Extracellular Matrix of the Human Lamina Cribrosa

Extracellular Matrix of the Human Lamina Cribrosa

AMERICAN VOLUME 104 JOURNAL OF OPHTHALMOLOGY® NUMBER 6 DECEMBER, 1987 Extracellular Matrix of the Human Lamina Cribrosa M. Rosario H e r n a n d...

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AMERICAN VOLUME 104

JOURNAL

OF

OPHTHALMOLOGY®

NUMBER 6

DECEMBER, 1987

Extracellular Matrix of the Human Lamina Cribrosa M. Rosario H e r n a n d e z , D . D . S . , Xing Xing Luo, M . D . , Frank Igoe, B . S . , and Arthur H . N e u f e l d , P h . D .

We used immunoperoxidase staining and double immunofluorescent staining to demon­ strate the macromolecular components of the extracellular matrix of the lamina cribrosa from young human donors. The cribriform plates were made up of a core of elastin fibers with a sparse, patchy distribution of collagen type III. The plates were coated with collagen type IV and laminin; these basement membrane components were presumably made by the astrocytes that were distributed on the surfaces of the plates. The insertion of the lamina cribrosa in the sclera was made up of concentric, circumferential elastin fibers that surrounded the lamina cribrosa and were con­ tinuous with the elastin in the cribriform plates. Astrocytic processes extended into the bundles of elastin fibers, whereas the base­ ment membrane components extended into the sclera. The mechanical properties of the macromolecules of the extracellular matrix of the lamina cribrosa may make this tissue compli­ ant and sensitive to intraocular pressure. Per­ haps individual differences in the macromo­ lecular components of this tissue contribute to the glaucomatous changes in the optic nerve head.

Accepted for publication Sept. 16, 1987. From the Ophthalmic Pharmacology Unit, Eye Research Institute, and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. This study was supported in part by United States Public Health Service grant EY-06416, New England Glaucoma Research Foundation, Inc., Boston, the Foundation for Glaucoma Research, San Francisco, and the Glaucoma Foundation, New York. Reprint requests to Arthur H. Neufeld, Ph.D., Eye Research Institute, 20 Staniford St., Boston, MA 02114.

I N PRIMARY OPEN-ANGLE GLAUCOMA, progressive, pathologic changes in the optic nerve head lead to a loss of vision. Any explanation of these changes must account for the apparent weakness of this tissue to withstand increased intraocular pressure as well as individual differences in the sensitivity of patients to intraocular pressure. We have focused attention on the components of the extracellular matrix making u p the lamina cribrosa, the tissue that supports the nerves as they leave the eye. A previous report 1 confirmed that the cribriform plates of the lamina cribrosa are rich in collagen type IV and laminin, and contain some collagen type III but relatively little collagen type I. Thus, the lamina cribrosa is not scleral tissue, but rather a specialized extracellular matrix within the central nervous system. To investigate further the cell biology of the lamina cribrosa, we grew cells from explanted human material in tissue culture and characterized their synthesis of extracellular matrix macromolecules. The cells grown from this tissue were flat, polygonal cells that did not resemble scleral fibroblasts or astrocytes. The lamina cribrosa cells synthesize collagen types III and IV, fibronectin, and elastin and are therefore capable of synthesis of the extracellular matrix macromolecules identified in vivo. 2 In this study, we confirmed, refined, and extended our characterizations of the extracellular matrix macromolecules of the lamina cribrosa. Using additional histochemical and double-labeling techniques, we demonstrated the major macromolecular components of this tissue and their relationship to each other, both in the cribriform plates a n d in the insertion of the lamina cribrosa into the sclera.

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Material and Methods Seven pairs of eyes from young donors (aged 5, 6, 7, 16, 16, 23, and 25 years) with no history of ocular disease were obtained from the New England Lions Eye Bank. The eyes were refrigerated and then dissected within 12 to 15 hours after enucleation. The optic nerve was cut so that it projected 2 mm from the surface of the globe. An incision was made in the sclera around the optic nerve and the retina was carefully cut around the optic cup. The optic cups were embedded in OCT compound, flash frozen in liquid nitrogen, and stored at - 8 0 C until needed. The optic cups were oriented for either sagittal or cross sections. Cryostat sections 6 n,m thick were placed on gelatin-coated microscope slides and incubated for 30 minutes with specific monoclonal or polyclonal antibodies against human antigens. The antibodies were diluted in phosphate buffered saline with 1.5% bovine serum albumin. The following antibodies (dilutions) were used: mouse monoclonal antibodies against human collagen type IV, I, and procollagen type III (1:100); rat monoclonal antibody against laminin (GP-2 subunit of laminin) (1:20); mouse monoclonal antibody against human laminin (1:50); mouse monoclonal antibody against human fibronectin (1:100); mouse monoclonal antibody against glial fibrillary acidic protein (1:10); and rabbit polyclonal antibody against pig a-elastin 3 (1:50). After 30 minutes' incubation with the primary antibody, the slides were washed in phosphate buffered saline with 1.5% bovine serum albumin and submitted for either immunofluorescence or immunoperoxidase staining. Immunofluorescence staining—After incubation with the primary antibody and washing, the sections were incubated with the appropriate fluorescein or rhodamine-conjugated second antibody (1:20) for 30 minutes. Double staining for two primary antibodies raised in different species was performed by incubating the sections with both primary antibodies, followed by fluorescein-labeled second antibody for 30 minutes, and, after washing, a rhodamine-labeled second antibody for 30 minutes. Double staining for two primary antibodies raised in the same species was performed by adding the first primary antibody followed by the fluorescein-labeled second antibody and then the second primary antibody followed by rhodamine-labeled second antibody. After in-

cubation with the fluorescent second antibodies, the slides were washed and mounted in glycerol with phosphate buffered saline (1:5). The slides were observed and photographed using a microscope equipped with epifluorescent illumination and appropriate filter systems. The exposure time was set automatically. To determine the level of nonspecific fluorescence, the primary antibody was replaced by either phosphate buffered saline alone or the appropriate nonimmune serum followed by the second fluorescent-labeled antibody. For the specificity of the primary antibody, we checked positive staining of structures present in the sections known to contain the antigen we were studying (for example, Bruch's membrane, blood vessel walls, and the like). Immunoperoxidase staining—The sections were incubated with primary antibody for 30 minutes, washed, and incubated with biotinylated second antibody for 30 minutes. After washing, the sections were incubated with streptavidin-peroxidase for 30 minutes, washed, and incubated with the substrate mixture 3,3'-diaminobenzidine tetrahydrochloride, 1% hydrogen peroxide in 0.1 M Tris buffer (pH 7.6). The sections were reacted in the dark until intense staining appeared (15 to 30 minutes). In control specimens, the primary antibody was replaced by either phosphate buffered saline or nonimmune rabbit serum.

Results Immunoperoxidase staining for collagen type IV was intense in the lamina cribrosa (Fig. 1). The cribriform plates appeared coated by collagen type IV, indicating fine, lamellar structures. As an internal control for collagen type IV-specific staining, the blood vessels throughout the region were also positively stained. The insertion of the lamina cribrosa in the adjacent sclera stained heavily for collagen type IV, whereas the sclera was unstained. In the insertion region, the collagen type IV-positive material was in the form of a fibrillar network that penetrated toward the sclera (Fig. 1). Similar results were obtained when the tissue was stained for laminin, another major component of basement membranes (data not shown). Immunoperoxidase staining for collagen types I and III was slightly positive in the lamina cribrosa and its insertion. As an internal control, the blood vessels in the region were intensely, positively stained (Fig. 2). Fibro-

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Fig. 1 (Hernandez and associates). Immunoperoxidase staining for human collagen type IV. Top left, Positive staining is present in the cribriform plates (CP) of the lamina cribrosa, blood vessels (BV), and insertion region (In). Note that the nerve bundles (Nb) and sclera (S) do not show positive staining. Pre L, prelaminar region (x40). Top right, Cribriform plates show lamellar pattern of staining for human collagen type IV. Post L, postlaminar region (x 100). Middle right, Detail of cribriform plates showing fine linear positive staining within the plates (x250). Bottom right, Detail of the insertion region (x200). Bottom left, Cross section of the lamina cribrosa. Note positive staining in the cribriform plates and insertion region (x40).

nectin w a s n o t d e t e c t e d in t h e cribriform p l a t e s of t h e l a m i n a cribrosa or in t h e i n s e r t i o n r e g i o n ; however, blood vessels t h r o u g h o u t the optic n e r v e h e a d a n d adjacent t i s s u e s w e r e s t r o n g l y

p o s i t i v e (Fig. 2). T h e c o n n e c t i v e t i s s u e s e p t a of t h e o p t i c n e r v e p r o p e r w e r e slightly p o s i t i v e for fibronectin a n d t h e a r a c h n o i d w a s well s t a i n e d (Fig. 2).

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Fig. 2 (Hernandez and associates). Immunoperoxidase staining for human collagen type III and human fibronectin. Top left, Staining for collagen type III is positive in the blood vessels (BV); the cribriform plates (CP) show faint, diffuse staining. Nb, nerve bundles (x 100). Top right, Positive staining is seen in the arachnoid (A) (x 100). Bottom left, Positive staining for human fibronectin in the arachnoid and blood vessels (x 100). Bottom right, Positive staining for human fibronectin in the blood vessels of the lamina cribrosa; the cribriform plates are not stained (x 100). Staining for a-elastin showed that the lamina cribrosa and its insertion in the sclera have large amounts of this macromolecule (Fig. 3). Elastin was in the form of fibers that ran parallel to the cribriform plates. In the anterior portion of the lamina cribrosa, only a few elastin fibers were found between the glial columns. The insertion of the lamina cribrosa in the sclera was intensely positive for elastin. In this region, the elastin fibers were cut in cross section in the sagittal view of the optic nerve head, indicating a circumferential pattern. In cross sections of the optic nerve head, the elastin fibers appeared circular and formed concentric rings around the lamina cribrosa, with fibers leaving the circumference and en-

tering the cribriform plates centripetally (Fig. 3). Double immunofluorescent staining for elastin and glial fibrillary acidic protein showed that the cribriform plates were positive for both antigens, but the nerve bundles were only positive for glial fibrillary acidic protein (Fig. 4). The astrocytes, glial fibrillary acidic proteinpositive cells, were dense in the prelaminar region where no staining for elastin was observed (Fig. 4). In the cribriform plates, the astrocytes were parallel to the plates, between layers of elastin fibers (Fig. 4). In the insertion region, positive glial fibrillary acidic protein staining of the astrocytes was observed between the bundles of elastin fibers (Fig. 4). In a

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Fig. 3 (Hernandez and associates). Immunoperoxidase staining for elastin. Top left, Sagittal section of the optic nerve head showing positive staining for elastin in the insertion region (In) and cribriform plates (CP). B, Bruch's membrane; Pre L, prelaminar region; Nb, nerve bundles; S, sclera (x 40). Top right, Elastin fibers in the cribriform plates (x 100). Middle right, Cross section of the lamina cribrosa. Elastin fibers are present in the cribriform plates and are continuous with those in the insertion region. P, pigment (x40). Bottom right, Control section incubated with nonimmune rabbit serum. Note the absence of positive staining (x40).

cross section of the insertion area, the processes of the astrocytes extended between layers of concentric elastin fibers (Fig. 4). Double immunofluorescent staining for collagen type IV and elastin showed that these

macromolecules were the major components of the lamina cribrosa and its insertion in the sclera. Alternating layers of collagen type IV and elastin formed the bulk of the lamina cribrosa (Fig. 5). Higher magnification of the cribri-

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PreL

e im­

post L Fig. 4 (Hernandez and associates). Double immunofluorescent staining for elastin (fluorescein label, green) and human glial acidic fibrillary protein (rhodamine label, orange-yellow). Left, Sagittal section of the optic nerve head. Pre L, prelaminar region; B, Bruch's membrane; CP, cribriform plates; In, insertion region; Post L, postlaminar region; Nb, nerve bundles (x 50). Top right, Detail of the cribriform plates in a sagittal section. Note the alternating layers of astrocytes and elastic material (x 100). Bottom right, Detail of the insertion region in a cross section of the lamina cribrosa. Note the astrocytic processes in between bundles of elastin fibers. S, sclera (xlOO). form plates showed that the elastin fibers formed part of the core and that the plates were coated by collagen type IV (Fig. 5). The insertion of the lamina cribrosa in the sclera had abundant collagen type IV material irregularly distributed between the elastin fibers (Fig. 5). By contrast, the sclera appeared devoid of collagen type IV and there were few elastin fibers. In a sagittal section of the optic nerve proper (posterior to the lamina cribrosa) positive staining for collagen type IV was seen coating the pial septa and blood vessels only. Few elastin fibers ran parallel to the septa (Fig. 5). Double staining for collagen type IV and glial fibrillary acidic protein showed that astrocytes lay on lamellae of collagen type IV, which coated the cribriform plates. In the insertion region, the limit of the staining for both anti-

gens did not correspond exactly: collagen type IV extended into the adjacent sclera, beyond the glial fibrillary acidic protein-positive cell processes (Fig. 6). Double staining for collagen type III and elastin showed the presence of both antigens in the core of the cribriform plates. However, in the lamina cribrosa from this age group of donors, staining for collagen type III was slight and in a diffuse, patchy pattern (Fig. 7). Nevertheless, the blood vessels throughout the region showed intense staining for collagen type III. Figure 7 is a diagram of the lamina cribrosa in sagittal and transverse views in which the extracellular matrix components are represented in relationship to the neural, vascular, and neighboring tissues. The schematic drawing is

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Fig. 5 (Hernandez and associates). Double immunofluorescent staining for human collagen type IV (rhodamine label, orange-yellow) and elastin (fluorescent label, green). Top left, Sagittal section of the optic nerve head. Pre L, prelaminar region; B, Bruch's membrane; BV, blood vessel; Nb, nerve bundles; In, insertion region; CP, cribriform plates (x40). Top right, Detail of the cribriform plates in a sagittal section. Note the linear staining for elastin and collagen type IV (x 100). Middle right, Detail of the insertion region in a sagittal section. Note that collagen type IV-stained material extends into the sclera. Arrowheads point to elastin fibers in cross section. S, sclera (xlOO). Bottom right, Pial septa (PS) in the postlaminar region in a sagittal section (xlOO).

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Fig. 6 (Hernandez and associates). Left, Double staining for human collagen type IV (rhodamine label, orange-yellow) and glial fibrillary acidic protein (fluorescein label, green-yellow). Astrocytes and collagen type IV alternate in the cribriform plates (CP). Note that collagen type IV material penetrates into the sclera (S) in the insertion region (In). BV, blood vessels; Nb, nerve bundles (x 100). Right, Double staining for human collagen type III (rhodamine label, orange-yellow) and elastin (fluorescein label, green). Both antigens are present in the cribriform plates, but the staining for collagen type III is faint and diffuse. Note that blood vessels are intensely stained for collagen type III (x 100).

based on the histochemical observations m a d e in this s t u d y a n d o n p r e v i o u s o b s e r v a t i o n s b y A n d e r s o n a n d Hoyt. 4

Discussion O u r r e s u l t s confirm, b y u s i n g i m m u n o p e r o x i dase staining, and expand, by using double

53SS Fig. 7 (Hernandez and associates). Schematic drawing of the lamina cribrosa showing the different extracellular matrix components and their relationships with nerve bundles, astrocytes, and surrounding tissues. Left, Sagittal section of the lamina cribrosa. Right, cross section of the lamina cribrosa. 1, cribriform plates; 2, nerve bundles; 3, blood vessels; 4, insertion region; 5, sclera. Color code: red, basement membranes; lavender, astrocytes; blue, elastin fibers.

i m m u n o f l u o r e s c e n t s t a i n i n g , t h e findings in o u r p r e v i o u s s t u d y . 1 This s t u d y d e m o n s t r a t e s t h e m a c r o m o l e c u l a r c o m p o n e n t s of t h e lamina cribrosa from y o u n g , n o r m a l h u m a n d o n o r s a n d forms t h e basis for f u r t h e r w o r k o n aged and glaucomatous tissue. T h e core of t h e cribriform p l a t e s forming t h e l a m i n a cribrosa h a s s u b s t a n t i a l a m o u n t s of elastin in t h e form of l o n g fibers. Collagen t y p e III c o d i s t r i b u t e s w i t h elastin in t h e core of t h e

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cribriform plates, but the appearance of the fibrillary collagen is patchy and not that of a major component. The. cribriform plates are coated with collagen type IV and laminin, the two macromolecules present in all basement membranes as the major components. Astrocytes, presumably the glial fibrillary acidic protein-positive cells, 5 cover the cribriform plates and are perhaps partially responsible for synthesis of the basement membrane components. Previous ultrastructural descriptions of the human lamina cribrosa showed striated collagen fibers in the core of the cribriform plates. 6 Collagen types I and III form striated fibrils in connective tissues. 7 The lamina cribrosa stained weakly for collagen types I and III using specific monoclonal antibodies and two different immunologic detection systems, whereas blood vessels and other tissues in the same specimens stained positively. We interpret this to mean that the cribriform plates of young, healthy humans have relatively little collagen types I and III. However, the relatively poor staining of the fibrillary types of collagens in our specimens could be a result of masking of the antigenic sites by other macromolecules (proteoglycans in the lamina cribrosa) or to the relatively young age of the specimens we used. More intense staining may be apparent in older eyes, 8 which may have increased amounts of fibrillary collagen in the lamina cribrosa. The insertion of the lamina cribrosa in the sclera is a specialized structure. Concentric, circumferential, tightly packed elastin fibers surround the laminar and prelaminar region of the optic nerve head. Furthermore, the elastin fibers of the cribriform plates, running perpendicular to the nerve bundles, are continuous with, and appear to originate from, those of the insertion. In the adjacent sclera, the elastin fibers are short and sparse and do not show any special orientation. Glial cell processes, which are covered by basement membranes, form an anchoring network through the bundles of elastin fibers in the insertion region, with the basement membrane components extending beyond the glial cell processes into the sclera. The presence of concentric elastin fibers surrounding the lamina cribrosa as it inserts into the sclera may explain the reversibility of the enlargement of the scleral canal observed when hypertensive eyes return to normal intraocular pressure. 9 Although some relaxation of the scleral wall follows deformation, scleral tissue

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is poorly resilient and elastin only accounts for a small percentage of the dry weight of sclera. 10 Thus, the presence of elastin may allow the insertion tissue to regain its previous shape when intraocular pressure is lowered. In the optic nerve proper, the extracellular matrix of the pial septa is continuous and its characteristics are similar to those of the pia mater. In the pial septa, basement membranes and astrocytes form a tissue that is continuous with the cribriform plates. The difference between the extracellular matrix in the lamina cribrosa and postlaminar region is that in the lamina cribrosa, the concentration of macromolecular elements is higher, appears lamellar, and is oriented perpendicularly to the nerve bundles. The macromolecules of the extracellular matrix in the lamina cribrosa form a tissue that, although lacking the tensile strength of sclera, is nevertheless compliant and may be resilient to fluctuations in intraocular pressure. The collagenous component of basement membranes, collagen type IV, forms a loose macromolecular network optimally adapted to the elastic and mechanically stable sheet-like organization of basement membranes. 11 Elastin is a unique macromolecule, a biologic rubber that absorbs stress with perfect recoil. Elastin, abundant in the walls of arteries and arterioles forming elastic lamellae, throughout the lung, and in Bruch's membrane, serves to store and to return mechanical energy. Elastin codistributes with interstitial collagen types I and III to provide tensile strength to connective tissues. 12 In the lamina cribrosa, the combination of elastin and collagen type IV may adapt the tissue to resist the distention caused by pressure changes in the eye or the distortion from ocular movements, and may provide the ability to recover the original structure. The long-term glaucomatous process causes the optic disk to excavate. This is apparently produced by a compressive rearrangement of the laminar sheets, 13 causing degeneration of the nerves. The mechanical properties of the extracellular matrix making u p the cribriform plates must be important in maintaining the compliance and resiliency of the tissue to resist or to respond to compression. Individual differences in age-related changes in the macromolecular components forming this tissue, in type, in amount, or in the architectural distribution of these components, may contribute to the glaucomatous process in the optic nerve head.

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ACKNOWLEDGMENTS

N i r m a l a S u n d a r - R a j , P h . D . , U n i v e r s i t y of Pittsburgh, provided the monoclonal antibodies a g a i n s t h u m a n collagen t y p e s I, III, a n d IV. Jeffrey D a v i d s o n , M . D . , Vanderbilt U n i v e r s i t y , p r o v i d e d t h e a n t i b o d y a g a i n s t p i g ot-elastuu

References 1. Hernandez, M. R., Igoe, F., and Neufeld, A. H.: Extracellular matrix of the human optic nerve head. Am. J. Ophthalmol. 102:139, 1986. 2. Hernandez, M. R., Igoe, F., and Neufeld, A. H.: Cell culture of the lamina cribrosa. Invest. Ophthalmol. Vis. Sci. In press. 3. Giro, M. G., Hill, K. E., Sanberg, L. B., and Davidson, J. M : Quantitation of elastin production in cultured vascular smooth muscle cells by a sensitive and specific enzyme-linked immunoassay. Coll. Relat. Res. 4:21, 1984. 4. Anderson, D. R., and Hoyt, W. F.: infrastructure of intraorbital portion of human, and monkey optic nerve. Arch. Ophthalmol. 82:506, 1969. 5. Bignami, A., Eng, L. F., Dahl, D., and Uyeda, C. T.: Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43:429, 1972. 6. Anderson, D. R.: Ultrastructure of human and

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monkey lamina cribrosa and optic nerve head. Arch. Ophthalmol. 82:800, 1969. 7. Linsenmayer, T. F.: Collagen. In Hay, E. D. (ed.): Cell Biology of the Extracellular Matrix. New York, Plenum Press, 1981, p. 15. 8. Jeng, S., Goldbaum, M. H., Logemann, R. B., and Weinreb, R. N.: Extracellular matrix of the optic nerve. ARVO Abstracts. Supplement to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1987, p. 61. 9. Pederson, J. E., and Herschler, J.: Reversal of glaucomatous cupping in adults. Arch. Ophthalmol. 100:426, 1982. 10. Moses, R. A., Grodzki, W. J., Jr., Starcher, B. C , and Gailione, M. J.: Elastin content of the scleral spur, trabecular meshwork, and sclera. Invest. Ophthalmol. Vis. Sci. 17:817, 1978. . 11. Kuhn, K., Glanvilie, R. W., Babel, W., Qiah, R. Q., Dieringer, H., Tilman, V., Siebold, B., Oberbaumer, I., Schwarz, V., and Yamada, Y.: The structure of type IV collagen. Ann. N.Y. Acad. Sci. 460:14, 1985. 12. Davidson, J. M., and Giro, M. G.: Control of elastin synthesis. Molecular and cellular aspects. In Mecham, R. P. (ed.): Regulation of Matrix Accumulation. Orlando, Fla., Academic Press Inc., 1986, p. 182. 13. Quigley, H. A., Hohman, R. M., Addicks, E. M., Massof, R. W., and Green W. R.: Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am. J. Ophthalmol. 95:673, 1983.