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The role of müller cells in the formation of the blood-retinal barrier
0306-4522/93$6.00+ 0.00 PergamonPress Ltd © 1993IBRO
Neuroscience Vol. 55, No. 1, pp. 291-301, 1993
Printed in Great Britain
THE ROLE OF MOLLER CELLS IN THE FORMATION OF THE BLOOD-RETINAL BARRIER S. TOUT,* T. CHAN-LING,* H. HOLL.~.NDERt and J. STONE*~ *Department of Anatomy FI3, University of Sydney, NSW 2006, Australia i'Department of Neuromorphology, Max Planck Institute for Psychiatry, 8033 Martinsried, Germany Abstract--We have compared the ability of Miiller cells and astrocytes to induce the formation of barrier properties in blood vessels. Miiller cells cultured from the rabbit retina, and astrocytes and meningealcells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Miiller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Mfillercells play a major role in the formation of barrier properties in retinal vessels.
The characteristics of the blood-brain barrier are determined by the junctions between the endothelial cells of cerebral capillaries and by the level and polarity of transport of molecules across these cells (for reviews see Refs 5 and 8). Evidence from recent studies L3'4,12,19,23,2s'39,44 suggests that the formation of barrier properties in the endothelial cells of cerebral capillaries is induced by the astrocytes which form their gila limitans. The retina is an extension of the brain and its vessels have the same barrier properties as other cerebral vessels. It is likely, therefore, that the barrier properties of retinal vessels are induced by the cells which form their glia limitans, and there is strong evidence that astrocytes play a role in this induction. Astrocytes have been found in all vascularized vertebrate retinas so far investigated, are absent from retinas which lack vessels and, in partially vascularized retinas such as those of the rabbit and horse, are found only in vascular regions. 35'36'43The migration of astrocytes across the retina during development closely matches the spread of patent vessels 26,27,46and the processes of these astrocytes wrap retinal vessels closely,35'43 contributing to their glia limitans.22 Further, in conditions in which retinal astrocytes degenerate, the barrier properties of retinal vessels fail to form. 14A5 It is also clear, however, that astrocytes provide only part of the glia limitans of retinal vessels. In the retinas of the human, rat and cat, for example, vessels :~To whom correspondence should be addressed. CMF, calcium-magnesium-free Hanks Buffered Saline Solution; EDTA, ethylenediaminetetraacetate; GFAP, glial fibrillary acidic protein; HBSS, Hanks Buffered Saline Solution; HEPES, N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid; HRP, horseradish peroxidase.
Abbreviations:
form two distinct layers, of which only the inner plexus, located in the axon and ganglion cell layers, is associated with astrocytesJ 1,j3.~4,22,35,43M/iller cells are the major contributor to the gila limitans of the outer vascular plexus22"24and, at least in the cat, 22also contribute to the gila limitans of the inner layer. These observations imply that Mfiller cells share the ability, so far demonstrated only in astrocytes, 7'9'2°'~ to induce endothelial cells to form barrier properties. To test this idea, we implanted cultured M/iller cells, and for comparison astrocytes and meningeal cells, into the anterior chamber of the rat eye. The implanted cells formed aggregates on the anterior surface of the iris and became associated with its vessels. 23,3° We have tested the barrier properties of the vessels in the aggregates, using the intravascular perfusion of Evans Blue to visualize serum albumin by light microscopy, and of horseradish peroxidase (HRP) for electron microscopic assessment of vessel properties.
EXPERIMENTAL PROCEDURES
Cell culture
All cells were grown in Dulbeeco's Modified Eagle's Medium (Commonwealth Serum Laboratories, Australia) supplemented with 2 mM glutamine, 4.5 g/1 glucose, 20 mM HEPES, 50 U/ml penicillin, 50 #g/ml streptomycin and 10% heat-inactivated fetal bovine serum (Commonwealth Serum Laboratories, Australia). Cultures were maintained at 37°C in a humidified atmosphere containing 5% COz. Medium was changed every three days. Mailer cells. Miiller cells were obtained from the periphery of rabbit retinas, a source free of both astroeytes and blood vessels.33,34,43,46Retinas from embryonic day 23-28 New Zealand White rabbit embryos were used (Laboratory Animal Services, University of Sydney, Australia). The superior half of each retina, which contains the medullary ray zone (the region to which vessels and astrocytes are restricted) was discarded.33The inferior halves of the retinas
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of six to eight fetuses were pooled and incubated for 15 rain at 3 T C in a trypsin solution [0.125% trypsin (Type III, Sigma U.S.A.), 0.02% E D T A and 20 m M HEPES in calcium-magnesium-free-Hanks Buffered Saline Solution (CMF)]. The trypsin solution was inactivated with a solution of trypsin inhibitor [CMF containing 0.3% bovine serum albumin, 2 5 0 g g / m l soybean trypsin inhibitor (Sigma) and 4 0 # g / m l D N A a s e Type I (Sigma)] and the retinal fragments were dissociated into a single cell suspension by gentle trituration. The cell suspension was centrifuged and resuspended in medium. Primary cultures were plated at I x 107 cells per 180cm 2 tissue culture flask (Coming, U.S.A.). Astroo'tes. Astrocyte cultures were prepared by a modification of established techniques. 29 Wistar rat pups (Laboratory Animal Services, University of Sydney, Australia) between 24 and 48 h old were anaesthetized by cooling, decapitated and the heads dipped briefly in 70% ethanol. The cortical hemispheres were removed, other regions of the brain were trimmed and discarded and the meninges were removed. The isolated cortical tissue was chopped finely with a scalpel and the fragments gently teased through a sterile nylon mesh (pore size 120 ~ m ) to generate a suspension of single cells and small aggregates of cells. Suspensions containing 4 6 x 106 cells were plated into 75-cm 2 tissue culture flasks. Seven to nine days after the cultures were established, thc medium was changed and the flasks were agitated on a rotary shaking table for two periods of 18 h (200 r.p.m., stroke diameter 25 m m , 37'C). The cells which becamc detached as a result of the agitation (predominantly processbearing astrocytes and oligodendrocytes) were discarded, leaving a culture of predominantly "flat" astrocytes attached to the flask surface. Meningeal cells. Cultures of meningeal cells were generated from meninges isolated from postnatal day 2 rat cortices. The meninges were dissociated into a single cell suspension by trypsin treatment as described above and plated at 2 x 106 cells per 80cm 2 flask. Cells were subcullured three times before transplantation.
Implantation o / cultured cells into the anterior chamber Cells were harvested from the culture flask by a brief incubation with trypsin/EDTA solution followed by gentle agitation. An equal volume of trypsin inhibitor solution was added to the cell suspension, and the cells centrifuged at 450g at 4 ~C in 1.5 ml conical centrifuge tubes, to generate a packed cell pellet. Cell pellets were stored briefly on ice while the rat eyes were prepared for implantation. Adult Wistar rats were anaesthetized with Halothane in a 2:1 mix of nitrous oxide/oxygen, and Pilocarpine drops 11'% aqueous solution) were applied to the rat's eye to constrict the pupil. Five to ten microtitres of celt pellet were drawn into a flame-polished glass capillary connected by catheter tubing to a 20-#1 Hamilton syringe. A small incision was made in the cornea with a beaver blade or 24-guage syringe needle l 2 mm from the limbus, and thc tip of the capillary tubing was inserted into the anterior chamber. The tip was positioned above the iris and the cells expelled gently, z''3° A quantity of aqueous h u m o u r was allowed to leak out to normalize the pressure in the anterior chamber, Chloromycetin ointment was applied to the eyes after the implantation procedure. The barrier properties of vessels within cell aggregates on the iris were examined two to four weeks after implantation.
temperature with 2% paraformatdehydc m O. 1 M phosphate buffer (pH 7.3). Cells were made permeable by incubating them for 3 rain in HBSS containing 0.1% Triton X-I00 and 10% fetal bovine serum. Cultures of astrocytes and M/iller cells were incubated with a monoclonal antibody to glial fibrillary acidic protein (GFAP: 1 200: Labsystems, Finland, using a protocol described prevmusly4=) to determine the proportion of the cells which were astrocytc,;. Additionally, Mfiller celt cultures were labelled with the monoclonal antibody 4H 11 (1 : 501, '" a, determine the p r o portion of cells which were M/tiler celis. Each culture was also examined for the presence of microglia and vascular endothelial cells which bound fluorescein isothiocyanateconjugated Griffbnia simplici/olia Ba i'~olectin (100 ,ug,'ml; Sigma, U.S.A.). :'2~ Following antibod) or lectin labelling, cell nuclei were labelled with a fluorescent bisbenzamide dye (Hoechst Stain H33258: Calbiochem, U S A . ) by incubating the cultures for 5 min in a solution of the dye m saline 120#g/ml) and rinsing them briefly m saline. Labelled cutures were mounted in phosphate buffer/glycerol I I :21, coverslipped and examined using an incident lighl f l u o f escence microscope. In all the Mfiller cell cultures cxamincd, over 97%, of Ihc cells were labelled with the 4HII antibody. None ,~cre labelled by a n t i - G F A P and only a small (<:1%) proportmn labelled with G. ~impliciJblia lectin: the~ resembled microglia in morphology. In all astrocyte culture~ -xamined, over 80% of the cells were labelled by a n t i - G F A P and none were labelled by 4 H l l . Lectin-labelled cells with the morphology of vascular endothelial cells were not found in any of the cultures examined.
Evam#tation of ~,essels in cell aggregate,/,y light micro.~cop~ Rats in which cells had been implanted were anaesthetized deeply with an intraperitoneal injection of sodium penlobarbitone ( 6 m g / 1 0 0 g body weight). One millilitre o f Evans Blue solution (1% Evans Blue dissolved in H a r t m a n n ' s solution, filtered through a 0 . 2 2 # m Millipore filter just prior to use) was injected intracardically m'er 30 s Evans Blue binds strongly to serum albumin/~ and its distribution after intravascular injection reflects the distribution of serum albumin. ~w Five to ten minutes later, the eyes were fixed either b y t i m m e r s i o n for 30ram i~ fixative (4% paraformaldehyde in 0. l M phosphate-bufli:red saline) or by perfusion, firstly with 25 ml of H a r t m a n n ' s solution followed by Ill0 ml of fixative at room temperature, after which lhe eyes were removed and postfixed by immersion fi~r lit 15 rain. Irises with cell aggregates wcre dissected tree from the e'yc, divided into quadrants, mounted on to ~lides with phosphate-buffered glycerol and coverstipped Samples of retina. choroid and ciliary process were also mounted on to slides and examined. The distribution of Evans Blue in these tissues was examined and photographed using an incident light fluorescence microscope, with an excitation wavelength of 465 550 n m In selected cases, the irises bearing the aggregates ~ere lifted from the slides and washed for 20 min in HBSS to remove the glycerol mounting medium, t'he aggregates were divided into two or more parts, of which one was labelled with a n t i - G F A P and another with 41-111, to allow assessment o f their boundaries and composition, The tissue was made permeable to antibodies by a 30-rain incubation in 0.25°/3 Triton X-100 and 10% fetal bovine serum in ttBSS. The iris pieces were incubated for 2 h in anIi-GFAP or 41-I I 1 and processed as described above.
Assessment q f culture purity A small quantity of each cell pellet was put aside to be examined for cell purity. Cells from the pellet were resuspended in medium and transferred to 35-ram plastic Petri dishes, where they were grown for a further four days. The cultured cells were then rinsed briefly with H a n k s Buffered Saline Solution (HBSS) and fixed for 10min at room
Examination by electron microwop~ The permeability of vessels was assessed by etecmm microscopy, following perfusion with HRP, a longestablished tracer in studies of the blood-brain barrier (reviewed in Broadwellm). Rats were anaesthetized with an overdose of sodium pentobarbitone 16 mg/kg, i.p.). Once
Miiller cells and the blood-retinal barrier anaesthetized they were injected intramuscularly with promethazine (0.1ml of 1% in distilled water/100g body weight). Ten minutes later they were injected intraeardially with Type II HRP (10 mg/100 g body weight) dissolved in 1 ml of Hartmann's solution. Ten minutes later the thorax was opened and a further intracardiac injection of 0.1 ml Heparin solution was made. The animal was then perfused briefly with ice-cold Hartmann's solution, and then for 15 min with fixative (2% paraformaldehyde, 4% glutaraldehyde, 2mM CaC12 and 3.4% sucrose in 0.1 M sodium cacodylate :at 37°C). The eyes were dissected free and postfixed at room temperature for 30 rain. The irises were dissected from the eyes and rinsed in buffer for 45 rain. They were then placed for 30 rain in buffer containing 0.1 mg/ml of diaminobenzidine, then for 6 rain in the same solution with 0.1ml 3% H202 added per 10rnl. They were then rinsed in buffer and fixed in 1% osmium tetroxide in 0.05 M buffer at 4°C for 2 h, rinsed overnight in 0.1 M buffer at 4°C, dehydrated in ascending alcohols and embedded in Spurr's resin. To enable more certain identification of HRP reaction product, tissue contrast was limited by not block-staining the tissue with uranyl acetate. Semithin (1 #m) sections were mounted on conventional slides for light microscopy and stained with Richardson's stain. Ultrathin sections were mounted onto butvar-coated slot grids and stained with uranyl acetate/lead citrate with an LKB Ultrostainer. Stained sections were examined under the electron microscope at 60kV. Blocks were prepared from irises with implants of Miiller cells, astrocytes and meningeal cells, and from normal irises, in all cases perfused with HRP.
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Three animals were used in this series. In one, astrocytes had been implanted in the anterior chamber, in another Miiller cells had been implanted and in the third, meningeal cells. For each tissue examined (normal iris, choriod, ciliary body, retina and iris with astrocyte, Mfiller cell or meningeal aggregate) several blocks were prepared, cut and examined, and in each block several vessels were examined.
RESULTS
Composition o f cell aggregates The aggregates of cells implanted into the anterior chamber adhered to the iris, in most cases to its anterior surface. In a minority of cases the aggregates extended to the iris-corneal angle or to the margin of the pupil. When the irises were examined by dissecting microscope, the aggregates were apparent as raised, vascularized regions. CcUs in aggregates formed after the implantation of astrocytes were labelled strongly by a n t i - G F A P (Fig. 1A) and were not labelled by 4 H l l . In both experimental and normal irises, the a n t i - G F A P antibody labelled a network of fine fibres (Fig. 1B), previously described by Bj6rklund et al.6 The labelled fibres did not extend into the aggregates. Cells in aggregates formed after implantation of Miiller cells
Fig. 1. Appearance of cell aggregates. (A) A region of an aggregate of astrocytes, labelled with anti-GFAP; the concentration of GFAP-immunoreactive somas is apparent. (B) GFAP immunoreactivity found in rat irises, on fibrous structures quite distinct from the cell aggregates. (C) In an aggregate of Mtller cells, the antibody 4H11 labels the concentration of cell bodies. (D) In the iris, 4HI 1 labels circumferentially oriented fibrous structures. Scale bar = 50/~m (A,C); 100 ~m (B, D).
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Table 1. The middle three columns provide a qualitative representation of the strength of Evans Blue fluorescence in ocular tissues after perfusion fixation Tissue Iris Choroid Ciliary processes Meningeal aggregate Astrocyte aggregate Mfiller cell aggregate Retina
Halo
Tissue fluorescence
+ + +/+ + + + + + + + + + ----
+ + + + + + + + + + + + + --
N 10 10 10 5 5 5 10
HRP leakage + + + + + + + + ----
A dash indicates no fluorescence; the number of + signs indicates the strength of fluorescence, from weak (+) to strong ( + + +). The two values presented for iris tissue represent the observation that most but not all of the iris vessels appeared to leak Evans Blue. N indicates the number of animals in which each tissue was examined. The two columns on the right provide a schematic representation of HRP leakage from vessels, as assessed by electron microscopy. were not labelled by anti-GFAP, but were labelled by 4H11 (Fig. 1C), a monoclonal antibody with specificity for Miiller cells. 32 In the normal iris, 4 H l l labelled circumferentially oriented fibrillar structures (Fig. 1D).
Assessment o f barrier properties o f vessels in normal tissues' o f the eye by perfusion o f Evans Blue In tissue fixed by immersion, Evans Blue remaining in the vessels made vascular trees readily visible. Vessels impermeable to the Evans Blue-albumin were sharply outlined, because the tissue between the vessels was free of fluorescence. By contrast, the outlines of permeable vessels appeared blurred by fluorescence in the tissue between vessels. In tissue fixed by perfusion, the perfusate washed Evans Blue from the vessel lumen, leaving impermeable vessels almost invisible. Permeable vessels then stood out, marked by a halo of fluorescence around their circumference and in the surrounding tissue. The present observations of the distribution of Evans Blue fluorescence within the ocular tissues are summarized in Table 1. Retina. We found no evidence that Evans Blue-albumin left the retinal vessels to enter the retinal neuropil. When the retina was immersionfixed the images in Fig. 2A and B were obtained. Evans Blue remaining in the vessels defined the lumen of vessels sharply in both the superficial (A) and deep plexus (B), and the neuropil between vessels lacked fluorescence. When fixative was perfused through the vasculature after the Evans Blue, no fluorescent material was apparent in the retinal vessels, and they were not visible. Ciliary processes. The vessels of the ciliary processes are fenestrated and lack barrier properties (reviewed in Ref. 16); correspondingly, they were seen to leak Evans Blue. In immersion-fixed tissue, for example, the profiles of vessels within the ciliary processes were unclear due to the high level of Evans Blue fluorescence in the surrounding stroma. Similarly, in tissue fixed by perfusion, the outlines of vessels were marked by a bright halo of fluorescence,
along with a high level of fluorescence in the ciliary process stroma (Fig. 2C). In perfused material, however, the vessel lumen was free of fluorescence. Iris. The barrier properties of the vessels of the iris vary between the species (reviewed in Ref. 16), the barrier being, for example, tighter in primates than in the rat. In our rat material, many vessels of the iris appeared to leak Evans Blue. When fixed by immersion, the fluorescent outline of iris vessels appeared less sharp than that of retinal vessels. In tissue fixed by perfusion, the vessel lumen was free of fluorescence but the outline of the vessels was marked by a bright fluorescent halo (Fig. 2D). Fluorescence in the stroma of the iris was weaker than in the ciliary processes, perhaps because the iris is thin and bathed on both sides by fluids able to carry away extravascular Evans Blue. Overall, the tendency to leak Evans Blue-albumin was more variable in vessels of the iris than in those of the ciliary processes
Evans Blue filling o f vessels within cell aggregates Within a week of implantation, aggregates of implanted cells had stabilized on the anterior surface of the iris. During perfusion, Evans Blue was seen to rapidly fill vessels in the iris and aggregates and in some cases to spread from the vessels into the surrounding tissue. These patterns of spread would have provided a simple basis for assessing the permeability of the vessels in the aggregates, but for two slower and initially unanticipated movements of Evans Blue. First, Evans Blue was seen in both normal and experimental eyes to spread from vessels in the iris and ciliary processes into the aqueous humour. This spread began during the perfusion of the dye and continued even after fixation, and tended to reduce the concentration of dye in the stroma of the ciliary processes and iris. Second, Evans Blue in the aqueous humour spread into surrounding tissue, including cell aggregates on the iris. This effect was reduced, but could not be eliminated, by dissecting the irises and attached aggregates from the other tissues of the eye and preparing them for fluorescence microscopy with as little delay as possible.
Miiller cells and the blood-retinal barrier
Assessment of barrierproperties of vessels in aggregates by Evans Blue perfusion Since each agg~gate formed as a raised area on the anterior surface of the iris, the irises were mounted with the anterior surface uppermost. The vessels of the aggregate were often distinct in their shape, forming small, disordered capillary loops, which lacked the radial orientation typical of iris vessels. Evans Blue fluorescence was consistently stronger in the tissue of cell aggregates (astrocyte, Miiller cell and meningeal) than in the surrounding iris tissue. Characteristic differences were noted, however, in the fluorescence associated with vessels within each of the aggregates. Meningeal aggregates. Vessels within meningeal aggregates appeared to be as permeable as the vessels of the iris proper. Evans Blue fluorescence formed a distinct halo around vessels, both as they approached the aggregate and as they spread within the aggregate. Typically, for example in Fig. 3A, a clear boundary could be seen between the limited fluorescence of the iris and the diffuse, brighter fluorescence within the meningeal aggregate.
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Astrocyte and bl~ller cell aggregates. Following immersion fixation, the outlines of vessels within the aggregates of both Mfiller cells and astrocytes appeared sharp. The absence of a fluorcsocat halo around vessels in the aggregates was men clearly ~ following perfusion fixation (Fig. 3B--F). Vessels could be traced from the iris, where the halo made them visible, to the edge of the aggregate, where the halo disappeared, and the vessels could no longer be clearly traced. Vessels appeared to end blindly (arrows); in fact they continued into the aggregate but, like the vessels of the retina, could not be seen using fluorescence optics. This absence of the fluorescent halo around their vessels was a consistent feature of MOiler cell and astrocyte aggregates. Assessment of barrierproperties by horseradishperoxidase perfusion The material perfused with H R P was observed by both light and electron microscopy. In the light microscopy, HRP reaction product appeared outside most vessels of the normal iris (arrows in Fig. 4A), in
Fig. 2. Distribution of Evans Blue in tissue wholemounts. (A, B) Retinal wholemount with Evans Blue-filled vessels, following immersion fixation. A shows the superficial plexus of vessels, B the deep plexus. The sharp outline of the vessels is due to the lack of fluorescence in the retinal neuropil and indicates that Evans Blue has not leaked from these vessels into the retina. (C) Evans Blue in the ciliary processes following perfusion fixation. Vessels within the ciliary processes are empty of fluorescent material and a bright halo of Evans Blue has formed around them and in the tissue between them. The large vessel pictured lies outside the ciliary processes. (D) Evans Blue in a normal iris following perfusion fixation. The vessels are empty of fluorescence, but Evans Blue has passed out of the vessels to form a bright halo around them and in the tissue between them. Scale bar = 200pro (A,B); 100/Jm (C,D).
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Fig. 3. Distribution of Evans Blue in cell aggregates. (A) Vessels extending from the iris (left) into an aggregate of meningeal cells (right) following peffusion fixation. The level of background fluorescence is high in the aggregate. Vessels within the aggregate have a similar fluorescent halo to vessels outside the aggregate. Arrows mark the approximate position of the aggregate border. (B-D) Vessels extending into an aggregate of astrocytes. The aggregate is apparent as the area of diffuse fluorescence. Vessels entering the aggregate cannot be traced within it; the vessels become visible at the point of entry (arrows), because Evans Blue has been washed from their lumen by perfusate, and within the aggregate the vessels lack a fluorescent halo. The point of entry of one vessel into the aggregate in B is shown with greater magnification in D. (E, F) Vessels extending into aggregates of Mfiller oetts~ As with astrocyte aggregates, vessels entering an aggregate cannot be traced within it, but become invisible at the point of entry (arrows). Scale bar = 200 #m (A~:?,E); 100 #m (D,F). some cases spreading well away from the vessel. In the retina, by contrast, there was no evidence of reaction product outside the vessel (Fig. 4B). Figure 4C shows a length of iris from an eye implanted with Miiller cells. When the iris was examined by dissecting microscope before sectioning, a cell aggregate was apparent in the tissue on the left side of Fig. 4C. in
this section, a population of cells with dark nuclei (arrowheads) could be distinguished there. H R P appeared not to have escaped from the vessels on the left, but had clearly leaked from the vessels on the right (arrow), in the region from which cells with dark nuclei are lacking. The vessels at the posterior surface of the iris (bottom) all appeared to have leaked HRP.
MOiler cells and the blood-retinal barrier Figure 4D shows part of an iris from an eye implanted with astrocytes. HRP reaction product was apparent within the vessels, but could not be detected outside the vessels. The walls of vessels from similar tissues, as observed in the electron microscope, are shown in Fig. 5. The low contrast of the tissue, a result of the deliberate omission of block-staining with uranyl acetate, allows ready identification of the electrondense HRP reaction product. In the normal iris (Fig. 5A), HRP reaction product is prominent in the extracellular space around vessels, in apparent yes-
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icles within the cytoplasm of endothelial cells, and in membrane "pits" which form in the abluminal plasma membrane.9'~° In a vessel in the retina, by contrast (Fig. 5B), reaction product was apparent in the lumen (top) but was not apparent external to the vessels, nor in an interendothelial cell junction (which runs between the arrows). Vesicles and membrane pits were not prominent in the endothelial cell. In a vessel from the choroid (Fig. 5C), HRP reaction product was very prominent in the extracellular space around the vessels, and in a wide interendothelial cleft. Membranous pits containing
D Fig. 4. Light microscopy of HRP-pcrfused material. (A) Section of a normal iris, with the anterior surface uppermost. HRP reaction product is evident (arrows) in the space around the larger vessels. (B) Section of retina from an eye peffused with HRP. No HRP reaction product is apparent in the tissue. (C) Section of iris from an eye in which Mftller cells were implanted. The aggregate formed in the anterior tissue of the iris is soon on the left of this panel. HRP reaction product is prominent (arrows) around the vessels at the anterior surface (riOt) and around vessels at the posterior surface 0ower). In contrast, reaction product is not apparoat around the vesselsat the anterior surface (left). These latter vesselsare surrounded by a population of cells with dark nuclei (arrowheads). The scale bar in B also applies to C. (D) Section of iris from an eye in which astrocytes were implanted. HRP was not detected outside the vessels.
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Miiller cells and the blood-retinal barrier reaction product were numerous. In a vessel from an iris implanted with meningeal cells, H R P was prominent in the extracellular space and in vesicles within the endothelial cells (Fig. 5D). In a vessel from an iris implanted with astrocytes (Fig. 5E), H R P reaction product was observed in the lumen of the vessel and in the luminal side of an interendothelial cleft (fight arrow), but not within the cleft or in the extracellular space surrounding the vessel. Similarly, in vessels from an iris implanted with Mfiller cells (Fig. 5F-H), reaction product was apparent in the lumen of the vessels and in the luminal end of interendothelial junctions, but not in the outer part of the cleft or in the perivascular space. In summary, the vessels of the normal choroid and iris appear to allow the movement of HRP out of the vessels, and the vessels of the iris of eyes implanted with meningeal cells appeared particularly permeable. Retinal vessels, and iris vessels associated with implanted astrocytes or Miiller cells, appeared impermeable to HRP. DISCUSSION
Summary of findings The present results provide evidence that Miiller cells share the ability of astrocytes to induce properties of the blood-brain barrier in vascular endothelial cells. Specifically, they confirm a previous finding23 that vessels associated with aggregates of astrocytes on the anterior surface of the iris are impermeable to albumin and HRP, while vessels associated with aggregates of meningeal cells are permeable; they also provide evidence that vessels associated with aggregates of Mfiller cells are similarly impermeable. The principal difficulty of interpretation encountered in this analysis was the level of fluorescence within the cell aggregates after perfusion with Evans Blue. If that level was determined only by the permeability of the vessels of the aggregate, it should be high in the meningeal aggregates and low in the aggregates of Mfiller cells and astrocytes. In fact, considerable background fluorescence was observed in all cell aggregates. We argue that this observation
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does not contradict our principal conclusion, for two reasons. First, it became clear that the level of fluorescence in the aggregates is not determined only by leakage from their vessels, but that Evans Bluebound albumin moves from the cilliary processes and iris into the aqueous humour and thence into the extracellular space of the aggregates. Second, the fluorescence in the aggregates may appear more prominent because the glial cells within the aggregates actively accumulate Evans Blue-albumin from the extracellular space~ and because the aggregates increase the thickness of the iris. We therefore relied on the presence or absence of a halo of fluoresence around perfused vessels to assess their permeability. It was consistently observed (Table 1) that the halo was absent from vessels known from previous work to be impermeable (retina), present in vessels known to be permeable (choroid, ciliary processes, iris, aggregates of meningeal cells) and absent from vessels in the two tissues under test, aggregates of Mfiller cells and astrocytes. Our interpretation of the material perfused with Evans Blue received strong support from the light and electron microscopic observations of the movement of HRP from vessels in normal irises, and in irises from eyes implanted with astrocytes, Miiller cells or meningeal cells (summarized in Table 1). Two recent studies report results at variance with those just presented. Small et al. 3s report that Miiller cells transplanted into the anterior chamber formed aggregates on the ciliary processes rather than the iris, and did not reduce the normally high permeability of ciliary vessels. Stewart and Holash, 41 in a report available to us only in brief form, attempted to establish gratis of astrocytes in the anterior chamber of the rat eye, and concluded that the gratis were not sufficiently pure to test the ability of astrocytes to induce barrier properties.
The plasicity of barrier properties Since Stewart and Wiley42 first provided evidence that the expression of barrier properties by blood vessels is influenced by their environment, evidence has accumulated that astrocytes induce endothelial
Fig. 5. Electron microscopy of HRP-perfused material. In each of A-H, the lumen of the vessel is at the top. (A) Section of the endothelium of a vessel and surrounding tissue from a normal iris. Dark HRP granules are prominent in vesicles and membrane pits in the cytoplasm of the endothelial cell and with the perivascular basement membrane. (B) Section of the endothelium of a retinal vessel. HRP granules are apparent in the lumen of a vessel, but not in an interendothelial junction (arrows) or in surrounding neuropil. (C) Section of the endothelium of a choroidal vessel. HRP is prominent in vesicles and pits in endothelial cells, in the wide cleft between two endothelial cells, and in the basal lamina of the endothelium. (D) Section of the endothelium of a vessel from an iris implanted with meningeal cells. HRP is profuse in intercellular regions around vessels. (E) Section of the endothelium of a vessel in an iris implanted with astrocytes. HRP reaction product is apparent in the lumen and adherent to the luminal surface of the endotlielium, but does not penetrate an interendothelial cell junction (arrowed) or appear outside the lumen. (F-H) Sections of endothelial cells in three vessels from an iris implanted with MiiUer cells. HRP reaction product is apparent in the lumen, adherent to the luminal surface of the endothelium, and at the inner end of interendothelial junctions. It does not penetrate the junctions (arrowed) or appear anywhere outside the lumen of the vessel. Scale bar = 0.4/am (A,D,F-H); 0.6/am (B,C,E). l, lumen of the vessel.
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cells to form barrier properties, specifically to form complex tight junctions with their neighbours, and to express various barrier-specific enzymes and molecular t r a n s p o r t e r s . 3"4A9"2°23'28'39"4°'4445 The dependence of barrier properties of capillaries on their local environment is emphasized by the tissue model employed here. The arteries of the retina, iris and ciliary processes are all branches of the ophthalmic artery and the vessels which become associated with the cell aggregates are branches of the iris vessels. Yet their barrier properties vary from being highly permeable, in the choroid, ciliary processes and meningeal aggregates; to less permeable, in the iris, to tight, in the retina, astrocyte and Mfiller cell aggregates. Studies already referred to 3'4"19"20"23"2s'39"40"44~45 indicate that vessels in central nervous tissue are induced to form barrier properties by their glia limitans. Though we have yet to identify the structural and metabolic specializations which constitute the barrier in the present model, it seems likely that mechanisms identified in earlier studies, such as the formation of complex zonula occludens and the closure of junctional clefts between vascular endothelial cells? ~74"44 are involvedY Implications
Four implications of the present results deserve to be mentioned. Elsewhere 22 we have noted extensive similarities between astrocytes and Mfiller cells in their functional specializations. We add here that these two classes of macroglia share an ability to induce the formation of blood-brain barrier properties in vascular endothelial cells. To our knowledge this study is the first description of the ability of a cell class other than astrocytes to induce blood-brain barrier properties in vessels, although the in ritro induction of barrier-specific enzymes in vascular endothelial cells by cortical neurons 45 has been described.
Second, we note that the mechanism by which macroglial cells induce the formation of barrier properties in endothelial cells is not species- or tissuespecific. In our experiments, and in the work of Janzer and Raft, 23 astrocytes from the cerebral cortex of the rat induced barrier properties in capillaries originating from the iris. Janzer and Raft have also demonstrated that rat cortical astrocytes induce barrier properties in vessels growing fiom the chorioallantoic membrane of the chick, and Tao-Cheng et a l Y have shown that cortical astrocytes from the rat could induce barrier properties in bovine microvascular endothelial cells. We have shown here that Miiller cells from the retina of the rabbit induce barrier properties in vessels in the iris of the rat. Third. we note that the ability w, induce barrier properties is retained by the Mfiller cells of lhc periphery of the rabbit retina, which remains avascular throughout life. 18'34-37"46These cells do not. therefore, normally function to induce barrier properties in vessels. Their ability to induce bar,~cr properties is nevertheless conserved. Finally. we note an important ciimcat implication of the present results: that Mfiller cells should be recognized ;ts a major factor in the formation and maintenance of the blood- retinal barrier. This conclusion may be of clinical relevance, since breaks in that barrier are a feature of several diseases of the retina, including retinopathy of prematurily and major causes of adull blindness and diabetic retinopathy. Acknowledgements --We wish to thank Mrs Z. Dreher lbr her expert assistance with tissue culture and the development of the 4HII monoclonal antibody, and Dr Stephen Robinson for his assistance and suggestions, This work was supported by grants from the National Health and Medical Research Council of Australia, the Government Employees" Medical Research Fund and the Ramaciotti Foundation. T.C.-L is an R. Douglas Wright Fellow of the National Health and Medical Research Council ~l' Australia.
REFERENCES
1. Arthur F. E., Shivers R. R. and Bowman P. D. (1987) Astrocyte-mediated induction of tight junctions m brain capillar~ endothelium: an efficient in vitro model. Derl Brain Res. 36, 155 159. 2. Ashwell K., Holl~inder H., Streit W. and Stone J. (1989) The appearance and distribution of microglia in the developing retina of the rat. Vis. Neurosci. 2, 437 448. 3. Beck D. W., Roberts R. L. and Olson J. J. (1986) Gtial cells influence membrane-associated enzyme activity at the blood-brain barrier. Brain Res. 381, 131 137. 4. Beck D. W., Vinters H. V., Hart M. N. and Cancilla P. A. (1984) Glial cells influence the polarity ot lhe blood-brain barrier. J. Neuropath. exp. Neurol. 43, 219-234. 5. Betz A. L. and Goldstein G. W. (1986) Specialized properties and solute transport in brain capillaries. ,.1. Rot:. t'h n~ird. 48, 241-250. 6. Bj6rklund H., Dahl D., Olson L. and Seiger A. (1984) Glial fibrillary acidic protein-like immunoreactivity in the iris: development, distribution, and reactive changes following transplantation. J. Neurosci. 4, 978-988. 7. Bouchard C., Lebert M. and Dupouey P. (1989) Are close contacts between astrocytes and endothelial cells a prerequisite condition of a blood brain barrier--the rat subfornical organ as an example. Biol. (¥1t 67, I59-165, 8. Bradbury M. W. B. (1985) The blood-brain barrier~ Transport across the cerebral endothelium, Circulatlon Res. 57, 213--222. 9. Brightman M. W. (1989) The anatomical basis of the blood-brain barrier. In Implications 0[' the Blood-Brain Barrier and its Man~ulation (ed. Neuwelt E. A.), Vol. 1, pp. 53 -83. Plenum, New York. 10. Broadwell R. D. (1989) Transcytosis of macromolecules through the blood-brain barrier: a cell biological perspective and critical appraisal, Acta neuropath., Berlin 79, 117- 128. 1I. Biissow H. (1980) The astrocytes in the retina and optic nerve head of mammals: a special glia fo r the ganglion cells axons. Cell Tiss. Res. 21~i, 367 378.
Miiller cells and the blood-retinal barrier
301
12. Cancilla P. A. and DeBault L. E. (1983) Neutral amino acid transport properties of cerebral endothelial cells/n vitro. J. Neuropath. exp. Neurol. 42, 191-199. 13. Chan-Ling T., Halasz P. and Stone J. (1990) Development of retinal vasculature in the cat: processes and mechanisms. Curr. Eye Res. 9, 459-478. 14. Chart-Ling T. and Stone J. (1992) Degeneration of astrocytes in feline retinopathy of prematurity causes failure of blood-retinal barrier. Invest. Ophthal. vis. Sci. 33, 2148-2159. 15. Chan-Ling T., Tout S., Holl/inder H. and Stone J. (1992) Vascular changes and their mechanisms in the feline model of retinopathy of prematurity. Invest. Ophthal. vis. Sci. 33, 2128-2147. 16. Cunha-Vaz J. (1979) The blood-ocular barriers. Surv. Ophthal. 23, 279-296. 17. Cunha-Vaz J. G., Shakib M. and Ashton N. (1966) Studies on the permeability of the blood-retinal barrier. I. On the existence, development, and site of a blood-retinal barrier. Br. J. Ophthal. 50, 441453. 18. Davis F. A. (1929) The anatomy and histology of the eye and orbit of the rabbit. Trans. Am. ophthal. Soc. 27, 401~141. 19. Debault L. E. and Cancilla P. A. (1980)),-Glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells/n vitro. Science 207, 653-655. 20. Dehouck M.-P., M~resse S., Delorme P., Fruchart J.-C. and Cecchelli R. (1990) An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. J. Neurochem. 54, 1798-1801. 21. del Cerro M., Gashi D. M., Rao G. N., Notter M. F., Wiegand S. J., Sathi S. and del Cerro C. (1987) Retinal transplants into the anterior chamber of the rat eye. Neuroscience 21, 707-723. 22. Holl~inder H., Makarov F., Dreher Z., van Driel D., Chart-Ling T. and Stone J. (1991) Structure of the macrogiia of the retina: sharing and division of labour between astrocytes and Miiller cells. J. comp. Neurol. 313, 587-603. 23. Janzer R. C. and Raft M. C. (1987) Astrocytes induce blood-brain properties in endothelial cells. Nature 325, 253-257. 24. Kondo H., Takahashi H. and Takahashi Y. (1984) Immunohistochemical study of S-100 protein in the postnatal development of Miiller cells and astrocytes in the rat retina. Cell Tiss. Res. 238, 503-508. 25. Laitinen L. (1987) Griffania simplicifolia lectin binds specifically to endothelial cells and some epithelial cells in mouse tissue. Histochem J. 19, 225-234. 26. Ling T., Mitrofanis J. and Stone J. (1989) Origin of retinal astrocytes in the rat: evidence of migration from the optic nerve. J. comp. Neurol. 286, 345-352. 27. Ling T. and Stone J. (1988) The development of astrocytes in the cat retina: evidence of migration from the optic nerve. Devl Brain Res. 44, 73-85. 28. Maxwell K., Berliner J. A. and Cancilla P. A. (1987) Induction of ),-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes. Brain Res. 410, 309-314. 29. McCarthy K. D. and de Vellis J. 0980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890-902. 30. Olson L., Seiger A. and Strfmberg I. (1983) Intraocular transplantation in rodents: a detailed account of the procedure and examples of its use in neurobiology with special reference to brain tissue grafting. Adv. cell. Neurobiol. 4, 407-442. 31. Radius R. L. and Anderson D. R. (1980) Distribution of albumin in the normal monkey eye as revealed by Evans Blue fluorescence microscopy. Invest. Ophthal. vis. Sci. 19, 238-243. 32. Robinson S. R. and Dreher Z. (1990) MiiUer cells in adult rabbit retinae: morphology, distribution and implications for function and development. J. comp. Neurol. 292, 178-192. 33. Scherer J. and Schnitzer J. (1989) The rabbit retina: a suitable mammalian tissue for obtaining astroglia-free MfiUer cultures. Neurosci. Letr 97, 51-56. 34. Schnitzer J. (1985) Distribution and immunoreactivity of glia in the retina of the rabbit. J. comp. Neurol. 240, 128-142. 35. Schnitzer J. (1988) Astrocytes in mammalian retina. Prog. Rer Res. 7, 209-231. 36. Schnitzer J. (1988) Astrocytes in the guinea pig, horse and monkey retina: their occurrence coincides with the presence of blood vessels. Glia 1, 74-89. 37. Schnitzer J. and Karschin A. (1986) The shape and distribution of astrocytes in the retina of the adult rabbit. Cell Tiss. Res. 246, 91-102. 38. Small R. K., Watkins B., Munro P. M. and Liu D. (1993) Functional properties of Miiller cells following transplantation into the anterior eye chamber. Glia 7, 158-169. 39. Stewart P. A. and Coomber B. L. (1986) Astrocytes and the blood-brain barrier. In Astrocytes (eds Fedoroff S. and Vernadakis A.), Vol. 1, Development, Morphology, and Regional Specialization o f Astrocytes, pp. 311-328. Academic Press, Orlando, FL. 40. Stewart P. A. and Hayakawa E. M. (1987) Interendothelial junctional changes underlie the developmental 'tightening' of the blood-brain barrier. Devl Brain Res. 32, 271-281. 41. Stewart P. and Holash J. A. (1991) Induction of the blood-brain barrier. Proceedings o f Castle Ringberg Symposium. 42. Stewart P. A. and Wiley M. J. (1981) Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: a study using quail-chick transplantation chimeras. Devl Biol. 84, 183-192. 43. Stone J. and Dreher Z. (1987) Relationship between astrocytes, ganglion cells and vasculature of the retina. J. comp. Neurol. 255, 35-49. 44. Tao-Cheng J.-H., Nagy Z. and Brightman M. W. (1987) Tight junctions of brain endothelium in vitro are enhanced by astrogiia. J. Neurosci. 7, 3293-3299. 45. Tonsch U. and Bauer H.-C. (1991) Glial cells and neurons induce blood-brain barrier related enzymes in cultured cerebral endothelial cells. Brain Res. 539, 247-253. 46. Tout S., Ashwell K. and Stone J. (1988) The development of astrocytes in the albino rabbit retina and their relationship to retinal vasculature. Neurosci. Let. 90, 241-247. 47. Tout S., Dreher Z., Chart-Ling T. and Stone J. (1993) Contact-spacing among astrocytes is independent of neighbouring structures: /n vivo and in vitro evidence. J. comp. Neurol. (in press). 48. Wolman M., Klatzo I., Chui E., Wilmes F., Nishimoto K., Fujiwara K. and Spatz M. (1981) Evaluation of the dye-protein tracers in pathophysiology of the blood-brain barrier. Acta neuropath., Berlin 54, 55-61. (Accepted 2 February 1993)
Neuroscience Vol. 55, No. 1, pp. 291-301, 1993
Printed in Great Britain
THE ROLE OF MOLLER CELLS IN THE FORMATION OF THE BLOOD-RETINAL BARRIER S. TOUT,* T. CHAN-LING,* H. HOLL.~.NDERt and J. STONE*~ *Department of Anatomy FI3, University of Sydney, NSW 2006, Australia i'Department of Neuromorphology, Max Planck Institute for Psychiatry, 8033 Martinsried, Germany Abstract--We have compared the ability of Miiller cells and astrocytes to induce the formation of barrier properties in blood vessels. Miiller cells cultured from the rabbit retina, and astrocytes and meningealcells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Miiller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Mfillercells play a major role in the formation of barrier properties in retinal vessels.
The characteristics of the blood-brain barrier are determined by the junctions between the endothelial cells of cerebral capillaries and by the level and polarity of transport of molecules across these cells (for reviews see Refs 5 and 8). Evidence from recent studies L3'4,12,19,23,2s'39,44 suggests that the formation of barrier properties in the endothelial cells of cerebral capillaries is induced by the astrocytes which form their gila limitans. The retina is an extension of the brain and its vessels have the same barrier properties as other cerebral vessels. It is likely, therefore, that the barrier properties of retinal vessels are induced by the cells which form their glia limitans, and there is strong evidence that astrocytes play a role in this induction. Astrocytes have been found in all vascularized vertebrate retinas so far investigated, are absent from retinas which lack vessels and, in partially vascularized retinas such as those of the rabbit and horse, are found only in vascular regions. 35'36'43The migration of astrocytes across the retina during development closely matches the spread of patent vessels 26,27,46and the processes of these astrocytes wrap retinal vessels closely,35'43 contributing to their glia limitans.22 Further, in conditions in which retinal astrocytes degenerate, the barrier properties of retinal vessels fail to form. 14A5 It is also clear, however, that astrocytes provide only part of the glia limitans of retinal vessels. In the retinas of the human, rat and cat, for example, vessels :~To whom correspondence should be addressed. CMF, calcium-magnesium-free Hanks Buffered Saline Solution; EDTA, ethylenediaminetetraacetate; GFAP, glial fibrillary acidic protein; HBSS, Hanks Buffered Saline Solution; HEPES, N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid; HRP, horseradish peroxidase.
Abbreviations:
form two distinct layers, of which only the inner plexus, located in the axon and ganglion cell layers, is associated with astrocytesJ 1,j3.~4,22,35,43M/iller cells are the major contributor to the gila limitans of the outer vascular plexus22"24and, at least in the cat, 22also contribute to the gila limitans of the inner layer. These observations imply that Mfiller cells share the ability, so far demonstrated only in astrocytes, 7'9'2°'~ to induce endothelial cells to form barrier properties. To test this idea, we implanted cultured M/iller cells, and for comparison astrocytes and meningeal cells, into the anterior chamber of the rat eye. The implanted cells formed aggregates on the anterior surface of the iris and became associated with its vessels. 23,3° We have tested the barrier properties of the vessels in the aggregates, using the intravascular perfusion of Evans Blue to visualize serum albumin by light microscopy, and of horseradish peroxidase (HRP) for electron microscopic assessment of vessel properties.
EXPERIMENTAL PROCEDURES
Cell culture
All cells were grown in Dulbeeco's Modified Eagle's Medium (Commonwealth Serum Laboratories, Australia) supplemented with 2 mM glutamine, 4.5 g/1 glucose, 20 mM HEPES, 50 U/ml penicillin, 50 #g/ml streptomycin and 10% heat-inactivated fetal bovine serum (Commonwealth Serum Laboratories, Australia). Cultures were maintained at 37°C in a humidified atmosphere containing 5% COz. Medium was changed every three days. Mailer cells. Miiller cells were obtained from the periphery of rabbit retinas, a source free of both astroeytes and blood vessels.33,34,43,46Retinas from embryonic day 23-28 New Zealand White rabbit embryos were used (Laboratory Animal Services, University of Sydney, Australia). The superior half of each retina, which contains the medullary ray zone (the region to which vessels and astrocytes are restricted) was discarded.33The inferior halves of the retinas
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of six to eight fetuses were pooled and incubated for 15 rain at 3 T C in a trypsin solution [0.125% trypsin (Type III, Sigma U.S.A.), 0.02% E D T A and 20 m M HEPES in calcium-magnesium-free-Hanks Buffered Saline Solution (CMF)]. The trypsin solution was inactivated with a solution of trypsin inhibitor [CMF containing 0.3% bovine serum albumin, 2 5 0 g g / m l soybean trypsin inhibitor (Sigma) and 4 0 # g / m l D N A a s e Type I (Sigma)] and the retinal fragments were dissociated into a single cell suspension by gentle trituration. The cell suspension was centrifuged and resuspended in medium. Primary cultures were plated at I x 107 cells per 180cm 2 tissue culture flask (Coming, U.S.A.). Astroo'tes. Astrocyte cultures were prepared by a modification of established techniques. 29 Wistar rat pups (Laboratory Animal Services, University of Sydney, Australia) between 24 and 48 h old were anaesthetized by cooling, decapitated and the heads dipped briefly in 70% ethanol. The cortical hemispheres were removed, other regions of the brain were trimmed and discarded and the meninges were removed. The isolated cortical tissue was chopped finely with a scalpel and the fragments gently teased through a sterile nylon mesh (pore size 120 ~ m ) to generate a suspension of single cells and small aggregates of cells. Suspensions containing 4 6 x 106 cells were plated into 75-cm 2 tissue culture flasks. Seven to nine days after the cultures were established, thc medium was changed and the flasks were agitated on a rotary shaking table for two periods of 18 h (200 r.p.m., stroke diameter 25 m m , 37'C). The cells which becamc detached as a result of the agitation (predominantly processbearing astrocytes and oligodendrocytes) were discarded, leaving a culture of predominantly "flat" astrocytes attached to the flask surface. Meningeal cells. Cultures of meningeal cells were generated from meninges isolated from postnatal day 2 rat cortices. The meninges were dissociated into a single cell suspension by trypsin treatment as described above and plated at 2 x 106 cells per 80cm 2 flask. Cells were subcullured three times before transplantation.
Implantation o / cultured cells into the anterior chamber Cells were harvested from the culture flask by a brief incubation with trypsin/EDTA solution followed by gentle agitation. An equal volume of trypsin inhibitor solution was added to the cell suspension, and the cells centrifuged at 450g at 4 ~C in 1.5 ml conical centrifuge tubes, to generate a packed cell pellet. Cell pellets were stored briefly on ice while the rat eyes were prepared for implantation. Adult Wistar rats were anaesthetized with Halothane in a 2:1 mix of nitrous oxide/oxygen, and Pilocarpine drops 11'% aqueous solution) were applied to the rat's eye to constrict the pupil. Five to ten microtitres of celt pellet were drawn into a flame-polished glass capillary connected by catheter tubing to a 20-#1 Hamilton syringe. A small incision was made in the cornea with a beaver blade or 24-guage syringe needle l 2 mm from the limbus, and thc tip of the capillary tubing was inserted into the anterior chamber. The tip was positioned above the iris and the cells expelled gently, z''3° A quantity of aqueous h u m o u r was allowed to leak out to normalize the pressure in the anterior chamber, Chloromycetin ointment was applied to the eyes after the implantation procedure. The barrier properties of vessels within cell aggregates on the iris were examined two to four weeks after implantation.
temperature with 2% paraformatdehydc m O. 1 M phosphate buffer (pH 7.3). Cells were made permeable by incubating them for 3 rain in HBSS containing 0.1% Triton X-I00 and 10% fetal bovine serum. Cultures of astrocytes and M/iller cells were incubated with a monoclonal antibody to glial fibrillary acidic protein (GFAP: 1 200: Labsystems, Finland, using a protocol described prevmusly4=) to determine the proportion of the cells which were astrocytc,;. Additionally, Mfiller celt cultures were labelled with the monoclonal antibody 4H 11 (1 : 501, '" a, determine the p r o portion of cells which were M/tiler celis. Each culture was also examined for the presence of microglia and vascular endothelial cells which bound fluorescein isothiocyanateconjugated Griffbnia simplici/olia Ba i'~olectin (100 ,ug,'ml; Sigma, U.S.A.). :'2~ Following antibod) or lectin labelling, cell nuclei were labelled with a fluorescent bisbenzamide dye (Hoechst Stain H33258: Calbiochem, U S A . ) by incubating the cultures for 5 min in a solution of the dye m saline 120#g/ml) and rinsing them briefly m saline. Labelled cutures were mounted in phosphate buffer/glycerol I I :21, coverslipped and examined using an incident lighl f l u o f escence microscope. In all the Mfiller cell cultures cxamincd, over 97%, of Ihc cells were labelled with the 4HII antibody. None ,~cre labelled by a n t i - G F A P and only a small (<:1%) proportmn labelled with G. ~impliciJblia lectin: the~ resembled microglia in morphology. In all astrocyte culture~ -xamined, over 80% of the cells were labelled by a n t i - G F A P and none were labelled by 4 H l l . Lectin-labelled cells with the morphology of vascular endothelial cells were not found in any of the cultures examined.
Evam#tation of ~,essels in cell aggregate,/,y light micro.~cop~ Rats in which cells had been implanted were anaesthetized deeply with an intraperitoneal injection of sodium penlobarbitone ( 6 m g / 1 0 0 g body weight). One millilitre o f Evans Blue solution (1% Evans Blue dissolved in H a r t m a n n ' s solution, filtered through a 0 . 2 2 # m Millipore filter just prior to use) was injected intracardically m'er 30 s Evans Blue binds strongly to serum albumin/~ and its distribution after intravascular injection reflects the distribution of serum albumin. ~w Five to ten minutes later, the eyes were fixed either b y t i m m e r s i o n for 30ram i~ fixative (4% paraformaldehyde in 0. l M phosphate-bufli:red saline) or by perfusion, firstly with 25 ml of H a r t m a n n ' s solution followed by Ill0 ml of fixative at room temperature, after which lhe eyes were removed and postfixed by immersion fi~r lit 15 rain. Irises with cell aggregates wcre dissected tree from the e'yc, divided into quadrants, mounted on to ~lides with phosphate-buffered glycerol and coverstipped Samples of retina. choroid and ciliary process were also mounted on to slides and examined. The distribution of Evans Blue in these tissues was examined and photographed using an incident light fluorescence microscope, with an excitation wavelength of 465 550 n m In selected cases, the irises bearing the aggregates ~ere lifted from the slides and washed for 20 min in HBSS to remove the glycerol mounting medium, t'he aggregates were divided into two or more parts, of which one was labelled with a n t i - G F A P and another with 41-111, to allow assessment o f their boundaries and composition, The tissue was made permeable to antibodies by a 30-rain incubation in 0.25°/3 Triton X-100 and 10% fetal bovine serum in ttBSS. The iris pieces were incubated for 2 h in anIi-GFAP or 41-I I 1 and processed as described above.
Assessment q f culture purity A small quantity of each cell pellet was put aside to be examined for cell purity. Cells from the pellet were resuspended in medium and transferred to 35-ram plastic Petri dishes, where they were grown for a further four days. The cultured cells were then rinsed briefly with H a n k s Buffered Saline Solution (HBSS) and fixed for 10min at room
Examination by electron microwop~ The permeability of vessels was assessed by etecmm microscopy, following perfusion with HRP, a longestablished tracer in studies of the blood-brain barrier (reviewed in Broadwellm). Rats were anaesthetized with an overdose of sodium pentobarbitone 16 mg/kg, i.p.). Once
Miiller cells and the blood-retinal barrier anaesthetized they were injected intramuscularly with promethazine (0.1ml of 1% in distilled water/100g body weight). Ten minutes later they were injected intraeardially with Type II HRP (10 mg/100 g body weight) dissolved in 1 ml of Hartmann's solution. Ten minutes later the thorax was opened and a further intracardiac injection of 0.1 ml Heparin solution was made. The animal was then perfused briefly with ice-cold Hartmann's solution, and then for 15 min with fixative (2% paraformaldehyde, 4% glutaraldehyde, 2mM CaC12 and 3.4% sucrose in 0.1 M sodium cacodylate :at 37°C). The eyes were dissected free and postfixed at room temperature for 30 rain. The irises were dissected from the eyes and rinsed in buffer for 45 rain. They were then placed for 30 rain in buffer containing 0.1 mg/ml of diaminobenzidine, then for 6 rain in the same solution with 0.1ml 3% H202 added per 10rnl. They were then rinsed in buffer and fixed in 1% osmium tetroxide in 0.05 M buffer at 4°C for 2 h, rinsed overnight in 0.1 M buffer at 4°C, dehydrated in ascending alcohols and embedded in Spurr's resin. To enable more certain identification of HRP reaction product, tissue contrast was limited by not block-staining the tissue with uranyl acetate. Semithin (1 #m) sections were mounted on conventional slides for light microscopy and stained with Richardson's stain. Ultrathin sections were mounted onto butvar-coated slot grids and stained with uranyl acetate/lead citrate with an LKB Ultrostainer. Stained sections were examined under the electron microscope at 60kV. Blocks were prepared from irises with implants of Miiller cells, astrocytes and meningeal cells, and from normal irises, in all cases perfused with HRP.
293
Three animals were used in this series. In one, astrocytes had been implanted in the anterior chamber, in another Miiller cells had been implanted and in the third, meningeal cells. For each tissue examined (normal iris, choriod, ciliary body, retina and iris with astrocyte, Mfiller cell or meningeal aggregate) several blocks were prepared, cut and examined, and in each block several vessels were examined.
RESULTS
Composition o f cell aggregates The aggregates of cells implanted into the anterior chamber adhered to the iris, in most cases to its anterior surface. In a minority of cases the aggregates extended to the iris-corneal angle or to the margin of the pupil. When the irises were examined by dissecting microscope, the aggregates were apparent as raised, vascularized regions. CcUs in aggregates formed after the implantation of astrocytes were labelled strongly by a n t i - G F A P (Fig. 1A) and were not labelled by 4 H l l . In both experimental and normal irises, the a n t i - G F A P antibody labelled a network of fine fibres (Fig. 1B), previously described by Bj6rklund et al.6 The labelled fibres did not extend into the aggregates. Cells in aggregates formed after implantation of Miiller cells
Fig. 1. Appearance of cell aggregates. (A) A region of an aggregate of astrocytes, labelled with anti-GFAP; the concentration of GFAP-immunoreactive somas is apparent. (B) GFAP immunoreactivity found in rat irises, on fibrous structures quite distinct from the cell aggregates. (C) In an aggregate of Mtller cells, the antibody 4H11 labels the concentration of cell bodies. (D) In the iris, 4HI 1 labels circumferentially oriented fibrous structures. Scale bar = 50/~m (A,C); 100 ~m (B, D).
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Table 1. The middle three columns provide a qualitative representation of the strength of Evans Blue fluorescence in ocular tissues after perfusion fixation Tissue Iris Choroid Ciliary processes Meningeal aggregate Astrocyte aggregate Mfiller cell aggregate Retina
Halo
Tissue fluorescence
+ + +/+ + + + + + + + + + ----
+ + + + + + + + + + + + + --
N 10 10 10 5 5 5 10
HRP leakage + + + + + + + + ----
A dash indicates no fluorescence; the number of + signs indicates the strength of fluorescence, from weak (+) to strong ( + + +). The two values presented for iris tissue represent the observation that most but not all of the iris vessels appeared to leak Evans Blue. N indicates the number of animals in which each tissue was examined. The two columns on the right provide a schematic representation of HRP leakage from vessels, as assessed by electron microscopy. were not labelled by anti-GFAP, but were labelled by 4H11 (Fig. 1C), a monoclonal antibody with specificity for Miiller cells. 32 In the normal iris, 4 H l l labelled circumferentially oriented fibrillar structures (Fig. 1D).
Assessment o f barrier properties o f vessels in normal tissues' o f the eye by perfusion o f Evans Blue In tissue fixed by immersion, Evans Blue remaining in the vessels made vascular trees readily visible. Vessels impermeable to the Evans Blue-albumin were sharply outlined, because the tissue between the vessels was free of fluorescence. By contrast, the outlines of permeable vessels appeared blurred by fluorescence in the tissue between vessels. In tissue fixed by perfusion, the perfusate washed Evans Blue from the vessel lumen, leaving impermeable vessels almost invisible. Permeable vessels then stood out, marked by a halo of fluorescence around their circumference and in the surrounding tissue. The present observations of the distribution of Evans Blue fluorescence within the ocular tissues are summarized in Table 1. Retina. We found no evidence that Evans Blue-albumin left the retinal vessels to enter the retinal neuropil. When the retina was immersionfixed the images in Fig. 2A and B were obtained. Evans Blue remaining in the vessels defined the lumen of vessels sharply in both the superficial (A) and deep plexus (B), and the neuropil between vessels lacked fluorescence. When fixative was perfused through the vasculature after the Evans Blue, no fluorescent material was apparent in the retinal vessels, and they were not visible. Ciliary processes. The vessels of the ciliary processes are fenestrated and lack barrier properties (reviewed in Ref. 16); correspondingly, they were seen to leak Evans Blue. In immersion-fixed tissue, for example, the profiles of vessels within the ciliary processes were unclear due to the high level of Evans Blue fluorescence in the surrounding stroma. Similarly, in tissue fixed by perfusion, the outlines of vessels were marked by a bright halo of fluorescence,
along with a high level of fluorescence in the ciliary process stroma (Fig. 2C). In perfused material, however, the vessel lumen was free of fluorescence. Iris. The barrier properties of the vessels of the iris vary between the species (reviewed in Ref. 16), the barrier being, for example, tighter in primates than in the rat. In our rat material, many vessels of the iris appeared to leak Evans Blue. When fixed by immersion, the fluorescent outline of iris vessels appeared less sharp than that of retinal vessels. In tissue fixed by perfusion, the vessel lumen was free of fluorescence but the outline of the vessels was marked by a bright fluorescent halo (Fig. 2D). Fluorescence in the stroma of the iris was weaker than in the ciliary processes, perhaps because the iris is thin and bathed on both sides by fluids able to carry away extravascular Evans Blue. Overall, the tendency to leak Evans Blue-albumin was more variable in vessels of the iris than in those of the ciliary processes
Evans Blue filling o f vessels within cell aggregates Within a week of implantation, aggregates of implanted cells had stabilized on the anterior surface of the iris. During perfusion, Evans Blue was seen to rapidly fill vessels in the iris and aggregates and in some cases to spread from the vessels into the surrounding tissue. These patterns of spread would have provided a simple basis for assessing the permeability of the vessels in the aggregates, but for two slower and initially unanticipated movements of Evans Blue. First, Evans Blue was seen in both normal and experimental eyes to spread from vessels in the iris and ciliary processes into the aqueous humour. This spread began during the perfusion of the dye and continued even after fixation, and tended to reduce the concentration of dye in the stroma of the ciliary processes and iris. Second, Evans Blue in the aqueous humour spread into surrounding tissue, including cell aggregates on the iris. This effect was reduced, but could not be eliminated, by dissecting the irises and attached aggregates from the other tissues of the eye and preparing them for fluorescence microscopy with as little delay as possible.
Miiller cells and the blood-retinal barrier
Assessment of barrierproperties of vessels in aggregates by Evans Blue perfusion Since each agg~gate formed as a raised area on the anterior surface of the iris, the irises were mounted with the anterior surface uppermost. The vessels of the aggregate were often distinct in their shape, forming small, disordered capillary loops, which lacked the radial orientation typical of iris vessels. Evans Blue fluorescence was consistently stronger in the tissue of cell aggregates (astrocyte, Miiller cell and meningeal) than in the surrounding iris tissue. Characteristic differences were noted, however, in the fluorescence associated with vessels within each of the aggregates. Meningeal aggregates. Vessels within meningeal aggregates appeared to be as permeable as the vessels of the iris proper. Evans Blue fluorescence formed a distinct halo around vessels, both as they approached the aggregate and as they spread within the aggregate. Typically, for example in Fig. 3A, a clear boundary could be seen between the limited fluorescence of the iris and the diffuse, brighter fluorescence within the meningeal aggregate.
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Astrocyte and bl~ller cell aggregates. Following immersion fixation, the outlines of vessels within the aggregates of both Mfiller cells and astrocytes appeared sharp. The absence of a fluorcsocat halo around vessels in the aggregates was men clearly ~ following perfusion fixation (Fig. 3B--F). Vessels could be traced from the iris, where the halo made them visible, to the edge of the aggregate, where the halo disappeared, and the vessels could no longer be clearly traced. Vessels appeared to end blindly (arrows); in fact they continued into the aggregate but, like the vessels of the retina, could not be seen using fluorescence optics. This absence of the fluorescent halo around their vessels was a consistent feature of MOiler cell and astrocyte aggregates. Assessment of barrierproperties by horseradishperoxidase perfusion The material perfused with H R P was observed by both light and electron microscopy. In the light microscopy, HRP reaction product appeared outside most vessels of the normal iris (arrows in Fig. 4A), in
Fig. 2. Distribution of Evans Blue in tissue wholemounts. (A, B) Retinal wholemount with Evans Blue-filled vessels, following immersion fixation. A shows the superficial plexus of vessels, B the deep plexus. The sharp outline of the vessels is due to the lack of fluorescence in the retinal neuropil and indicates that Evans Blue has not leaked from these vessels into the retina. (C) Evans Blue in the ciliary processes following perfusion fixation. Vessels within the ciliary processes are empty of fluorescent material and a bright halo of Evans Blue has formed around them and in the tissue between them. The large vessel pictured lies outside the ciliary processes. (D) Evans Blue in a normal iris following perfusion fixation. The vessels are empty of fluorescence, but Evans Blue has passed out of the vessels to form a bright halo around them and in the tissue between them. Scale bar = 200pro (A,B); 100/Jm (C,D).
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Fig. 3. Distribution of Evans Blue in cell aggregates. (A) Vessels extending from the iris (left) into an aggregate of meningeal cells (right) following peffusion fixation. The level of background fluorescence is high in the aggregate. Vessels within the aggregate have a similar fluorescent halo to vessels outside the aggregate. Arrows mark the approximate position of the aggregate border. (B-D) Vessels extending into an aggregate of astrocytes. The aggregate is apparent as the area of diffuse fluorescence. Vessels entering the aggregate cannot be traced within it; the vessels become visible at the point of entry (arrows), because Evans Blue has been washed from their lumen by perfusate, and within the aggregate the vessels lack a fluorescent halo. The point of entry of one vessel into the aggregate in B is shown with greater magnification in D. (E, F) Vessels extending into aggregates of Mfiller oetts~ As with astrocyte aggregates, vessels entering an aggregate cannot be traced within it, but become invisible at the point of entry (arrows). Scale bar = 200 #m (A~:?,E); 100 #m (D,F). some cases spreading well away from the vessel. In the retina, by contrast, there was no evidence of reaction product outside the vessel (Fig. 4B). Figure 4C shows a length of iris from an eye implanted with Miiller cells. When the iris was examined by dissecting microscope before sectioning, a cell aggregate was apparent in the tissue on the left side of Fig. 4C. in
this section, a population of cells with dark nuclei (arrowheads) could be distinguished there. H R P appeared not to have escaped from the vessels on the left, but had clearly leaked from the vessels on the right (arrow), in the region from which cells with dark nuclei are lacking. The vessels at the posterior surface of the iris (bottom) all appeared to have leaked HRP.
MOiler cells and the blood-retinal barrier Figure 4D shows part of an iris from an eye implanted with astrocytes. HRP reaction product was apparent within the vessels, but could not be detected outside the vessels. The walls of vessels from similar tissues, as observed in the electron microscope, are shown in Fig. 5. The low contrast of the tissue, a result of the deliberate omission of block-staining with uranyl acetate, allows ready identification of the electrondense HRP reaction product. In the normal iris (Fig. 5A), HRP reaction product is prominent in the extracellular space around vessels, in apparent yes-
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icles within the cytoplasm of endothelial cells, and in membrane "pits" which form in the abluminal plasma membrane.9'~° In a vessel in the retina, by contrast (Fig. 5B), reaction product was apparent in the lumen (top) but was not apparent external to the vessels, nor in an interendothelial cell junction (which runs between the arrows). Vesicles and membrane pits were not prominent in the endothelial cell. In a vessel from the choroid (Fig. 5C), HRP reaction product was very prominent in the extracellular space around the vessels, and in a wide interendothelial cleft. Membranous pits containing
D Fig. 4. Light microscopy of HRP-pcrfused material. (A) Section of a normal iris, with the anterior surface uppermost. HRP reaction product is evident (arrows) in the space around the larger vessels. (B) Section of retina from an eye peffused with HRP. No HRP reaction product is apparent in the tissue. (C) Section of iris from an eye in which Mftller cells were implanted. The aggregate formed in the anterior tissue of the iris is soon on the left of this panel. HRP reaction product is prominent (arrows) around the vessels at the anterior surface (riOt) and around vessels at the posterior surface 0ower). In contrast, reaction product is not apparoat around the vesselsat the anterior surface (left). These latter vesselsare surrounded by a population of cells with dark nuclei (arrowheads). The scale bar in B also applies to C. (D) Section of iris from an eye in which astrocytes were implanted. HRP was not detected outside the vessels.
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Miiller cells and the blood-retinal barrier reaction product were numerous. In a vessel from an iris implanted with meningeal cells, H R P was prominent in the extracellular space and in vesicles within the endothelial cells (Fig. 5D). In a vessel from an iris implanted with astrocytes (Fig. 5E), H R P reaction product was observed in the lumen of the vessel and in the luminal side of an interendothelial cleft (fight arrow), but not within the cleft or in the extracellular space surrounding the vessel. Similarly, in vessels from an iris implanted with Mfiller cells (Fig. 5F-H), reaction product was apparent in the lumen of the vessels and in the luminal end of interendothelial junctions, but not in the outer part of the cleft or in the perivascular space. In summary, the vessels of the normal choroid and iris appear to allow the movement of HRP out of the vessels, and the vessels of the iris of eyes implanted with meningeal cells appeared particularly permeable. Retinal vessels, and iris vessels associated with implanted astrocytes or Miiller cells, appeared impermeable to HRP. DISCUSSION
Summary of findings The present results provide evidence that Miiller cells share the ability of astrocytes to induce properties of the blood-brain barrier in vascular endothelial cells. Specifically, they confirm a previous finding23 that vessels associated with aggregates of astrocytes on the anterior surface of the iris are impermeable to albumin and HRP, while vessels associated with aggregates of meningeal cells are permeable; they also provide evidence that vessels associated with aggregates of Mfiller cells are similarly impermeable. The principal difficulty of interpretation encountered in this analysis was the level of fluorescence within the cell aggregates after perfusion with Evans Blue. If that level was determined only by the permeability of the vessels of the aggregate, it should be high in the meningeal aggregates and low in the aggregates of Mfiller cells and astrocytes. In fact, considerable background fluorescence was observed in all cell aggregates. We argue that this observation
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does not contradict our principal conclusion, for two reasons. First, it became clear that the level of fluorescence in the aggregates is not determined only by leakage from their vessels, but that Evans Bluebound albumin moves from the cilliary processes and iris into the aqueous humour and thence into the extracellular space of the aggregates. Second, the fluorescence in the aggregates may appear more prominent because the glial cells within the aggregates actively accumulate Evans Blue-albumin from the extracellular space~ and because the aggregates increase the thickness of the iris. We therefore relied on the presence or absence of a halo of fluoresence around perfused vessels to assess their permeability. It was consistently observed (Table 1) that the halo was absent from vessels known from previous work to be impermeable (retina), present in vessels known to be permeable (choroid, ciliary processes, iris, aggregates of meningeal cells) and absent from vessels in the two tissues under test, aggregates of Mfiller cells and astrocytes. Our interpretation of the material perfused with Evans Blue received strong support from the light and electron microscopic observations of the movement of HRP from vessels in normal irises, and in irises from eyes implanted with astrocytes, Miiller cells or meningeal cells (summarized in Table 1). Two recent studies report results at variance with those just presented. Small et al. 3s report that Miiller cells transplanted into the anterior chamber formed aggregates on the ciliary processes rather than the iris, and did not reduce the normally high permeability of ciliary vessels. Stewart and Holash, 41 in a report available to us only in brief form, attempted to establish gratis of astrocytes in the anterior chamber of the rat eye, and concluded that the gratis were not sufficiently pure to test the ability of astrocytes to induce barrier properties.
The plasicity of barrier properties Since Stewart and Wiley42 first provided evidence that the expression of barrier properties by blood vessels is influenced by their environment, evidence has accumulated that astrocytes induce endothelial
Fig. 5. Electron microscopy of HRP-perfused material. In each of A-H, the lumen of the vessel is at the top. (A) Section of the endothelium of a vessel and surrounding tissue from a normal iris. Dark HRP granules are prominent in vesicles and membrane pits in the cytoplasm of the endothelial cell and with the perivascular basement membrane. (B) Section of the endothelium of a retinal vessel. HRP granules are apparent in the lumen of a vessel, but not in an interendothelial junction (arrows) or in surrounding neuropil. (C) Section of the endothelium of a choroidal vessel. HRP is prominent in vesicles and pits in endothelial cells, in the wide cleft between two endothelial cells, and in the basal lamina of the endothelium. (D) Section of the endothelium of a vessel from an iris implanted with meningeal cells. HRP is profuse in intercellular regions around vessels. (E) Section of the endothelium of a vessel in an iris implanted with astrocytes. HRP reaction product is apparent in the lumen and adherent to the luminal surface of the endotlielium, but does not penetrate an interendothelial cell junction (arrowed) or appear outside the lumen. (F-H) Sections of endothelial cells in three vessels from an iris implanted with MiiUer cells. HRP reaction product is apparent in the lumen, adherent to the luminal surface of the endothelium, and at the inner end of interendothelial junctions. It does not penetrate the junctions (arrowed) or appear anywhere outside the lumen of the vessel. Scale bar = 0.4/am (A,D,F-H); 0.6/am (B,C,E). l, lumen of the vessel.
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cells to form barrier properties, specifically to form complex tight junctions with their neighbours, and to express various barrier-specific enzymes and molecular t r a n s p o r t e r s . 3"4A9"2°23'28'39"4°'4445 The dependence of barrier properties of capillaries on their local environment is emphasized by the tissue model employed here. The arteries of the retina, iris and ciliary processes are all branches of the ophthalmic artery and the vessels which become associated with the cell aggregates are branches of the iris vessels. Yet their barrier properties vary from being highly permeable, in the choroid, ciliary processes and meningeal aggregates; to less permeable, in the iris, to tight, in the retina, astrocyte and Mfiller cell aggregates. Studies already referred to 3'4"19"20"23"2s'39"40"44~45 indicate that vessels in central nervous tissue are induced to form barrier properties by their glia limitans. Though we have yet to identify the structural and metabolic specializations which constitute the barrier in the present model, it seems likely that mechanisms identified in earlier studies, such as the formation of complex zonula occludens and the closure of junctional clefts between vascular endothelial cells? ~74"44 are involvedY Implications
Four implications of the present results deserve to be mentioned. Elsewhere 22 we have noted extensive similarities between astrocytes and Mfiller cells in their functional specializations. We add here that these two classes of macroglia share an ability to induce the formation of blood-brain barrier properties in vascular endothelial cells. To our knowledge this study is the first description of the ability of a cell class other than astrocytes to induce blood-brain barrier properties in vessels, although the in ritro induction of barrier-specific enzymes in vascular endothelial cells by cortical neurons 45 has been described.
Second, we note that the mechanism by which macroglial cells induce the formation of barrier properties in endothelial cells is not species- or tissuespecific. In our experiments, and in the work of Janzer and Raft, 23 astrocytes from the cerebral cortex of the rat induced barrier properties in capillaries originating from the iris. Janzer and Raft have also demonstrated that rat cortical astrocytes induce barrier properties in vessels growing fiom the chorioallantoic membrane of the chick, and Tao-Cheng et a l Y have shown that cortical astrocytes from the rat could induce barrier properties in bovine microvascular endothelial cells. We have shown here that Miiller cells from the retina of the rabbit induce barrier properties in vessels in the iris of the rat. Third. we note that the ability w, induce barrier properties is retained by the Mfiller cells of lhc periphery of the rabbit retina, which remains avascular throughout life. 18'34-37"46These cells do not. therefore, normally function to induce barrier properties in vessels. Their ability to induce bar,~cr properties is nevertheless conserved. Finally. we note an important ciimcat implication of the present results: that Mfiller cells should be recognized ;ts a major factor in the formation and maintenance of the blood- retinal barrier. This conclusion may be of clinical relevance, since breaks in that barrier are a feature of several diseases of the retina, including retinopathy of prematurily and major causes of adull blindness and diabetic retinopathy. Acknowledgements --We wish to thank Mrs Z. Dreher lbr her expert assistance with tissue culture and the development of the 4HII monoclonal antibody, and Dr Stephen Robinson for his assistance and suggestions, This work was supported by grants from the National Health and Medical Research Council of Australia, the Government Employees" Medical Research Fund and the Ramaciotti Foundation. T.C.-L is an R. Douglas Wright Fellow of the National Health and Medical Research Council ~l' Australia.
REFERENCES
1. Arthur F. E., Shivers R. R. and Bowman P. D. (1987) Astrocyte-mediated induction of tight junctions m brain capillar~ endothelium: an efficient in vitro model. Derl Brain Res. 36, 155 159. 2. Ashwell K., Holl~inder H., Streit W. and Stone J. (1989) The appearance and distribution of microglia in the developing retina of the rat. Vis. Neurosci. 2, 437 448. 3. Beck D. W., Roberts R. L. and Olson J. J. (1986) Gtial cells influence membrane-associated enzyme activity at the blood-brain barrier. Brain Res. 381, 131 137. 4. Beck D. W., Vinters H. V., Hart M. N. and Cancilla P. A. (1984) Glial cells influence the polarity ot lhe blood-brain barrier. J. Neuropath. exp. Neurol. 43, 219-234. 5. Betz A. L. and Goldstein G. W. (1986) Specialized properties and solute transport in brain capillaries. ,.1. Rot:. t'h n~ird. 48, 241-250. 6. Bj6rklund H., Dahl D., Olson L. and Seiger A. (1984) Glial fibrillary acidic protein-like immunoreactivity in the iris: development, distribution, and reactive changes following transplantation. J. Neurosci. 4, 978-988. 7. Bouchard C., Lebert M. and Dupouey P. (1989) Are close contacts between astrocytes and endothelial cells a prerequisite condition of a blood brain barrier--the rat subfornical organ as an example. Biol. (¥1t 67, I59-165, 8. Bradbury M. W. B. (1985) The blood-brain barrier~ Transport across the cerebral endothelium, Circulatlon Res. 57, 213--222. 9. Brightman M. W. (1989) The anatomical basis of the blood-brain barrier. In Implications 0[' the Blood-Brain Barrier and its Man~ulation (ed. Neuwelt E. A.), Vol. 1, pp. 53 -83. Plenum, New York. 10. Broadwell R. D. (1989) Transcytosis of macromolecules through the blood-brain barrier: a cell biological perspective and critical appraisal, Acta neuropath., Berlin 79, 117- 128. 1I. Biissow H. (1980) The astrocytes in the retina and optic nerve head of mammals: a special glia fo r the ganglion cells axons. Cell Tiss. Res. 21~i, 367 378.
Miiller cells and the blood-retinal barrier
301
12. Cancilla P. A. and DeBault L. E. (1983) Neutral amino acid transport properties of cerebral endothelial cells/n vitro. J. Neuropath. exp. Neurol. 42, 191-199. 13. Chan-Ling T., Halasz P. and Stone J. (1990) Development of retinal vasculature in the cat: processes and mechanisms. Curr. Eye Res. 9, 459-478. 14. Chart-Ling T. and Stone J. (1992) Degeneration of astrocytes in feline retinopathy of prematurity causes failure of blood-retinal barrier. Invest. Ophthal. vis. Sci. 33, 2148-2159. 15. Chan-Ling T., Tout S., Holl/inder H. and Stone J. (1992) Vascular changes and their mechanisms in the feline model of retinopathy of prematurity. Invest. Ophthal. vis. Sci. 33, 2128-2147. 16. Cunha-Vaz J. (1979) The blood-ocular barriers. Surv. Ophthal. 23, 279-296. 17. Cunha-Vaz J. G., Shakib M. and Ashton N. (1966) Studies on the permeability of the blood-retinal barrier. I. On the existence, development, and site of a blood-retinal barrier. Br. J. Ophthal. 50, 441453. 18. Davis F. A. (1929) The anatomy and histology of the eye and orbit of the rabbit. Trans. Am. ophthal. Soc. 27, 401~141. 19. Debault L. E. and Cancilla P. A. (1980)),-Glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells/n vitro. Science 207, 653-655. 20. Dehouck M.-P., M~resse S., Delorme P., Fruchart J.-C. and Cecchelli R. (1990) An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. J. Neurochem. 54, 1798-1801. 21. del Cerro M., Gashi D. M., Rao G. N., Notter M. F., Wiegand S. J., Sathi S. and del Cerro C. (1987) Retinal transplants into the anterior chamber of the rat eye. Neuroscience 21, 707-723. 22. Holl~inder H., Makarov F., Dreher Z., van Driel D., Chart-Ling T. and Stone J. (1991) Structure of the macrogiia of the retina: sharing and division of labour between astrocytes and Miiller cells. J. comp. Neurol. 313, 587-603. 23. Janzer R. C. and Raft M. C. (1987) Astrocytes induce blood-brain properties in endothelial cells. Nature 325, 253-257. 24. Kondo H., Takahashi H. and Takahashi Y. (1984) Immunohistochemical study of S-100 protein in the postnatal development of Miiller cells and astrocytes in the rat retina. Cell Tiss. Res. 238, 503-508. 25. Laitinen L. (1987) Griffania simplicifolia lectin binds specifically to endothelial cells and some epithelial cells in mouse tissue. Histochem J. 19, 225-234. 26. Ling T., Mitrofanis J. and Stone J. (1989) Origin of retinal astrocytes in the rat: evidence of migration from the optic nerve. J. comp. Neurol. 286, 345-352. 27. Ling T. and Stone J. (1988) The development of astrocytes in the cat retina: evidence of migration from the optic nerve. Devl Brain Res. 44, 73-85. 28. Maxwell K., Berliner J. A. and Cancilla P. A. (1987) Induction of ),-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes. Brain Res. 410, 309-314. 29. McCarthy K. D. and de Vellis J. 0980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890-902. 30. Olson L., Seiger A. and Strfmberg I. (1983) Intraocular transplantation in rodents: a detailed account of the procedure and examples of its use in neurobiology with special reference to brain tissue grafting. Adv. cell. Neurobiol. 4, 407-442. 31. Radius R. L. and Anderson D. R. (1980) Distribution of albumin in the normal monkey eye as revealed by Evans Blue fluorescence microscopy. Invest. Ophthal. vis. Sci. 19, 238-243. 32. Robinson S. R. and Dreher Z. (1990) MiiUer cells in adult rabbit retinae: morphology, distribution and implications for function and development. J. comp. Neurol. 292, 178-192. 33. Scherer J. and Schnitzer J. (1989) The rabbit retina: a suitable mammalian tissue for obtaining astroglia-free MfiUer cultures. Neurosci. Letr 97, 51-56. 34. Schnitzer J. (1985) Distribution and immunoreactivity of glia in the retina of the rabbit. J. comp. Neurol. 240, 128-142. 35. Schnitzer J. (1988) Astrocytes in mammalian retina. Prog. Rer Res. 7, 209-231. 36. Schnitzer J. (1988) Astrocytes in the guinea pig, horse and monkey retina: their occurrence coincides with the presence of blood vessels. Glia 1, 74-89. 37. Schnitzer J. and Karschin A. (1986) The shape and distribution of astrocytes in the retina of the adult rabbit. Cell Tiss. Res. 246, 91-102. 38. Small R. K., Watkins B., Munro P. M. and Liu D. (1993) Functional properties of Miiller cells following transplantation into the anterior eye chamber. Glia 7, 158-169. 39. Stewart P. A. and Coomber B. L. (1986) Astrocytes and the blood-brain barrier. In Astrocytes (eds Fedoroff S. and Vernadakis A.), Vol. 1, Development, Morphology, and Regional Specialization o f Astrocytes, pp. 311-328. Academic Press, Orlando, FL. 40. Stewart P. A. and Hayakawa E. M. (1987) Interendothelial junctional changes underlie the developmental 'tightening' of the blood-brain barrier. Devl Brain Res. 32, 271-281. 41. Stewart P. and Holash J. A. (1991) Induction of the blood-brain barrier. Proceedings o f Castle Ringberg Symposium. 42. Stewart P. A. and Wiley M. J. (1981) Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: a study using quail-chick transplantation chimeras. Devl Biol. 84, 183-192. 43. Stone J. and Dreher Z. (1987) Relationship between astrocytes, ganglion cells and vasculature of the retina. J. comp. Neurol. 255, 35-49. 44. Tao-Cheng J.-H., Nagy Z. and Brightman M. W. (1987) Tight junctions of brain endothelium in vitro are enhanced by astrogiia. J. Neurosci. 7, 3293-3299. 45. Tonsch U. and Bauer H.-C. (1991) Glial cells and neurons induce blood-brain barrier related enzymes in cultured cerebral endothelial cells. Brain Res. 539, 247-253. 46. Tout S., Ashwell K. and Stone J. (1988) The development of astrocytes in the albino rabbit retina and their relationship to retinal vasculature. Neurosci. Let. 90, 241-247. 47. Tout S., Dreher Z., Chart-Ling T. and Stone J. (1993) Contact-spacing among astrocytes is independent of neighbouring structures: /n vivo and in vitro evidence. J. comp. Neurol. (in press). 48. Wolman M., Klatzo I., Chui E., Wilmes F., Nishimoto K., Fujiwara K. and Spatz M. (1981) Evaluation of the dye-protein tracers in pathophysiology of the blood-brain barrier. Acta neuropath., Berlin 54, 55-61. (Accepted 2 February 1993)