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Sequential appearance of anionic domains in the developing blood-brain barrier
Developmental Brain Research, 52 (1990) 31-37 Elsevier
31
BRESD 51019
Sequential appearance of anionic domains in the developing blood-brain barrier Andrzej W. Vorbrodt, Albert S. Lossinsky, Danuta H. Dobrogowska and Henryk M. Wisniewski Department of Pathological Neurobiology, New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314 (U.S.A.) (Accepted 29 August 1989)
Key words: Anionic site; Cationic colloidal gold; Blood-brain barrier; Brain development; Barrier maturation
The distribution of anionic sites in the walls of mouse brain micro-blood vessels (MBVs) during development and maturation of the blood-brain barrier (BBB) was studied by electron microscopy. Cationic colloidal gold (CCG) and Lowicryl K4M-embedded brain samples obtained from mouse fetuses (13th and 19th days) and from 1-, 5-, 12- and 24-day-old and adult mice were used. The labeling of anionic sites with CCG was more intense on the abluminal than on the luminal front of the endothelial cells (ECs) in fetuses and in newborn mice. Only a few anionic sites appear on the luminal front of the ECs of proliferating blood vessels invading the neural tissue in 13-day-old fetuses. They become slightly, although steadily, more abundant during further stages of development, and their number rapidly increases between the 12th and 24th day of life at which time they attain the density typical for mature animals. The maturation of the basement membrane (BM), which occurs during the myelinization period (12th-24th day of life), also coincides with an increasing concentration of anionic sites. These observations suggest that the gradual appearance of anionic sites on both fronts of the endothelium, as well as in the developing and maturing BM, represents one of the mechanisms responsible for differentiation of cerebral microvasculature into BBB-type MBVs.
INTRODUCTION In the course of o u r previous studies, we have noted that the functional m a t u r a t i o n of the b l o o d - b r a i n barrier ( B B B ) , as evidenced by a 'closure' of brain micro-blood vessels ( M B V s ) to intravenously injected m a c r o m o l e c ular tracer ( H R P ) , takes place b e t w e e n the 12th and 24th day o f life in the mouse 7'z1'22. This m a t u r a t i o n coincides with the a p p e a r a n c e of alkaline p h o s p h a t a s e activity and with the f o r m a t i o n and r e m o d e l i n g of the glycoprotein layer on the surfaces of endothelial cells (ECs) 22'23. S o m e of the m o n o s a c c h a r i d e residues present in this glycoprotein coat maintain the negative charge on both surfaces of the E C s in the non-BBB 1°'11 as well as in the B B B type 17-2° microvasculature. It is believed that the negatively charged surface layer (anionic sites) helps restrict the m o v e m e n t of various molecules across the vessel wall 1"1° and also helps maintain the BBB 3-6'9"15. C o n s e q u e n t l y , one can expect that the functional and structural m a t u r a t i o n of the BBB is a c c o m p a n i e d by the f o r m a t i o n of anionic domains in the wall of brain MBVs. A l t h o u g h in our previous study the main attention was on the distribution of various glycoconjugates in deve-
loping mouse brain M B V s , we have also m a d e an a t t e m p t to detect the distribution of anionic sites using cationized ferritin (CF) applied to tissue sections o b t a i n e d from a tissue sectioner 23. This p r e - e m b e d d i n g technique, similar to those described by T h ~ r a u f et al. 16, enables one to label the anionic sites located only on the easily accessible luminal surface of the endothelium. Thus, the results o b t a i n e d are incomplete and do not show the distribution of negatively charged domains in the hardly accessible constituents of the vessel wall, such as the abluminal front of the ECs and the basal lamina (BM). Fortunately, in the m e a n t i m e , a new, m o r e reliable technique for the visualization of anionic sites with cationic calloidal gold ( C C G ) was e l a b o r a t e d by Skutelsky and R o t h 13. In o u r previous studies, this new cationic p r o b e was successfully a p p l i e d to ultrathin sections of brain samples e m b e d d e d in hydrophilic resin Lowicryl K4M ~7,18. W e were able to detect anionic sites in the entire cross-section of the vessel wall, and o t h e r cellular c o m p o n e n t s such as smooth muscle cells, pericytes, and glial perivascular endfeet. O u r present work is an a t t e m p t to apply this new technique to study the a p p e a r a n c e and distribution of
Correspondence: A.W. Vorbrodt, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
32 a n i o n i c d o m a i n s in the brain m i c r o v a s c u l a t u r e of m i c e
TABLE 1
d u r i n g the d e v e l o p m e n t and m a t u r a t i o n of the B B B .
The intensity of labeling of anionic s'ite,~on the lutrunal and abluminal fronts of the endothelial cells (ECs) ~ brain MBI "s during develop, ment and maturation of the mouse blood-brain barrier
MATERIALS AND METHODS
Animals All experiments were performed on IM or BALBc/J mice of both sexes. Brain samples were taken from mouse fetuses (13th and 19th day of gestation), newborn mice (lst day of life) or 5-, 12- and 24-day-old animals. For comparative purposes, adult animals (6-10 months old) were also used. Under sodium-pentobarbital anesthesia, animals (newborn, infants or adult) were fixed by intracardiac perfusion with a mixture of freshly prepared 2% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, supplemented with 3% polyvinyl pyrrolidone. Time of perfusion was 15 rain followed by immersion fixation up to 2 h in ice-cold fixative. Brains of fetuses were fixed by immersion without prior perfusion. After fixation, brain samples were cut into small blocks (1-2 mm), immersed for 4 h in ice-cold 0.1 M NH4CI to block free aldehyde groups and embedded at low temperature in Lowicryl K4M. After polymerization at low temperature under a UV lamp, specimens were cut with a diamond knife on a SorvaU MT 5000 (DuPont) microtome and picked up on 100-mesh, formvar-carboncoated nickel grids. Preparation of CCG Particles of colloidal gold with a mean diameter of 14 nm were prepared according to Frens 2. CCG was prepared according to the method of Skutelsky and Roth 13 by complexing poly-L-lysine (mol. wt. 306,000, Miles Labs., Naperville, IL) and colloidal gold; the preparation was similar to the one described previouslyaT. Demonstration of anionic sites Ultrathin sections attached to nickel grids were placed in a moist chamber at room temperature (22 °C) on a drop of 0.05 M Tris-HCI buffer-saline (TBS). After 5 min, they were transferred to 0.1 M glycine in TBS, pH 7, for 30 min and then to TBS, pH 2.1, supplemented with 0.02% polyethylene glycol, mol. wt. 20,000 (TBS-PEG) for 5 min. Finally, they were incubated at room temperature on a drop of solution of CCG in TBS-PEG containing 0.1% bovine serum albumin. The final concentration of CCG was approximately 50/tg/ml, giving an absorbance of 0.70 at 525 nm wavelength. The solution was prepared immediately before incubation and was centrifuged for 10 min at 400 g for removal of the large aggregates of gold complexes. The incubation time was 45-60 min. After labeling, the sections were washed in TBS and stained with 4% aqueous uranyl acetate (3 min) and with lead acetate (MiUonigs) for 45 s. The sections were examined in a Philips 420 electron microscope (EM). In the control sections, the anionic sites were blocked by
The intensity of the labeling was graded according to the number ot gold particles per l u m of the EC surface: -, no labeling; +-, less than 1 gold particle: +, 1-5 particles: + + . 6.--1(1 particles: + + + , 11-15 particles; + + + +, more than 16 particles per 1 um
Stage of development Intensity of labeling (age of the mouse) of EC surfaces
Additional comments
Luminal Abluminal 13-day-old fetus
+-
+
19-day-old fetus Newborn (lst day of life)
+
++
+
++
5 days old 12daysold 24 days old
++ +++ ++++
++ ++ +++
Adult(6months old)
++++
+++
The BM is not visible (structureless)
The BM becomes recognizable and labeled with CCG
The BM is well,developed and labeled with CCG
incubation in a solution of non-labeled poly-L-lysine (1 mg/ml of TBS, pH 2.1) for 1 h at room temperature before incubation with CCG.
RESULTS T h e results of o u r o b s e r v a t i o n s are s u m m a r i z e d in T a b l e I. A l t h o u g h o u r study has a d e s c r i p t i v e and m o r p h o logical c h a r a c t e r ,
t h e density o f t h e l a b e l i n g of b o t h
fronts o f t h e E C is p r e s e n t e d w i t h t h e a r b i t r a r y s y m b o l s d e s c r i b e d in T a b l e I. F o r this p u r p o s e , in brain sections, 10 r a n d o m l y c h o s e n t r a n s v e r s e l y s e c t i o n e d M B V s w e r e p h o t o g r a p h e d , and t h e n u m b e r o f g o l d particles associa t e d with b o t h l u m i n a l a n d a b l u m i n a l p l a s m a m e m b r a n e s of t h e E C s was c o u n t e d .
The mean values obtained
(calculated as the n u m b e r of g o l d p a r t i c l e s p e r 1 /~m)
Fig. 1. A section of the wall of a brain MBV of 13-day-old mouse fetus is shown Few aniomc sites are labeled with CCG on the abluminal front (arrowheads), whereas they are absent from the luminal surface of the EC. In the cytoplasm of the EC hardly noticeable plasmalemmal vesicles (curved arrows) are present. The abluminal surface of attenuated cells adjacent to the EC (presumably a primordial perieyte) is labeled with numerous gold particles (arrows). B, basement membrane; E, endothelial cell; J, intercellular junction; L, vessel lumen; N, cell nucleus; S, smooth muscle cell (all figures). All figures represent ultrathin sections of mouse brain cortex embedded in Lowicryl K4M and incubated with poly-L-lysine-gold complex (CCG) for the demonstration of anionic sites in the wall of micro-blood vessels (MBVs). x31.500. Fig. 2. Another, slightly larger MBV than shown in Fig. 1 of 13-day-old mouse fetus. The luminal surface of the EC is weakly labeled with few irregularly scattered gold particles except the terminal foldings near the junctional areas (arrows) which are decorated with several gold particles. On the contrary, the abluminal front of the EC is labeled with more numerous CCG particles scattered With some regularity (arrowheads). The surface of a primitive blood cell (curved arrows) is evidently more intensely labeled than the luminal surface of the EC. x31,500. Fig. 3. Localization of anionic sites labeled with CCG in the wall of MBV of 19-day-old mouse fetus is shown. The EC body is of uneven thickness, and few pseudopodial processes protrude into the vessel lumen. The labeling of the luminal surface of the EC is more intense than in the 13-day fetuses, although it is still discontinuous and irregular (arrows). The labeling of the abluminal front of the EC (arrowheads) is more intense. The surface of primordial pericyte facing the adjacent neural tissue is labeled with numerous gold particles. ×40,500.
33 were used for presenting the intensity of labeling with minur or plus signs. The presented values are approximations because in the Lowicryl K4M-embedded sam-
pies, the exact localization of gold particles in the hardly visible endothelial plasma membranes is frequently rather difficult due to omission of the osmium tetroxide.
34 In 13-day-old mouse fetuses, the neural tube becomes infolded into the ventricle to form the telencephalon. Perineural vessels invade the neural tissue where matrix
cells differentiate into neuroblasts, A1 this stage ~ development, the ECs of invading vessels are characterized by uneven thickness of their cytoplasm
35 The wall of these vessels is composed of a single layer of the endothelium accompanied by attenuated processes of cells presumably representing primordial pericytes. Present between these two types of cells is a narrow space filled with a structureless material labeled with a few gold particles. This narrow space is the area where the BM is ultimately to be formed during the further stages of development (Fig. 1). In the majority of blood vessels, numerous plasmalemmal pits and vesicles appear in the EC cytoplasm. Their outlines are only rarely recognizable because of rather poor staining of membranous structures resulting from the omission of the osmium tetroxide as a fixative. The luminal surface of the ECs of some vessels is not labeled (Fig. 1), whereas in other vessels a few irregularly scattered gold particles are present (Fig. 2). As a rule, the filopodia protruding into the vessel lumen in the junctional areas of the ECs, known as the terminal foldings, are more intensely labeled than the remaining fiat segments of the endothelial surface (Fig. 2, arrows). Evidently, in all MBVs, the abluminal front of the ECs is more intensely, although irregularly, labeled with CCG particles (Figs. 1 and 2, arrowheads). In 19-day-old fetuses, the endothelium is of uneven thickness, with numerous protrusions that are especially conspicuous in the junctional areas. The labeling of the luminal surface is irregular, i.e. some segments of the EC surface remain unlabeled, whereas others, especially the undulated segments, are decorated with single, numerous or clustered gold particles (Fig. 3). The abluminal front of the EC and the subendothelial BM-like space are labeled with irregularly scattered gold particles. The surface of primitive pericytes, facing the adjacent brain parenchyma, shows a similar labeling (Fig. 3). In newborn mice (1-day-old), the endothelium is of uneven thickness with numerous finger-like processes protruding into the vessel lumen. The labeling of the luminal surface is scanty and irregular, whereas the abluminal front of the EC is decorated more intensely (Fig. 4). Numerous MBVs have a rather flat luminal surface (Fig. 5) that is also less intensely labeled than the
abluminal front of the EC. In these vessels, the subendothelial BM-like space is strongly labeled with numerous CCG particles (Fig. 5). At this stage, the tight junctions between adjacent ECs are well-developed and easily discernible. In the 5-day-old mouse brain, the labeling of both luminal and abluminal fronts of the ECs becomes almost of the same intensity, although some variations between particular vessels can be noted (Figs. 6 and 7). The subendothelial, BM-like space is also labeled with numerous, irregularly scattered gold particles. In the EC cytoplasm, numerous plasmalemmal vesicles are present, although their delimiting membranes are hardly visible. In the vast majority of brain MBVs of the 12-day-old mouse, the localization of anionic sites is essentially similar to that observed in 24-day-old and in adult animals, although in the last two groups, the density of the labeling is slightly higher (see Table I). In capillaries, the luminal surface is uniformly decorated with an almost continuous row of CCG particles, whereas the labeling of abluminal front of the EC is less regular and less intense (Fig. 8). The easily recognizable BM is labeled with numerous, relatively uniformly scattered gold particles. The labeling of anionic sites is almost completely abolished in control sections exposed to unlabeled polyL-lysine prior to incubation with CCG. In these sections, only a few accidentally scattered gold particles are present, adhering unspecifically to the sectioned tissue as well as to the tissue-free areas of polymerized resin. DISCUSSON
In the present study, we were able to observe the distribution of anionic sites in such hardly accessible sites of the vessel wall as the abluminal surface of the EC, the adjacent space with basal lamina-like material, primordial pericytes and smooth muscle cells. Our results indicate that in 13-day-old mouse fetuses, the luminal surface of the endothelium of proliferating blood vessels contains only a few sparsely scattered
Fig. 4. A cross-section of a brain MBV of a newborn (1-day-old) mouse is shown. The undulated luminal surface of the EC is irregularly labeled with few gold particles (arrows). On the contrary, the labeling of the abluminal front of the EC is more regular and intense (arrowheads).
x55,500. Fig. 5. Another cross-sectioned brain MBV of 1-day-old mouse is shown. Although the labeling of the luminal surface is irregular and discontinuous (arrows), the labeling of the abluminal surface of the EC (arrowheads) as well as of the subendothelial basal lamina (B) is much more intense. Gold particles scattered throughout the EC cytoplasm indicate the presence of some not recognizable anionic domains which are accessible for the cationic probe, x55,500. Fig. 6. A cross-sectioned brain capillary of a 5-day-old mouse is shown. In this vessel, the EC cytoplasm is of irregular thickness and shows numerous indentations and protrusions. Labeling of the luminal surface (arrows) is slightly less regular and less intense than of the abluminal EC surface (arrowheads) x55,500. Fig. 7. Another, presumably larger brain MBV of a 5-day-old mouse is shown. In this vessel, the labeling of a relatively flat luminal surface of the plasmalemma proper (arrows) is less intense and less regular than of the abluminal front of the EC (arrowheads). A weakly stained subendothelial basal lamina-like material is also labeled with numerous gold particles, x31,500.
36
Fig. 8. A cross-sectioned brain capillary of a 12-day-old mouse is shown. The labeling of the luminal surface of the E(" is regular and almost continuous (arrows). The labeling of abluminal front of the EC (arrowheadsl and of the basemen! membrane (B) is also conspicuous. Some undefined structures in the EC cytoplasm are also labeled, indicating the presence of anionic microdomains. ~40,500. anionic domains. These domains become slightly, although steadily, more abundant during further stages of development, i.e. in 19-day-old fetuses as well as in newborn and 5-day-old animals. At this stage of development (5th day) the density of the labeling of both fronts of the EC becomes almost equal. They rapidly increase between the 12th and 24th days of life, attaining a high level typical for mature animals with a fully developed BBB (see Table I). It is worth noting that the anionic sites appear earlier and are more abundant on the abluminai than on the luminal front of the EC in the early stages of vascularization of the neural tube by proliferating MBVs. The faintly stained narrow space adjoining the abluminal surface of the ECs, where the BM is ultimately formed, also shows an increasing concentration of anionic domains considerably exceeding those located on the luminal surface of the ECs in newborn mice. Our previous studies performed on both developing 23 and adult mouse brain MBVs 19"2° indicate that it is terminal sialic acid residues that are mainly responsible for the maintenance of anionic domains on the luminal front of the ECs. Indeed, the gradual appearance and the distribution pattern of the binding sites for Limax flavus agglutinin, which is specific for sialic acid, observed by us previously 23, are very similar to the localization of anionic sites demonstrated in the present study with CCG. On the other hand, heparan and chondroitin sulfatesrich glycosaminoglycans 19 contribute to a substantial degree to the maintenance of the anionic domains located on the abluminal front on the EC and in the BM. The early appearance of anionic domains on the abluminal front of the ECs observed in invading MBVs in 13day-old mouse fetuses suggests that the synthesis of such
a type of proteoglycans does occur early during vascularization of the neural tissue. It also indicates that the gradual accumulation of negatively charged material occurs in the subendothelial space early before the final elaboration and formation of the BM in this area; according to Simionescu et a1.12 the BM attains maturity only after birth, during the process of myelinization. The early appearance of negatively charged domains on the abluminal side of the wall of newly formed MBVs seems to be functionally significant. Most probably, these domains constitute a negatively charged screen or filter that helps control the movement of various negatively charged solutes between the blood and brain parenchyma ~1. Our data suggest that the gradual appearance of anionic domains on both fronts of the endothelium, as well as in the area of the BM, represents one of the developmental mechanisms that cause differentiation of cerebral capillaries into the BBB-type character. It was suggested by Yoshida et al. 24 that developing neural cells (neuroblasts and glial cells) influence the ECs in such a way as to change their morphology and surface properties. The results of our studies indicate that the functional maturation of the mouse BBB, as evidenced previously by sealing of brain MBVs to injected H R P 7'14'21, is paralleled by the formation of negatively charged surface layers on both fronts of the endothelium and also in the BM. These observations also suggest the elaboration of the structural, and probably functional, polarity of brain endothelia as evidenced by the uneven concentration of anionic sites on both fronts of the ECs. Stronger labeling with CCG of the abluminal front occurred during early stages of development, followed finally by a more intense labeling of the luminal than abluminal front of the ECs
37 which we observed in 12-day-old mice and m a i n t a i n e d in
Further experimental studies are n e e d e d to elucidate
brain capillaries of adult animals. Since the tightening of
the problem of the mechanisms responsible for differen-
brain microvessels during d e v e l o p m e n t is accompanied by the d e v e l o p m e n t of tight junctionsTM and by the formation and m a t u r a t i o n of the B M 12'22, our present
tiation of the developing brain MBVs into the BBB-type vessels.
results suggest that the formation of anionic microdomains represents only one of several events accompany-
Acknowledgements. The authors wish to express their appreciation to Ms. Janis Kay for excellent secretarial assistance and to Ms. Maureen Stoddard Marlow for editorial suggestions. This study is supported in part by Grant 17271-08 from NINCDS.
ing the elaboration and m a t u r a t i o n of the BBB. REFERENCES 1 Danon, D. and Skutelsky, E., Endothelial surface charge and its possible relationship to thrombogenesis, Ann. N.Y. Acad. Sci., 275 (1976) 47-63. 2 Frens, G., Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nature Physiol. Sci., 241 (1973) 20-22. 3 Hardebo, J.E. and Khhrstrrm, J., Endothelial negative surface charge areas and blood-brain barrier function, Acta Physiol. Scand., 125 (1985) 495-499. 4 Hart, M.N., Van Dyk, L.E, Moore, S.A., Shasby, D.M. and Cancilla, P.A., Differential opening of the brain endothelial barrier following neutralization of the endothelial luminal anionic charge in vitro, J. Neuropathol. Exp. Neurol., 46 (1987) 141-153. 5 Houthoff, H.J., Moretz, R.C., Rennke, H.G. and Wisniewski, H.M., The role of molecular charge in the extravasation and clearance of protein tracers in blood-brain barrier impairment and cerebral edema. In K.G. Go and A. Baethman (Eds.), Recent Progress in the Study and Therapy of Brain Edema, Plenum, New York, 1984, pp. 67-79. 6 Jo6, E, Current aspects of the development of the blood-brain barrier, Int. J. Dev. Neurosci., 5 (1987) 369-372. 7 Lossinsky, A.S., Vorbrodt, A.W. and Wisniewski, H.M., Characterization of endothelial cell transport in the developing mouse blood-brain barrier, Dev. Neurosci., 8 (1986) 61-75. 8 MiUonig, G., A modified procedure for lead staining of thin sections, J. Biophys. Biochem. Cytol., 11 (1961) 736-739. 9 Nagy, Z., Peters, M. and Hfittner, I., Endothelial surface charge: blood-brain barrier opening to horseradish peroxidase induced by the polycation protamine sulfate, Acta Neuropathol., Suppl. 7 (1981) 7-9. 10 Simionescu, M., Simionescu, N., Silbert, J.E. and Palade, G.E., Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites, J. Cell Biol., 90 (1981) 614-621. 11 Simionescu, M., Simionescu, N. and Palade, G.E., Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries, J. Cell Biol., 95 (1982) 425-434. 12 Simionescu, M., Ghinea, N., Fixman, A., Lasser, M., Kukes, L., Simionescu, N. and Palade, G.E., The cerebral microvascu-
13 14 15 16 17 18 19 20
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24
lature of the rat: structure and luminal surface properties during early development, J. Submicrosc. Cytol. Pathol., 20 (1988) 243-261. Skutelsky, E. and Roth, J., Cationic colloidal gold - a new probe for the detection of anionic cell surface sites by electron microscopy, J. Histochem. Cytochem., 34 (1986) 693-699. Stewart, P.A. and Hayakawa, E.M., Interendothelial junctional changes underlie the developmental 'tightening' of the bloodbrain barrier, Brain Res., 429 (1987) 271-281. Strausbaugh, L.J., Intracarotid infusions of protamine sulfate disrupt the blood-brain barrier of rabbits, Brain Res., 409 (1987) 221-226. Thfirauf, N., Dermietzel, R. and Kalweit, P., Surface charges associated with fenestrated brain capillaries. I. In vitro labeling of anionic sites, J. Ultrastruct. Res., 84 (1983) 103-110. Vorbrodt, A.W., Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex, J. Histochem. Cytochem., 35 (1987) 1261-1266. Vorbrodt, A.W., Ultrastructural cytochemistry of blood-brain barrier endothelia, Progr. Histochem. Cytochem., Vol. 18, no. 3, Gustav Fischer, Stuttgart, 1988, 99 pp. Vorbrodt, A.W., Ultracytochemicai characterization of anionic sites in the wall of brain capillaries, J. Neurocytol., 18 (1989) 359-368. Vorbrodt, A.W., Dobrogowska, D.H., Lossinksy, A.S. and Wisniewski, H.M., Ultrastructural localization of iectin receptors on the luminal and abluminal aspects of brain micro-blood vessels, J. Histochem. Cytochem., 34 (1986) 251-261. Vorbrodt, A.W., Lossinsky, A.S. and Wisniewski, H.M., Cytochemical and ultrastructural characterization of developing blood-brain barrier (BBB), J. Neuropathol. Exp. Neurol., 43 (1984) 350. Vorbrodt, A.W., Lossinsky, A.S. and Wisniewski, H.M., Localization of alkaline phosphatase activity in endothelia of developing and mature mouse blood-brain barrier, Dev. Neurosci., 8 (1986) 1-13. Vorbrodt, A.W., Lossinsky, A.S., Dobrogowska, D.H. and Wisniewski, H.M., Distribution of anionic sites and glycoconjugates on the endothelial surfaces of the developing bloodbrain barrier, Dev. Brain Res., 29 (1986) 69-79. Yoshida, Y., Yamada, M., Wakabayashi, K. and Ikuta, E, Endothelial fenestrae in the rat fetal cerebrum, Dev. Brain Res., 44 (1988) 211-219.
31
BRESD 51019
Sequential appearance of anionic domains in the developing blood-brain barrier Andrzej W. Vorbrodt, Albert S. Lossinsky, Danuta H. Dobrogowska and Henryk M. Wisniewski Department of Pathological Neurobiology, New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314 (U.S.A.) (Accepted 29 August 1989)
Key words: Anionic site; Cationic colloidal gold; Blood-brain barrier; Brain development; Barrier maturation
The distribution of anionic sites in the walls of mouse brain micro-blood vessels (MBVs) during development and maturation of the blood-brain barrier (BBB) was studied by electron microscopy. Cationic colloidal gold (CCG) and Lowicryl K4M-embedded brain samples obtained from mouse fetuses (13th and 19th days) and from 1-, 5-, 12- and 24-day-old and adult mice were used. The labeling of anionic sites with CCG was more intense on the abluminal than on the luminal front of the endothelial cells (ECs) in fetuses and in newborn mice. Only a few anionic sites appear on the luminal front of the ECs of proliferating blood vessels invading the neural tissue in 13-day-old fetuses. They become slightly, although steadily, more abundant during further stages of development, and their number rapidly increases between the 12th and 24th day of life at which time they attain the density typical for mature animals. The maturation of the basement membrane (BM), which occurs during the myelinization period (12th-24th day of life), also coincides with an increasing concentration of anionic sites. These observations suggest that the gradual appearance of anionic sites on both fronts of the endothelium, as well as in the developing and maturing BM, represents one of the mechanisms responsible for differentiation of cerebral microvasculature into BBB-type MBVs.
INTRODUCTION In the course of o u r previous studies, we have noted that the functional m a t u r a t i o n of the b l o o d - b r a i n barrier ( B B B ) , as evidenced by a 'closure' of brain micro-blood vessels ( M B V s ) to intravenously injected m a c r o m o l e c ular tracer ( H R P ) , takes place b e t w e e n the 12th and 24th day o f life in the mouse 7'z1'22. This m a t u r a t i o n coincides with the a p p e a r a n c e of alkaline p h o s p h a t a s e activity and with the f o r m a t i o n and r e m o d e l i n g of the glycoprotein layer on the surfaces of endothelial cells (ECs) 22'23. S o m e of the m o n o s a c c h a r i d e residues present in this glycoprotein coat maintain the negative charge on both surfaces of the E C s in the non-BBB 1°'11 as well as in the B B B type 17-2° microvasculature. It is believed that the negatively charged surface layer (anionic sites) helps restrict the m o v e m e n t of various molecules across the vessel wall 1"1° and also helps maintain the BBB 3-6'9"15. C o n s e q u e n t l y , one can expect that the functional and structural m a t u r a t i o n of the BBB is a c c o m p a n i e d by the f o r m a t i o n of anionic domains in the wall of brain MBVs. A l t h o u g h in our previous study the main attention was on the distribution of various glycoconjugates in deve-
loping mouse brain M B V s , we have also m a d e an a t t e m p t to detect the distribution of anionic sites using cationized ferritin (CF) applied to tissue sections o b t a i n e d from a tissue sectioner 23. This p r e - e m b e d d i n g technique, similar to those described by T h ~ r a u f et al. 16, enables one to label the anionic sites located only on the easily accessible luminal surface of the endothelium. Thus, the results o b t a i n e d are incomplete and do not show the distribution of negatively charged domains in the hardly accessible constituents of the vessel wall, such as the abluminal front of the ECs and the basal lamina (BM). Fortunately, in the m e a n t i m e , a new, m o r e reliable technique for the visualization of anionic sites with cationic calloidal gold ( C C G ) was e l a b o r a t e d by Skutelsky and R o t h 13. In o u r previous studies, this new cationic p r o b e was successfully a p p l i e d to ultrathin sections of brain samples e m b e d d e d in hydrophilic resin Lowicryl K4M ~7,18. W e were able to detect anionic sites in the entire cross-section of the vessel wall, and o t h e r cellular c o m p o n e n t s such as smooth muscle cells, pericytes, and glial perivascular endfeet. O u r present work is an a t t e m p t to apply this new technique to study the a p p e a r a n c e and distribution of
Correspondence: A.W. Vorbrodt, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
32 a n i o n i c d o m a i n s in the brain m i c r o v a s c u l a t u r e of m i c e
TABLE 1
d u r i n g the d e v e l o p m e n t and m a t u r a t i o n of the B B B .
The intensity of labeling of anionic s'ite,~on the lutrunal and abluminal fronts of the endothelial cells (ECs) ~ brain MBI "s during develop, ment and maturation of the mouse blood-brain barrier
MATERIALS AND METHODS
Animals All experiments were performed on IM or BALBc/J mice of both sexes. Brain samples were taken from mouse fetuses (13th and 19th day of gestation), newborn mice (lst day of life) or 5-, 12- and 24-day-old animals. For comparative purposes, adult animals (6-10 months old) were also used. Under sodium-pentobarbital anesthesia, animals (newborn, infants or adult) were fixed by intracardiac perfusion with a mixture of freshly prepared 2% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, supplemented with 3% polyvinyl pyrrolidone. Time of perfusion was 15 rain followed by immersion fixation up to 2 h in ice-cold fixative. Brains of fetuses were fixed by immersion without prior perfusion. After fixation, brain samples were cut into small blocks (1-2 mm), immersed for 4 h in ice-cold 0.1 M NH4CI to block free aldehyde groups and embedded at low temperature in Lowicryl K4M. After polymerization at low temperature under a UV lamp, specimens were cut with a diamond knife on a SorvaU MT 5000 (DuPont) microtome and picked up on 100-mesh, formvar-carboncoated nickel grids. Preparation of CCG Particles of colloidal gold with a mean diameter of 14 nm were prepared according to Frens 2. CCG was prepared according to the method of Skutelsky and Roth 13 by complexing poly-L-lysine (mol. wt. 306,000, Miles Labs., Naperville, IL) and colloidal gold; the preparation was similar to the one described previouslyaT. Demonstration of anionic sites Ultrathin sections attached to nickel grids were placed in a moist chamber at room temperature (22 °C) on a drop of 0.05 M Tris-HCI buffer-saline (TBS). After 5 min, they were transferred to 0.1 M glycine in TBS, pH 7, for 30 min and then to TBS, pH 2.1, supplemented with 0.02% polyethylene glycol, mol. wt. 20,000 (TBS-PEG) for 5 min. Finally, they were incubated at room temperature on a drop of solution of CCG in TBS-PEG containing 0.1% bovine serum albumin. The final concentration of CCG was approximately 50/tg/ml, giving an absorbance of 0.70 at 525 nm wavelength. The solution was prepared immediately before incubation and was centrifuged for 10 min at 400 g for removal of the large aggregates of gold complexes. The incubation time was 45-60 min. After labeling, the sections were washed in TBS and stained with 4% aqueous uranyl acetate (3 min) and with lead acetate (MiUonigs) for 45 s. The sections were examined in a Philips 420 electron microscope (EM). In the control sections, the anionic sites were blocked by
The intensity of the labeling was graded according to the number ot gold particles per l u m of the EC surface: -, no labeling; +-, less than 1 gold particle: +, 1-5 particles: + + . 6.--1(1 particles: + + + , 11-15 particles; + + + +, more than 16 particles per 1 um
Stage of development Intensity of labeling (age of the mouse) of EC surfaces
Additional comments
Luminal Abluminal 13-day-old fetus
+-
+
19-day-old fetus Newborn (lst day of life)
+
++
+
++
5 days old 12daysold 24 days old
++ +++ ++++
++ ++ +++
Adult(6months old)
++++
+++
The BM is not visible (structureless)
The BM becomes recognizable and labeled with CCG
The BM is well,developed and labeled with CCG
incubation in a solution of non-labeled poly-L-lysine (1 mg/ml of TBS, pH 2.1) for 1 h at room temperature before incubation with CCG.
RESULTS T h e results of o u r o b s e r v a t i o n s are s u m m a r i z e d in T a b l e I. A l t h o u g h o u r study has a d e s c r i p t i v e and m o r p h o logical c h a r a c t e r ,
t h e density o f t h e l a b e l i n g of b o t h
fronts o f t h e E C is p r e s e n t e d w i t h t h e a r b i t r a r y s y m b o l s d e s c r i b e d in T a b l e I. F o r this p u r p o s e , in brain sections, 10 r a n d o m l y c h o s e n t r a n s v e r s e l y s e c t i o n e d M B V s w e r e p h o t o g r a p h e d , and t h e n u m b e r o f g o l d particles associa t e d with b o t h l u m i n a l a n d a b l u m i n a l p l a s m a m e m b r a n e s of t h e E C s was c o u n t e d .
The mean values obtained
(calculated as the n u m b e r of g o l d p a r t i c l e s p e r 1 /~m)
Fig. 1. A section of the wall of a brain MBV of 13-day-old mouse fetus is shown Few aniomc sites are labeled with CCG on the abluminal front (arrowheads), whereas they are absent from the luminal surface of the EC. In the cytoplasm of the EC hardly noticeable plasmalemmal vesicles (curved arrows) are present. The abluminal surface of attenuated cells adjacent to the EC (presumably a primordial perieyte) is labeled with numerous gold particles (arrows). B, basement membrane; E, endothelial cell; J, intercellular junction; L, vessel lumen; N, cell nucleus; S, smooth muscle cell (all figures). All figures represent ultrathin sections of mouse brain cortex embedded in Lowicryl K4M and incubated with poly-L-lysine-gold complex (CCG) for the demonstration of anionic sites in the wall of micro-blood vessels (MBVs). x31.500. Fig. 2. Another, slightly larger MBV than shown in Fig. 1 of 13-day-old mouse fetus. The luminal surface of the EC is weakly labeled with few irregularly scattered gold particles except the terminal foldings near the junctional areas (arrows) which are decorated with several gold particles. On the contrary, the abluminal front of the EC is labeled with more numerous CCG particles scattered With some regularity (arrowheads). The surface of a primitive blood cell (curved arrows) is evidently more intensely labeled than the luminal surface of the EC. x31,500. Fig. 3. Localization of anionic sites labeled with CCG in the wall of MBV of 19-day-old mouse fetus is shown. The EC body is of uneven thickness, and few pseudopodial processes protrude into the vessel lumen. The labeling of the luminal surface of the EC is more intense than in the 13-day fetuses, although it is still discontinuous and irregular (arrows). The labeling of the abluminal front of the EC (arrowheads) is more intense. The surface of primordial pericyte facing the adjacent neural tissue is labeled with numerous gold particles. ×40,500.
33 were used for presenting the intensity of labeling with minur or plus signs. The presented values are approximations because in the Lowicryl K4M-embedded sam-
pies, the exact localization of gold particles in the hardly visible endothelial plasma membranes is frequently rather difficult due to omission of the osmium tetroxide.
34 In 13-day-old mouse fetuses, the neural tube becomes infolded into the ventricle to form the telencephalon. Perineural vessels invade the neural tissue where matrix
cells differentiate into neuroblasts, A1 this stage ~ development, the ECs of invading vessels are characterized by uneven thickness of their cytoplasm
35 The wall of these vessels is composed of a single layer of the endothelium accompanied by attenuated processes of cells presumably representing primordial pericytes. Present between these two types of cells is a narrow space filled with a structureless material labeled with a few gold particles. This narrow space is the area where the BM is ultimately to be formed during the further stages of development (Fig. 1). In the majority of blood vessels, numerous plasmalemmal pits and vesicles appear in the EC cytoplasm. Their outlines are only rarely recognizable because of rather poor staining of membranous structures resulting from the omission of the osmium tetroxide as a fixative. The luminal surface of the ECs of some vessels is not labeled (Fig. 1), whereas in other vessels a few irregularly scattered gold particles are present (Fig. 2). As a rule, the filopodia protruding into the vessel lumen in the junctional areas of the ECs, known as the terminal foldings, are more intensely labeled than the remaining fiat segments of the endothelial surface (Fig. 2, arrows). Evidently, in all MBVs, the abluminal front of the ECs is more intensely, although irregularly, labeled with CCG particles (Figs. 1 and 2, arrowheads). In 19-day-old fetuses, the endothelium is of uneven thickness, with numerous protrusions that are especially conspicuous in the junctional areas. The labeling of the luminal surface is irregular, i.e. some segments of the EC surface remain unlabeled, whereas others, especially the undulated segments, are decorated with single, numerous or clustered gold particles (Fig. 3). The abluminal front of the EC and the subendothelial BM-like space are labeled with irregularly scattered gold particles. The surface of primitive pericytes, facing the adjacent brain parenchyma, shows a similar labeling (Fig. 3). In newborn mice (1-day-old), the endothelium is of uneven thickness with numerous finger-like processes protruding into the vessel lumen. The labeling of the luminal surface is scanty and irregular, whereas the abluminal front of the EC is decorated more intensely (Fig. 4). Numerous MBVs have a rather flat luminal surface (Fig. 5) that is also less intensely labeled than the
abluminal front of the EC. In these vessels, the subendothelial BM-like space is strongly labeled with numerous CCG particles (Fig. 5). At this stage, the tight junctions between adjacent ECs are well-developed and easily discernible. In the 5-day-old mouse brain, the labeling of both luminal and abluminal fronts of the ECs becomes almost of the same intensity, although some variations between particular vessels can be noted (Figs. 6 and 7). The subendothelial, BM-like space is also labeled with numerous, irregularly scattered gold particles. In the EC cytoplasm, numerous plasmalemmal vesicles are present, although their delimiting membranes are hardly visible. In the vast majority of brain MBVs of the 12-day-old mouse, the localization of anionic sites is essentially similar to that observed in 24-day-old and in adult animals, although in the last two groups, the density of the labeling is slightly higher (see Table I). In capillaries, the luminal surface is uniformly decorated with an almost continuous row of CCG particles, whereas the labeling of abluminal front of the EC is less regular and less intense (Fig. 8). The easily recognizable BM is labeled with numerous, relatively uniformly scattered gold particles. The labeling of anionic sites is almost completely abolished in control sections exposed to unlabeled polyL-lysine prior to incubation with CCG. In these sections, only a few accidentally scattered gold particles are present, adhering unspecifically to the sectioned tissue as well as to the tissue-free areas of polymerized resin. DISCUSSON
In the present study, we were able to observe the distribution of anionic sites in such hardly accessible sites of the vessel wall as the abluminal surface of the EC, the adjacent space with basal lamina-like material, primordial pericytes and smooth muscle cells. Our results indicate that in 13-day-old mouse fetuses, the luminal surface of the endothelium of proliferating blood vessels contains only a few sparsely scattered
Fig. 4. A cross-section of a brain MBV of a newborn (1-day-old) mouse is shown. The undulated luminal surface of the EC is irregularly labeled with few gold particles (arrows). On the contrary, the labeling of the abluminal front of the EC is more regular and intense (arrowheads).
x55,500. Fig. 5. Another cross-sectioned brain MBV of 1-day-old mouse is shown. Although the labeling of the luminal surface is irregular and discontinuous (arrows), the labeling of the abluminal surface of the EC (arrowheads) as well as of the subendothelial basal lamina (B) is much more intense. Gold particles scattered throughout the EC cytoplasm indicate the presence of some not recognizable anionic domains which are accessible for the cationic probe, x55,500. Fig. 6. A cross-sectioned brain capillary of a 5-day-old mouse is shown. In this vessel, the EC cytoplasm is of irregular thickness and shows numerous indentations and protrusions. Labeling of the luminal surface (arrows) is slightly less regular and less intense than of the abluminal EC surface (arrowheads) x55,500. Fig. 7. Another, presumably larger brain MBV of a 5-day-old mouse is shown. In this vessel, the labeling of a relatively flat luminal surface of the plasmalemma proper (arrows) is less intense and less regular than of the abluminal front of the EC (arrowheads). A weakly stained subendothelial basal lamina-like material is also labeled with numerous gold particles, x31,500.
36
Fig. 8. A cross-sectioned brain capillary of a 12-day-old mouse is shown. The labeling of the luminal surface of the E(" is regular and almost continuous (arrows). The labeling of abluminal front of the EC (arrowheadsl and of the basemen! membrane (B) is also conspicuous. Some undefined structures in the EC cytoplasm are also labeled, indicating the presence of anionic microdomains. ~40,500. anionic domains. These domains become slightly, although steadily, more abundant during further stages of development, i.e. in 19-day-old fetuses as well as in newborn and 5-day-old animals. At this stage of development (5th day) the density of the labeling of both fronts of the EC becomes almost equal. They rapidly increase between the 12th and 24th days of life, attaining a high level typical for mature animals with a fully developed BBB (see Table I). It is worth noting that the anionic sites appear earlier and are more abundant on the abluminai than on the luminal front of the EC in the early stages of vascularization of the neural tube by proliferating MBVs. The faintly stained narrow space adjoining the abluminal surface of the ECs, where the BM is ultimately formed, also shows an increasing concentration of anionic domains considerably exceeding those located on the luminal surface of the ECs in newborn mice. Our previous studies performed on both developing 23 and adult mouse brain MBVs 19"2° indicate that it is terminal sialic acid residues that are mainly responsible for the maintenance of anionic domains on the luminal front of the ECs. Indeed, the gradual appearance and the distribution pattern of the binding sites for Limax flavus agglutinin, which is specific for sialic acid, observed by us previously 23, are very similar to the localization of anionic sites demonstrated in the present study with CCG. On the other hand, heparan and chondroitin sulfatesrich glycosaminoglycans 19 contribute to a substantial degree to the maintenance of the anionic domains located on the abluminal front on the EC and in the BM. The early appearance of anionic domains on the abluminal front of the ECs observed in invading MBVs in 13day-old mouse fetuses suggests that the synthesis of such
a type of proteoglycans does occur early during vascularization of the neural tissue. It also indicates that the gradual accumulation of negatively charged material occurs in the subendothelial space early before the final elaboration and formation of the BM in this area; according to Simionescu et a1.12 the BM attains maturity only after birth, during the process of myelinization. The early appearance of negatively charged domains on the abluminal side of the wall of newly formed MBVs seems to be functionally significant. Most probably, these domains constitute a negatively charged screen or filter that helps control the movement of various negatively charged solutes between the blood and brain parenchyma ~1. Our data suggest that the gradual appearance of anionic domains on both fronts of the endothelium, as well as in the area of the BM, represents one of the developmental mechanisms that cause differentiation of cerebral capillaries into the BBB-type character. It was suggested by Yoshida et al. 24 that developing neural cells (neuroblasts and glial cells) influence the ECs in such a way as to change their morphology and surface properties. The results of our studies indicate that the functional maturation of the mouse BBB, as evidenced previously by sealing of brain MBVs to injected H R P 7'14'21, is paralleled by the formation of negatively charged surface layers on both fronts of the endothelium and also in the BM. These observations also suggest the elaboration of the structural, and probably functional, polarity of brain endothelia as evidenced by the uneven concentration of anionic sites on both fronts of the ECs. Stronger labeling with CCG of the abluminal front occurred during early stages of development, followed finally by a more intense labeling of the luminal than abluminal front of the ECs
37 which we observed in 12-day-old mice and m a i n t a i n e d in
Further experimental studies are n e e d e d to elucidate
brain capillaries of adult animals. Since the tightening of
the problem of the mechanisms responsible for differen-
brain microvessels during d e v e l o p m e n t is accompanied by the d e v e l o p m e n t of tight junctionsTM and by the formation and m a t u r a t i o n of the B M 12'22, our present
tiation of the developing brain MBVs into the BBB-type vessels.
results suggest that the formation of anionic microdomains represents only one of several events accompany-
Acknowledgements. The authors wish to express their appreciation to Ms. Janis Kay for excellent secretarial assistance and to Ms. Maureen Stoddard Marlow for editorial suggestions. This study is supported in part by Grant 17271-08 from NINCDS.
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