The Effect of Chronically Elevated Intraocular Pressure on the Rat Optic Nerve Head Extracellular Matrix

Exp. Eye Res. (1996) 62, 663–674

The Effect of Chronically Elevated Intraocular Pressure on the Rat Optic Nerve Head Extracellular Matrix E L A I N E C. J O H N S O Na*, J O H N C. M O R R I S ONa, b, S U S A N F A R R E L La, L I S A D E P P M E I E Ra, C. G. M O O R Ea    M. R. M C G I N T Ya a

Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Department of Ophthalmology, Oregon Health Sciences University and b The Portland Veterans Administration Hospital and Medical Center, Portland, OR, U.S.A. (Received Columbia 28 July 1995 and accepted in revised form 11 September 1995) The extracellular matrix of the optic nerve head is altered in both human glaucoma and in experimental primate models of this disease. However, the relationship of this change to glaucomatous optic nerve degeneration is unknown. This report describes similar matrix alterations in rats with unilateral elevated intraocular pressure. Brown Norway rats received episcleral vein injections of hypertonic saline to produce prolonged elevations of intraocular pressure. After up to 6 months of pressure elevation, optic nerve head sections from the rats were evaluated by light microscopic immunohistochemistry using antibodies to collagens I, III, IV and VI, laminin, elastin and chondroitin and dermatan sulfate proteoglycans. In experimental eyes with 11 days or more of pressure elevation, depositions of collagen IV, collagen VI and laminin were found within regions of the optic nerve head that, in normal eyes, are occupied solely by nerve bundles. Collagen I and III deposition appeared to be more dependent on the level and duration of the pressure rise. Eyes with lower mean intraocular pressures showed deposits of interstitial collagens primarily at the level of the sclera, while eyes with higher mean pressure elevations had depositions in the neck regions as well. Chondroitin and dermatan sulfate proteoglycans were deposited in a pattern similar to that of collagen I. No extracellular matrix deposition was seen in the orbital optic nerve in any experimental eye. These extracellular matrix changes in rats replicate previous findings in human glaucomatous eyes and monkey eyes with experimentally elevated pressures. They also suggest a sequence of extracellular matrix protein deposition in response to pressure elevation. The optic nerve head deposition of matrix materials in response to elevated intraocular pressures may affect the susceptibility of remaining axons to pressure by changing the physical properties of their support tissues, by affecting the support functions of astrocytes and by changing the microenvironment of injured axons. This model may be useful for studying these and other aspects of the process of axonal injury resulting from elevated intraocular pressure. # 1996 Academic Press Limited Key words : rat model ; astrocyte ; glaucoma ; intraocular pressure ; optic nerve head ; lamina cribrosa ; extracellular matrix ; collagen ; proteoglycan.

1. Introduction The lamina cribrosa of the optic nerve head has been identified as the site of axonal injury in human glaucoma (Quigley et al., 1981). Tengroth originally reported alterations in the collagenous composition of the optic nerve head in human eyes with glaucoma (Tengroth and Ammitzboll, 1984). This prompted speculation that alterations in the biochemical properties of the glaucomatous lamina cribrosa may affect its function and, consequently, axonal susceptibility to intraocular pressure. Studies of the composition of the lamina cribrosa in normal human and monkey eyes demonstrate a very close similarity between the two species. (Hernandez, Igoe and Neufeld, 1986 ; Hernandez, Luo and Igoe, 1987 ; Morrison, Jerdan and Presented in part at the Association for Research in Vision and Ophthalmology Spring Meeting, Ft. Lauderdale, Florida, 1995. * For correspondence at : Casey Eye Institute, Oregon Health Sciences University, 3375 SW Terwilliger Blvd., Portland, OR 97201, U.S.A.

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L’Hernault, 1988 ; Morrison, L’Hernault, and Jerdan, 1988 ; Goldbaum, Jeng and Logemann, 1989 ; Morrison, et al., 1994). In both, the lamina cribrosa consists of collagenous beams with interspersed elastin fibrils. The beams label for collagens I, III, VI and VII, and proteoglycans. Basement membranes, associated with beam exteriors and internal capillaries, label with collagen IV, laminin and basement membrane proteoglycans. Studies of glaucomatous human eyes demonstrate that the architecture of the human lamina cribrosa and distribution its extracellular matrix (ECM) components are altered in glaucoma (Hernandez, Ye and Roy, 1994 ; Tengroth and Ammitzboll, 1984 ; Hernandez, Andrzejewska, and Neufeld, 1990 ; Fukuchi et al., 1994 ; Hernandez, 1992 ; Sawaguchi et al., 1992). Analyses of nerve heads from primates with experimentally elevated intraocular pressure (W IOP) show that similar ECM abnormalities occur in otherwise normal eyes exposed to W IOP. These abnormalities include altered collagen and elastin fibrils, as # 1996 Academic Press Limited

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well as deposition of ECM components within the pores of the lamina cribrosa (Morrison, Dorman-Pease and Dunkleberger, 1990 ; Quigley, Dorman-Pease and Brown, 1991 ; Quigley, Brown and Dorman-Pease, 1991 ; Fukuchi et al., 1992). The study of pressure-induced ECM changes in animal models may improve our understanding of the possible roles these depositions may play in axonal susceptibility to W IOP in human glaucoma. Some of these roles include altered biomechanical properties of the glaucomatous optic nerve head, compromised glial neuronal-support functions and a less permissive extracellular matrix microenvironment for the vulnerable or injured axon. A rat model of W IOP, neuropathy has been developed (Morrison et al., submitted ; Moore, Johnson and Morrison, 1994), in part, to study the evolution and consequences of pressure-induced alterations in the optic nerve head ECM. We have recently demonstrated that the composition of connective tissues in the normal rat optic nerve head is nearly identical to that of the primate (Morrison, et al., 1995a). Here we report the ECM alterations in rat optic nerve heads following exposure to chronically W IOP.

2. Materials and Methods Unilateral IOP elevation All experiments were performed in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Male Brown Norway retired breeder rats (Rattus norvegicus) weighing between 250 and 400 g were used in this study. A complete description of our method for producing W IOP based on the anatomy of the rat limbal microvasculature (Morrison et al., 1995b) appears elsewhere (Morrison et al., submitted ; Moore, Johnson and Morrison, 1994) and is described in brief here. Rats were anesthetized by intraperitoneal injection of standard rat cocktail, consisting of 1±5 ml kg−" of a solution of 5 ml ketamine (100 mg ml−"), 2±5 ml xylazine (20 mg ml−"), 1 ml acepromazine (10 mg ml−"), and 1±5 ml sterile water. A polypropylene ring, with a 1 mm groove cut into its inner surface, was fitted around the equator of the globe to temporarily occlude other aqueous veins and confine injected sclerosing agents to the limbus. A gap in the ring was oriented over one radial aqueous vein (draining the limbal venous plexus). After exposing the aqueous vein under magnification, a specially designed microneedle, pointing toward the limbus, was inserted into the vessel lumen. Immediately after cannulation, 50 µl of 1±75  to 1±80  hypertonic saline solution was injected into the limbal vascular plexus, Schlemm’s canal and the trabecular meshwork. The force of injection was sufficient to just barely blanch the limbal artery, thus avoiding excessive pressure. Polysporin ophthalmic ointment

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(Burroughs Wellcome Co., Research Triangle Park, NC, U.S.A.) was applied to the eye, and the animal allowed to recover from anesthesia.

IOP determination In the initial stages of this study, preinjection and periodic post-injection IOP was determined with the Tono Pen 2 tonometer (BioRad, Santa Ana) on rats anesthetized with inhalational isoflurane (Ohmeda, Liberty Corner) as previously described (Moore, Milne and Morrison, 1993). As the study progressed, we began to determine IOP on awake rats, using a drop of 0±5 % proparacaine hydrochloride for each eye and gentle restraint (Moore, Milne and Morrison, 1993 ; Moore et al., 1995). We determined that IOP readings determined under isoflurane were consistently depressed relative to awake readings taken in the same animals immediately prior to anesthesia. The relationship between awake and anesthetized IOP values was evaluated in a group of ten rats with unilateral W IOP. Awake IOP was plotted against anesthetized IOP and the relationship was found by linear regression to be : awake IOP ¯ [1±3887(isoflurane IOP)]®3±0285. In order to more accurately summarize pressure history data (containing both awake and isoflurane-anesthetized pressure readings) for this study, IOP values obtained under anesthesia were converted to awake IOP values using the above formula. Uninjected, awake rats have a daytime IOP of 19±3³1±9 mmHg. (Moore, Johnson and Morrison, 1996). Eyes were routinely examined under a dissecting microscope for inflammation and corneal changes. Experimental eye Tono-Pen 2 IOP determinations were compared to those of the uninjected fellow eye to determine the W IOP at each IOP determination. The mean difference³standard error of the mean was used to express the level of IOP elevation and the variability of the IOP determinations over the time course of pressure elevation. Following durations of W IOP ranging from 1 day to 6 months, 13 rats were killed, eyes immediately enucleated, lanced at the superior equator, cryopreserved by soaking overnight in 10 % sucrose at 4°C, immersed in OCT mounting media and frozen in 2-methyl butane chilled in liquid nitrogen.

Immunohistochemistry Most samples were cut to produce vertical longitudinal cryosections (3 µm) of the optic nerve head and retrolaminar optic nerve. Two specimens were cut in cross-section through the optic nerve head, at the level of the sclera. All sections were collected on 3-aminopropyltriethoxysilane-coated (Sigma) glass slides and stored at ®80°C until use for immunohistochemistry.

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F. 1. (A) Overview of the normal rat optic nerve head region. The nerve head consists of the unmyelinated neck (N) and transition (T) region with distinct laminar beams (arrowheads) at the level of the posterior sclera (S), illustrated by immunolabeling with antibodies to collagen VI, an interstitial collagen. The retina (R) is oriented at the top and the fully myelinated optic nerve (ON) at the bottom of the figure. Small, pale glial nuclei (hematoxylin counterstain) are oriented in columns which parallel the course of the nerve fiber bundles. Cross-sections of transition area nerve heads immunolabeled for collagen I (B) and IV (C), reveal the normal distribution patterns of interstitial and basement membrane proteins. The laminar beams (arrowheads) are primarily oriented to span the scleral opening vertically. In (C), blood vessels (arrows) are apparent within the laminar beams. [Original magnification (A) 95¬, (B) and (C) 280¬].

Sections were first fixed in methanol for 5 min at 4°C and then washed in phosphate buffered saline (PBS). Sections were incubated for 30 min in PBS with 1 % bovine serum albumin (PBS–BSA) and 20 % normal serum from an appropriate species to block non-specific binding. Excess blocking serum was then removed and the sections were overlaid with primary antibodies diluted as indicated below in PBS–BSA overnight at 4°C. Sections were then washed with PBS with 0±01 % Triton X-100 and overlaid for 30 min with biotinylated secondary antibodies (Vector laboratories, Burlingame, CA, U.S.A.) diluted 1 : 200 in PBS–BSA with 20 % appropriate serum. Following a

second wash, the slides were exposed to avidin biotin peroxidase complex (Vector Laboratories, Burlingame, CA, U.S.A.) in PBS–BSA (1 : 140 for rabbit primary antibodies, and 1 : 50 for mouse primary antibodies) for an additional 45 min and then developed in 0±05 % 3,3-diaminobenzidine with 0±02 % hydrogen peroxide in 20 m TRIS buffer, pH 7±2, for 3 min. They were then counterstained lightly with hematoxylin, dehydrated and coverslipped for viewing with a Zeiss Axiofot light microscope. Primary antibodies consisted of goat IgG antibodies to collagen I (diluted 1 : 100), III (1 : 500) and VI (1 : 100) from Southern Biotechnologies, rabbit IgG

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antibodies to collagen IV (1 : 5000, BioDesign Kennebunkport, ME, U.S.A.), goat IgG antibodies to rat α-elastin (1 : 5000, Elastin Products, Owensville, MD, U.S.A.) and rabbit IgG antibodies to purified laminin (1 : 5000, Telios, San Diego, CA, U.S.A.). Phosphorylated neurofilament antibodies (1 : 15000, SMI 34, Sternberger Monoclonals, Baltimore, MD, U.S.A.) were also used to estimate the axonal loss in each eye. Use of all of these antibodies has been documented and published previously in several organ systems, including the eye (Morrison et al., 1988a, 1995a ; Demarchez, Hartmann and Prunieras, 1987 ; Engvall et al., 1986 ; Wrenn and Mecham, 1987 ; Sternberger and Sternberger, 1983). For chondroitin and dermatan sulfate proteoglycan studies, sections were incubated for 30 min at 37°C in enzyme buffer (pH 7±2) consisting of 20 m Tris, 50 m sodium acetate, 100 m sodium chloride, 0±01 % bovine serum albumin (fraction V, Sigma, St. Louis, MO, U.S.A.), and 0±1 U ml−" chondroitinase ABC (EC 4\2\2\4, from Proteus vulgaris, Sigma) or chondroitinase ACII (EC 4\2\2\5, from Arthrobacter aurescens, Sigma). All slides were then rinsed in PBS and processed for light microscopic immunohistochemistry using mouse monoclonal IgG antibodies to chondroitin-4 sulfate (4S) and mouse monoclonal IgM antibodies to chondroitin-6 sulfate (6S) (both 1 : 7000, ICN Immunobiologicals, Costa Mesa, CA, U.S.A.). These antibodies are well characterized and specifically recognize the carbohydrate stubs that remain attached to the proteoglycan core protein after enzymatic removal of dermatan and chondroitin sulfate side chains (Caterson, Christner and Baker, 1985 ; Porello and LaVail, 1986 ; Hageman and Johnson, 1987 ; Porello, Yasumura, and LaVail, 1987). Incubation with 4S antibodies following chondroitinase ABC digestion labels core proteins with both dermatan and chondroitin 4-sulfate glycosaminoglycans ; while only chondroitin 4-sulfate containing proteoglycans are labeled after chondroitinase AC digestion. Patterns of labeling in experimental eyes were compared to those of their control, fellow eyes. Antibody controls consisted of substituting corresponding dilutions of appropriate, purified immunoglobins for primary antibodies, both with and without enzyme pretreatment, where indicated. 3. Results Histologically, the rat anterior optic nerve has a bottle-neck configuration, and can be divided into three regions (Hildebrand, Remahl and Waxman,

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F. 2. Specificity of deposition of ECM in the optic nerve head following W IOP. Illustration shows collagen VI labeling following W IOP of 10³3 for 193 days. Compare the nerve head appearance to the normal distribution pattern illustrated in Fig. 1(A). Deposition of ECM proteins is seen in the nerve head neck (N) and transition (T) regions, not in the retrolaminar optic nerve (ON). (Nuclear counterstain. Original magnification 95¬.)

1985) [Fig. 1(A)]. The neck is most anterior. It is bordered by the choroid and sclera and is devoid of myelinated axons. A conical transition zone begins near the posterior edge of the sclera and gradually expands with increased myelination of the axons. Posteriorly, the optic nerve proper begins where the nerve is widest and is fully myelinated. Control tissues and eyes examined after 1 day of W IOP showed a distribution of all ECM components that was identical to that previously described for the normal rat optic nerve head (Morrison et al., 1995a), as summarized in Fig. 1. In all other experimental eyes, deposition of ECM was detected in the optic nerve head, but not in the optic nerve (Fig. 2). This deposition appeared to be in

F. 3. Pattern of ECM deposition following long term, relatively low pressure elevation. A specimen with a mean W IOP of 10³3 mmHg for 193 days illustrates dense, brown deposition of collagen type IV (A) between the laminar beams (arrowheads) throughout the neck (N) and transition (T) region of the optic nerve head. In contrast, collagen type I (B) deposition is limited to the transition region of the nerve, with relative sparing of the neck regions. A restricted deposition of collagen type III (C) is seen in the transition region. The laminar beam (arrowheads) labeling pattern is identical to that seen in fellow, untreated eyes. The retinal surface is oriented to top of the figure. (Hematoxylin nuclear counterstain. Original magnification 190¬.)

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F. 3. For legend see opposite.

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F. 4. Detail of ECM deposition in an eye with mean W IOP of 16³5 mmHg for 11 days shows the posterior neck (N), as well as the transition (T) region, of the optic nerve head. (A) Extensive deposition of collagen IV is seen in both regions. (B) In contrast, collagen I deposition is only faintly apparent between laminar beams (arrowheads). (Hematoxylin nuclear counterstain. Original magnification 280¬.)

regions formerly occupied by nerve fiber bundles. There was no apparent alteration in distribution or density of ECM components within the laminar beams, themselves. While the protein composition of the depositions varied, depending upon the pressure history of the specimen, elastin was not found in these depositions in any specimen. We found specific patterns of ECM deposition within the degenerating nerve fiber bundles of the optic nerve head. In eyes with extended periods of relative low mean W IOPs (less 12 mmHg), laminin, collagen IV and VI deposition predominated, with labeling distributed throughout the optic nerve head, including the neck region [Fig. 3(A)]. In these same tissues, depositions of interstitial collagens I and III were more limited to the transitional region of the optic nerve head [Fig. 3(B) and 3(C)]. A similar pattern of ECM deposition was seen in eyes with short duration, higher mean W IOPs (greater than 14 mmHg). For example, after 11 days of 16³5 mmHg W IOP, deposition of laminin, collagen IV and VI appear heavy in the neck and transition region of the optic nerve head (Fig. 4). Only faint deposition of collagen I and III was evident, limited to the transition regions.

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F. 5. Longer periods of higher pressure elevation, as in this rat eye with W IOP of 18³6 mmHg for 36 days, result in more collagen type I deposition both in the neck (N) and between the laminar beams (arrowheads) in the transition (T) region of the optic nerve head. (Hematoxylin nuclear counterstain. Original magnification 190¬.)

Longer periods of exposure to higher W IOP values resulted in deposition of all ECM proteins throughout the nerve head, including the neck region (Fig. 5). However, the depositions of laminin, collagen IV and collagen VI always appeared more extensive than collagen I ; collagen III deposition was always the least extensive. Cross-sections of nerve from rat no. 10 at the level of the sclera displayed collagen IV and VI depositions in nerve fiber bundles between laminar beams, along with less extensive collagen I and III labeling (Fig. 6). In rats with more than 1 month of W IOP, the ECM depositions within degenerating nerve fiber bundles also labeled for proteoglycan glycosaminoglycans, resembling the pattern of collagen I deposition. Deposition was most intense in the transition region, but could also be detected in the neck region of the optic nerve head in some specimens. Enzymatic digestion with chondroitinase AC or ABC failed to reveal a differential distribution of chondroitin-4 sulfate and dermatan sulfate proteoglycan within the ECM depositions (Fig. 7). Chondroitin-6 sulfate proteoglycans appeared to be only a very minor component of the nerve head fiber bundle ECM depositions.

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F. 6. Detail of ECM deposition in optic nerve head cross sections at the transition level from an eye with mean IOP elevation of 20³3 mmHg for 54 days. Brown deposits of collagen I (A) and collagen IV (B) appear within the nerve bundles, between the laminar beams (arrowheads). (Hematoxylin nuclear counterstain. Original magnification 950¬.)

Experimental eyes with 11 days or more of W IOP demonstrated an apparent loss of nerve fibers, as illustrated by reduced axonal labeling with phosphorylated neurofilament antibodies (Fig. 8). No alteration from normal axonal density was apparent in the optic nerve head sampled after 1 day of W IOP. Laminar beam elastin fibers are scarce in the rat optic nerve head (Morrison et al., 1995a) and no alteration in the distribution of elastin labeling was detectable in any experimental eye. 4. Discussion While the lamina cribrosa of the optic nerve head has been identified as the site of axonal injury in glaucoma (Quigley et al., 1981), the mechanism of axonal injury in response to elevated pressure is not understood. The ECM of the optic nerve head is known to be altered in human glaucoma. Hernandez, Andrzejewska and Neufeld (1990) showed that human eyes with primary open angle glaucoma have deposits of collagen IV extending throughout the optic nerve head. These deposits were heavier within nerve fiber bundles in the lamina cribrosa of more damaged eyes. Also, collagen I and VI showed increased density in the laminar beams in all glaucoma specimens. In addition, collagen IV mRNA expression is increased in

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astrocytes and lamina cribrosa cells in eyes with primary open angle glaucoma (Hernandez, Ye and Roy, 1994). W IOP alone can alter the ECM of otherwise normal monkey eyes. Morrison et al. (1990), demonstrated ECM material within the pores between the laminar beams from experimentally W IOP monkey eyes labeled with antibodies to collagens I, III and IV. Similarly, Fukuchi et al. (1992) reported laminar pore deposition of laminin and collagens III, IV and VI in cynomolgus monkeys with experimental glaucoma. Working with this same animal model, this same group has recently shown deposits of enlarged or elongated chondroitin and dermatan sulfate proteoglycans fibrils between the laminar beam collagen fibers and an abnormal heparin-containing filament associated with basal laminae (Fukuchi et al., 1994). Although the rat lamina cribrosa is less extensive than the primate, it is identifiable and appears as capillary-filled connective tissue bands spanning the scleral opening and separated from axons by an astroglial layer (Hildebrand, Remahl and Waxmam, 1985). The bands are most concentrated in the transition region of the nerve head. We have recently shown that the composition of these bands is identical to that of primate laminar beams (Morrison et al., 1995a). They consist of collagen I, III, and VI and dermatan}chondroitin-sulfate proteoglycans, with small amounts of elastin. Basement membrane components collagen IV and laminin show a typical distribution, arising from astrocytes along the margins of the lamina, and from vascular endothelial cells within the bands. In the nerve head transition region of rats as well as in the primate lamina cribrosa, astrocytes are oriented transversely across the scleral opening and perpendicular to the nerve fibers. (ffrench-Constant et al., 1988 ; Anderson, 1969). Our current findings indicate that the normal rat optic nerve head responds to chronic W IOP in a manner similar to that observed in primates. In general, we found depositions of collagen IV, collagen VI and laminin to be more sensitive to W IOP than the interstitial collagens : their distribution was more extensive in animals with mild W IOPs and appeared earlier in response to higher IOP elevation than either collagen I or III. Human glaucoma specimens have also primarily shown abnormalities of collagen IV and VI (Hernandez, Andrzejewska and Neufeld, 1990). Monkey eyes with severe damage from experimental elevations of IOP to 40 mmHg showed deposits of collagens I and III within nerve bundles, along with collagen IV (Morrison et al., 1990), similar to our current findings in rats with higher W IOPs. The apparent dependency of collagen I and III deposition on the level of IOP may explain why similar deposits were not seen in human eyes with primary open angle glaucoma (Hernandez, Andrzejewska and Neufeld, 1990), which probably had received treatment to minimize IOP elevation.

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F. 7. Label for chondroitin-4 and dermatan sulfate proteoglycans (A) in eye from Fig. 3 shows deposition similar to that of the interstitial collagens [see Fig. 3, (B) and (C)]. Labeling for chondroitin-4 sulfate alone (B) reveals that the deposition patterns for the two glycosaminoglycan types are very similar. (Hematoxylin nuclear counterstain. Original magnification 190¬.)

Deposition of ECM proteins in pressure-injured nerves could represent a non-specific response to axonal loss. However, optic nerve transection in primates did not show the ECM deposits found in the glaucomatous monkeys (Morrison et al., 1990). Similarly, our specimens with mild W IOP for prolonged periods of time showed much less extensive collagen I and III deposition than rats with higher levels of IOP, even though both had extensive reduction of neurofilament immunolabeling. These observations, plus the fact that all of these changes are restricted to the optic nerve head regardless of species, suggest that the changes represent a specific response by cells of the optic nerve head to W IOP. The appearance of changes in the optic nerve head ECM in otherwise normal eyes following experimental IOP elevation suggests that their presence in glaucoma eyes may be secondary to the glaucomatous process, rather than a primary abnormality. However, these alterations may influence the survival of remaining axons in several ways. First, abnormal ECM deposits are likely to alter the physical behavior of the optic nerve head and could explain loss of resiliency and compliance noted in both human and experimental glaucoma (Zeimer and Ogura, 1989). Elastin fibers may be decreased or abnormal in human glau-

comatous or experimental monkey laminar beams (Hernandez, Andrzejewska, and Neufeld, 1990 ; Hernandez, 1992 ; Quigley, Brown and Dorman-Pease, 1991) and elastin has not been found in W IOP-induced ECM depositions in either experimental monkeys (Morrison et al., 1990) or in the current study. Thus, W IOP results in a reduction of the elastic component relative to an increase in the collagen component of the optic nerve head in all species studied. The resulting changes in the physical behavior of the optic nerve head could affect its ability to support and protect remaining axons. Second, although the cells responsible for producing these ECM proteins are currently unknown, their location within the nerve fiber bundles suggests that they may be astrocytes. Astrocytes have been reported to proliferate within the optic nerve head in human glaucoma (Minckler and Spaeth, 1981) and have thickened basement membranes in monkeys with experimental IOP elevation (Fukuchi et al., 1994, Morrison, Dorman-Pease and Dunkleberger, 1990). In our current studies of W IOP ECM deposition in rats, cross-sections show that the interstitial collagen deposits appear primarily within axon bundle regions in areas normally occupied by astrocytes and their processes, rather than adjacent to laminar beams.

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T I Elevated eye intraocular pressure histories* Rat no.

Days

Mean W IOP³...

23 8 65 38 20 29 10 26† 22 53‡ 30 35 40

1 11 36 45 54 54 54 65 82 86 173 193 202

20 mmHg 16³5 mmHg 18³6 mmHg 19³5 mmHg 11³8 mmHg 15³8 mmHg 20³3 mmHg 5³5 mmHg 15³5 mmHg 17³8 mmHg 11³8 mmHg 10³3 mmHg 14³5 mmHg

* Reported as mean difference (W IOP) between injected and fellow eye intraocular pressure determinations. Average fellow eye IOP in the awake rat is 19³3³1±9 mmHg. Unless otherwise indicated, days indicates the number of days between the first pressure determination at which the injected eye IOP was 5 mmHg above the fellow eye and the day of the death of the animal. In addition, it includes no more than two IOP determinations per month in which the experimental eye IOP was within 5 mmHg of the fellow eye. † Mean includes an initial 8 day W IOP (mean W IOP of 7³0±1 mmHg), a period of pressure normalization (IOP within 5 mmHg of the fellow eye) and a 33 day second period of W IOP (mean 9³4 mmHg) following reinjection. ‡ Mean excludes a 20 day period of pressure normalization prior to death.

F. 8. Relative nerve fiber density illustrated by brown immunolabelling for phosphorylated neurofilament protein in the neck region of the optic nerve head. Longitudinal section of a control nerve head (A) illustrates normal axonal density. Reduced axonal density is seen following W IOP of 16³5 mmHg for 11 days (B) with axonal swellings (arrows) indicative of ongoing degeneration. A few scattered, enlarged axons remain following W IOP of 10³3 mmHg for 193 days (C). (Asterisk indicates blood vessel, hematoxylin nuclear counterstain. Original magnification 190¬.)

Astrocytes have been shown in vitro to produce laminin (Chiu et al., 1991) and in situ (Webersinke et al., 1992) to produce collagen IV, as well as other ECM components. Similarly, rat glioma and human glioblastoma cells synthesize collagens I and VI respectively (Ghahary et al., 1992, Han et al., 1994). If W IOP induces optic nerve head astrocytes to synthesize ECM materials, this change in cellular programming may, either directly or indirectly via feedback mechanisms, affect astrocytic support of the remaining axons and increase axonal vulnerability to pressure. Finally, the deposition of ECM materials within the axon bundles may have direct detrimental effects on the axons themselves. Normally, axons are separated from ECM components in the optic nerve head by glial cell processes (Anderson 1969). The deposition of these materials, including chondroitin and dermatan sulfate, within the axon bundles may put these proteins into direct contact with remaining axons. Collagens IV and VI (Carri et al., 1992) and chondroitin sulfate proteoglycans (Snow et al., 1991 ; Snow and Letourneau, 1992) have been shown to inhibit retinal neurite outgrowth in vitro and may also have inhibitory effects in vivo. In addition, collagen I is not a permissive substrate for adult rat retinal neurites (Hopkins and Bunge, 1991). The presence of these ECM proteins within the nerve fiber bundles may diminish the already limited capacity for regeneration of injured retinal ganglion cells and affect their ability to repair axolemmal damage. Continued studies of the

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responses of optic nerve head astrocytes to W IOP, including analysis of the temporal and spatial relationships of these responses to axonal degeneration, are necessary to understand the process of glaucomatous optic nerve damage. The similarities between rats and primates with regard to the composition of the lamina cribrosa (Morrison et al., 1995a), as well as the ECM response to W IOP shown here, support the use of this rat model to investigate these important relationships. Acknowledgements Supported by NIH grant EY10145 and unrestricted funds from Research to Prevent Blindness. Dr Morrison is a 1990 RPB Miriam and Benedict Wolfe Scholar.

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