Altered Antigenicify of Keraton Sulfare Proreoglycan in Selected Corneal Diseases

James L. Funderburgh,* Martha L. Funderburgh,* Merlyn M. Rodrigues,t Jay H. Krachmer4 and Gary W. Conrad*

Monoclonal antibody against keratan sulfate (KS) was used for immunofluorescent staining of sections of human corneas from 8 normal eyes, 19 with keratoconus, 4 with pellucid marginal degeneration, 5 with primary macular corneal dystrophy, and 1 with recurrent macular corneal dystrophy. The anti-KS monoclonal antibody did not stain the corneas with primary macular corneal dystrophy, but stained ail other corneas to varying degrees. Staining intensity was weaker than normal in most keratoconus and pellucid marginal degeneration corneas, and was very weak in a case of macular corneal dystrophy that had recurred in a transplanted normal cornea. In several corneas with keratoconus, normal staining was seen at the periphery, and staining intensity decreased in the thinned central portion of the stroma. The decreased KS staining was not localized in stromal scar tissue found in the keratoconus and pellucid marginal degeneration corneas. Quantitation of relative staining intensity found keratoconus and pellucid marginal degeneration corneas to be 49% and 40% as intensely stained, respectively, as normal corneas, a statistically significant decrease (P < 0.01). Distribution of staining intensities of the keratoconus corneas demonstrated a single modality. These results are in agreement with findings of previous biochemical studies, which show reduction of highly sulfated keratan sulfate epitopes in corneas from keratoconus and pellucid marginal degeneration, and absence of sulfated keratan sulfate epitopes in macular corneal dystrophy., Invest Ophthalmol Vis Sci 31:419-428,1990

Keratoconus and pellucid marginal degeneration the stroma are reduced.4"6 Keratoconus corneas also are disorders of the cornea involving progressive contain increased levels of hexosamine and uronic thinning of the stroma.1'2 In keratoconus, stromal acid,57 and stain abnormally with a cationic dye, Saf- thinning is central and accompanied by corneal ecta- ranin O, suggesting an increase of glycosaminogly- sia, whereas in the less frequent condition of pellucid cans in the stroma.4 marginal degeneration, thinning occurs in the infe- Glycosaminoglycans are the carbohydrate moieties rior peripheral stroma.12 In both cases, disruption of of proteoglycans, components of the stroma which Bowman's layer and stromal scarring occur. Stromal are second only to collagen in abundance in the cor- thinning may involve modification of stromal extra- nea. Proteoglycans appear to be important in the cellular matrix metabolism, and even though no bio- maintenance of the ultrastructural integrity of the chemical characteristic of the stroma has been linked stroma, and alterations of corneal proteoglycans have to the etiology of these diseases, abnormalities in ker- been identified in several corneal abnormalities. In atoconus have been identified in both protein and macular corneal dystrophy, a defect in synthesis of carbohydrate components of the stromal extracellu- keratan sulfate (KS) results in production of an un- lar matrix. Work on this subject has been reviewed.3 sulfated KS proteoglycan (KSPG)-related glycopro- In some keratoconus corneas, protein and collagen in tein in place of fully sulfated KSPG.8 Corneas with lattice dystrophy exhibit an unusual, highly sulfated dermatan sulfate,9 and stromal scar tissue formed From the *Division of Biology, Kansas State University, Man- after penetrating wounds contains alterations in both hattan, Kansas; the fDepartment of Ophthalmology, University of KSPG and dermatan sulfate proteoglycans.10"12 De- Maryland, Baltimore, Maryland; and the ^Department of Ophthal- mology, University Hospitals, Iowa City, Iowa. spite the apparent importance of proteoglycans, nei- Supported by NIH Grant EY-00952 (GWC) and by an unre- ther the role of the altered proteoglycans in the pa- stricted grant from Research to Prevent Blindness, Inc., New York thology of these conditions nor the metabolic changes (JHK). involved in producing altered proteoglycans has been Submitted for publication: December 27, 1988; accepted June elucidated. 30, 1989. Reprint requests: James L. Funderburgh, Division of Biology, Development of antibodies to proteoglycans has Ackert Hall, Kansas State University, Manhattan, KS 66506. provided a sensitive and accurate means of identify-

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ing these major components of connective tissues in standardized protocol with a Zeiss (Oberkochen, extracts and in histologic sections. Monoclonal anti- West Germany) epifluorescence microscope. Phy- bodies against KS have been useful in identifying KS coerythrin fluorescence was detected with an excita- during normal corneal embryonic development13 and tion filter of 530-560 nm and an emission filter of in healing corneal wounds.10 A recent study found 565-580 nm. Bisbenzimide fluorescence was de- that pooled extracts of keratoconus stromas showed a tected with excitation < 360 nm and emission at reduced ability to bind anti-KS monoclonal antibod- >485 nm. Negatives were produced on T-max film ies.14 In the current study, individual sections of nor- (Eastman Kodak, Rochester, NY) and developed at mal corneas and corneas from patients with kerato- ASA 400 with Kodak D-76 developer, 22-24°C, for conus, pellucid marginal degeneration, and macular lengths of time recommended by the manufacturer. corneal dystrophy were examined with an anti-KS Negatives were printed on Agfa BEH 310 PE contrast monoclonal antibody using immunofluorescence. A 5 paper, using exposure times matched to produce significant reduction in the binding of anti-KS mono- equal brightness for the corneal control section in clonal antibody to the diseased corneas was found. A each group. preliminary report of this work has been presented.15 Quantitation of Staining Intensity Materials and Methods The optical density of negatives taken through a Tissue 25X objective was determined at 450 nm using 96- well microplate spectrophotometer (model EL 307; Corneal buttons from patients with keratoconus, Bio-Tek Instruments, Winsooki, VT), illuminating a pellucid marginal degeneration, and macular corneal portion of the negative with a diameter equivalent to dystrophy were removed during keratoplasty and 40 /im of the corneal section. The average optical were fixed immediately in 10% formalin (3.7% w/v density for a negative was found to be a linear func- formaldehyde), 0.04 M sodium phosphate, pH 7.2, at tion of the exposure time for optical densities in the room temperature for 48-72 hr, before dehydration range 0-0.8. Intensity of stromal fluorescence was and embedding in paraffin. Normal corneas of eyes estimated by averaging three optical density measure- obtained from the University of Iowa eye bank were ments randomly chosen from the central stromal re- processed in a manner similar to that for the diseased gion of each negative. Variation in the optical density tissue. within a single stroma typically gave standard devia- tions of less than 10% of the mean. The relative Staining staining intensity of each experimental cornea was normalized by comparison with the appropriate con- Staining was carried out in batches, with sections trol cornea. Interbatch variation, assessed by optical (8-/i) of two to five experimental corneas and one densities of the negatives from sections of control control cornea, each on separate albuminized slides. cornea stained over a period of several months, gave a The control was one of a series of sections from the standard deviation of 14% of the mean. central region of a single normal cornea, and was included with each staining. After deparaffinization, Results the sections were immediately blocked, and then were incubated with monoclonal antibody 122 at 20 Monoclonal antibody 122 has been found to be a jtg/ml for 2 hr, as described previously.13 After incu- highly specific reagent for immunohistochemical bation with primary antibody, sections were incu- staining of corneal KS in formalin-fixed, paraffin- bated with phycoerythrin-labeled goat anti-mouse embedded sections of chicken tissues.13 Figure 1 IgG (Accurate Chemical and Scientific, Westbury, demonstrates the effectiveness of this antibody in NY) at 100 Mg/ml, in 1% (w/v) bovine serum albu- staining KS in paraffin sections of formalin-fixed min, 0.02 M Tris-HCl, pH 7.2, 0.15 M NaCl, 0.01% human corneas. The corneal stroma, including Bow- (w/v) sodium azide for 60 min at room temperature man's layer and Descemet's membrane, was brightly in the dark. These staining conditions maximized stained. Occasional light staining of the epithelial antibody-specific to nonspecific staining. Slides were cells, as well as moderate variation in staining inten- rinsed and counterstained with bisbenzimide to stain sity within the stroma (Figure IF) was observed. nuclei,13 and then were mounted in 20% (w/v) poly vi- Staining was eliminated by substitution of nonspe- nyl alcohol, 10% (v/v) glycerol in 0.05 M Tris-HCl, cific mouse IgG (Figure IE) or by preincubation of pH 8, containing 100 fig/m\ p-phenylenediamine and the antibody with an excess of KS (Figure 1G). 10 Mg/ml ascorbic acid. The slides were stored 4°C for Preincubation with heparin, a similar sulfated glycos- 14-18 hr in the dark, and then photographed under a aminoglycan (Figure 1H), had no effect on staining.

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Fig. 1. Specificity of KS staining in human corneas with monoclonal antibody 122. Four serial sections of a normal human cornea were stained with anti-KS monoclonal antibody 122 (E-H) and counterstained with bisbenzimide to visualize cell nuclei (A-D), as described in Materials and Methods. (A, E) Nonspecific mouse IgG was substituted for antibody 122. (B, F) Stained with antibody 122. (C, G) Antibody 122 was preincubated with 1 mg/ml bovine corneal KS, 25°C, 30 min before staining. (D, H) Antibody 122 was preincubated with I mg/ml heparin as in (C) and (G). Bar, 100 /xm.

Staining was eliminated also by incubation of the tis- than that of normal corneas, ranging from normal sue sections with endo-/?-galactosidase, an enzyme intensity to virtually no staining (2C, D). In several that digests KS (data not shown). keratoconus sections, a gradient of staining intensity Antibody 122 was used to stain sections of 37 was present: intensity was nearly normal at the pe- human corneas, both normal and those from patients riphery and decreased in the thinned central region of with keratoconus, pellucid marginal degeneration, the stroma. Figure 3 presents montage photographs and macular corneal dystrophy, and revealed a vari- illustrating sections of two corneas in which this gra- ety of staining patterns. Normal corneas (Figures 2 A, dient of staining was readily apparent. E, I-L) exhibited bright uniform stromal staining, In Figure 4, staining of pellucid marginal degener- occasionally more intense in separations between ation corneas is shown. Like keratoconus, these cor- stromal lamellae, Keratoconus corneas (Figure neas exhibited a noticeable decrease in staining in- 2B-D, F-H) typically were much less intense in their tensity compared to normal corneas. Unlike kerato- staining, which often was limited to anomalously conus, however, regions of stromal thinning were not bright interlamellar staining, giving these corneas a observed to stain in a manner different from the re- striped or spotted look (Figure 2F, G). The intensity mainder of the stroma; rather, the staining was con- of staining in these corneas was more heterogeneous sistently weak throughout the stroma (not shown).

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•4-

Fig. 2. Comparison of KS staining in normal and keratoconus corneas. Four batches of slides containing three experimental sections and one control cornea section were stained with monoclonal antibody 122, as described in Materials and Methods. Experimental corneas are the second, third, and fourth figures in each horizontal row. On the left (A, E, I) are sections of the same normal cornea, included in each batch as v an internal control. The top two rows show staining of keratoconus corneas (B-D, F-H) and the bottom row (J-L) shows three normal corneas. Bar, 100 ^m.

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Fig. 3. Association of reduced KS staining with the thinned region of keratoconus corneas. Montage photographs of two keratoconus corneas (A, B) show staining with anti-KS antibody 122 {upper sections) and nuclear staining (lower sections) of the same sections. Bar, 500 /am.

Figure 5 illustrates the staining of corneas from mac- anti-KS antibody present in the epithelium at these ular corneal dystrophy. Primary macular corneal locations (Fig. 6C, F). Staining in the scarred regions dystrophy (Figure 5C, F) had no detectable staining of the stroma was not significantly reduced compared in sections of the five corneas tested. A corneal graft to surrounding nonscarred stroma. removed after recurrence of the stromal deposits The absence of staining in macular corneal dys- characteristic of macular corneal dystrophy (Figure trophy corneas by anti-KS antibody was unambig- 5B, E) showed weak positive staining. uous. On the other hand, the major observation re- Keratoconus corneas develop ruptures in Bow- garding staining of keratoconus and pellucid mar- man's layer, leading to stromal disruption and scar- ginal degeneration was that the stroma in these ring.13 As shown in Figure 6, for keratoconus, the corneas generally had a reduced intensity of staining. stroma under the breaks in Bowman's layer was char- This conclusion was documented quantitatively by acterized by reduced cellularity (Fig. 6B, E), disrup- measurement of the optical density of photographic tion of the lamellar regularity (Fig. 6A, D), and an negatives taken of the corneas under carefully stan- uneven staining with anti-KS antibody (Fig. 6C, F). It dardized conditions. These data, summarized in Fig- was not unusual to find material staining with the ure 7, support the initial assessment that corneal sec-

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Fig. 4. Staining of KS in corneas from pellucid marginal degeneration. Two corneas with pellucid marginal degeneration (B, E; and C, F) were compared to the normal control (A, D) for KS staining. (A-C) Bis- benzimide counterstain for nuclei. (D-F) Anti-KS antibody 122 staining for KS, as described in Materials and Methods. Bar, 100 fim.

tions from keratoconus and pellucid marginal degen- reduced binding of anti-KS monoclonal antibody eration exhibited a significant decrease in staining compared to a group of normal corneas, as assayed by intensity with monoclonal antibody 122 compared to immunohistochemical staining. Corneas from macu- controls. Keratoconus had a mean staining intensity lar corneal dystrophy showed no binding of this anti- of 49% of that of controls (P < 0.001) and pellucid body. The reduced staining in the keratoconus cor- marginal degeneration corneas were 40% as intensely neas appeared to be associated with the central stained as controls (P < 0.01). The numeric distribu- thinned region in sections where both normal thick- tion of the staining intensities (Figure 8) suggests a ness and thinned regions were present. Irregular single modality for both keratoconus and normal staining was found in scar tissue near disruptions in corneas. Keratoconus corneas cannot therefore be Bowman's layer, and KS-antigenic material was classified into two groups by the criterion of staining sometimes found in the epithelium near these points. intensity for KS. The staining intensity did not show Biosynthetic studies have demonstrated that cor- correlation with age or sex of the patients from whom neas from macular corneal dystrophy patients do not the corneas were removed (data not shown). produce normally sulfated KSPG.8 These patients appear to lack sulfated KS in their serum as well.16 In a previous study, immunohistology of a large number Discussion of corneas from macular corneal dystrophy patients The data presented here show that corneas from showed that corneas from 58% of cases reacted with keratoconus and pellucid marginal degeneration had at least one of five different anti-KS monoclonal anti-

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Fig. 5. Staining of corneas with macular corneal dystrophy. A normal control (A, D), a case in which macular corneal dystrophy recurred in a corneal graft (B, E), and a case of primary macular corneal dystrophy (C, F) were stained for KS (D-F), as described in Materials and Methods. The upper row (A-Q shows phase contrast of the corneal sections. Bar, 100 /*m.

bodies.17 In the current study, 11 total primary cases which macular corneal dystrophy had recurred in a of macular corneal dystrophy were examined (5 as normal graft suggests that the sulfated KS present in reported in Results and 6 more corneal sections pro- the grafted tissue was degraded, and was not replaced vided by Drs. J Baum and N Panjwani, Tufts Univer- by host keratocytes which presumably repopulated sity) with 122, a well characterized monoclonal anti- the stroma. Our results support the conclusion of the body against sulfated KS, different from the antibod- original biosynthetic studies8 that sulfated KS (de- ies used in the earlier study. None of the cases in the fined here by the sulfated epitope recognized by anti- current study showed reactivity with 122, suggesting body 122) is not present in corneas with macular cor- that the epitope recognized by this antibody may be neal dystrophy. In addition, the results with a recur- more consistently absent in macular corneal dys- rent case support the idea that this disease results trophy than are those of antibodies used in the earlier from an error in synthesis, and not from degradation study.17 The extremely weak staining of the cornea in of sulfated KS.

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Fig. 6. Staining of KS at breaks in Bowman's layer. Two keratoconus corneas with disruptions in Bowman's layer were stained for KS with monoclonal antibody 122 {C, F) and counterstained with bisbenzimide (B, E), as described in Materials and Methods. (A, D) Phase contrast staining of the same sections. Bar, 100 pm.

Keratoconus and pellucid marginal degeneration mal corneas by using a quantitative immunoassay.14 corneas present a pattern distinctly different from The 51% decrease in staining intensity of keratoconus that of macular corneal dystrophy, in that staining corneas found in the current study is in close agree- with monoclonal antibody 122 was not absent but ment with the quantitative immunoassay, suggesting instead was present in reduced amounts compared that changes in staining reflect actual changes in with normals. This conclusion arises from simple ob- abundance of KS epitope in the stroma. servation of the photomicrographs in Figures 2, 3, Monoclonal antibody 122 has been shown to bind and 4, as well as from the quantitative (diseased vs sulfated epitopes in the KS chain and to be almost normal) comparisons of the photographic negatives inactive with unsulfated KS.18 The reduction in 122 produced under standardized conditions (Figs. 7, 8). binding may result from KSPG that has reduced sul- The quantitative technique measures fluorescencein - fation or that contains a pattern of sulfation not rec- directly and was used to determine the relative degree ognized by the antibody. Reduced antigenicity may to which the fluorescence of the diseased corneas dif- result also from KSPG molecules with fewer sulfated fered from that of normals. In a previous study, KS KS chains per protein core, or with KS chain lengths antigen in pooled extracts of keratoconus corneas was shorter than normal. Anseth found no change in the found to be reduced by 52% compared to KS in nor- abundance of keratan sulfate sugar residues in kera-

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which the loss of KS was more pronounced in the 150- thinned region (Figure 3). Accumulation of an antigenically altered KSPG in keratoconus and pellucid marginal degeneration would not seem to be a likely primary event in the m pathologic process of these diseases. Rather, this al- c 100-- a> teration may reflect a response of the keratocytes to an abnormal extracellular environment. Secretion of abundant, highly sulfated KS appears to be a tightly regulated function carried out by keratocytes only 'c under conditions present in the normal stroma. In 'o healing wounds, viral keratitis, and lattice dystrophy, and in corneal grafts undergoing rejection, the stroma contains KS with reduced sulfation and dermatan sulfate with increased sulfation.9"1219 A similar pattern of altered glycosaminoglycan synthesis occurs in Normal Keratoconus Pellucid Fig. 7. Intensity of KS staining of normal and diseased corneas. Relative staining intensity with anti-KS monoclonal antibody 122 was calculated as described in Materials and Methods for normal (squares), keratoconus (circles), and pellucid marginal degeneration (triangles) corneas. Data are presented as a scatter diagram showing each value (open symbols) and as the mean and standard deviation of the group (solid symbols). The student t-test showed a significant decrease from normal of both keratoconus (P < 0.001) and pellucid marginal degeneration (P < 0.01).

toconus corneas;9 therefore, it would appear that a change in KS sulfation is the most likely cause for our current findings. CJorneal scar tissue resulting from penetrating wounds contains a reduction in sulfated KS antigen10 similar to that of keratoconus.14 This similarity raises the possibility that stromal scar tissue present in many keratoconus corneas is responsible for the altered KS content in this disease. In scarred regions of the cornea, as seen in Figure 6, KS immunofluorescence was irregular, but binding of antibody 122 was not reduced compared to the surrounding nonscarred stromal tissue, suggesting that the loss of sulfated KS antigen in keratoconus was not limited to the scarred regions of these corneas. Pellucid marginal degeneration has similarities to keratoconus, but stromal scarring is infrequent and stromal thinning is restricted to a portion of the inferior peripheral stroma in pellucid marginal degeneration.1-2 All four pellucid marginal degeneration corneas showed significantly decreased KS staining, but the intensity of staining had no ap- 40 80 120 parent relationship to the region of stromal thinning. The loss of KS antigen in keratoconus and pellucid Relative Staining Intensity degeneration therefore does not appear to be a phe- nomenon associated with localized stromal scarring, Fig. 8. Frequency distribution of staining intensities. Data from but instead appears to occur throughout cornea. The all normal corneas (A) and from keratoconus corneas (B) as shown only pattern of staining localization which could be in Figure 7, are displayed on a histogram comparing frequency distribution of the relative staining intensities. Intensity given by identified was in 3 of the 19 keratoconus corneas, in the normal control is denned as 100.

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cultured stromal fibroblasts and in corneal organ cul- Macular corneal dystrophy: Failure to synthesize a mature tures in which the epithelium has been damaged.20-21 keratan sulfate proteoglycan. Proc Natl Acad Sci USA The alteration of KSPG documented in the current 77:3705, 1980. 9. Anseth A: Studies on corneal polysaccharides: VIII. Changes in study may be a similar response of keratocytes to an the glycosaminoglycans in some human corneal disorders. Exp abnormal stromal extracellular environment. The re- Eye Res 8:438, 1969. cent demonstration by Matsuda et al22 that irregular- 10. Funderburgh JL, Cintron CC, Covington HI, and Conrad GW: ities in endothelial cell shape precede all other symp- Immunoanalysis of keratan sulfate proteoglycan from corneal toms of keratoconus, suggests a source of the altered scars. Invest Ophthalmol Vis Sci 26:1116, 1988. 11. Hassell JR, Cintron C, and Kublin C: Proteoglycan changes stromal environment. The idea that keratoconus during restoration of transparency in corneal scars. Arch Bio- (and possibly pellucid marginal degeneration) are chem Biophys 222:362, 1983. metabolic consequences of endothelial abnormalities 12. Anseth A and Fransson L-A: Studies on corneal polysaccha- is compelling but as yet unsupported by specific ex- rides: IV. Isolation of dermatan sulfate from corneal scar tis- perimental data. To address the implications of the sue. Exp Eye Res 8:302, 1969. 13. Funderburgh JL, Caterson B, and Conrad GW: Keratan sul- data presented in the current study, better under- fate proteoglycan during embryonic development of the standing is needed of the factors regulating proteo- chicken cornea. Dev Biol 116:267, 1986. glycan biosynthesis, accumulation, and turnover in 14. Funderburgh JL, Panjwani N, Conrad GW, and Baum J: Al- both normal and diseased corneas. tered keratan sulfate epitopes in keratoconus. Invest Ophthal- mol Vis Sci 30:2278, fc989. Key words: cornea, keratoconus, keratan sulfate, macular 15. Conrad GW, Funderburgh JL, Funderburgh ML, and Rodri- gues M: Keratan sulfate in keratoconus, pellucid degeneration, corneal dystrophy, pellucid marginal degeneration and macular corneal dystrophy. ARVO Abstracts. Invest Ophthalmol Vis Sci 29(Suppl):215, 1988. 16. Thonar EJ-MA, Meyer RF, Dennis RF, Lenz ME, Maldonado References B, Hassell JR, Hewitt AT, Stark WJ, Stock EL, Kuettner KE, and Klintworth GK: Absence of normal keratan sulfate in the 1. Krachmer JH, Feder RS, and Belin ME: Keratoconus and blood of patients with macular corneal dystrophy. Am J related noninflammatory corneal thinning disorders. Surv Ophthalmol 102:561, 1986. Ophthalmol 28:293, 1984. 17. Yang CJ, SundarRaj N, Thonar EJ-MA, and Klintworth GK: 2. Rodrigues MM, Newsome DA, Krachmer JH, and Eiferman Immunohistochemical evidence of heterogeneity in macular RA: Pellucid marginal degeneration: A clinicopathologic study corneal dystrophy. Am J Ophthalmol 106:65, 1988. of two cases. Exp Eye Res 33:277, 1981. 18. Funderburgh JL, Caterson B, and Conrad GW: Distribution of 3. Bron AJ: Keratoconus. Cornea 7:163, 1988. proteoglycans antigenically related to corneal keratan sulfate 4. Yue BYJT, Sugar J, and Schrode K: Histochemical studies of proteoglycan. J Biol Chem 262:11634, 1987. keratoconus. Curr Eye Res 7:81, 1988. 19. Anseth A: Studies on corneal polysaccharides: VII. Changes in 5. Critchfield JW, Calandra AJ, Nesburn AB, and Kenney MC: glycosaminoglycans in penetrating corneal grafts. Exp Eye Res Keratoconus: I. Biochemical studies. Exp Eye Res 46:953, 8:310, 1969. 1988. 20. Funderburgh JL, Stenzel-Johnson PR, and Chandler JW: 6. Yue BYJT, Sugar J, and Benveniste K: Heterogeneity in kera- Corneal glycosaminoglycan synthesis in long-term organ cul- toconus: Possible biochemical basis. Proc Soc Exp Biol Med ture. Invest Ophthalmol Vis Sci 24:208, 1983. 175:335, 1984. 21. Dahl I-M and Coster L: Proteoglycan biosynthesis in cultures 7. Buddecke E and Wollensak J: Saure und Mucopolysaccharide of corneas and corneal stroma cells from adult rabbits. Exp Eye und glykoproteine der Menschlichen Cornea in Abhangigkeit Res 27:175, 1978. vom hebensalter und bei keratonconus. Graefes Arch Clin Exp 22. Matsuda M, Suda T, and Manabe R: Quantitative analysis of Ophthalmol 171:929, 1966. endothelial mosaic pattern changes in anterior keratoconus. 8. Hassell JR, Newsome DA, Krachmer J, and Rodrigues MM: Am J Ophthalmol 98:43, 1984.

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