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582 Reports
in volume, excision of the control eye (untouched)
would allow gravimetric estimation of mean
radius in the experimental eye to 0.1 mm. Other
linear dimensional parameters also could be estimated with an accuracy acceptable for most purposes by this rapid and simple method.
From The Wilmer Institute, W. K. Kellogg
Foundation Laboratories, The Johns Hopkins University School of Medicine, Baltimore, Md. This
investigation was conducted under a Post-Doctoral Research Fellowship supported by a Bob
Hope-Fight For Sight Award of the National
Council to Combat Blindness, Inc., New York,
N. Y. Submitted for publication Nov. 30, 1976.
Reprint requests: Dr. Roger C. Wales, 156 Glen
Castle Road, Kingston, Ontario, Canada K7M
4N6. ^Present address: Department of Ophthalmology, Queen's University, Kingston, Ontario,
Canada K7L 3N6.
Key words: ocular rigidity, surface area, gravimetric measurement, Archimedes' principle.
REFERENCES
1. Kearns, J. P.: Elastic modulus data on rabbit
eye tissue from vibration tests, Report No.
MCS-0-042, Applied Physics Laboratory, The
Johns Hopikins University, 1966.
2. Green, K., and House, C. R.: Ion and water
movement across isolated intestine of a marine
teleost, Cottus scorpius, J. Exp. Biol. 42:177,
1965.
3. Land, R. E.: Effects of non-absorbable intrascleral sutures on the growing albino rabbit
eye, Am. J. Ophthalmol. 43:611, 1957.
Gap junctions between optic nerve head
astrocytes. HARRY A. QUIGLEY.
Astrocytes of the primate and human optic nerve
head are joined to each other by the gap junction type of intercellular membrane specialization.
Although the precise function of these contacts
is not fully determined, they may serve such
diverse roles as adhesive bonding and intercellular
electrical and chemical coupling.
Many types of cells are interconnected through
specializations of their plasma membranes. Farquhar and Palade1 described three types of cell
junctions: (1) the desmosome (macula adherens),
(2) the zonula adherens, and (3) the zonula
occludens. The three kinds of contacts are found
in series in many epithelia and are collectively
called the terminal bar. The first two types of
contact have been assumed to serve as microscopic welds, mechanically cementing a cell to
its neighbor. The zonula occludens, or tight junction, seemed to represent a fusion of the outer
leaflets of adjoining cell membranes, acting as
a barrier to the passage of molecules between
the adjacent cells which are bonded together.
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Invest. Ophthalmol. Visual Sci.
June 1977
Such tight junction between capillaries in the
central nervous system,2 including the retina and
optic nerve head, are the ultrastructural basis
of the blood-brain barrier.
Although some tight junctions indeed act as
if the intercellular space is obliterated completely
within them, recent observations have shown that
others, which are similar in appearance, have a
minute gap between the outer membrane leaflets,3
allowing the passage of material between cells.
Moreover, these gap junctions are found intermittently along adjoining cell borders rather than
forming complete encircling bands as do true
tight junctions. In the brain, the gap junction is
the only specialized contact between astrocytes.4
In the optic nerve head of primate and human
eyes, I have found frequent gap junctions between
astrocytes. This report describes the location, the
morphology, and the possible roles of these astrocyte gap junctions.
Methods. The optic nerve heads examined were
from 26 owl monkeys (Aotes trivirgatus), 14 squirrel monkeys (Saimiri sciurea), and 26 human eyes
obtained 2 to 24 hr. post-mortem. Among the
human eyes, 13 were from premature infants,
10 from normal adults, and three from persons
known to have open-angle glaucoma. All eyes
were part of other experimental and histopathological studies.
Monkey eyes were primarily fixed by retrograde perfusion through the aorta of either 4%
paraformaldehyde or 5% glutaraldehyde buffered
with phosphate. Human eyes were fixed by immersion in 5% glutaraldehyde with division into
small optic disk segments in fixative. All tissues
were post-fixed in 2% osmium tetroxide, dehydrated in alcohols, and embedded in epoxy resin.
Some specimens of both human and monkey
disks were stained in block with a saturated
solution of uranium in 70% ethanol for 1 hr.
during dehydration to facilitate the identification
of gap junctions.3' ' Thin sections of this material
were not stained further. Thin sections of specimens which were not treated with uranium in
block were stained with uranyl acetate and lead
citrate for 10 min. each. Observations were made
with an electron microscope (Model 7B; JEOL
USA, Electron Optics, Medford, Mass.)
Results. Despite the variety of fixation techniques, gap junctions between optic nerve head
astrocytes have a similar ultrastructural appearance in primates and in human fetal and adult
tissues. With uranium and lead staining of thin
sections, the junctions have a pentalaminar structure composed of three dense lines separated by
two light bands (Fig. 1). When tissues are
stained wirh uranium in block, a gap can be seen
in the central dense line (Fig. 2). The gap diameter varies from 2 to 6 nm., as compared to the
normal intermembrane distance of at least 10 nm.
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Reports 583
Fig. 1. Prelaminar optic nerve head of a squirrel monkey eye with experimental optic atrophy,
6 months after orbital optic nerve transection. An astrocyte cell body (G) is included in the
field. All the cell processes belong to astrocytes, since all axons have degenerated, highlighting the large number of gap junctions between astrocytes (arrows). A capillary (C) seen
at upper left has a tight junction between endothelial cell membranes (white arrow). Inset,
High-power view of gap junction between astrocytes, illustrating pentalaminar appearance
after uranyl acetate and lead citrate staining of thin section. (4% paraformaldehyde and 2%
osmium fixation; x21,000 and inset xl57,000.)
in areas of noncontact. In some sections, the gap
is bridged by regularly occurring fusions of the
outer membrane leaflets. The gap junctions vary
considerably in length, from tiny contacts to segments up to 1 fi in length. There is sometimes an
amorphous densification of the cell cytoplasm adjoining the gap junction.
The astrocytes which physically separate axons
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from contact with capillary basement membranes5
have frequent gap junctions (Fig. 3). However,
gap junctions are not found between all astrocyte
processes which line capillaries. Because of the
extensive arborization of nerve head astrocytes, it
is difficult to determine the frequency of occurrence of gap junctions under normal circumstances. In eyes with experimental descending
584
Reports
Invest. Ophthalmol. Visual Sci.
June 1977
A
Fig. 2. Gap junctions between disk astrocytes with 5-layered structure after uranium and lead
grid staining (A) and 7-layered structure with gap between outer membrane leaflets after
uranium in block staining (B to D). In D, intermittent fusions of the outer membrane leaflets
are suggested (circled). A, Human; B to D, squirrel monkey. (5% glutaraldehyde and 2%
osmium fixation; A xl47,000, B xl80,000, C x290,000, and D x370,000.)
Fig. 3. Nerve head astrocytes (G) separate axons (A) from the basement membrane of a
capillary (C). The astrocyte processes in this area are joined by a gap junction (arrow).
(Squirrel monkey.) (5% glutaraldehyde and 2% osmium fixation; uranyl acetate and lead
citrate grid staining; x63,000.)
axonal degeneration, only astrocytes and capillaries
remain in the nerve head. Under these conditions,
a majority of the astrocyte processes in a given
section have at least one gap junction along their
course (Fig. 1).
No intercellular junctions are seen between
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axons or between astrocytes and axons in the
nerve head. No typical desmosomes or zonulae
adherentes are found between astrocytes.
Discussion. It is not surprising that optic nerve
head astrocytes are joined by gap junctions, since
astrocytes in the brain are connected exclusively
Volume 16
Number 6
by similar structures.4 The functions of gap junctions have been discussed extensively." First, it
seems likely that they hold adjacent cells together.
Even under severe osmotic forces, gap junctions
remain intact,1 suggesting that they have considerable adhesive strength. In the nerve head,
this would facilitate the maintenance of an astrocytic framework through which axons pass.
Second, gap junctions have been found in cells
which exhibit the property of electrical and
metabolic coupling.11 In electrical coupling, a current applied to one cell spreads to its coupled
neighboring cell to a greater degree than would
be expected from their physical closeness. Ions
seem to pass from one cell to the next via lowresistance pathways. Since all cells with electrical
coupling possess gap junctions, it is assumed that
the intermittent membrane fusions within gap
junctions contain small pathways between the
cytoplasm of adjoining cells. In addition to small
ions, larger molecules such as amino acids and
fluorescein have been shown to spread rapidly
from one cell to another when the cells are joined
by gap junctions.'1 Thus gap junctions allow disk
astrocytes to act as an electrical and chemical
syncytium. It has been noticed that when :1Hproline is injected intravitreally, astrocytes are
sequentially labeled in a wave which passes from
the superficial disk to the retrobulbar nerve.7 It
is possible that the proline may be moving from
one cell to another by means of the passages in
gap junctions.
Third, the extracellular movement of fluid and
molecules in the disk area is influenced by the
cell junctions between astrocytes. Gap junctions
do not form complete encircling bands around
cells, and extracellular material may pass through
them. Where the optic nerve adjoins the choroid
and sclera (the border tissue of Elschnig), the
lining astrocytes have been shown to permit the
entry of extracellular protein into the nerve head.s
This physiological leak in the blood-brain barrier
is likely the cause of fluorescein leakage into the
disk during angiography. It is important to distinguish these astrocytes from the cells which
join the retinal pigment epithelium to the retina
at the nerve head (the border tissue of Kuhnt).
In the latter area, the cells are not typical astrocytes. They resemble pigment epithelial cells by
having full terminal bar junctions and they prevent
molecular passage from the optic nerve extracellular space to the subretinal space.8- n At the
surface of the nerve head, the lack of occluding
junctions between astrocytes allows passage of
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Reports 585
molecules such as lanthanum from the vitreous
compartment into the extracellular space of the
nerve head.0 The physiological significance of
molecular and fluid movement between the vitreous and choroid via the extracellular space of
the optic nerve head deserves future experimental
study.
From the Department of Ophthalmology (Bascom Palmer Eye Institute), University of Miami
School of Medicine, Miami, Fla. This investigation
was supported in part by United States Public
Health Service Research Fellowship EY 05042,
awarded by the National Eye Institute, Bethesda,
Md. Submitted for publication Dec. 21, 1976.
Reprint requests: Glaucoma Service, Wilmer Institute, Johns Hopkins Hospital, Baltimore, Md.
21205.
Key words: optic nerve head, astrocyte, gap junction, intercellular junctional complex, optic nerve.
REFERENCES
1. Farquhar, M. G., and Palade, G. E.: Junctional complexes in various epithelia, J. Cell
Biol. 17:375, 1963.
2. Reese, T. S., and Karnovsky, M. J.: Fine
structural localization of a blood-brain barrier
to exogenous peroxidase, J. Cell Biol. 34:207,
1967.
3. Revel, J. P., and Karnovsky, M. J.: Hexagonal
array of subunits in intercellular junctions of
the mouse heart and liver, J. Cell Biol. 33:C7,
1967.
4. Brightman, M. W., and Reese, T. S.: Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol.
40:648, 1969.
5. Anderson, D. R., Hoyt, W. F., and Hogan,
M. J.: The fine structure of the astroglia in the
human optic nerve and optic nerve head,
Trans. Am. Ophthalmol. Soc. 65:275, 1967.
6. Bennet, M. V. L.: Function of electrotonic
junctions in embryonic and adult tissues, Fed.
Proc. 32:65, 1973.
7. Minckler, D. S., and T'so, M. O. M.: A light
microscopic, autoradiographic study of axoplasmic transport in the normal rhesus optic
nerve head, Am. J. Ophthalmol. 82:1, 1976.
8. T'so, M. O. M., Shih, C-Y., and McLean,
I. W.: Is there a blood-brain barrier at the
optic nerve head? Arch. Ophthalmol. 93:815,
1975.
9. Okinami, S., Ohkuma, M., and Tsukahara, I.:
Kuhnt intermediary tissue as a barrier between
the optic nerve and retina, Albrecht v. Graefes
Arch. Klin. Exp. Ophthalmol. 201:57, 1976.