yields an electron-dense precipitate, has become widely used in the study of cerebral vascular permeability (Reese and Karnovsky, 1967). While peroxidase easily passes through the vascular endothelium of striated and cardiac muscles, it is withheld within the capillary lumen in the brain. The capillary endothelium of muscle shows open slits between cells and many pinocytotic vesicles: these are the routes by which peroxidase leaves the circulation and enters the parenchyma. In contrast, the vascular endothelium of the brain has neither high pinocytotic activity nor patent intercellular contacts: they form tight junctions (zonulae occludentes) which prevent the passage of peroxidase (Reese and Karnovsky, 1967). It was therefore thought that these specialized junctions between endothelial cells were responsible for the phenomenon of the blood-brain barrier: these pentalaminar structures constitute a morphological barrier to the passage of material between the cerebral blood vessels and the parenchyma. It was, and still is, disputed whether the tight junctions are solely responsible for the protection of the brain from certain circulating agents or whether other constituents of the vessel wall also contributed to this effect. Basement membranes and astrocytic foot processes were both implicated in the maintenance of the blood-brain barrier (Tani and Evans, 1965). It was, however, Steinwall and Klatzo (1966) who visualized the barrier not as a simple morphological structure but as a complex control mechanism in the transport of materials. It was early appreciated that the breakdown of this barrier must play an important, occasionally central, role in pathological conditions of the brain. Brightman et al. (1970), studying the


cerebral vascular permeability in normal and pathological conditions-including oedema, hypertension, concussion and hypoxia-thought that the disturbance of the blood-brain barrier may be more than an ephemeral phenomenon in the pathogenesis of disease: it may constitute the crucial pathogenetic factor which, setting a chain of events into motion, determines the final pathological lesion. Cerebral neoplasms are important and frequent lesions in which the blood-brain barrier is altered. Vascular permeability can be abnormally increased by various routes via the endothelial cells: intercellular passage, vesicular transport and gross endothelial cell damage. In ENUinduced neural tumours all these changes were encountered: opening of the tight junctions, increased pinocytotic activity and fenestrated endothelial cells were all present. In addition, irregularities of the basement membranes and loss of astrocytic foot processes have further contributed to the disturbance of the bloodbrain barrier. The widened extracellular space, particularly around capillaries, is a clear indication of the breakdown: it contained escaped plasma and extravasated blood cells. The resulting oedema is mainly vasogenic in type (Klatzo, 1967): it is caused by increased vascular permeability with the consequent escape of serum constituents. The morphological changes of blood vessels in cerebral tumours are associated with alterations in the distribution of various enzymes. O'Connor and Laws (1969) found the reduction or disappearance of those enzymes which take part in the energy transport within endothelial cells. Moreover, these authors suggested that the biochemical and histochemical alterations underlie, or even precede, the obvious morphological changes. An interesting feature of the blood vessels of cerebral tumours in man is the pronounced endothelial hyperplasia leading to the formation of many new capillaries (Russell and Rubinstein, 1971). Although endothelial cells were seen to

429

divide in ENU-induced neural tumours, this mitotic activity was lower than that observed in human tumours. No dense meshwork of newly-formed capillaries, as is seen in gliomas of man, was found in rat tumours. It is now widely ac7cepted that the survival of large, solid tumours is dependent on their vascular supply-the facility by which new blood vessels are formed. This phenomenon was once regarded as a secondary reaction to the tumour, now it is considered to play a crucial role in the neoplastic growth. Folkman (1975) has suggested that the tumour stimulates angiogenesis by specific humoral factors and that the newly formed vessels, in turn, have a controlling influence on the rate of growth of the tumour. He has envisaged avascular and vascular phases in the neoplastic growth: the latter, he postulates, is initiated by a diffusible angiogenetic factor produced by the neoplasm. Without this factor the avascular phase is prolonged and the tumour fails to grow. In ENU-induced neural tumours the moderate degree of angiogenesis may be responsible for the necrotic areas found in larger gliomas and schwannomas. These 2 phenomena, the breakdown of the blood-brain barrier and the formation of new blood vessels, are those changes which most influence the progression of neural tumours. We thank Dr Helen C. Grant for her help in the preparation of this manuscript. The secretarial help of Miss Kathleen Woolf is gratefully acknowledged. REFERENCES BRIGHTMAN, M. W., KLATZO, I., OLSSON, Y. & REESE, T. S. (1970) The Blood-Brain Barrier to Proteins under Normal and Pathological Conditions. J. neurol. Sci., 10, 215. DRUCKREY, H., IVANKOVIC, S. & PREUSSMANN, R. (1966) Teratogenic and Carcinogenic Effects in the Offspring after Single Injection of Ethylnitrosourea to Pregnant Rats. Nature, Lond., 210, 1378. FOLKMAN, J. (1975) Tumor Angiogenesis: A Possible Control Point in Tumor Growth. Ann. intern. Med., 82, 96.

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Blood-Brain Barrier in Human Malignant Brain Tumors. J. Neuro8urg., 32, 127. LUSE, S. A. (1960) Electron Microscopic Studies of Brain Tumors. Neurology, Minneapoli8, 10, 881. O'CONNOR, J. S. & LAWS, E. R., JR. (1969) Changes in Histochemical Staining of Brain Tumor Blood Vessels Associated with Increasing Malignancy. Acta neuropathol., 14, 161. RAIMONDI, A. J. (1966) Ultrastructure and the Biology of Human Brain Tumors. In Progre88 in Neurological Surgery, 1, Ed. H. Krayenbuhl, P. E. Maspes and W. H. Sweet. Basel: Karger. p. 1. REESE, T. S. & KARNOVSKY, M. J. (1967) Fine Structural Localization of a Blood-Brain Barrier to Exogenous Peroxidase. J. Cell Biol., 34, 207. RUSSELL, D. S. & RUBINSTEIN, L. J. (1971) Pathology of Tumours of the Nervous System. London: Edward Arnold. STEINWALL, 0. & KLATZO, I. (1966) Selective Vulnerability of the Blood-Brain Barrier in Chemically Induced Lesions. J. Neuropath. exp. Neurol., 25, 542. TANI, E. & EVANS, J. P. (1965) Electron Microscope Studies of Cerebral Swelling. Acta neuropathol., 4, 507. VICK, N. A. & BIGNER, D. D. (1972) Microvascular Abnormalities in Virally-induced Canine Brain Tumors. J. neurol. Sci., 17, 29.