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Ultrastructural studies of bovine retinal microvascular basement membranes with the cationic dye ruthenium red.

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THE ANATOMICAL RECORD 219:363-368 (1987)
Ultrastructural Studies of Bovine Retinal
Microvascular Basement Membranes With the
Cationic Dye Ruthenium Red
Department of Neurology, School of Medicine, Case Western Reserve University,
Cleveland, Ohio 44106-5000
Our recent observation that the basement membranes of brain
microvessels do not stain with the cationic dye ruthenium red has raised the
question of whether the basement membranes of this and other vascular beds
functioning as barriers between blood and neural tissues are deficient in the polyanionic macromolecules, such as glycosaminoglycans, which are responsible for the
ruthenium red staining of other vascular basement membranes. We therefore attempted to produce staining in the only barrier-type microvascular basement membrane known to contain heparan sulfate. Bovine retinas were fixed by immersion in
aldehyde fixatives containing ruthenium red, buffered with either 10 mM or 100
mM sodium cacodylate. We found discrete, electron-dense deposits of ruthenium red
in vascular basement membranes, quite similar to those seen in vascular basement
membranes of nonneural tissues after exposure to ruthenium red. These deposits
were more distinct and more frequent in tissue exposed to ruthenium red-aldehyde
solutions buffered with 10 mM cacodylate. They were not seen if ruthenium red was
omitted from the fixative. The results demonstrate that anionic macromolecules in
basement membranes of barrier-type microvessels can be stained with cationic dyes,
and suggest that the failure of brain microvessels to stain with ruthenium red may
be the result of a relative or total lack of polyanion in this basement membrane, or
of other unique properties.
After exposure to fixatives containing ruthenium red
(RR), the basement membranes (BM) of capillaries exhibit regularly spaced, discrete, electron-dense deposits
of this cationic dye, which binds electrostatically to anionic macromolecules (for reviews, see Charonis and
Wissig, 1983; Schmidley and Wissig, 1986). Biochemical
and immunohistochemical studies further suggest that
in some vascular BM these sites of RR accumulation
represent the glycosaminoglycan (GAG) chains of proteoglycans (PG) (Charonis et al., 1983; Farquhar, 1981;
Kanwar and Farquar, 197913; Simionescu et al., 1984).
The BM of the continuous capillaries forming the
blood-brain barrier CBBB) is the only vascular BM studied thus far that does not exhibit such discrete electrondense deposits following fixation in RR, either in situ
(following vascular perfusion) or in vitro (following isolation from cerebral cortex) (Schmidley and Wissig, 1986;
Schmidley, in press). The restrictive properties of the
BBB are probably not responsible for this lack of staining, because attempts to circumvent the BBB by including detergents or dimethylsulfoxide in the perfusion
solutions (Schmidley and Wissig, 1986), and by immersing thin slices of cerebral cortex (Schmidley and Wissig,
1986) or isolated cerebral microvessels (Schmidley, in
press) in RR-aldehyde solutions, did not produce discrete deposits of RR in the blood-brain barrier BM. The
lack of discrete staining in the BM of isolated cerebral
0 1987 ALAN R. LISS, INC.
microvessels cannot be attributed to loss of PGs during
isolation, since anionic macromolecules in other BMs
survive disruptive isolation procedures (Kanwar and
Farquhar, 1979a,b). These findings contrast sharply with
those in other vascular and nonvascular BM (including
the endothelial and epithelia1 BM of the choroid plexus,
and circumventricular organs, regions of the CNS with
fenestrated capillaries, lacking a BBB), which are
readily stained with RR (Charonis and Wissig, 1983;
Thurauf et al., 1983; Schmidley and Wissig, 1986). Possible explanations for the failure to demonstrate discrete
sites of RR deposition in blood-brain barrier BM might
include 1)a relative or absolute absence of polyanionic
macromolecules, such a s the GAG component of PGs, or
2) a difference in the structure of this BM that would
render the GAG molecules inaccessible to RR or would
prevent them from collapsing, after dehydration, to form
aggregates of sufficient electron density to be detectable
in thin sections (Hascall, 1980). Against explanation (1)
is the immunohistochemical evidence for a HSPG core
protein in blood-brain barrier BM of the rat (Laurie et
al., 1983)and the mouse (Schmidley and Wissig, unpublished observations).
Retina1 capillaries, like brain capillaries, are interposed between the circulation and a neuronal tissue
Received February 19, 1987; accepted June 22, 1987
requiring a precisely regulated extracellular environment for optimal function. They have structural and
functional characteristics very similar to, if not identical
with, those of BBB capillaries, which enable them to
serve a similar barrier function (Raviola, 1977). The BM
of microvessels in the bovine retina is the only “barrier
type” microvessel BM that has been shown, by direct
biochemical analysis, to contain GAG (heparan sulfate)
(Kennedy et al., 1986; Cohen and Ciborowski, 1981). It
thus might reasonably be expected to show, after exposure to fixatives containing RR, discrete electron-dense
accumulations of this cationic dye similar to those found
in the BM of nonneural capillaries. A failure to produce
this discrete pattern of staining in a BM known to contain polyanionic macromolecules would support the idea
that the architecture of the BM of barrier vessels differs
from that of nonbarrier vessels, and that this different
structure, rather than a n absence of polyanionic macromolecules, is responsible for the failure of staining with
RR (i.e., explanation 2 above). On the other hand, demonstration of discrete RR deposits in BM of retina1 capillaries would suggest that our failure to produce similar
staining in rat BBB capillary BM is the result of a
relative or total deficiency of polyanionic macromolecules in this BM.
Fig. 1. Deposits of RR in the interphotoreceptor matrix. In this and
all subsequent figures, except Figure 6, the retinas were fixed and
Cow eyes were obtained from a local slaughterhouse processed
according to the high-ionic-strength protocol. 63,000 x .
and transported on ice. Within 45 min, the retinas were
removed, minced with a razor blade, and immersed in
fixative overnight. Two fixatives were used: The first,
referred to as high-ionic-strength fixative, consisted of
2% glutaraldehyde (GA), 1% paraformaldehyde (PF),in iments, because it is possible that the CEC for RR stain0.1 M Na-cacodylate buffer, pH 7.4, with 0.2% RR. The ing of anionic sites in bovine microvessel BM is
low-ionic-strength fixative consisted of the same concen- approximately the same as that of buffers customarily
trations of GA, PF, and RR, but was buffered with 0.01 used for electron microscopy (that is, approximately 0.1
M cacodylate, and contained 6.8% sucrose. The following M Na?.
day, the tissues were washed in solutions containing
6.8% sucrose and 0.1% RR, buffered with either 0.15 M
or 0.015 M cacodylate for 1 hr, then postfixed in 1%
Os04, 0.05% RR, buffered with either 0.1 M, or 0.01 M
Because of the delay before fixation and the trauma
cacodylate. The tissues were dehydrated in ethanol and
embedded in Spurr. Additional fragments of retina were caused by mincing the tissue while it is still fresh, the
immersed in high-ionic-strength fixative, but without ultrastructural appearance of the retinas was not as
RR. These tissues were subsequently processed in solu- good as that of either freshly immersion-fixed retina or
tions of high ionic strength that did not contain RR. perfused tissue. There was myelin figure formation, and
Ultrathin sections were stained with lead citrate and/or some swelling of cellular processes. Preservation was
ethanolic uranyl acetate and examined with a Zeiss EM more than adequate, however, for identification and ultrastructural study of capillaries and the components of
109 electron microscope.
According to Scott and Dorling (1965), the staining of their walls.
The internal limiting membranes ( E M ) and interphoanionic micromolecules by cationic dyes can be reversibly inhibited by increasing concentrations of inorganic toreceptor matrices (IPM) of retinas fixed and processed
cations. The concentration of cation at which this occurs according to both the high-ionic-strength and the lowis termed the critical electrolyte concentration (CEC). ionic-strength protocols were diffusely electron-dense,
Ultrastructural studies by Charonis and Wissig (1983) without any suggestion of discrete or punctate deposihave recently shown the CEC to be in the range of 0.5- tion of the RR (Fig. 1). These findings are in accord with
1.3 M for capillary BM in nonneural organs. Although I previous observations on the ILM (Matsusaka, 19711,
have recently demonstrated (Schmidley, in press) that and with the presence of GAGS in the IPM (Hewitt,
BM of isolated rat cerebral cortical microvessels do not 1986).
exhibit RR-staining sites even after immersion in RRThe BMs of capillaries, small arterioles, and veins in
aldehyde solutions containing only 0.01 M Na+, the low- retinas fixed and processed in solutions of low ionic
ionic-strength fixative protocol was used in these exper- strength exhibited discrete, electron-dense sites along
Figs. 2, 3. Deposits of RR in the BM of retina1 capillaries. A few of these are indicated by
arrows. In this and subsequent figures, N=nucleus, P=pericyte, E=endothelial cell, L=lumen.
Fig. 4.Where the BM splits to enclose a pericyte, RR-staining sites (some of which are marked
by arrows) are seen along the endothelial and retinal surfaces of the BM, as well as in the BM
on both sides of the pericyte. 61,000 X .
their inner and outer surfaces; as in other BMs, these
sites were 10-20 nm in diameter and arranged in a more
or less regular fashion at intervals of 20-70 nm (Figs. 2,
3) (Charonis and Wissig, 1983). Where the BM split to
enclose a pericyte or smooth muscle cell, sites could be
seen along both surfaces of the enclosed cell (Fig. 4).
Where the BM was sectioned tangentially, the electrondense sites could be seen in a more or less regular array
(Fig. 5). The sites were seen much less frequently, and
when seen were less distinct, in BM of capillaries of
retinas fixed and processed in solutions of high ionic
strength (not illustrated). Electron-dense sites were
never observed in BM of retinal microvessels fixed and
processed in solutions not containing RR (Fig. 6).
The results suggest that anionic macromolecules in
the BM of “barrier-type” microvessels can be stained in
a discrete fashion with the cationic dye RR. The sites of
RR deposition in BM are thought to represent accumulation of this cationic molecule bound to the polyanionic
GAG chains of PG molecules (Farquhar, 1981).Heparan
sulfate is a known constituent of bovine retinal microvessel BM (Cohen and Ciborowski, 1981; Kennedy et al.,
1986), and it is very likely responsible for RR accumulation. However, in vitro RR staining is not specific for
GAGs (Luft, 1971),and therefore it cannot be considered
as proven that the RR deposits observed in these experiments represent individual PG molecules. The pattern
of staining is very similar to that seen in the BM of
nonneural capillaries when they are fixed in aldehydes
containing RR (see, for example, Charonis and Wissig,
1983). The only difference between retinal and nonneural capillaries seems to be that in the former, staining is more frequent and more distinct after exposure to
fixatives of low ionic strength, suggesting that the molecule(s) stained are relatively less anionic than those of
other BM. If the stained molecules were GAGs, for example, they might be less sulfated than GAGs in other
BMs. The latter hypothesis is testable by direct biochemical analysis (Robinson et al., 1984).
With regard to the original observations that stimulated these experiments (Schmidley and Wissig, 1986),
the findings suggest that the failure t o produce discrete
RR-staining sites in the BBB of rat brain microvessels
is not the result of a unique or universal ultrastructural
characteristic of the BM of microvessels forming interfaces between blood and neural tissue, which would
Fig. 6. Bovine retina fixed in aldehydes, without RR. No electrondense sites are seen in the capillary BM. 62,000X .
Fig. 5. Tangential section of BM. RR staining sites are arranged in
a quasi-regular array. 61,000 X .
possible that, the immunohistochemical studies (Laurie
et al., 1983) notwithstanding, PG molecules, or at least
their GAG components, are absent from rat BBB capillary BM. Alternatively, the GAGS could be undersulprevent the formation of discrete, electron-dense depos- fated (Robinson et al., 1984). This possibility is also
its of RR. Of course, it is still possible that the structures testable by direct biochemical analyses of BM, which
of the two “barrier-type” BM are different, such that the are underway in our laboratory.
structure of the BM of BBB capillaries in the rat proACKNOWLEDGMENTS
hibits, while that of the BM of bovine retina1 capillaries
permits, the formation of discrete RR deposits in associIt is a pleasure to thank Patricia Blue for excellent
ation with GAG molecules or other polyanions. It is also technical assistance, and Sandy D’Amico for help in
preparing the manuscript. This study was supported by
grants NS-22524 and HL-35617 from the U S . Public
Health Service, and by Clinician-Scientist Award 81428 from the American Heart Association.
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