JOURNAL OF ULTRASTRUCTURE RESEARCH 56, 6 5 - 7 7
(1976)
Observations on the Blood-Brain Barrier in Hypertensive Rats, with Particular Reference to Phagocytic Pericytes B. VAN DEURS Anatomy Department A, University of Copenhagen, 71 Rddmandsgade, DK 2200 Copenhagen N, Denmark Received January 21, 1976 The endothelial and periendothelial parts of the blood-brain barrier in the r a t have been studied by electron microscopy. Extravasation of horseradish peroxidase (HRP) occurred after Aramine-induced acute hypertension. It was exclusively due to a local vesicular transport. This was most p r o m i n e n t in arterioles, but also occurred in segments of capillaries and venules. The t i g h t junctions between adjacent endothelial cells were never penetrated by HRP. Following extravasation, HRP reached the endothelial b a s e m e n t membrane, from where it often spread into the extracellular space of the neuropil. A phagocytic pericyte was observed, exhibiting a well-developed vacuolar apparatus and a pronounced uptake of HRP. It is suggested t h a t the phagocytic pericytes in the r a t b r a i n represent "microglial" cells, and t h a t they play a n i m p o r t a n t role in the periendothelial part of the blood-brain barrier.
It is well established that a barrier exists, preventing an exchange of protein, between the bloodstream and the brain parenchyma (10, 11). This blood-brain barrier (BBB) is due to two features of the cerebral endothelium (4, 34): the tight junctions (zonulae occludentes; 42) between adjacent endothelial cells effectively sealing the intercellular spaces, and the sparse number of endothelial vesicles ruling out any significant vesicular transport. However, also outside the endothelium a barrier may exist (4, 10, 11,24, 27, 55) protecting the neuropil against extravasated protein. The endothelial basement membrane, and the sheath of astrocytic end-feet surrounding the endothelium, may be elements of such a barrier. Furthermore, pericytal phagocytes have been described in the cerebral microvasculature (5, 24, 25, 27, 30, 47), and these are also likely to play a role in a periendothelial barrier. The endothelial part of the BBB is susceptible to experimental intervention, and various methods of breaking down the barrier have been applied. Certain vasotoxic agents (nickel chloride, mercuric chloride, and lysolecitin) induce extravasation of protein in the brain (20, 30), and the
transport of intravenously injected horseradish peroxidase (HRP) across the endothelium increases significantly when serotonin is perfused through the cerebral ventricles (49). Acute hypertension induced by intravenously injected Aramine (metaraminol bitartrate) also increases the transfer of H R P across cerebral endothelia (16, 19, 30, 51). Hence, under various experimental conditions it is possible to obtain a considerable amount of a tracer protein outside the endothelium of the cerebral microvasculature. The aim of the present study was therefore to investigate, at the ultrastructural level, by which mechanism the HRP extravasation following acute hypertension takes place, and the significance of the endothelial basement membrane, the perivascular space, the pericytal phagocytes, and the astrocytic end-feet in the extravascular distribution of HRP. MATERIALS AND METHODS
Experiments. E i g h t rats ~ were anesthetized with N e m b u t a l (sodium pentobarbitone) and the blood pressure (BP) monitored in the femoral artery. After The hypertensive rats used in this study were a part of the material in a study by Dr. E. Westergaard, Dr. H. E. Br0ndsted, and the present author (see refs. 51, 52). 65
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
B. VAN DEURS
30-60 min with a mean BP between 110 and 120 mm Hg, Aramine (metaraminol bitartrate), 0.07 mg/kg/ rain, was intravenously infused over a period of 1-14 min. This resulted in a rise of the mean BP to about 170 mm Hg. Two-hundred milligrams of horseradish peroxidase (HRP; Sigma type II) in 1 ml of phosphate-buffered saline (PBS) were intravenously infused during 4-12 min, and allowed to circulate for a further 0-15 min before fixation. To study the "normal" exchange of HRP between blood and parenchyma, three rats received 200 mg of HRP without any Aramine treatment. The HRP was allowed to circulate for 3, 10, and 30 min. As a control for endogenous peroxidase, three untreated rats were included in the material. Fixation procedure. All the animals were fixed by a two-step perfusion with aldehydes through the left ventricle. The initial fixative contained 2% formaldehyde (freshly prepared from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.3, and the perfusion time was 3 min. The second fixative contained 4% formaldehyde and 5% glutaraldehyde in the same buffer. This fixative was perfused for 10-15 min. Brains were kept overnight in situ at 4°C. Incubation for peroxidase activity. Brain slices (frontal sections) 0.5-1.0-mm thick were rinsed in sodium cacodylate buffer and incubated for peroxidase activity in the following way: for 1 hr at room temperature in Tris-HC1 buffer (600 mg of Trizmabase, Sigma, in 100 ml of water; pH adjusted to 7.6) containing 0.4 ml of glucose oxidase (Sigma type V, 10,000 units) per 100 ml of buffer, and thereafter for 3 hr at 0°C in the above solution to which were added 50 mg of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 36 mg of anhydrous glucose (Merck) per 100 ml of buffer. Preparation for electron microscopy. After incubation, the brain slices were rinsed in 0.1 M sodium cacodylate buffer, cut into blocks, and treated at room temperature with 2% OsO4 in 0.1 M sodium cacodylate buffer, pH 7.4, for 3 hr, followed by impregnation in 2% uranyl acetate in maleate buffer for 2 hr at room temperature. The blocks were dehydrated in graded series of ethanols and embedded in Spurr epoxy plastic (41). Ultrathin sections were contrasted with uranyl acetate and lead citrate. OBSERVATIONS
The B l o o d - B r a i n Barrier in N o r m a l R a t s After i n t r a v e n o u s injection of H R P i n n o r m a l rats, only v e r y little reaction product was observed in the brain. W i t h the exception of certain ~leaky" regions such as the choroid plexus, H R P was in general confined to vesicles w i t h i n the endothelial
cells a n d to the b a s e m e n t m e m b r a n e a n d the p e r i v a s c u l a r space of a few short segm e n t s of arterioles. However, the walls of most arterioles as well as of capillaries a n d venules did not exhibit a n y HRP-labeling. N e i t h e r could a n y H R P be detected in the extracellular space of the neuropil outside the vessels. Pericytal cells with a welldeveloped v a c u o l a r a p p a r a t u s were somet i m e s seen.
The Blood-Brain Barrier in Hypertensive Rats E n d o t h e l i a l cells a n d basement m e m brane. In the A r a m i n e - t r e a t e d r a t s H R P was p r e s e n t in the b a s e m e n t m e m b r a n e of most p a r t s of the cerebral microvasculat u r e (exceptions are shown in Figs. 2 a n d 13). The t i g h t junctions (zonulae occludentes), seen as points of m e m b r a n e fusion b e t w e e n adjacent endothelial cells, were n e v e r p e n e t r a t e d by H R P (Fig. 1). Reaction product was p r e s e n t from the basem e n t m e m b r a n e into the intercellular cleft only up to the most a b l u m i n a l point of m e m b r a n e fusion (Figs. 1 a n d 6). A t the l u m i n a l aspect of the most l u m i n a l m e m b r a n e fusion the t r a c e r was u s u a l l y w a s h e d out, as seen in Fig. 6. However, in vessels imperfectly perfused d u r i n g fixation, in which labeling could be seen also on the l u m i n a l surface of the endothelial cells or in the entire v a s c u l a r l u m e n (a situation t h u s similar to t h a t obtained after i m m e r s i o n fixation), the H R P h a d evidently not p e n e t r a t e d the m o s t l u m i n a l point of fusion (Fig. 1). HRP-labeled vesicles were n u m e r o u s in the endothelial cells of certain s e g m e n t s of the microvasculatt/re. These s e g m e n t s were in p a r t i c u l a r arterioles (Figs. 2-4), b u t sometimes also capillaries a n d venules exhibited a considerable n u m b e r of labeled vesicles (Figs. 5 a n d 6). The vesicles were m o s t l y u n c o a t e d and m e a s u r e d a b o u t 70 n m in diameter, a l t h o u g h l a r g e r ones occurred, probably r e p r e s e n t i n g fusions of the smaller vesicles. Labeling of the basem e n t m e m b r a n e , especially of capillaries,
HRP EXTRAVASATION IN THE BRAIN
67
FIGs. 1-4. Different parts of the same small arteriole, photographed from a single section. Although perfusion fixation has been used, reaction product is seen on the luminal surface of the endothelium (imperfect perfusion). FIG. 1. Shown is a tight junction between two adjacent endothelial cells: reaction product is absent between the most luminal point of membrane fusion and the most abluminal fusion point. Arrows indicate the fusion points. Note t h a t HRP is also seen in the basement membrane although no HRP-labeled vesicles are present in the endothelium, x 130 000. FIG. 2. Only luminal vesicles are labeled; the basement membrane is unlabeled, x 80 000. FIG. 3. Intraendothelial vesicles also exhibit labeling, as does the basement membrane, x 80 000. FIG. 4. Both luminal and abluminal vesicles are labeled, as is the basement membrane, x 80 000.
was frequently seen, although the endothelium above was almost or completely devoid of labeled vesicles (Figs. 7 and 15).
In vessels imperfectly perfused during fixation, labeling was sometimes confined to the luminal vesicles. In other speci-
68
B. VAN DEURS
FIG. 5. Part of a venule. The endothelium contains labeled vesicles, some of which are opening at the basement membrane, which is also labeled. A, Astrocytic end-feet; P, pericyte, x 57 000. FIGS. 6 and 7. Different parts of the same capillary; in Fig. 6 many vesicles are labeled, while vesicles are almost completely absent in Fig. 7. The arrow in Fig. 6 indicates a point of membrane fusion between two adjacent cells, above which HRP has been washed out during perfusion fixation. The arrow in Fig. 7 indicates a labeled vesicle in a pericyte (P). A, Astrocytic end-feet, x 57 000. mens, some of w h i c h r e p r e s e n t e d longer H R P - c i r c u l a t i o n t i m e s , a n a l m o s t cornp l e t e l a b e l i n g o f v e s i c l e s f r o m t h e l u m e n to
t h e b a s e m e n t m e m b r a n e , as Well as o f t h e b a s e m e n t m e m b r a n e , could be obtained. H o w e v e r , the v a r i a t i o n in p a t t e r n of label-
HRP EXTRAVASATION IN THE BRAIN
ing could also be seen in various parts of the same vascular segment obtained from the same specimen as shown in Figs. 1-4, no matter what the HRP-circulation time of the particular experiment. Hence, the number and distribution of labeled vesicles seem independent of the circulation time in the present study. It is assumed that the extravasation, where and when it occurs, is a very rapid process, the degree of extravasation, however, varying along a vascular segment. Pericytes. The reaction product clearly outlined the periendothelial cells in the different parts of the microvasculature. Thus, in arterioles the layer of smooth muscle cells and the surrounding pericytes typically were outlined, and likewise the pericytes in the basement membrane of venules and larger capillaries (in the smallest capillaries pericytes were absent) (Figs. 5-8). The pericytes mostly appeared as flattened, branched cells embedded in the endothelial basement membrane (Fig. 8, P). The content of organelles in these cells was in general sparse; some microtubules, and a few mitochondria, strands of endoplasmic reticulum, and small Golgi complexes were noticed. In a few cases HRP-labeled, round profiles were seen (Fig. 7, arrow). Phagocytes. Some cells embedded in expansions of the endothelial basement membrane, thus being pericytal, contained many HRP-labeled vesicles or vacuoles of various size (Figs. 8-12). In addition, many very large membrane-bound bodies were present in these cells. The smallest of the HRP-labeled vesicles, measuring about 70-120 nm in diameter, was sometimes connected with the cell surface (Fig. 12, arrows). Most of these vesicles were distinctly coated, although uncoated ones appeared (Fig. 10). The larger HRP-labeled vesicles or vacuoles measured from about 120 nm up to about 1 tLm in diameter, and the reaction product was present as a peripheral rim rather than a homogeneous labeling (Fig. 9, PH).
69
The membrane-bound cytoplasmic bodies (Figs. 9, 11, 12, LY) measured from about 120 nm up to about 2 t~m in diameter. Their content was of moderate density. Sometimes rounded structures of very high density were seen, often more or less incorporated into the cytoplasmic bodies mentioned above (Figs. 9, 11, 12). It could not be determined whether or not these very dark structures contained HRP, since, in the controls for endogenous peroxidase, similar very dark structures could sometimes be found in pericytal cells. Fusion between typical HRP-labeled vesicles and the large bodies of moderate density was also observed (Fig. 9, double arrow). Compared with these dominant components, other organelles were sparse. The HRP-labeled vesicles as well as the other cytoplasmic bodies were found in the relatively cytoplasm-rich perinuclear region, from which flattened branches with only a few or no labeled vesicles protruded, surrounding the endothelium (Fig. 8, arrows). The degree of HRP-labeling varied greatly: from only a few or no labeled vesicles as in a typical pericyte, to near repletion with large vesicles or vacuoles. In many sections "ordinary" pericytes could be seen in addition to the phagocytes, the latter then being the outermost (Fig. 8). The phagocytes were not present in all the sections of cerebral vessels; but they appeared around all parts of the microvasculature, especially arterioles. They were never seen to extend across the astrocytic end-feet lining, migrating into the neuropil. Perivascular space. Some arterioles, especially the larger ones (30 t~m or more in diameter), were surrounded by a distinct perivascular space (Fig. 13, PVS). The space was lined at its inner border by the outermost basement membrane of the arteriolar smooth muscle layer, and collagen microfibrils. At its outer border it was separated from the astrocytic end-feet by another basement membrane. The space contained free cells and cell processes. The
FIG. 8. The endothelium (E) of this small vessel (venule) is surrounded by pericytes (P), a phagocytic pericyte (PP), and processes from this (arrows) and by astrocytic end-feet (A). Reaction product is present in the basement membrane surrounding the pericytes and pericyte processes between the endothelium and the astrocytic end-feet, as well as in the extracellular space of the neuropil, z 6300. FIG. 9. Part ofaphagocyti~ pericyte, showingthe well-developed vacuolar apparatus. A, Astrocytic endfeet; LY, lysosome; M, mitochondrion; P, pericyte; PH, phagosome. The double arrow indicates fusion between a large heterophagosome and a lysosome. × 19 000. 70
71
HRP EXTRAVASATION IN THE BRAIN
/
FIGS. 10-12. Details of phagocytic pericytes, FIG. 10. Shown are m a n y coated micropinocytic vesicles containing HRP, a m o n g which uncoated vesicles occur (arrow). T h i s section was t a n g e n t i a l to t h e cell surface, x 78 000. FIGS. 11 a n d 12. Shown are large and small lysosomes (LY), in some of which very dense bodies are more or less embedded. The arrows in Fig. 12 indicate the formation of the micropinocytic vesicles. Fig. 11. x 31 000; Fig. 12. x 57 000.
cells were either fibroblastlike or clearly phagocytic (Fig. 13), similar to those described above. Although the perivascular space in a few cases, only, contained "free"
HRP, the collagen microfibrils, the limiting basement membrane, and the many vesicles of the phagocytes were mostly clearly labeled, sometimes also in cases
Fro. 13. An arteriole (ART) surrounded by the perivascular space (PVS). In this, a phagocyte is present. LY, lysosome; PH, phagosome, x 13 000. Fro. 14, The neuropil immediately outside the endothelium (E), the pericyte process (P), and the incomplete lining of astrocytic end-feet (A) exhibit a pronounced labeling of the extracellular space, x 25 000. 72
HRP EXTRAVASATION IN THE BRAIN
when no labeling of the endothelial vesicles or of the endothelial basement membrane was seen (Fig. 13).
The extracellular space of the neuropil. The "sheaths" of astrocytic end-feet around the vessels mostly appeared insufficient as a barrier preventing the extravasated H R P in the basement membrane from penetrating into the extracellular space of the neuropil (Figs. 8 and 14), but the degree of labeling of the extracellular space varied considerably. In some cases only very little or no labeling at all could be detected (Fig. 15). Cells in the neuropil. The astrocytes or astrocytic end-feet, the oligodendrocytes, and the neurons rarely showed uptake of H R P (except for a few small vesicles in the neurons). In some instances cells filled with HRP-labeled vesi'cles and vacuoles as well as cytoplasmic bodies were observed. These cells closely resembled the above described phagocytes (Fig. 16), and in most cases they were clearly surrounded by astrocytic end-feet; they were therefore likely to be pericytal rather than intraparenchymal. DISCUSSION
Extravasation of HRP. The observations demonstrate that acute hypertension provoked by intravenously injected Aramine induces a pronounced extravasation of HRP in the brain of rats. Further morphologic and physiologic details of this acute hypertension model will be given elsewhere (52). In experiments with hypertension induced by partially constricting renal artery (15), it was found that in cerebral precapillary vessels an extravasation could mainly be attributed to intercellular passage of H R P due to broken tight junctions. Acute hypertension caused by Aramine has previously been reported to increase the permeability of the cerebral microvasculature to dye-albumin complexes (18, 19). Extravasation of HRP following Ara-
73
mine-induced hypertension has also been reported (16, 30), but in these studies evidence for the pathway across the endothelium being vesicular rather than intercellular was not given. However, as it appears from the present study the extravasation of HRP resulting from Aramineinduced acute hypertension is exclusively due to vesicular transport, whereas the junctions between the endothelial cells are not penetrated by the tracer protein. Recently it has been demonstrated that vesicular transport also occurs in a few segments of the cerebral microvasculature in normal animals (28, 48, 50). In permeability studies immersion fixation (21, 38, 40, 54) and short time sequence analysis (38, 40) have often been applied, in contrast to the present study, A brief discussion of the different techniqt/es is therefore warranted. Perfusion fixation gives in general a better preservation of brain tissue than immersion fixation (31, 53), but it has the disadvantage that tracers are usually washed out of the vascular lumen. However, as shown in the present study, perfusions are often incomplete, leaving blood-filled vessels scattered through the tissue, with no apparent decrease in fixation quality. In such cases the tracer protein will be observed at the luminal front of the endothelial cells. Perfusion fixation may therefore be the best fixation method, also in permeability studies, regarding the brain. A labeling of vesicles in t h e endothelium, from the lumen towards the basement membrane, could be observed in different tissue blocks representing an increasing HRP-circulation time. However, this could also be seen within the same tissue block. Therefore, to make a further, detailed time-sequence study of the events during the first few seconds or minutes after administration of the tracer protein did not appear justified. As it appears in the literature, a timesequence study comprises very short circulation times after which only a few lu-
FIG. 15. P a r t of a capillary endothelium (E), astrocytic end-feet (A), and the neuropil. HRP-labeling is only distinct in the endothelial b a s e m e n t membrane, x 50 000. Fia. 16. A phagocyte apparently situated within the neuropil. Note the astrocytic end-feet (A) surrounding the phagocyte, x 12 000. 74
HRP EXTRAVASATION IN THE BRAIN minal vesicles should be labeled, followed by slightly longer circulation times with increasing labeling of abluminal vesicles and finally also of the basement membrahe (38, 40). This simplified model has, however, some conspicuous disadvantages. First, it is impossible within the limit of seconds, and probably minutes, to determine the exact time of fixation of a particular region in the tissue block (especially after immersion fixation). Furthermore, the transfer time for a vesicle across an endothelial cell should be measured in seconds (6, 23, 36, 37). It is therefore obvious that comparison between parts of the vascular endothelium from tissue blocks obtained from experiments with slightly different circulation times should be interpretated with the utmost care. Second, the degree of vesicular transport at a given time may not be the same all along the endothelial lining of a particular segment of the microvasculature, as shown in the present study, and also suggested by other authors (38, 39). In the literature, micrographs of segments of muscular vessels, for instance, in which the tracer (HRP) is confined to the luminal aspect of the endothelium even after several minutes of circulation, also occur (21, 23, 54). Furthermore, it is likely that the vesicular transport across vascular endothelia is bidirectional; that is, that a transport of protein and other macromolecules also takes place from the basement membrane to the bloodstream (22, 37). With respect to vesicle labeling, it therefore seems very difficult to choose a "representative" micrograph from a single experiment (which of Figs. 1-4 are ~representative" for that particular experiment ?).
Basement membrane, perivascular space, and astrocytic end-feet. In the present study the vesicular transport appeared to be focal. The labeling of basement membranes below parts of the endothelium devoid of labeled vesicles could be explained by a vesicular transport having occurred
75
before fixation of the tissue. This is in agreement with the above considerations. However, the phenomenon may also be explained by some spread of HRP in the basement membrane to segments without any vesicular transport at all (/6). A spread of H R P along the basement membrane has previously been suggested after intraventricular injection of the tracer (47), and to some extent also after intraparenchymal injection of HRP (3). Sometimes labeling was confined to the basement membrane. In most cases, however, HRP spread into the extracellular space of the neuropil. The latter situation could be due to an %verloading" of the basement membrane, which may act as a more efficient ~'trap" or ~filter" when exposed to smaller amounts of extravasated protein. In addition, it may be that larger molecules than HRP (MW 40 000) are more completely retained in the basement membrane. This has been shown to be the case with the glomerular basement membrane (7, 46). A transport of HRP m a y also occur along the perivascular space (the Virchow-Robin space) of the arterioles, since HRP-labeling of phagocytes and extracellular structures of the space was sometimes noticed in the absence of extravasation in the particular vessel. However, HRP may also reach this position by diffusion through the extracellular space of the neuropil. In conclusion, it seems difficult to evaluate the role of the endothelial basement membrane and the perivascular space in the periendothelial diffusion barrier, but they may play some role for the barrier. On the other hand, the astrocytic end-feet did not constitute a barrier between the endothelial basement membrane and the neuropil. Perivascular phagocytes. It seems reasonable to assume that the perivascular phagocytes play the most important role in the periendothelial part of the BBB. In the thymic cortex a blood-tissue barrier also exists, which is partly due to the presence
76
B. VAN DEURS
of macrophages immediately outside the brain. At some distance from the subendothelium phagocytosing the small arachnoid space, usually at the level of the amounts of protein that are carried across smallest arterioles, the basement memthe endothelium by vesicles (33). The cere- branes bordering the perivascular space bral phagocytic pericytes were provided fuse, and the phagocytes thus become periwith a well-developed vacuolar apparatus cytes, embedded in the basement mem(9, 12). The small, often coated vesicles brane. It is uncertain, however, whether containing HRP are typical micropinocytic two different line of pericytes exist, a vesicles similar to those described in mac- phagocytic and a nonphagocytic, or rophages (8) and absorptive epithelial cells whether phagocytic cells develop directly (13, 14, 44). The micropinocytic vesicles from th.e nonphagocytic ones. may fuse with each other forming larger The presence of pericytal phagocytes in heterophagosomes, or with primary lyso- the brain deserves some comment in the somes forming secondary lysosomes. The light of previous discussions on the origin other kinds of cytoplasmic bodies or vacu- of microglia. A development of nonphagooles probably represent primary and sec- cytic pericytes into phagocytic microglial ondary lysosomes. Acid phosphatase activ- cells has been suggested by some authors ity has been described in cerebral pericytes (1, 2, 26, 47), whereas other investigators (27). have not found any relationship between The vacuolar apparatus appeared simi- microglia and pericytes (32, 43, 45). In the lar to that previously observed in cerebral present study, no phagocytes were obpericytes after provoked protein extrava- served within the neuropil, and neither sation (5, 30). Furthermore, Cancillaet al. the present study nor a previous long-time (5) found that, after increased HRP-expo- tracer-study of cerebral pericytes (5) demsure time the protein was no longer pres- onstrated any migration of pericytes, ent in the extracellular space of the neuro- phagocytic or nonphagocytic, into the neupil, the degree of HRP accumulation in the ropil. The present observations therefore pericytes had increased considerably. The indicate, as was also suggested by Maxendothelial part of the BBB in immature well and Kruger (25), that some pericytes animals is less efficient than in adults. It are themselves, in fact, the ~'microglial" has been shown that in the brain of suck- cells. ling mice, pericytes endocytose extravaREFERENCES sated HRP to a considerable extent, while no tracer appears in the neuropil; it has 1. BALDWIN, F., WENDELL-SMITH, C. P., AND BLUNT, M. J., J. Anat. 104, 401 (1969). been concluded, therefore, that the peri2. BARON,M., AND GALLEGO,A., Z. Zellforsch. 128, cytes constitute a barrier between blood 42 (1972). and neuropil in the immature cerebral tis3. BECKER,N. H., HIRANO, A., AND ZIMMERMAN, sue (24). In modiolar arterioles extravaH. M., J. Neuropathol. Exp. Neurol. 27, 439 sated HRP becomes likewise endocytosed (1968). 4. BRIGHTMAN,M. W., KLATZO,I., OLSSON,Y., AND by perivascular phagocytes (17). In dorsal REESE, T. S., J. Neurol. Sci. 10, 215 (1970). root ganglia which apparently lack an en-5. CANCILLA,P. A., BAKER,R. N., POLLOCK,P. S., dothelial BBB (27, 29), pericytes accumuAND FROMMES, S. P., Lab. Invest. 26, 376 late extravasated protein (27). Also after (1972). intraventricular administration of HRP, 6. CASLEY-SMITH,J. R . , J . Microsc. 90, 25 (1969). 7. CAULFIELD,J. P., AND FARQUHAR,M. G., J. Cell pericytal phagocytes have been demonBiol. 63, 883 (1975). strated (47). 8. DAEMS, W. TH., AND BREDEROO, P., Z. ZellThe phagocytes are probably ~pial" cells forsch. 144, 247 (1973). which migrate from the subarachnoid 9. DAEMS, W. TH., WISSE, E., AND BREDEROO,P., in space along the perivasular space into the DINGLE, J. T. (Ed.), Lysosomes, a Laboratory
HRP EXTRAVASATION IN THE BRAIN
10. 11.
12. 13. 14. 15.
16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30. 31.
32.
77
Handbook, p. 150. North-Holland Publishing 33. RAVIOLA,E., AND KARNOVSKY,M. J., d. Exp. Company, Amsterdam, London, 1972. Med. 136, 466 (1972). DAVSON, H., Physiology of the Cerebrospinal 34. REESE, T. S., AND KARNOVSKY, M. J., J. Cell Fluid. J. & A. Churchill, London, 1967. Biol. 34, 207 (1967). DAVSON,H., in BOURNE,G. H. (Ed.), The Struc- 35. SHEA, S. M., AND BOSSERT, W. H., Microvasc. ture and Function of the Nervous Tissue, Vol. Res. 6, 305 (1973). IV, p. 321. Academic Press, New York and 36. SHEA, S. M., AND KARNOVSKY, M. J., Nature London, 1972. (London) 212, 353 (1966). DUVE, C. DE, AND WATTIAUX, R., Ann. Rev. 37. SHEA, S. M., KARNOVSKY,M. J., AND BOSSERT, Physiol. 28, 435 (1966). W. H., J. Theoret. Biol. 24, 30 (1969). FRIEND,D. S., J. Cell Biol. 41, 269 (1969). 38. SIMIONESCU,N., SIMINOESCU,M., AND PALADE, FRIEND, D. S., AND FARQUHAR,M. G., J. Cell G. E., J. Cell Biol. 57, 424 (1973). Biol. 35, 357 (1967). 39. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, GIACOMELLI, F., WIENER, J., AND SPIRO, D., G. E., J. Cell Biol. 60, 128 (1974). Arner. J. Pathol. 59, 133 (1970). 40. SIMIONESCU,N., SIMIONESCU,M., AND PALADE, HANSSON, n . A., JOHANSSON, B., AND BLOMG. E., J. Cell Biol. 64, 586 (1975). STRAND, C., Acta Neuropathol. 32, 187 (1975). 41. SPURR,A. R., J. Ultrastruct. Res. 26, 31 (1969). JAHNKE, K., AND GORGAS, K., Anat. Embryol. 42. STAEHLIN,L. A., Int. Rev. Cytol. 39, 191 (1974). 146, 21 (1974). 43. STENSAAS,L. J., Cell Tiss. Res. 158, 517 (1975). JOHANSSON, B., Thesis. Gotab, Kung~ilv 44. VAN DEURS, B., J. Ultrastruct. Res. 55, 400 (1976). 74.12707 Sweden, 1974. JOHANSSON, B., LI, C. L., OLSSON, Y., AND 45. VAUGHAN,D. W., AND PETERS, A., J. Neurocytol. 3, 405 (1974). KLATZO, I., Acta Neuropathol. 16, 117 (1970). 46. VENKATACHALAM,M. A., KARNOVSgV, M. J., Job, F., Brit. J. Exp. Pathol. 52, 646 (1971). FAHIMI, H., D., AND COTRAN, R. S., J. Exp. KARNOVSKY,M. J.,J. CellBiol. 35, 213 (1967). Med. 132, 1153 (1970). KARNOVSKY, M. J., J. Gen. Physiol. 52, 64S 47. WAGNER,H. J., PILGRIM,CH., AND BRANDL,J., (1968). Acta Neuropathol. 27, 299 (1974). KARNOVSKY,M. J., AND SHEA, S. M., Microvasc. 48. WESTERGAARD, E., in CERVOS-NAVARRO, J. Res. 2, 353 (1970). (Ed.), Pathology of Cerebral Microcirculation, KRISTENSSON,K., AND OLSSON,Y., Acta Neurol. p. 218. Walter de Gruyter & Co., Berlin, New Scandinav. 49, 189 (1973). York, 1974. MAXWELL,D. S., ANDKRUGER,L., Exp. Neurol. 49. WESTERGAARD, E., Acta Neuropathol. 32, 27 12, 33 (1965). (1975). MORI, S., AND LEBLOND,C. P., J. Comp. Neur. 50. WESTERGAARD,E., AND BRIGHTMAN,M. W., J. 135, 57 (1969). Comp. Neurol. 152, 17 (1973). NOVIKOFF,A. B., in HYD~N, H. (Ed.), The Neu- 51. WESTERGAARD, E., VAN DEURS, B., AND ron, p. 319. Elsevier Publishing Company, BRONDSTED,H. E., in MOSSAKOWSKI,M. (Ed.), Amsterdam, 1967. Pathophysiological, Biochemical and MorphoNORTVED,J., ANDWESTERGAARD,E., Anat. Rec. logical Aspects of Cerebral Ischemia and Ar178, 428 (1974). terial Hypertension. Warsaw, in press. OLSSON,Y., Acta Neuropathol. 10, 26 (1968). 52. WESTERGAARD, E., VAN DEURS, B., AND OLSSON,Y., AND HOSSMANN,K. A., A cta NeuroBRONDSTED, H. E., submitted for publication. pathol. 16, 103 (1970). 53. WILLIAMS,T. H., ANDJEW, J. Y., Tissue & Cell PALAY,S. L., McGEE-RuSSELL, S. M., GORDON, 7, 407 (1975). S., AND GRILLO, M., J. Cell Biol. 12, 385 54. WILLIAMS,M. C., ANDWISSIG,S. L.,J. CellBiol. (1962). 66, 531 (1975). PHILLIPS,D. E., Z. Zellforsch. 140, 145 (19~3). 55. WOLFF,J., Z. Zellforsch. 60, 409 (1963).
(1976)
Observations on the Blood-Brain Barrier in Hypertensive Rats, with Particular Reference to Phagocytic Pericytes B. VAN DEURS Anatomy Department A, University of Copenhagen, 71 Rddmandsgade, DK 2200 Copenhagen N, Denmark Received January 21, 1976 The endothelial and periendothelial parts of the blood-brain barrier in the r a t have been studied by electron microscopy. Extravasation of horseradish peroxidase (HRP) occurred after Aramine-induced acute hypertension. It was exclusively due to a local vesicular transport. This was most p r o m i n e n t in arterioles, but also occurred in segments of capillaries and venules. The t i g h t junctions between adjacent endothelial cells were never penetrated by HRP. Following extravasation, HRP reached the endothelial b a s e m e n t membrane, from where it often spread into the extracellular space of the neuropil. A phagocytic pericyte was observed, exhibiting a well-developed vacuolar apparatus and a pronounced uptake of HRP. It is suggested t h a t the phagocytic pericytes in the r a t b r a i n represent "microglial" cells, and t h a t they play a n i m p o r t a n t role in the periendothelial part of the blood-brain barrier.
It is well established that a barrier exists, preventing an exchange of protein, between the bloodstream and the brain parenchyma (10, 11). This blood-brain barrier (BBB) is due to two features of the cerebral endothelium (4, 34): the tight junctions (zonulae occludentes; 42) between adjacent endothelial cells effectively sealing the intercellular spaces, and the sparse number of endothelial vesicles ruling out any significant vesicular transport. However, also outside the endothelium a barrier may exist (4, 10, 11,24, 27, 55) protecting the neuropil against extravasated protein. The endothelial basement membrane, and the sheath of astrocytic end-feet surrounding the endothelium, may be elements of such a barrier. Furthermore, pericytal phagocytes have been described in the cerebral microvasculature (5, 24, 25, 27, 30, 47), and these are also likely to play a role in a periendothelial barrier. The endothelial part of the BBB is susceptible to experimental intervention, and various methods of breaking down the barrier have been applied. Certain vasotoxic agents (nickel chloride, mercuric chloride, and lysolecitin) induce extravasation of protein in the brain (20, 30), and the
transport of intravenously injected horseradish peroxidase (HRP) across the endothelium increases significantly when serotonin is perfused through the cerebral ventricles (49). Acute hypertension induced by intravenously injected Aramine (metaraminol bitartrate) also increases the transfer of H R P across cerebral endothelia (16, 19, 30, 51). Hence, under various experimental conditions it is possible to obtain a considerable amount of a tracer protein outside the endothelium of the cerebral microvasculature. The aim of the present study was therefore to investigate, at the ultrastructural level, by which mechanism the HRP extravasation following acute hypertension takes place, and the significance of the endothelial basement membrane, the perivascular space, the pericytal phagocytes, and the astrocytic end-feet in the extravascular distribution of HRP. MATERIALS AND METHODS
Experiments. E i g h t rats ~ were anesthetized with N e m b u t a l (sodium pentobarbitone) and the blood pressure (BP) monitored in the femoral artery. After The hypertensive rats used in this study were a part of the material in a study by Dr. E. Westergaard, Dr. H. E. Br0ndsted, and the present author (see refs. 51, 52). 65
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
B. VAN DEURS
30-60 min with a mean BP between 110 and 120 mm Hg, Aramine (metaraminol bitartrate), 0.07 mg/kg/ rain, was intravenously infused over a period of 1-14 min. This resulted in a rise of the mean BP to about 170 mm Hg. Two-hundred milligrams of horseradish peroxidase (HRP; Sigma type II) in 1 ml of phosphate-buffered saline (PBS) were intravenously infused during 4-12 min, and allowed to circulate for a further 0-15 min before fixation. To study the "normal" exchange of HRP between blood and parenchyma, three rats received 200 mg of HRP without any Aramine treatment. The HRP was allowed to circulate for 3, 10, and 30 min. As a control for endogenous peroxidase, three untreated rats were included in the material. Fixation procedure. All the animals were fixed by a two-step perfusion with aldehydes through the left ventricle. The initial fixative contained 2% formaldehyde (freshly prepared from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.3, and the perfusion time was 3 min. The second fixative contained 4% formaldehyde and 5% glutaraldehyde in the same buffer. This fixative was perfused for 10-15 min. Brains were kept overnight in situ at 4°C. Incubation for peroxidase activity. Brain slices (frontal sections) 0.5-1.0-mm thick were rinsed in sodium cacodylate buffer and incubated for peroxidase activity in the following way: for 1 hr at room temperature in Tris-HC1 buffer (600 mg of Trizmabase, Sigma, in 100 ml of water; pH adjusted to 7.6) containing 0.4 ml of glucose oxidase (Sigma type V, 10,000 units) per 100 ml of buffer, and thereafter for 3 hr at 0°C in the above solution to which were added 50 mg of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 36 mg of anhydrous glucose (Merck) per 100 ml of buffer. Preparation for electron microscopy. After incubation, the brain slices were rinsed in 0.1 M sodium cacodylate buffer, cut into blocks, and treated at room temperature with 2% OsO4 in 0.1 M sodium cacodylate buffer, pH 7.4, for 3 hr, followed by impregnation in 2% uranyl acetate in maleate buffer for 2 hr at room temperature. The blocks were dehydrated in graded series of ethanols and embedded in Spurr epoxy plastic (41). Ultrathin sections were contrasted with uranyl acetate and lead citrate. OBSERVATIONS
The B l o o d - B r a i n Barrier in N o r m a l R a t s After i n t r a v e n o u s injection of H R P i n n o r m a l rats, only v e r y little reaction product was observed in the brain. W i t h the exception of certain ~leaky" regions such as the choroid plexus, H R P was in general confined to vesicles w i t h i n the endothelial
cells a n d to the b a s e m e n t m e m b r a n e a n d the p e r i v a s c u l a r space of a few short segm e n t s of arterioles. However, the walls of most arterioles as well as of capillaries a n d venules did not exhibit a n y HRP-labeling. N e i t h e r could a n y H R P be detected in the extracellular space of the neuropil outside the vessels. Pericytal cells with a welldeveloped v a c u o l a r a p p a r a t u s were somet i m e s seen.
The Blood-Brain Barrier in Hypertensive Rats E n d o t h e l i a l cells a n d basement m e m brane. In the A r a m i n e - t r e a t e d r a t s H R P was p r e s e n t in the b a s e m e n t m e m b r a n e of most p a r t s of the cerebral microvasculat u r e (exceptions are shown in Figs. 2 a n d 13). The t i g h t junctions (zonulae occludentes), seen as points of m e m b r a n e fusion b e t w e e n adjacent endothelial cells, were n e v e r p e n e t r a t e d by H R P (Fig. 1). Reaction product was p r e s e n t from the basem e n t m e m b r a n e into the intercellular cleft only up to the most a b l u m i n a l point of m e m b r a n e fusion (Figs. 1 a n d 6). A t the l u m i n a l aspect of the most l u m i n a l m e m b r a n e fusion the t r a c e r was u s u a l l y w a s h e d out, as seen in Fig. 6. However, in vessels imperfectly perfused d u r i n g fixation, in which labeling could be seen also on the l u m i n a l surface of the endothelial cells or in the entire v a s c u l a r l u m e n (a situation t h u s similar to t h a t obtained after i m m e r s i o n fixation), the H R P h a d evidently not p e n e t r a t e d the m o s t l u m i n a l point of fusion (Fig. 1). HRP-labeled vesicles were n u m e r o u s in the endothelial cells of certain s e g m e n t s of the microvasculatt/re. These s e g m e n t s were in p a r t i c u l a r arterioles (Figs. 2-4), b u t sometimes also capillaries a n d venules exhibited a considerable n u m b e r of labeled vesicles (Figs. 5 a n d 6). The vesicles were m o s t l y u n c o a t e d and m e a s u r e d a b o u t 70 n m in diameter, a l t h o u g h l a r g e r ones occurred, probably r e p r e s e n t i n g fusions of the smaller vesicles. Labeling of the basem e n t m e m b r a n e , especially of capillaries,
HRP EXTRAVASATION IN THE BRAIN
67
FIGs. 1-4. Different parts of the same small arteriole, photographed from a single section. Although perfusion fixation has been used, reaction product is seen on the luminal surface of the endothelium (imperfect perfusion). FIG. 1. Shown is a tight junction between two adjacent endothelial cells: reaction product is absent between the most luminal point of membrane fusion and the most abluminal fusion point. Arrows indicate the fusion points. Note t h a t HRP is also seen in the basement membrane although no HRP-labeled vesicles are present in the endothelium, x 130 000. FIG. 2. Only luminal vesicles are labeled; the basement membrane is unlabeled, x 80 000. FIG. 3. Intraendothelial vesicles also exhibit labeling, as does the basement membrane, x 80 000. FIG. 4. Both luminal and abluminal vesicles are labeled, as is the basement membrane, x 80 000.
was frequently seen, although the endothelium above was almost or completely devoid of labeled vesicles (Figs. 7 and 15).
In vessels imperfectly perfused during fixation, labeling was sometimes confined to the luminal vesicles. In other speci-
68
B. VAN DEURS
FIG. 5. Part of a venule. The endothelium contains labeled vesicles, some of which are opening at the basement membrane, which is also labeled. A, Astrocytic end-feet; P, pericyte, x 57 000. FIGS. 6 and 7. Different parts of the same capillary; in Fig. 6 many vesicles are labeled, while vesicles are almost completely absent in Fig. 7. The arrow in Fig. 6 indicates a point of membrane fusion between two adjacent cells, above which HRP has been washed out during perfusion fixation. The arrow in Fig. 7 indicates a labeled vesicle in a pericyte (P). A, Astrocytic end-feet, x 57 000. mens, some of w h i c h r e p r e s e n t e d longer H R P - c i r c u l a t i o n t i m e s , a n a l m o s t cornp l e t e l a b e l i n g o f v e s i c l e s f r o m t h e l u m e n to
t h e b a s e m e n t m e m b r a n e , as Well as o f t h e b a s e m e n t m e m b r a n e , could be obtained. H o w e v e r , the v a r i a t i o n in p a t t e r n of label-
HRP EXTRAVASATION IN THE BRAIN
ing could also be seen in various parts of the same vascular segment obtained from the same specimen as shown in Figs. 1-4, no matter what the HRP-circulation time of the particular experiment. Hence, the number and distribution of labeled vesicles seem independent of the circulation time in the present study. It is assumed that the extravasation, where and when it occurs, is a very rapid process, the degree of extravasation, however, varying along a vascular segment. Pericytes. The reaction product clearly outlined the periendothelial cells in the different parts of the microvasculature. Thus, in arterioles the layer of smooth muscle cells and the surrounding pericytes typically were outlined, and likewise the pericytes in the basement membrane of venules and larger capillaries (in the smallest capillaries pericytes were absent) (Figs. 5-8). The pericytes mostly appeared as flattened, branched cells embedded in the endothelial basement membrane (Fig. 8, P). The content of organelles in these cells was in general sparse; some microtubules, and a few mitochondria, strands of endoplasmic reticulum, and small Golgi complexes were noticed. In a few cases HRP-labeled, round profiles were seen (Fig. 7, arrow). Phagocytes. Some cells embedded in expansions of the endothelial basement membrane, thus being pericytal, contained many HRP-labeled vesicles or vacuoles of various size (Figs. 8-12). In addition, many very large membrane-bound bodies were present in these cells. The smallest of the HRP-labeled vesicles, measuring about 70-120 nm in diameter, was sometimes connected with the cell surface (Fig. 12, arrows). Most of these vesicles were distinctly coated, although uncoated ones appeared (Fig. 10). The larger HRP-labeled vesicles or vacuoles measured from about 120 nm up to about 1 tLm in diameter, and the reaction product was present as a peripheral rim rather than a homogeneous labeling (Fig. 9, PH).
69
The membrane-bound cytoplasmic bodies (Figs. 9, 11, 12, LY) measured from about 120 nm up to about 2 t~m in diameter. Their content was of moderate density. Sometimes rounded structures of very high density were seen, often more or less incorporated into the cytoplasmic bodies mentioned above (Figs. 9, 11, 12). It could not be determined whether or not these very dark structures contained HRP, since, in the controls for endogenous peroxidase, similar very dark structures could sometimes be found in pericytal cells. Fusion between typical HRP-labeled vesicles and the large bodies of moderate density was also observed (Fig. 9, double arrow). Compared with these dominant components, other organelles were sparse. The HRP-labeled vesicles as well as the other cytoplasmic bodies were found in the relatively cytoplasm-rich perinuclear region, from which flattened branches with only a few or no labeled vesicles protruded, surrounding the endothelium (Fig. 8, arrows). The degree of HRP-labeling varied greatly: from only a few or no labeled vesicles as in a typical pericyte, to near repletion with large vesicles or vacuoles. In many sections "ordinary" pericytes could be seen in addition to the phagocytes, the latter then being the outermost (Fig. 8). The phagocytes were not present in all the sections of cerebral vessels; but they appeared around all parts of the microvasculature, especially arterioles. They were never seen to extend across the astrocytic end-feet lining, migrating into the neuropil. Perivascular space. Some arterioles, especially the larger ones (30 t~m or more in diameter), were surrounded by a distinct perivascular space (Fig. 13, PVS). The space was lined at its inner border by the outermost basement membrane of the arteriolar smooth muscle layer, and collagen microfibrils. At its outer border it was separated from the astrocytic end-feet by another basement membrane. The space contained free cells and cell processes. The
FIG. 8. The endothelium (E) of this small vessel (venule) is surrounded by pericytes (P), a phagocytic pericyte (PP), and processes from this (arrows) and by astrocytic end-feet (A). Reaction product is present in the basement membrane surrounding the pericytes and pericyte processes between the endothelium and the astrocytic end-feet, as well as in the extracellular space of the neuropil, z 6300. FIG. 9. Part ofaphagocyti~ pericyte, showingthe well-developed vacuolar apparatus. A, Astrocytic endfeet; LY, lysosome; M, mitochondrion; P, pericyte; PH, phagosome. The double arrow indicates fusion between a large heterophagosome and a lysosome. × 19 000. 70
71
HRP EXTRAVASATION IN THE BRAIN
/
FIGS. 10-12. Details of phagocytic pericytes, FIG. 10. Shown are m a n y coated micropinocytic vesicles containing HRP, a m o n g which uncoated vesicles occur (arrow). T h i s section was t a n g e n t i a l to t h e cell surface, x 78 000. FIGS. 11 a n d 12. Shown are large and small lysosomes (LY), in some of which very dense bodies are more or less embedded. The arrows in Fig. 12 indicate the formation of the micropinocytic vesicles. Fig. 11. x 31 000; Fig. 12. x 57 000.
cells were either fibroblastlike or clearly phagocytic (Fig. 13), similar to those described above. Although the perivascular space in a few cases, only, contained "free"
HRP, the collagen microfibrils, the limiting basement membrane, and the many vesicles of the phagocytes were mostly clearly labeled, sometimes also in cases
Fro. 13. An arteriole (ART) surrounded by the perivascular space (PVS). In this, a phagocyte is present. LY, lysosome; PH, phagosome, x 13 000. Fro. 14, The neuropil immediately outside the endothelium (E), the pericyte process (P), and the incomplete lining of astrocytic end-feet (A) exhibit a pronounced labeling of the extracellular space, x 25 000. 72
HRP EXTRAVASATION IN THE BRAIN
when no labeling of the endothelial vesicles or of the endothelial basement membrane was seen (Fig. 13).
The extracellular space of the neuropil. The "sheaths" of astrocytic end-feet around the vessels mostly appeared insufficient as a barrier preventing the extravasated H R P in the basement membrane from penetrating into the extracellular space of the neuropil (Figs. 8 and 14), but the degree of labeling of the extracellular space varied considerably. In some cases only very little or no labeling at all could be detected (Fig. 15). Cells in the neuropil. The astrocytes or astrocytic end-feet, the oligodendrocytes, and the neurons rarely showed uptake of H R P (except for a few small vesicles in the neurons). In some instances cells filled with HRP-labeled vesi'cles and vacuoles as well as cytoplasmic bodies were observed. These cells closely resembled the above described phagocytes (Fig. 16), and in most cases they were clearly surrounded by astrocytic end-feet; they were therefore likely to be pericytal rather than intraparenchymal. DISCUSSION
Extravasation of HRP. The observations demonstrate that acute hypertension provoked by intravenously injected Aramine induces a pronounced extravasation of HRP in the brain of rats. Further morphologic and physiologic details of this acute hypertension model will be given elsewhere (52). In experiments with hypertension induced by partially constricting renal artery (15), it was found that in cerebral precapillary vessels an extravasation could mainly be attributed to intercellular passage of H R P due to broken tight junctions. Acute hypertension caused by Aramine has previously been reported to increase the permeability of the cerebral microvasculature to dye-albumin complexes (18, 19). Extravasation of HRP following Ara-
73
mine-induced hypertension has also been reported (16, 30), but in these studies evidence for the pathway across the endothelium being vesicular rather than intercellular was not given. However, as it appears from the present study the extravasation of HRP resulting from Aramineinduced acute hypertension is exclusively due to vesicular transport, whereas the junctions between the endothelial cells are not penetrated by the tracer protein. Recently it has been demonstrated that vesicular transport also occurs in a few segments of the cerebral microvasculature in normal animals (28, 48, 50). In permeability studies immersion fixation (21, 38, 40, 54) and short time sequence analysis (38, 40) have often been applied, in contrast to the present study, A brief discussion of the different techniqt/es is therefore warranted. Perfusion fixation gives in general a better preservation of brain tissue than immersion fixation (31, 53), but it has the disadvantage that tracers are usually washed out of the vascular lumen. However, as shown in the present study, perfusions are often incomplete, leaving blood-filled vessels scattered through the tissue, with no apparent decrease in fixation quality. In such cases the tracer protein will be observed at the luminal front of the endothelial cells. Perfusion fixation may therefore be the best fixation method, also in permeability studies, regarding the brain. A labeling of vesicles in t h e endothelium, from the lumen towards the basement membrane, could be observed in different tissue blocks representing an increasing HRP-circulation time. However, this could also be seen within the same tissue block. Therefore, to make a further, detailed time-sequence study of the events during the first few seconds or minutes after administration of the tracer protein did not appear justified. As it appears in the literature, a timesequence study comprises very short circulation times after which only a few lu-
FIG. 15. P a r t of a capillary endothelium (E), astrocytic end-feet (A), and the neuropil. HRP-labeling is only distinct in the endothelial b a s e m e n t membrane, x 50 000. Fia. 16. A phagocyte apparently situated within the neuropil. Note the astrocytic end-feet (A) surrounding the phagocyte, x 12 000. 74
HRP EXTRAVASATION IN THE BRAIN minal vesicles should be labeled, followed by slightly longer circulation times with increasing labeling of abluminal vesicles and finally also of the basement membrahe (38, 40). This simplified model has, however, some conspicuous disadvantages. First, it is impossible within the limit of seconds, and probably minutes, to determine the exact time of fixation of a particular region in the tissue block (especially after immersion fixation). Furthermore, the transfer time for a vesicle across an endothelial cell should be measured in seconds (6, 23, 36, 37). It is therefore obvious that comparison between parts of the vascular endothelium from tissue blocks obtained from experiments with slightly different circulation times should be interpretated with the utmost care. Second, the degree of vesicular transport at a given time may not be the same all along the endothelial lining of a particular segment of the microvasculature, as shown in the present study, and also suggested by other authors (38, 39). In the literature, micrographs of segments of muscular vessels, for instance, in which the tracer (HRP) is confined to the luminal aspect of the endothelium even after several minutes of circulation, also occur (21, 23, 54). Furthermore, it is likely that the vesicular transport across vascular endothelia is bidirectional; that is, that a transport of protein and other macromolecules also takes place from the basement membrane to the bloodstream (22, 37). With respect to vesicle labeling, it therefore seems very difficult to choose a "representative" micrograph from a single experiment (which of Figs. 1-4 are ~representative" for that particular experiment ?).
Basement membrane, perivascular space, and astrocytic end-feet. In the present study the vesicular transport appeared to be focal. The labeling of basement membranes below parts of the endothelium devoid of labeled vesicles could be explained by a vesicular transport having occurred
75
before fixation of the tissue. This is in agreement with the above considerations. However, the phenomenon may also be explained by some spread of HRP in the basement membrane to segments without any vesicular transport at all (/6). A spread of H R P along the basement membrane has previously been suggested after intraventricular injection of the tracer (47), and to some extent also after intraparenchymal injection of HRP (3). Sometimes labeling was confined to the basement membrane. In most cases, however, HRP spread into the extracellular space of the neuropil. The latter situation could be due to an %verloading" of the basement membrane, which may act as a more efficient ~'trap" or ~filter" when exposed to smaller amounts of extravasated protein. In addition, it may be that larger molecules than HRP (MW 40 000) are more completely retained in the basement membrane. This has been shown to be the case with the glomerular basement membrane (7, 46). A transport of HRP m a y also occur along the perivascular space (the Virchow-Robin space) of the arterioles, since HRP-labeling of phagocytes and extracellular structures of the space was sometimes noticed in the absence of extravasation in the particular vessel. However, HRP may also reach this position by diffusion through the extracellular space of the neuropil. In conclusion, it seems difficult to evaluate the role of the endothelial basement membrane and the perivascular space in the periendothelial diffusion barrier, but they may play some role for the barrier. On the other hand, the astrocytic end-feet did not constitute a barrier between the endothelial basement membrane and the neuropil. Perivascular phagocytes. It seems reasonable to assume that the perivascular phagocytes play the most important role in the periendothelial part of the BBB. In the thymic cortex a blood-tissue barrier also exists, which is partly due to the presence
76
B. VAN DEURS
of macrophages immediately outside the brain. At some distance from the subendothelium phagocytosing the small arachnoid space, usually at the level of the amounts of protein that are carried across smallest arterioles, the basement memthe endothelium by vesicles (33). The cere- branes bordering the perivascular space bral phagocytic pericytes were provided fuse, and the phagocytes thus become periwith a well-developed vacuolar apparatus cytes, embedded in the basement mem(9, 12). The small, often coated vesicles brane. It is uncertain, however, whether containing HRP are typical micropinocytic two different line of pericytes exist, a vesicles similar to those described in mac- phagocytic and a nonphagocytic, or rophages (8) and absorptive epithelial cells whether phagocytic cells develop directly (13, 14, 44). The micropinocytic vesicles from th.e nonphagocytic ones. may fuse with each other forming larger The presence of pericytal phagocytes in heterophagosomes, or with primary lyso- the brain deserves some comment in the somes forming secondary lysosomes. The light of previous discussions on the origin other kinds of cytoplasmic bodies or vacu- of microglia. A development of nonphagooles probably represent primary and sec- cytic pericytes into phagocytic microglial ondary lysosomes. Acid phosphatase activ- cells has been suggested by some authors ity has been described in cerebral pericytes (1, 2, 26, 47), whereas other investigators (27). have not found any relationship between The vacuolar apparatus appeared simi- microglia and pericytes (32, 43, 45). In the lar to that previously observed in cerebral present study, no phagocytes were obpericytes after provoked protein extrava- served within the neuropil, and neither sation (5, 30). Furthermore, Cancillaet al. the present study nor a previous long-time (5) found that, after increased HRP-expo- tracer-study of cerebral pericytes (5) demsure time the protein was no longer pres- onstrated any migration of pericytes, ent in the extracellular space of the neuro- phagocytic or nonphagocytic, into the neupil, the degree of HRP accumulation in the ropil. The present observations therefore pericytes had increased considerably. The indicate, as was also suggested by Maxendothelial part of the BBB in immature well and Kruger (25), that some pericytes animals is less efficient than in adults. It are themselves, in fact, the ~'microglial" has been shown that in the brain of suck- cells. ling mice, pericytes endocytose extravaREFERENCES sated HRP to a considerable extent, while no tracer appears in the neuropil; it has 1. BALDWIN, F., WENDELL-SMITH, C. P., AND BLUNT, M. J., J. Anat. 104, 401 (1969). been concluded, therefore, that the peri2. BARON,M., AND GALLEGO,A., Z. Zellforsch. 128, cytes constitute a barrier between blood 42 (1972). and neuropil in the immature cerebral tis3. BECKER,N. H., HIRANO, A., AND ZIMMERMAN, sue (24). In modiolar arterioles extravaH. M., J. Neuropathol. Exp. Neurol. 27, 439 sated HRP becomes likewise endocytosed (1968). 4. BRIGHTMAN,M. W., KLATZO,I., OLSSON,Y., AND by perivascular phagocytes (17). In dorsal REESE, T. S., J. Neurol. Sci. 10, 215 (1970). root ganglia which apparently lack an en-5. CANCILLA,P. A., BAKER,R. N., POLLOCK,P. S., dothelial BBB (27, 29), pericytes accumuAND FROMMES, S. P., Lab. Invest. 26, 376 late extravasated protein (27). Also after (1972). intraventricular administration of HRP, 6. CASLEY-SMITH,J. R . , J . Microsc. 90, 25 (1969). 7. CAULFIELD,J. P., AND FARQUHAR,M. G., J. Cell pericytal phagocytes have been demonBiol. 63, 883 (1975). strated (47). 8. DAEMS, W. TH., AND BREDEROO, P., Z. ZellThe phagocytes are probably ~pial" cells forsch. 144, 247 (1973). which migrate from the subarachnoid 9. DAEMS, W. TH., WISSE, E., AND BREDEROO,P., in space along the perivasular space into the DINGLE, J. T. (Ed.), Lysosomes, a Laboratory
HRP EXTRAVASATION IN THE BRAIN
10. 11.
12. 13. 14. 15.
16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30. 31.
32.
77
Handbook, p. 150. North-Holland Publishing 33. RAVIOLA,E., AND KARNOVSKY,M. J., d. Exp. Company, Amsterdam, London, 1972. Med. 136, 466 (1972). DAVSON, H., Physiology of the Cerebrospinal 34. REESE, T. S., AND KARNOVSKY, M. J., J. Cell Fluid. J. & A. Churchill, London, 1967. Biol. 34, 207 (1967). DAVSON,H., in BOURNE,G. H. (Ed.), The Struc- 35. SHEA, S. M., AND BOSSERT, W. H., Microvasc. ture and Function of the Nervous Tissue, Vol. Res. 6, 305 (1973). IV, p. 321. Academic Press, New York and 36. SHEA, S. M., AND KARNOVSKY, M. J., Nature London, 1972. (London) 212, 353 (1966). DUVE, C. DE, AND WATTIAUX, R., Ann. Rev. 37. SHEA, S. M., KARNOVSKY,M. J., AND BOSSERT, Physiol. 28, 435 (1966). W. H., J. Theoret. Biol. 24, 30 (1969). FRIEND,D. S., J. Cell Biol. 41, 269 (1969). 38. SIMIONESCU,N., SIMINOESCU,M., AND PALADE, FRIEND, D. S., AND FARQUHAR,M. G., J. Cell G. E., J. Cell Biol. 57, 424 (1973). Biol. 35, 357 (1967). 39. SIMIONESCU,M., SIMIONESCU,N., AND PALADE, GIACOMELLI, F., WIENER, J., AND SPIRO, D., G. E., J. Cell Biol. 60, 128 (1974). Arner. J. Pathol. 59, 133 (1970). 40. SIMIONESCU,N., SIMIONESCU,M., AND PALADE, HANSSON, n . A., JOHANSSON, B., AND BLOMG. E., J. Cell Biol. 64, 586 (1975). STRAND, C., Acta Neuropathol. 32, 187 (1975). 41. SPURR,A. R., J. Ultrastruct. Res. 26, 31 (1969). JAHNKE, K., AND GORGAS, K., Anat. Embryol. 42. STAEHLIN,L. A., Int. Rev. Cytol. 39, 191 (1974). 146, 21 (1974). 43. STENSAAS,L. J., Cell Tiss. Res. 158, 517 (1975). JOHANSSON, B., Thesis. Gotab, Kung~ilv 44. VAN DEURS, B., J. Ultrastruct. Res. 55, 400 (1976). 74.12707 Sweden, 1974. JOHANSSON, B., LI, C. L., OLSSON, Y., AND 45. VAUGHAN,D. W., AND PETERS, A., J. Neurocytol. 3, 405 (1974). KLATZO, I., Acta Neuropathol. 16, 117 (1970). 46. VENKATACHALAM,M. A., KARNOVSgV, M. J., Job, F., Brit. J. Exp. Pathol. 52, 646 (1971). FAHIMI, H., D., AND COTRAN, R. S., J. Exp. KARNOVSKY,M. J.,J. CellBiol. 35, 213 (1967). Med. 132, 1153 (1970). KARNOVSKY, M. J., J. Gen. Physiol. 52, 64S 47. WAGNER,H. J., PILGRIM,CH., AND BRANDL,J., (1968). Acta Neuropathol. 27, 299 (1974). KARNOVSKY,M. J., AND SHEA, S. M., Microvasc. 48. WESTERGAARD, E., in CERVOS-NAVARRO, J. Res. 2, 353 (1970). (Ed.), Pathology of Cerebral Microcirculation, KRISTENSSON,K., AND OLSSON,Y., Acta Neurol. p. 218. Walter de Gruyter & Co., Berlin, New Scandinav. 49, 189 (1973). York, 1974. MAXWELL,D. S., ANDKRUGER,L., Exp. Neurol. 49. WESTERGAARD, E., Acta Neuropathol. 32, 27 12, 33 (1965). (1975). MORI, S., AND LEBLOND,C. P., J. Comp. Neur. 50. WESTERGAARD,E., AND BRIGHTMAN,M. W., J. 135, 57 (1969). Comp. Neurol. 152, 17 (1973). NOVIKOFF,A. B., in HYD~N, H. (Ed.), The Neu- 51. WESTERGAARD, E., VAN DEURS, B., AND ron, p. 319. Elsevier Publishing Company, BRONDSTED,H. E., in MOSSAKOWSKI,M. (Ed.), Amsterdam, 1967. Pathophysiological, Biochemical and MorphoNORTVED,J., ANDWESTERGAARD,E., Anat. Rec. logical Aspects of Cerebral Ischemia and Ar178, 428 (1974). terial Hypertension. Warsaw, in press. OLSSON,Y., Acta Neuropathol. 10, 26 (1968). 52. WESTERGAARD, E., VAN DEURS, B., AND OLSSON,Y., AND HOSSMANN,K. A., A cta NeuroBRONDSTED, H. E., submitted for publication. pathol. 16, 103 (1970). 53. WILLIAMS,T. H., ANDJEW, J. Y., Tissue & Cell PALAY,S. L., McGEE-RuSSELL, S. M., GORDON, 7, 407 (1975). S., AND GRILLO, M., J. Cell Biol. 12, 385 54. WILLIAMS,M. C., ANDWISSIG,S. L.,J. CellBiol. (1962). 66, 531 (1975). PHILLIPS,D. E., Z. Zellforsch. 140, 145 (19~3). 55. WOLFF,J., Z. Zellforsch. 60, 409 (1963).
