Age-Related Differences
in the Human Vitreoretinal Interface
J.
Sebag, MD
\s=b\ Age-related differences in vitreoretinal adhesion were investigated in 59 human eyes from donors aged from 33 weeks of gestation to 94 years with the use of dark-field and electron microscopy of whole vitreous. In all eyes from individuals aged 21 years or older, dissection of the retina off the vitreous resulted in cleavage between the vitreous cortex and the internal limiting lamina. However, in six (40%) of 15 eyes from individuals aged 20 years or younger, the internal limiting lamina remained adherent to the vitreous cortex in an area that encompassed the macula, temporal arcades, and peripapillary posterior pole. Ultrastructural studies revealed inner portions of M\l=u"\ller'scells that were attached to the posterior aspect of the internal limiting lamina. These results suggested that, in youth, there is adhesion between the vitreous cortex and internal limiting lamina that is stronger than M\l=u"\ller'scell itself and is topographically more extensive than previously
thought. (Arch Ophthalmol. 1991;109:966-971)
mportant pathologic processes
occur
at the interface between the vitre¬
and retina. Vitreous traction on the peripheral retina causes retinal tears' and retinal detachment.2,3 In the macula, axial4 and/or tangential' vitre¬ ous traction is believed to result in macular holes. Cell migration and pro¬ liferation in neovascularization, prolifous
erative vitreoretinopathy, and pre¬ macular membrane formation all occur at the vitreoretinal interface. The most common event at this interface is de¬ hiscence of the vitreous cortex from the internal limiting lamina (ILL) of the retina with collapse of the vitreous, a condition known clinically as posteri¬ or vitreous detachment (PVD). The two components of true PVD are de¬ hiscence at the vitreoretinal interface and liquefaction of the vitreous. If the vitreous is liquefied without dehis¬ cence at the interface, collapse of the vitreous can exert traction at sites of usual and/or pathologic vitreoretinal adhesion. Previous reports have em¬ phasized the importance of PVD (par¬ tial or complete) in the pathogenesis of rhegmatogenous retinal detachment,1-3 some macular disorders,4h'' and neo¬ vascularization. 810 The present study compares the mi¬ croscopic and ultrastructural features of the vitreoretinal interface in eyes from young individuals with eyes from older individuals. The specific ques¬ tions addressed are as follows: Does the strength of vitreoretinal adhesion differ at the equator as compared with the posterior pole? Does the strength of vitreoretinal adhesion vary with age? Is strong vitreoretinal adhesion focal (limited to the central macula, optic disc, and along blood vessels), or is the adhesion more diffusely distrib¬ uted in the posterior pole? MATERIALS AND METHODS
Accepted for publication February 27, 1991. From the Doheny Eye Institute, University of Southern California, Los Angeles, and the Eye
Research Institute of Retina Foundation, Harvard Medical School, Boston, Mass. Dr Sebag is now in private practice in Huntington Beach, Calif. Reprint requests to Pacifica Tower, 18800 Delaware St, Eleventh Floor Suite, Huntington Beach, CA 92648 (Dr Sebag).
Fifty-nine eyes from humans aged from 33 weeks of gestation to 94 years were obtained through the courtesy of the New England Eye Bank, Boston, Mass, and the New York Bank for Sight Restoration, New York City. The age distribution of this population was as follows: 0 to 10 years (n 7), 11 to 20 years (n 8), 21 to 40 years =
=
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
( 7), 41 to 60 years ( 10), 61 to 80 years ( =14), and 81 to 100 years (n=13). The =
=
donors had no history of ocular disease or trauma, intraocular surgery, or diabetes mellitus. The techniques for study of the macro¬ scopic structure of the vitreous have been In brief, previously described in detail. freshly enucleated human eyes that were kept in moist chambers at 4°C for less than 36 hours following death were dissected so that the vitreous remained attached to the anterior segment while the outer posterior layers (sclera, choroid, and retina) were dissected away. The retina was peeled off the vitreous by finding a surgical plane between the vitreous and retina at the equator (by using colibri forceps) and by peeling the retina forward to the posterior border of the vitreous base and posteriorly to the optic nerve. The entire fresh (un¬ fixed) specimen was examined by dark-field slit microscopy and photographed as previ¬ ously described.,''11'1" In all specimens, there was clean separation between the ILL of the retina and the vitreous cortex at the equator (confirmed histologicalry), demon¬ strating that dissection techniques did not artifactually influence the findings at the '
"
'
posterior pole. Specimens of dissected retina from the periphery and posterior pole and portions of
the vitreous cortex from these sites were cut with scissors, fixed overnight in a solu¬ tion of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1-mol/L cacodylate buffer (pH 7.4), and postfixed in 0.1-mol/L cacodylate buffer (pH 7.4) for 1 hour at 4°C. Specimens were dehydrated in a graded acetone series, followed by propylene ox¬ ide, and then embedded in epoxy resin (Epon)-araldite. Thick sections (1 µ ) were stained with toluidine blue, and suitable areas were chosen for study. Blocks were trimmed, thin sectioned, stained with 2% uranyl acetate-lead citrate, and photo¬ graphed with the use of a transmission electron microscope (Philips 200). For scanning electron microscopy, dis¬ sected portions of the posterior vitreous were mounted onto filter paper, with the vitreous cortex facing up (away from the
paper). Specimens on filter paper fixed overnight in 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1-mol/L cacodylate buffer (pH 7.4), and critical point dried in carbon dioxide. The tissue then was dehydrated in a graded alcohol series. Spec¬ imens and filter paper were mounted to¬ gether on aluminum stub mounts and coated with gold. Photographs were taken with a scanning electron microscope (AMR filter were
1000A).
RESULTS
By dark-field slit microscopy, the vitreous cortex in all eyes had a dense
appearance that scattered light more intensely than did the inner portions of the vitreous (Fig 1). In all eyes from individuals aged 21 years or older
(n 44; 37 eyes [84%]
from indi¬ viduals aged 41 years or older), the posterior vitreous had two "holes" in the vitreous cortex (Fig 1). The prepapillary hole (a true hole in situ) measured approximately 1.0 to 1.25 mm in diameter, and the premacular hole was about four times larger. Ul¬ trastructural studies demonstrated that the retina removed from the vitre=
were
Fig 1. —Posterior vitreous in a 59-year-old man. The cortex (arrows) surrounds the entire vitreous. The premacular hole (m) is four times larger than the prepapillary hole (p) (original magnification approximately 9).
ous was
intact in the eyes that had this
microscopic appearance. The ILL had an irregular posterior aspect and a
smooth anterior surface with no vitre¬ fibrils (Fig 2). The vitreous in these specimens had an outer layer of densely packed collagen fibrils (Fig 3) and no retinal elements. In six (40%) of the 15 eyes from individuals aged 20 years or younger (four eyes [66.7%] were from individ¬ uals aged younger than 10 years), the appearance of the peripheral vitreous was similar to that of the older individ¬ uals, but the appearance of the posteri¬ or vitreous was entirely different. Fig¬ ure 4 demonstrates the macroscopic structure observed in the vitreous of a 9-month-old female infant. Although the prepapillary hole is clearly seen, there appears to be no hole in the premacular vitreous cortex (Fig 4, top left and top right). The vitreous cortex in the posterior pole of this eye had more intense light-scattering proper¬ ties than did the equatorial vitreous cortex (Fig 4, bottom left and bottom right). Scanning electron microscopy of the posterior vitreous in this eye demonstrated a membranous structure adherent to the vitreous cortex in the posterior pole (Fig 5, left). Transmis¬ sion electron microscopy identified this structure as the ILL of the retina with vesicles of membranous material pre¬ sent on the posterior aspect (Fig 5, ous
right). Figure 6 demonstrates the macro¬ scopic appearance of the posterior vit-
Fig 2. Ultrastructure of the inner retina after dissection away from the vitreous. Left, Müllers cells (M) insert into the posterior aspect of the internal limiting lamina (arrows). The anterior surface is smooth, whereas the posterior aspect follows the contours of the inner retinal cells (original magnification 20 800). Right, Scanning electron microscopy of the anterior aspect of the internal limiting lamina, demonstrating the smooth surface and absence of vitreous collagen fibrils (original magnification 2250). —
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
in a 14-year-old boy. The pre¬ papillary hole in the vitreous cortex can be clearly identified, but there is no hole in the premacular region. The posterior pole appears to be covered by a "cap" of tissue that surrounds the
reous
prepapillary
hole. Within this tissue, of the fovea and linear patterns arising from branching the prepapillary hole (the former site of the optic disc) that follow the course of retinal blood vessels. The peripheral vitreous has the same appearance as the vitreous cortex of older individuals shown in Fig 1. Scanning electron mi¬ croscopy demonstrated that the vitre¬ ous in this specimen contained a mem¬ brane adherent to the posterior vitreous cortex. The posterior surface of this cap of membranous tissue was covered by numerous vesicular struc¬ tures (Fig 7). Transmission electron microscopy identified this tissue as the ILL of the retina (Fig 8). The vesicles on the posterior surface of this tissue were identified as the inner portions (or inner dense cytoplasm) of Muller's cells of the retina with "attachment plaques" present at the points of inserthere
Fig 3.—Scanning electron microscopy of the vitreous cortex after removal of the retina (same as Fig 2, right). Densely packed collagen fibrils are seen with no internal limiting lamina components. The compactness of the collagen fibrils is probably exaggerated by dehydration during processing (original magnification 3750). eye
are
"imprints"
Fig 4.—Macroscopic structure of posterior vitreous in a 9-month-old female infant. Top left, The prepapillary hole (p) in the vitreous cortex is seen, but no hole is present in the premacular region (arrow) (original magnification approximately 7). Top right, Cartoon companion of Fig 4, top left. Bottom left, Special illumination demonstrates the more intense light-scattering properties of the tissue on the premacular vitreous cortex (original magnification approximately 7). Bottom right, Cartoon companion of Fig 4, bottom left. Extruding Vitreous
Prepapillary Hole Premacular
Region
Tissue
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
on
Vitreous Cortex
tion into the ILL (Fig 8). Transmission electron microscopy of the retina in this region demonstrated marked dis¬ ruption of the plasma membranes of all inner retinal cells with cytoplasmic organelles dispersed into the preretinal space. COMMENT
study demonstrates topograph¬ age-related variations in the strength of human vitreoretinal adhe¬ sion. Although the technique of speci¬ men preparation did involve the creation of a ("surgical") plane of disThis ic and
section at the
equator, when the retina
peeled posteriorly, the adhesive forces between the ILL of the retina and the posterior vitreous cortex were the sole determinants (without artifactual influence) of the level of cleavage. In all eyes from individuals aged 21 years or older (the majority of whom were aged older than 41 years), adhe¬ sion between the ILL of the retina and the vitreous cortex in the posterior pole was weak, and the retina separat¬ ed cleanly from the vitreous between the ILL and the posterior vitreous cortex. However, in 40% of eyes from
was
individuals aged 20 years
or younger, adhesion between the ILL and the posterior vitreous cortex was stronger than Müller's cell itself. Consequently, the inner portions of Müller's cells tore away from the retina and were found adherent to the ILL-vitreous cortex complex. This topographic difference is consistent with clinical observations by Schachat and Sommer14 who sug¬ gested that stronger vitreoretinal ad¬ hesion at the macula accounted for a variety of vitreomaculopathies associ¬ ated with PVD. Moreover, these re¬ sults also demonstrate that this strong
Fig 5.—Ultrastructural appearance of the posterior vitreous cortex shown in Fig 4. Left, Scanning electron microscopy demonstrates a membranelike structure (M) that was adherent to the premacular region of the vitreous. The coarse fibers of the filter paper (F) are seen beneath the membrane (original magnification 600). Right, Transmission electron microscopy demonstrates that this structure consists of the internal limiting lamina (thick arrow) bordered by collagen fibrils (c) on its anterior aspect and membranous vesicles of inner retinal origin (m, thin arrows) on its posterior aspect (original magnification 21 300). Fig 6. —Posterior vitreous in a 14-year-old boy. Left, The prepapillary hole (p) is surrounded by tissue with lightscattering characteristics different from those of the vitreous cortex (c). This tissue contains what appears to be the "imprints" of the fovea (white arrow) and the retinal vessels (black arrows) (original magnification approxi¬ mately 9). Right, Cartoon companion of Fig 6, left. Foveal Retinal Vessel
Imprints.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
Imprint
Tissue
on
Vitreous Cortex
Fig 7.—Scanning electron microscopy demonstrates that the posteri¬ surface of the tissue shown in Fig 6 contains numerous vesicular structures. Top, Low original magnification ( 2400). Bottom, High original magnification ( 12 000). or
adhesion is not focal, ie, limited to the macula, the optic disc, and along reti¬ nal blood vessels. Rather, the phenom¬ enon is distributed throughout the pos¬ terior pole in a "sheetlike" configuration that encompasses the macula, as well as the peripapillary
posterior pole.
The exact source of the adhesive forces between the vitreous and retina is not known. Early studies18,16 re¬ ported the presence of vitreous colla¬ gen fibril insertion into the ILL. Other investigations17 found fine fibrillar at¬ tachments between the vitreous and retina, but these were neither identi¬ fied nor confirmed. Studies'" of frozen, resin-cracked, and enzyme-digested monkey retina demonstrated a dense fibrillar meshwork close to the base¬ ment membrane of Müller's cells, whereas there was a loose network of
Fig 8.—Transmission electron microscopy of the posterior vitreous shown in Figs 6 and 7 demonstrates the internal limiting lamina (thick arrows), collagen fibrils (c) of the posterior vitreous cortex, and inner portion of a Müllers cell (m) inserting into the internal limiting lamina with attachment plaques (p) at this insertion (original magnification 20 800). The space within the vitreous cortex is probably artifact, resulting from specimen preparation for electron microscopy.
fibrils that extended from this region into the vitreous. Other studies19 have shown that the ILL is composed of type IV collagen associated with glycoproteins. Laminin, fibronectin, and proteoglycans have been identified in the human ILL.2" The findings of the present study suggest that extracellu¬ lar matrix components between the ILL and vitreous cortex are likely to be responsible for the sheetlike adhe¬ sion observed in these experiments. Several studies18'21"2'5 have shown that the thickness of the ILL of the retina increases with age. Kohno et al21 found that in age-related thickening of the ILL, there is a bilaminar deposition of fibronectin and laminin in the ILL of the posterior pole but not of the equa¬ torial fundus. It is possible that this thickening adversely affects the ability of Müller's cells to synthesize and
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
maintain the components of the extra¬ cellular matrix at the ILL-vitreous cortex interface,26"30 thus weakening vitreoretinal adhesion.81,32 Such weak¬ ening leads to dehiscence at the ILLvitreous cortex interface and allows liquid vitreous to dissect a plane be¬ tween the vitreous cortex and retina.13 This results in true PVD.81 However, if the process of liquefaction occurs with¬ out adequate dehiscence between the vitreous cortex and the ILL, traction will be exerted at sites of persistent adhesion, and, as demonstrated in this study, retinal elements can tear away and remain adherent to the vitreous cortex. This may explain the severity of rhegmatogenous events in patients with high myopia and vitreous degen¬ erations31 where liquefaction is ad¬ vanced but there is no vitreoretinal dehiscence.
Elucidating the nature of the adhe¬ sive forces at the vitreoretinal inter¬ face would contribute greatly to our understanding of PVD, the most com¬ mon event in the life of the human vitreous. In particular, the composi¬ tion of the extracellular matrix that bonds the vitreous cortex to the ILL of
the retina should be investigated. Such information would improve our under¬ standing of the conditions that result in retinal tears and detachment and mac¬ ular holes.2"81 Characterizing the mech¬ anisms underlying vitreoretinal adhe¬ sion could also enable the development of means by which vitreoretinal adhe-
sion could be enhanced or weakened, thereby inhibiting or facilitating PVD.
This could be very useful since, de¬ pending on the clinical circumstances, it may be advantageous to be able to prevent or induce PVD. Steven J. Brennan performed all technical as¬ pects of electron microscopy.
References 1. Schepens CL. Retinal Detachment and Allied Diseases. Philadelphia, Pa: WB Saunders Co;
1983;1:37-67.
2. Foulds WS. The vitreous in retinal detachment. Eye. 1975;95:412-416. 3. Chaine G, Sebag J, Coscas G. The induction of retinal detachment. Eye. 1983;103:480-485. 4. Avila MP, Jalkh AE, Murakami K, Trempe CL, Schepens CL. Biomicroscopic study of the vitreous in macular breaks. Ophthalmology.
1983;90:1277-1283.
5. Johnson RN, Gass JDM. Idiopathic macular holes: observations, stages of formation, and implications for surgical intervention. Ophthalmology.
1988;85:917-924. 6. Sebag J, Balazs EA. Pathogenesis of cystoid
macular edema: an anatomic consideration of vitreoretinal adhesions. Surv Ophthalmol. 1984;
28(suppl):493-498. 7. Schepens CL,
Avila MP, Jalkh AE, Trempe CL. Role of the vitreous in cystoid macular edema. Surv Ophthalmol. 1984;28(suppl):499-504. 8. Faulborn J, Bowald S. Microproliferations in proliferative diabetic retinopathy and their relationship to the vitreous: corresponding light and electron microscopic studies. Graefes Arch Clin
Exp Ophthalmol. 1985;223:130-138. 9. Jalkh A, Takahashi M, Topilow HW, Trempe CL, McMeel JW. Prognostic value of vitreous findings in diabetic retinopathy. Arch Ophthalmol. 1982;100:432-434. 10. Trempe CL, Takahashi M, Topilow HM. Vit-
changes in retinal branch vein occlusion. Ophthalmology. 1981;88:681-687. 11. Sebag J, Balazs EA. Human vitreous fibres and vitreoretinal disease. Eye. 1985;104:123-128. 12. Sebag J. Age-related changes in human vitreous structure. Graefes Arch Clin Exp Ophthalreous
mol. 1987;225:89-93. 13. Sebag J, Balazs EA. Morphology and ultrastructure of human vitreous fibers. Invest Ophthalmol Vis Sci. 1989;30:1867-1871. 14. Schachat AP, Sommer A. Macular hemorrhages associated with posterior vitreous detachment. Am J Ophthalmol. 1986;102:647-649. 15. Fine BS, Tousimis AJ. The structure of the vitreous body and the suspensory ligaments of the lens. Arch Ophthalmol. 1961;65:95-110, 119-134. 16. Gartner J. Electron microscopic studies on the fine structure of the normal and pathologically changed vitreoretinal limiting membrane. Graefes Arch Clin Exp Ophthalmol. 1962;165:71-102. 17. Zimmerman LE, Straatsma BR. Anatomic relationships of the retina to the vitreous body and to the pigment epithelium. In: Schepens CL, ed. Importance ofthe Vitreous Body in Retina Surgery With Special Emphasis on Reoperations. St Louis, Mo: Mosby\p=m-\YearBook; 1960:15-28. 18. Heegaard S, Jensen OA, Prause JU. Structure and composition of the inner limiting membranes of the retina: SEM on frozen resin-cracked and enzyme-digested retinas of Macacca mulatta. Graefes Arch Clin Exp Ophthalmol. 1986;224:355\x=req-\ 360. 19. Kefalides NA. The biology and chemistry of basement membranes. In: Kefalides NA, ed. Biology and Chemistry of Basement Membranes. Orlando, Fla: Academic Press Inc; 1978:215-228. 20. Jerdan JA, Kao L, Glaser BM. The inner limiting membrane\p=m-\amodified basement membrane? Invest Ophthalmol Vis Sci. 1986;27(ARVO
suppl):230.
21. Kohno T, Sorgente N, Ishibashi T, et al. Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci. 1987;28:506-514.
22. Favre
M, Goldmann H.
teren Glaskorperabhebung.
132:87-97. 23. Fine BS, Yanoff M. Ocular Histology: A Text and Atlas. 2nd ed. New York, NY: Harper & Row Publishers Inc; 1979:98-99. 24. Hogan MJ, Zimmerman LE. Ophthalmic Pathology: An Atlas and Textbook. 2nd ed. Philadelphia, Pa: WB Saunders Co; 1962:473-476. 25. Balazs EA. Functional anatomy of the vitreous. In: Duane TD, Jaeger EA, eds. Biomedical Foundations of Ophthalmology. New York, NY: Harper & Row Publishers Inc; 1985:8-9. 26. Balazs EA. Die Mikrostruktur und Chimie des Glaskorpers. In: Bericht uber die 68: Zusammenkunft der deutschen ophthalmologischen Gesellschaft in Heidelberg, 1967. Munich, Federal Republic of Germany: JF Bergmann; 1968:537-571. 27. Foos RY. Vitreoretinal juncture: topographical variations. Invest Ophthalmol Vis Sci.
1972;11:801-808.
28. Sebag J. Vitreoretinal interface and the role of vitreous in macular disease. In: Brancato R, Coscas G, Lumbroso B, eds. Retinal Diseases: II. Proceedings of the Retina Workshop. Amsterdam, the Netherlands: Kugler & Ghendi; 1987:3-6. 29. Green WR. Vitreoretinal interface. In: Ryan SJ, ed. Retinal Disease. St Louis, Mo: Mosby\p=m-\Year
Book; 1989;3:13-69. 30. Newsome DA, Linsenmayer TF, Trelstad RL. Vitreous body collagen: evidence for a dual origin from the neural retina and hyalocytes. J Cell
Biol. 1976;71:59-67. 31. Sebag J. The Vitreous\p=m-\Structure,Function and Pathobiology. New York, NY: Springer-Verlag NY Inc; 1989:87-90. 32. Sebag J. Ageing of the vitreous. Eye.
1987;1:254-262.
Currently in Other AMA Journals ARCHIVES OF INTERNAL MEDICINE A Prospective Study of Maturity-Onset Diabetes Mellitus and Risk of Coronary Heart Disease and Stroke in Women JoAnn E. Manson, MD; Graham A. Colditz, MB, BS; Jeir J. Stampfer, MD; Walter C. Willett, MD; Andrzej S. Krolewski, MD; Bernard Rosner, PhD; Ronald A. Arky, MD; Frank E. Speizer, MD; Charles H. Hennekens, MD (Arch Intern Med. 1991;151:1141-1147)
Pilocarpine Treatment of Salivary Gland Hypofunction and Dry Mouth (Xerostomia) Philip C. Fox, DDS; Jane C. Atkinson, DDS; Alice A. Macynski, RN; Andy Wolff, DND; Daivd S. Kung, DDS; Ingrid H. Valdez, DMD; William Jackson, MD, PhD; Robert A. Delapenha, MD; Jeffrey Shiroky, MD; Bruce J. Baum, DMD, PhD (Arch InternMed. 1991;151:1149-1152)
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
Zur Genese der hin-
Ophthalmolgica. 1956;
in the Human Vitreoretinal Interface
J.
Sebag, MD
\s=b\ Age-related differences in vitreoretinal adhesion were investigated in 59 human eyes from donors aged from 33 weeks of gestation to 94 years with the use of dark-field and electron microscopy of whole vitreous. In all eyes from individuals aged 21 years or older, dissection of the retina off the vitreous resulted in cleavage between the vitreous cortex and the internal limiting lamina. However, in six (40%) of 15 eyes from individuals aged 20 years or younger, the internal limiting lamina remained adherent to the vitreous cortex in an area that encompassed the macula, temporal arcades, and peripapillary posterior pole. Ultrastructural studies revealed inner portions of M\l=u"\ller'scells that were attached to the posterior aspect of the internal limiting lamina. These results suggested that, in youth, there is adhesion between the vitreous cortex and internal limiting lamina that is stronger than M\l=u"\ller'scell itself and is topographically more extensive than previously
thought. (Arch Ophthalmol. 1991;109:966-971)
mportant pathologic processes
occur
at the interface between the vitre¬
and retina. Vitreous traction on the peripheral retina causes retinal tears' and retinal detachment.2,3 In the macula, axial4 and/or tangential' vitre¬ ous traction is believed to result in macular holes. Cell migration and pro¬ liferation in neovascularization, prolifous
erative vitreoretinopathy, and pre¬ macular membrane formation all occur at the vitreoretinal interface. The most common event at this interface is de¬ hiscence of the vitreous cortex from the internal limiting lamina (ILL) of the retina with collapse of the vitreous, a condition known clinically as posteri¬ or vitreous detachment (PVD). The two components of true PVD are de¬ hiscence at the vitreoretinal interface and liquefaction of the vitreous. If the vitreous is liquefied without dehis¬ cence at the interface, collapse of the vitreous can exert traction at sites of usual and/or pathologic vitreoretinal adhesion. Previous reports have em¬ phasized the importance of PVD (par¬ tial or complete) in the pathogenesis of rhegmatogenous retinal detachment,1-3 some macular disorders,4h'' and neo¬ vascularization. 810 The present study compares the mi¬ croscopic and ultrastructural features of the vitreoretinal interface in eyes from young individuals with eyes from older individuals. The specific ques¬ tions addressed are as follows: Does the strength of vitreoretinal adhesion differ at the equator as compared with the posterior pole? Does the strength of vitreoretinal adhesion vary with age? Is strong vitreoretinal adhesion focal (limited to the central macula, optic disc, and along blood vessels), or is the adhesion more diffusely distrib¬ uted in the posterior pole? MATERIALS AND METHODS
Accepted for publication February 27, 1991. From the Doheny Eye Institute, University of Southern California, Los Angeles, and the Eye
Research Institute of Retina Foundation, Harvard Medical School, Boston, Mass. Dr Sebag is now in private practice in Huntington Beach, Calif. Reprint requests to Pacifica Tower, 18800 Delaware St, Eleventh Floor Suite, Huntington Beach, CA 92648 (Dr Sebag).
Fifty-nine eyes from humans aged from 33 weeks of gestation to 94 years were obtained through the courtesy of the New England Eye Bank, Boston, Mass, and the New York Bank for Sight Restoration, New York City. The age distribution of this population was as follows: 0 to 10 years (n 7), 11 to 20 years (n 8), 21 to 40 years =
=
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
( 7), 41 to 60 years ( 10), 61 to 80 years ( =14), and 81 to 100 years (n=13). The =
=
donors had no history of ocular disease or trauma, intraocular surgery, or diabetes mellitus. The techniques for study of the macro¬ scopic structure of the vitreous have been In brief, previously described in detail. freshly enucleated human eyes that were kept in moist chambers at 4°C for less than 36 hours following death were dissected so that the vitreous remained attached to the anterior segment while the outer posterior layers (sclera, choroid, and retina) were dissected away. The retina was peeled off the vitreous by finding a surgical plane between the vitreous and retina at the equator (by using colibri forceps) and by peeling the retina forward to the posterior border of the vitreous base and posteriorly to the optic nerve. The entire fresh (un¬ fixed) specimen was examined by dark-field slit microscopy and photographed as previ¬ ously described.,''11'1" In all specimens, there was clean separation between the ILL of the retina and the vitreous cortex at the equator (confirmed histologicalry), demon¬ strating that dissection techniques did not artifactually influence the findings at the '
"
'
posterior pole. Specimens of dissected retina from the periphery and posterior pole and portions of
the vitreous cortex from these sites were cut with scissors, fixed overnight in a solu¬ tion of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1-mol/L cacodylate buffer (pH 7.4), and postfixed in 0.1-mol/L cacodylate buffer (pH 7.4) for 1 hour at 4°C. Specimens were dehydrated in a graded acetone series, followed by propylene ox¬ ide, and then embedded in epoxy resin (Epon)-araldite. Thick sections (1 µ ) were stained with toluidine blue, and suitable areas were chosen for study. Blocks were trimmed, thin sectioned, stained with 2% uranyl acetate-lead citrate, and photo¬ graphed with the use of a transmission electron microscope (Philips 200). For scanning electron microscopy, dis¬ sected portions of the posterior vitreous were mounted onto filter paper, with the vitreous cortex facing up (away from the
paper). Specimens on filter paper fixed overnight in 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1-mol/L cacodylate buffer (pH 7.4), and critical point dried in carbon dioxide. The tissue then was dehydrated in a graded alcohol series. Spec¬ imens and filter paper were mounted to¬ gether on aluminum stub mounts and coated with gold. Photographs were taken with a scanning electron microscope (AMR filter were
1000A).
RESULTS
By dark-field slit microscopy, the vitreous cortex in all eyes had a dense
appearance that scattered light more intensely than did the inner portions of the vitreous (Fig 1). In all eyes from individuals aged 21 years or older
(n 44; 37 eyes [84%]
from indi¬ viduals aged 41 years or older), the posterior vitreous had two "holes" in the vitreous cortex (Fig 1). The prepapillary hole (a true hole in situ) measured approximately 1.0 to 1.25 mm in diameter, and the premacular hole was about four times larger. Ul¬ trastructural studies demonstrated that the retina removed from the vitre=
were
Fig 1. —Posterior vitreous in a 59-year-old man. The cortex (arrows) surrounds the entire vitreous. The premacular hole (m) is four times larger than the prepapillary hole (p) (original magnification approximately 9).
ous was
intact in the eyes that had this
microscopic appearance. The ILL had an irregular posterior aspect and a
smooth anterior surface with no vitre¬ fibrils (Fig 2). The vitreous in these specimens had an outer layer of densely packed collagen fibrils (Fig 3) and no retinal elements. In six (40%) of the 15 eyes from individuals aged 20 years or younger (four eyes [66.7%] were from individ¬ uals aged younger than 10 years), the appearance of the peripheral vitreous was similar to that of the older individ¬ uals, but the appearance of the posteri¬ or vitreous was entirely different. Fig¬ ure 4 demonstrates the macroscopic structure observed in the vitreous of a 9-month-old female infant. Although the prepapillary hole is clearly seen, there appears to be no hole in the premacular vitreous cortex (Fig 4, top left and top right). The vitreous cortex in the posterior pole of this eye had more intense light-scattering proper¬ ties than did the equatorial vitreous cortex (Fig 4, bottom left and bottom right). Scanning electron microscopy of the posterior vitreous in this eye demonstrated a membranous structure adherent to the vitreous cortex in the posterior pole (Fig 5, left). Transmis¬ sion electron microscopy identified this structure as the ILL of the retina with vesicles of membranous material pre¬ sent on the posterior aspect (Fig 5, ous
right). Figure 6 demonstrates the macro¬ scopic appearance of the posterior vit-
Fig 2. Ultrastructure of the inner retina after dissection away from the vitreous. Left, Müllers cells (M) insert into the posterior aspect of the internal limiting lamina (arrows). The anterior surface is smooth, whereas the posterior aspect follows the contours of the inner retinal cells (original magnification 20 800). Right, Scanning electron microscopy of the anterior aspect of the internal limiting lamina, demonstrating the smooth surface and absence of vitreous collagen fibrils (original magnification 2250). —
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
in a 14-year-old boy. The pre¬ papillary hole in the vitreous cortex can be clearly identified, but there is no hole in the premacular region. The posterior pole appears to be covered by a "cap" of tissue that surrounds the
reous
prepapillary
hole. Within this tissue, of the fovea and linear patterns arising from branching the prepapillary hole (the former site of the optic disc) that follow the course of retinal blood vessels. The peripheral vitreous has the same appearance as the vitreous cortex of older individuals shown in Fig 1. Scanning electron mi¬ croscopy demonstrated that the vitre¬ ous in this specimen contained a mem¬ brane adherent to the posterior vitreous cortex. The posterior surface of this cap of membranous tissue was covered by numerous vesicular struc¬ tures (Fig 7). Transmission electron microscopy identified this tissue as the ILL of the retina (Fig 8). The vesicles on the posterior surface of this tissue were identified as the inner portions (or inner dense cytoplasm) of Muller's cells of the retina with "attachment plaques" present at the points of inserthere
Fig 3.—Scanning electron microscopy of the vitreous cortex after removal of the retina (same as Fig 2, right). Densely packed collagen fibrils are seen with no internal limiting lamina components. The compactness of the collagen fibrils is probably exaggerated by dehydration during processing (original magnification 3750). eye
are
"imprints"
Fig 4.—Macroscopic structure of posterior vitreous in a 9-month-old female infant. Top left, The prepapillary hole (p) in the vitreous cortex is seen, but no hole is present in the premacular region (arrow) (original magnification approximately 7). Top right, Cartoon companion of Fig 4, top left. Bottom left, Special illumination demonstrates the more intense light-scattering properties of the tissue on the premacular vitreous cortex (original magnification approximately 7). Bottom right, Cartoon companion of Fig 4, bottom left. Extruding Vitreous
Prepapillary Hole Premacular
Region
Tissue
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
on
Vitreous Cortex
tion into the ILL (Fig 8). Transmission electron microscopy of the retina in this region demonstrated marked dis¬ ruption of the plasma membranes of all inner retinal cells with cytoplasmic organelles dispersed into the preretinal space. COMMENT
study demonstrates topograph¬ age-related variations in the strength of human vitreoretinal adhe¬ sion. Although the technique of speci¬ men preparation did involve the creation of a ("surgical") plane of disThis ic and
section at the
equator, when the retina
peeled posteriorly, the adhesive forces between the ILL of the retina and the posterior vitreous cortex were the sole determinants (without artifactual influence) of the level of cleavage. In all eyes from individuals aged 21 years or older (the majority of whom were aged older than 41 years), adhe¬ sion between the ILL of the retina and the vitreous cortex in the posterior pole was weak, and the retina separat¬ ed cleanly from the vitreous between the ILL and the posterior vitreous cortex. However, in 40% of eyes from
was
individuals aged 20 years
or younger, adhesion between the ILL and the posterior vitreous cortex was stronger than Müller's cell itself. Consequently, the inner portions of Müller's cells tore away from the retina and were found adherent to the ILL-vitreous cortex complex. This topographic difference is consistent with clinical observations by Schachat and Sommer14 who sug¬ gested that stronger vitreoretinal ad¬ hesion at the macula accounted for a variety of vitreomaculopathies associ¬ ated with PVD. Moreover, these re¬ sults also demonstrate that this strong
Fig 5.—Ultrastructural appearance of the posterior vitreous cortex shown in Fig 4. Left, Scanning electron microscopy demonstrates a membranelike structure (M) that was adherent to the premacular region of the vitreous. The coarse fibers of the filter paper (F) are seen beneath the membrane (original magnification 600). Right, Transmission electron microscopy demonstrates that this structure consists of the internal limiting lamina (thick arrow) bordered by collagen fibrils (c) on its anterior aspect and membranous vesicles of inner retinal origin (m, thin arrows) on its posterior aspect (original magnification 21 300). Fig 6. —Posterior vitreous in a 14-year-old boy. Left, The prepapillary hole (p) is surrounded by tissue with lightscattering characteristics different from those of the vitreous cortex (c). This tissue contains what appears to be the "imprints" of the fovea (white arrow) and the retinal vessels (black arrows) (original magnification approxi¬ mately 9). Right, Cartoon companion of Fig 6, left. Foveal Retinal Vessel
Imprints.
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
Imprint
Tissue
on
Vitreous Cortex
Fig 7.—Scanning electron microscopy demonstrates that the posteri¬ surface of the tissue shown in Fig 6 contains numerous vesicular structures. Top, Low original magnification ( 2400). Bottom, High original magnification ( 12 000). or
adhesion is not focal, ie, limited to the macula, the optic disc, and along reti¬ nal blood vessels. Rather, the phenom¬ enon is distributed throughout the pos¬ terior pole in a "sheetlike" configuration that encompasses the macula, as well as the peripapillary
posterior pole.
The exact source of the adhesive forces between the vitreous and retina is not known. Early studies18,16 re¬ ported the presence of vitreous colla¬ gen fibril insertion into the ILL. Other investigations17 found fine fibrillar at¬ tachments between the vitreous and retina, but these were neither identi¬ fied nor confirmed. Studies'" of frozen, resin-cracked, and enzyme-digested monkey retina demonstrated a dense fibrillar meshwork close to the base¬ ment membrane of Müller's cells, whereas there was a loose network of
Fig 8.—Transmission electron microscopy of the posterior vitreous shown in Figs 6 and 7 demonstrates the internal limiting lamina (thick arrows), collagen fibrils (c) of the posterior vitreous cortex, and inner portion of a Müllers cell (m) inserting into the internal limiting lamina with attachment plaques (p) at this insertion (original magnification 20 800). The space within the vitreous cortex is probably artifact, resulting from specimen preparation for electron microscopy.
fibrils that extended from this region into the vitreous. Other studies19 have shown that the ILL is composed of type IV collagen associated with glycoproteins. Laminin, fibronectin, and proteoglycans have been identified in the human ILL.2" The findings of the present study suggest that extracellu¬ lar matrix components between the ILL and vitreous cortex are likely to be responsible for the sheetlike adhe¬ sion observed in these experiments. Several studies18'21"2'5 have shown that the thickness of the ILL of the retina increases with age. Kohno et al21 found that in age-related thickening of the ILL, there is a bilaminar deposition of fibronectin and laminin in the ILL of the posterior pole but not of the equa¬ torial fundus. It is possible that this thickening adversely affects the ability of Müller's cells to synthesize and
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
maintain the components of the extra¬ cellular matrix at the ILL-vitreous cortex interface,26"30 thus weakening vitreoretinal adhesion.81,32 Such weak¬ ening leads to dehiscence at the ILLvitreous cortex interface and allows liquid vitreous to dissect a plane be¬ tween the vitreous cortex and retina.13 This results in true PVD.81 However, if the process of liquefaction occurs with¬ out adequate dehiscence between the vitreous cortex and the ILL, traction will be exerted at sites of persistent adhesion, and, as demonstrated in this study, retinal elements can tear away and remain adherent to the vitreous cortex. This may explain the severity of rhegmatogenous events in patients with high myopia and vitreous degen¬ erations31 where liquefaction is ad¬ vanced but there is no vitreoretinal dehiscence.
Elucidating the nature of the adhe¬ sive forces at the vitreoretinal inter¬ face would contribute greatly to our understanding of PVD, the most com¬ mon event in the life of the human vitreous. In particular, the composi¬ tion of the extracellular matrix that bonds the vitreous cortex to the ILL of
the retina should be investigated. Such information would improve our under¬ standing of the conditions that result in retinal tears and detachment and mac¬ ular holes.2"81 Characterizing the mech¬ anisms underlying vitreoretinal adhe¬ sion could also enable the development of means by which vitreoretinal adhe-
sion could be enhanced or weakened, thereby inhibiting or facilitating PVD.
This could be very useful since, de¬ pending on the clinical circumstances, it may be advantageous to be able to prevent or induce PVD. Steven J. Brennan performed all technical as¬ pects of electron microscopy.
References 1. Schepens CL. Retinal Detachment and Allied Diseases. Philadelphia, Pa: WB Saunders Co;
1983;1:37-67.
2. Foulds WS. The vitreous in retinal detachment. Eye. 1975;95:412-416. 3. Chaine G, Sebag J, Coscas G. The induction of retinal detachment. Eye. 1983;103:480-485. 4. Avila MP, Jalkh AE, Murakami K, Trempe CL, Schepens CL. Biomicroscopic study of the vitreous in macular breaks. Ophthalmology.
1983;90:1277-1283.
5. Johnson RN, Gass JDM. Idiopathic macular holes: observations, stages of formation, and implications for surgical intervention. Ophthalmology.
1988;85:917-924. 6. Sebag J, Balazs EA. Pathogenesis of cystoid
macular edema: an anatomic consideration of vitreoretinal adhesions. Surv Ophthalmol. 1984;
28(suppl):493-498. 7. Schepens CL,
Avila MP, Jalkh AE, Trempe CL. Role of the vitreous in cystoid macular edema. Surv Ophthalmol. 1984;28(suppl):499-504. 8. Faulborn J, Bowald S. Microproliferations in proliferative diabetic retinopathy and their relationship to the vitreous: corresponding light and electron microscopic studies. Graefes Arch Clin
Exp Ophthalmol. 1985;223:130-138. 9. Jalkh A, Takahashi M, Topilow HW, Trempe CL, McMeel JW. Prognostic value of vitreous findings in diabetic retinopathy. Arch Ophthalmol. 1982;100:432-434. 10. Trempe CL, Takahashi M, Topilow HM. Vit-
changes in retinal branch vein occlusion. Ophthalmology. 1981;88:681-687. 11. Sebag J, Balazs EA. Human vitreous fibres and vitreoretinal disease. Eye. 1985;104:123-128. 12. Sebag J. Age-related changes in human vitreous structure. Graefes Arch Clin Exp Ophthalreous
mol. 1987;225:89-93. 13. Sebag J, Balazs EA. Morphology and ultrastructure of human vitreous fibers. Invest Ophthalmol Vis Sci. 1989;30:1867-1871. 14. Schachat AP, Sommer A. Macular hemorrhages associated with posterior vitreous detachment. Am J Ophthalmol. 1986;102:647-649. 15. Fine BS, Tousimis AJ. The structure of the vitreous body and the suspensory ligaments of the lens. Arch Ophthalmol. 1961;65:95-110, 119-134. 16. Gartner J. Electron microscopic studies on the fine structure of the normal and pathologically changed vitreoretinal limiting membrane. Graefes Arch Clin Exp Ophthalmol. 1962;165:71-102. 17. Zimmerman LE, Straatsma BR. Anatomic relationships of the retina to the vitreous body and to the pigment epithelium. In: Schepens CL, ed. Importance ofthe Vitreous Body in Retina Surgery With Special Emphasis on Reoperations. St Louis, Mo: Mosby\p=m-\YearBook; 1960:15-28. 18. Heegaard S, Jensen OA, Prause JU. Structure and composition of the inner limiting membranes of the retina: SEM on frozen resin-cracked and enzyme-digested retinas of Macacca mulatta. Graefes Arch Clin Exp Ophthalmol. 1986;224:355\x=req-\ 360. 19. Kefalides NA. The biology and chemistry of basement membranes. In: Kefalides NA, ed. Biology and Chemistry of Basement Membranes. Orlando, Fla: Academic Press Inc; 1978:215-228. 20. Jerdan JA, Kao L, Glaser BM. The inner limiting membrane\p=m-\amodified basement membrane? Invest Ophthalmol Vis Sci. 1986;27(ARVO
suppl):230.
21. Kohno T, Sorgente N, Ishibashi T, et al. Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci. 1987;28:506-514.
22. Favre
M, Goldmann H.
teren Glaskorperabhebung.
132:87-97. 23. Fine BS, Yanoff M. Ocular Histology: A Text and Atlas. 2nd ed. New York, NY: Harper & Row Publishers Inc; 1979:98-99. 24. Hogan MJ, Zimmerman LE. Ophthalmic Pathology: An Atlas and Textbook. 2nd ed. Philadelphia, Pa: WB Saunders Co; 1962:473-476. 25. Balazs EA. Functional anatomy of the vitreous. In: Duane TD, Jaeger EA, eds. Biomedical Foundations of Ophthalmology. New York, NY: Harper & Row Publishers Inc; 1985:8-9. 26. Balazs EA. Die Mikrostruktur und Chimie des Glaskorpers. In: Bericht uber die 68: Zusammenkunft der deutschen ophthalmologischen Gesellschaft in Heidelberg, 1967. Munich, Federal Republic of Germany: JF Bergmann; 1968:537-571. 27. Foos RY. Vitreoretinal juncture: topographical variations. Invest Ophthalmol Vis Sci.
1972;11:801-808.
28. Sebag J. Vitreoretinal interface and the role of vitreous in macular disease. In: Brancato R, Coscas G, Lumbroso B, eds. Retinal Diseases: II. Proceedings of the Retina Workshop. Amsterdam, the Netherlands: Kugler & Ghendi; 1987:3-6. 29. Green WR. Vitreoretinal interface. In: Ryan SJ, ed. Retinal Disease. St Louis, Mo: Mosby\p=m-\Year
Book; 1989;3:13-69. 30. Newsome DA, Linsenmayer TF, Trelstad RL. Vitreous body collagen: evidence for a dual origin from the neural retina and hyalocytes. J Cell
Biol. 1976;71:59-67. 31. Sebag J. The Vitreous\p=m-\Structure,Function and Pathobiology. New York, NY: Springer-Verlag NY Inc; 1989:87-90. 32. Sebag J. Ageing of the vitreous. Eye.
1987;1:254-262.
Currently in Other AMA Journals ARCHIVES OF INTERNAL MEDICINE A Prospective Study of Maturity-Onset Diabetes Mellitus and Risk of Coronary Heart Disease and Stroke in Women JoAnn E. Manson, MD; Graham A. Colditz, MB, BS; Jeir J. Stampfer, MD; Walter C. Willett, MD; Andrzej S. Krolewski, MD; Bernard Rosner, PhD; Ronald A. Arky, MD; Frank E. Speizer, MD; Charles H. Hennekens, MD (Arch Intern Med. 1991;151:1141-1147)
Pilocarpine Treatment of Salivary Gland Hypofunction and Dry Mouth (Xerostomia) Philip C. Fox, DDS; Jane C. Atkinson, DDS; Alice A. Macynski, RN; Andy Wolff, DND; Daivd S. Kung, DDS; Ingrid H. Valdez, DMD; William Jackson, MD, PhD; Robert A. Delapenha, MD; Jeffrey Shiroky, MD; Bruce J. Baum, DMD, PhD (Arch InternMed. 1991;151:1149-1152)
Downloaded From: http://archopht.jamanetwork.com/ by a University of Manitoba User on 06/10/2015
Zur Genese der hin-
Ophthalmolgica. 1956;
