Transport of colloidal particles from small
blood vessels correlated with cyclic
changes in permeability
George D. Pappas and E. Joan Blanchette
The passage of colloidal particles through the small blood vessels of the uterus and ovary
during periods of increased vascular permeability is primarily an extracellular one. In contrast
to conditions of pronounced inflammation, the intercellular junction of the endothelial cells
does not generally increase or open. These findings suggest that the passage of large molecules from the microcirculation occurs at the •junctions between endothelial cells.
TJLh
mation may greatly exaggerate vascular
changes in comparison with those that occur in response to endogenous agents.
Fluctuations of vascular permeability
under normal physiological conditions in
response to a variety of influences are well
established in the female reproductive system. Ovarian and uterine vessels are influenced by changes in the hormonal balance. In the ovary, the permeability of the
blood-liquor barrier of the Graafian follicle
has been studied by Zachariae0 and Burr
and Davies.7 They concluded that the preovulatory swelling of the follicle is a result,
at least in part, of an increased permeability of the vessels and a resulting transudate from the blood.
Periodic uterine edema was demonstrated by Astwoods in the normal estrus
cycle of the mature rat. It has been postulated that the imbibition of water results
from vasodilation and from changes in the
permeability of the blood vessels.0 Accumulation of fluid within the uterine lumen
is an effect which occurs in response to
endogenous estrogen. Spaziani and Szego10
suggested that estrogens induce the release
.he passage of marker particles through
the endothelial wall of small blood vessels
has been reported to occur via pinocytotic
vesicles1 or intracellular caveolae.2 On the
other hand, experiments which increase
the permeability of blood vessels by local
injection of histamine or serotonin3 show
that marker particles pass through the intercellular space between endothelial cells.
This junction or gap enlarges or opens
during locally induced inflammation.3'4
Systemic injection of endotoxin, causing
the development of a cloudy aqueous humor, produces similar changes in the blood
vessels of the ciliary body5 (see Fig. 1).
However, these types of induced inflam-
From the Departments of Anatomy and Obstetrics
and Cynecology, Columbia University College
of Physicians and Surgeons, New York, N. Y.
This investigation was supported in part by
Grants NB-02314-06 and NB-03448-04 from
the National Institutes of Neurological Diseases
and Blindness of the National Institutes of
Health, United States Public Health Service,
and from The Life Insurance Medical Research
Fund.
1026
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Transport of colloidal particles
1027
Fig. 1. Electron micrograph of a portion of a venous capillary of the ciliary body of an
albino rabbit treated with endotoxin so that a cloudy aqueous humor was present in the
anterior chamber of the eye. Thirty minutes before fixation, saccharated iron oxide was injected intravenously. While no particles remain in the lumen, there is a large accumulation
in the enlarged intercellular space between overlapping endothelial cells. (x42,000.)
of histamine in the uterus, which then alters the permeability.
Ovarian and uterine blood vessels of the
rabbit and rat were studied at various
stages of ovulation and the estrus cycle.31
Blood vessels of the preovulatory follicle
were examined with the electron microscope to determine: (a) if fine structural
changes can be associated with increased
permeability, and (b) the mode of transport of marker particles through the vessels.
Blood vessels just beneath the uterine
epithelium were studied in respect to
changes that take place during the estrus
cycle. Marker particles (thorium dioxide)
were used to elucidate the morphological
changes associated with the increased permeability of these vessels.
Rabbit ovarian tissue and rat uterine
tissue were fixed for two hours in 2 per
cent buffered OsO,t 15 to 30 minutes after
the injection of colloidal marker (ThCX,)
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into the marginal ear vein of the rabbit
and into the rat femoral vein. The tissue
was embedded in Epon 812.
Transport of particles during the
preovulatory period across the capillary
walls of the ovarian Graafian follicle
The capillaries of the theca interna of
the adult estrus rabbit are continuous without fenestrae or pores. Junctions between
endothelial cells maintain close contact
with each other. The endothelium is not
greatly attenuated and the peri nuclear area
usually bulges into the lumen. Numerous
vesicles are present throughout the cytoplasm of the endothelial cells. Thirty minutes after the injection of colloidal particles, only few particles are present in the
subendothelial area.
The vessels of the theca interna are
much more permeable to marker particles
in the rabbit after mating. During the preovulatory period 9 hours after mating or
Investigative Ophthalmology
December 1965
1028 Pappas and Blanchette
approximately 2 hours before follicle rupture and 30 minutes after the injection of
ThCX, particles are found immediately beneath the endothelial basement membrane
in the area of the junction between endothelial cells (Fig. 2). In addition, a few
particles can sometimes be found in membrane-bounded vesicles or vacuoles close
to the area of the endothelial junctions, indicating passage from the lumen. Particles
are also found at a distance from the capillary lumen and have been observed within
the follicle basement membrane, 30 minutes after injection. There are no differences noted in the fine structure of the
blood vessels of the theca interna of the
unmated or mated (Fig. 2) rabbits. The
passage of marker particles in the area of
the endothelial junctions is the distinguishing feature of the preovulatory follicles.
Passage of particles across the walls of
uterine blood vessels during the estrus
cycle
The subepithelial capillary plexus and
capillary baskets surrounding the endometrial glands of the uterus of the rat were
studied. The estrus stages were determined
by the Long-Evans method of vaginal
smears. Passage of marker particles from
the capillaries can be correlated with
stages of uterine endometrial proliferation
and with the known ovarian physiology
during these periods. From late diestrus to
*- v
V
BM
F i g .
2.
E l e c t r o n
m i c r o g i . ^ . n
n ia i.ijullaiA
w i t h i n
iii<-
im-v.i
liiii-m.i
\ i i >
.-.iiiiwiiiivling
a
m a t u r e
Graafian follicle. The rabbit was mated 9 hours before the intravenous injection of Thorotrast. Particles of ThO3 are found in the lumen (L), in a membrane-bounded vacuole (V)
in the endothelial cells and in the basement membrane area (BM), 30 minutes after injection.
Note that the subcndothclial accumulation of marker is present characteristically at the base
of the endothelial junction, suggesting transport at this site. (x29,000.)
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Transport of colloidal particles
proestrus, the blood vessels enlarge and a
dense plexiform arrangement of the subendothelial capillaries becomes prominent.12 During this period the uterine lumen accumulates fluid, resulting in a maximum distention in early estrus. Concomitantly the ovary is undergoing a preovulatory spurt of growth of the follicles,
resulting in estrogen secretion.
During diestrus, the passage of marker
particles from the lumen to the surrounding tissue is minimal (Fig. 3). However,
evidence exists for particles, during late
diestrus, to be in transit through the endothelial layers (Figs. 4 and 5). Marker is
found in the intercellular space between
adjacent cells, which is not increased in
1029
width (Figs. 4 and 5). Specialized junctional complexes, identified by the dense
cytoplasmic material at localized sites
along the apposing plasma membranes,
are devoid of particles. Marker is abundant, however, in the intercellular spaces
on both sides of the complexes (see Fig.
4). In addition, particles are present in the
cytoplasm in membrane-bounded vesicles
and vacuoles in the vicinity of these cell
junctions. The possibility exists that material is transferred by pinocytosis, thereby
bypassing the sites of the junctional complexes where the extracellular space may
be obliterated. On the other hand, during
periods of increased permeability the junctional complexes may be discontinuous,
Text continued on p. .1035.
BM
3
Fig. 3. Subepithelial capillary from the rat enclometrium during early diestrus. Marker particles
(ThCX) accumulate in the lumen but are not generally found in subendothelial areas at this
stage. BM=basement membrane. (x.10,000.)
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Investigative Ophthalmology
December 1965
1030 Pappas and Blanchette
^O ( *
'. \
V
r
••}
A
'••
•BM
\
•
Fig. 4. l.uigcnlial bcitiou i)t .i purliun ut a \unuus cupillary at tin.' junction of two cnclothclial
cells during late diestrus, 30 minutes after the injection of ThO ; . Particles in transit from
the lumen (L) to the basement membrane (BM) can be found in the intercellular space (IS)
on both the luminal and basal sides of a junctioiial complex (]C). In addition, vacuoles (V)
containing particles are found in the endothelial cells characteristically close to the junction.
(x23,000.)
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Transport of colloidal particles 1031
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Number 6
N
vc
sr JC
Fi^.
•">• A i l j . u - i l i t e n d o t h e l i a l
cells
from
a vessel
during
Lite
tliesims.
H) m i n u t e s
.liter t h e
injection of TI1O2. Particles (P) are present within the intercellular space. Junetional complexes (JC) at sites along apposing plasma membranes are devoid of particles. A vacuole
(V) close to the junction as well as deep caveolae at the luminal surface contain particles.
L, lumen, C, collagen fiber, N, nucleus of endothelial cell. (x40,000.)
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FT"
1032 Fappas and Blanchette
Investigatioe Ophthalmology
December 1965
N
15
I
6
Fig. 6. Portion ot a subepitlielial capillar\ Ironi tlie eiKloinetriuni (il a proestru.s rat. Thirtv
minutes after the injection of marker, particles are found in the collapsed lumen (L) as well
as along the irregular intercellular space (IS) indicating an extracellular transit of particles
from the vessel. N, nucleus of endothelial cell. (*32,500.)
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permitting a completely extracellular pathway through the vessel wall (see Fig. 6 of
late proestrus stage).
Continuing from advanced or late proestrus, as tissue edema increases to a maximum at midestrus, particles can be readily
found in transit in the vessel wall (see
Figs. 6, 7, and 8). At this time only occasionally an enlarged intercellular space
may be found containing marker (Fig. 7).
Particles are also found not only at the
intercellular space but also in vesicles and
vacuoles at sites close along the margins
of adjacent cells (Fig. 8).
In contrast, markers were infrequently
observed in transit or in the subendothelial
areas during late estrus (Fig. 9). This finding is well correlated with the fact that a
decreased edematous condition produces a
less distended uterus and a reduction of
fluid flow from the blood vessels in this
postovulatory period.
Conclusion
In the small blood vessels of the theca
intern a of the preovulatory vesicular follicle and during periods of uterine hyperemia, the passage of marker particles from
the vascular lumen occurs primarily at intercellular sites. The width of the intercellular space of the endothelial junctions
is not generally increased during periods
of tissue edema. The tight intercellular
junctions13-ll are usually present and no
markers are seen at these sites. However,
particles are present at both the luminal
and basal sides of the tight junctions.
Transport of colloidal particles
The extracellular passage of particles
can be accounted for by two pathways.
One pathway, an exclusively extracellular
one, would presuppose a discontinuous
tight junction. The zonula occludens™
may in fact not be continuous across the
entire zone of endothelial cell apposition,
especially at times of increased vascular
permeability. The term macula occludens
would more appropriately describe a discontinuous fusion of apposing endothelial
plasma membranes.
Since some particles are found in intracytoplasmic vacuoles and vesicles in the
area of the junctional complex, a combined
intra- and extracellular pathway may be
involved. This suggests that particles may
bypass this area by the process of pinocytosis as reported by Kaye and Pappas15 in
the corneal endothelium (mesothelium).
It has been well established that under
induced conditions of pronounced inflammation, marker particles, as well as leukocytes, migrate from the lumen through the
enlarged junctions between endothelial
cells.3"5'16 The present studies show that
in the absence of pronounced inflammation
and diapedesis but under conditions of increased vascular permeability, a primarily
intercellular passage of marker particles
also takes place. These findings support
the earlier views of Chambers and Zweifach17 that the passage of large molecules
from the lumen to the perivascular spaces
takes place by way of the junctions between endothelial cells.
Fig. 8. Tangential section of a portion of a small blood vessel from the rat endometrium during late proestrus, 30 minutes after the injection of ThO«. Particles are present in transit toward the subendothelial area in the intercellular space (IS) as well as in vacuoles (V) in
endothelial cells especially closely adjacent to the areas of the junctional complexes (JC).
(x25,000.)
Fig. 9. Electron micrograph of a portion of a small blood vessel from the rat endometrium
during late estrus. During this period, while marker particles (30 minutes after injection) are
present in the lumen (L) they are not generally found in the subendothelial space or in transit
in the endothelial wall. JC, endothelial junctional complex. (x24,000.)
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1035
1036 Pappas and Blanchette
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Investigative Ophthalmology
December 1965
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