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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. Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017 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. Volume 16 Number 6 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 Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017 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 Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017 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 Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017 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.