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Human Trabecular Mesh Work Organ Culture: Morphology and Glycosaminoglycan Synthesis

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Human Trabecular Mesh Work Organ Culture: Morphology and Glycosaminoglycan Synthesis Investigative Ophthalmology & Visual Science, Vol. 29, No. 1, January 1988 Copyright © Association for Research in Vision and Ophthalmology Human Trabecular Mesh work Organ Culture: Morphology and Glycosaminoglycan Synthesis Ted 5. Acott,* Paul D. Kingsley, John R. Samples, and E. Michael Van Buskirk Human corneoscleral explants were maintained for several weeks in defined, serum-free media. Tra- becular cell vitality, as judged by vital stain exclusion, is high for at least one month. Trabecular ultrastructure, as compared to that of fresh eyes, first shows minor cellular and extracellular matrix degradation after 3 weeks in culture. The biosynthetic profiles of trabecular glycosaminoglycans (GAGs) change significantly by 3 weeks in culture. Eyes that are stored at 5°C for up to 48 hr postmortem exhibit changes in trabecular ultrastructure and in GAG profiles; both characteristics return to normal by 7 days in culture. The incorporation pattern of 35S-sulfate and 3H-glucosamine into the GAGs of the trabecular meshwork (TM) is distinct from corneal or scleral incorporation. The relative incorporation of 3H-glucosamine into trabecular GAGs, as determined by sequential enzy- matic degradation, is: 22.3% hyaluronic acid (HA), 27.9% chondroitin sulfate (CS), 21.3% dermatan sulfate (DS), 5.9% keratan sulfate (KS), 17.7% heparan sulfate (HS) and 4.9% unidentified material. The relative incorporation of 35S-sulfate into trabecular GAGs is: 0% HA, 32.9% CS, 34.8% DS, 7.7% KS, 13.8% HS and 11.1% into unidentified material. This profile is in good agreement with the profile that was previously obtained for human and nonhuman primate meshworks prior to culture. We conclude that corneoscleral explant organ culture is a useful tool for extracellular matrix studies within a time window from 7 to at least 14 days in culture. Invest Ophthalmol Vis Sci 29:90- 100,1988 Glycosaminoglycans (GAGs), which are the major Because of its small size, the trabecular extracellu- carbohydrate portion of proteoglycans, may play an lar matrix does not lend itself easily to direct bio- important role in maintaining intraocular pressure by chemical analysis.3 One method of overcoming this acting as the aqueous humor outflow resistance bar- difficulty is to study extracellular matrix components rier.1"9 In direct biochemical studies, hyaluronic acid that have been radiolabeled in trabecular cell or organ (HA), chondroitin sulfate (CS), dermatan sulfate culture. Cell culture7-18"20 and organ culture1'27'21"23 (DS), keratan sulfate (KS) and heparan sulfate (HS) have been used to study the biosynthesis of trabecular have been identified in the human trabecular mesh- GAGs. We have used corneoscleral explants since work.1"37"10 In histochemical studies with Alcian they can be maintained for several weeks in culture Blue and Ruthenium Red, staining that is removed without serum or growth factors1'2'7'20'24"27 and be- by GAG-degrading enzymes has been identified in cause they do not require disruption of trabecular several regions of the meshwork.6"12 Fibronectin, structure. laminin, heparan sulfate proteoglycan and types I, III In this report, we describe the conditions used to and IV collagen have been identified in the TM by maintain corneoscleral explants in organ culture for immunohistochemical methods.13"15 Enzymatic per- several weeks and present the ultrastructural mor- fusion studies have been used to implicate GAGs in phology of these explants as viewed with transmission the maintenance of resistance to aqueous humor out- electron microscopy (TEM). We also present the re- flow.4'5-16'17 sults from biochemical studies of the dynamics of radiolabel incorporation and the distribution of this label into the individual GAGs of the TM and the From the Departments of Ophthalmology and *Biochemistry, adjacent structures of the explant. School of Medicine, Oregon Health Sciences University, Portland, Oregon. Supported in part by grants from PHS (NIH EY-03279), Ameri- Materials and Methods can Health Assistance Foundation: National Glaucoma Research Program and National Society for Research to Prevent Blindness. Submitted for publication: October 24, 1986; accepted July 31, Materials 1987. Reprint requests: Ted S. Acott, Department of Ophthalmology, Human eyes, which were enucleated within 3 hr LI05, Oregon Health Sciences University, 3181 S.W. Sam Jackson postmortem (mean time 1.7 hr) and stored at 5°C, Park Road, Portland, OR 97201. were obtained from local eye banks and used within 90 Downloaded from iovs.arvojournals.org on 10/01/2021 No. 1 HUMAN TRABECULAR GLYCOSAMINOGLYCANS / Acorr er ol. 91 48 hr postmortem. The average donor was between Fungizone) in Falcon 60 X 15 mm culture 50 and 75 years of age, although some were younger dishes. Explants were then transferred to 10 ml of or older. A total of 102 eyes were used for the bio- fresh DMEM containing IX antibiotic-antimycotic chemical analysis and 12 for the morphologic studies. and 0.292 mg/ml additional L-glutamine in Falcon The paired eye of each of the latter 12 was included in 60 X 15 mm culture dishes and incubated, concave the former group. In all of the studies reported herein, side up, with gentle shaking at 37°C under a humidi- eyes from donors with any history of major eye sur- fied 5% to 95%, carbon dioxide to air atmosphere. gery or disease, diabetes, liver failure, sepsis, immune Cultures were changed to fresh media every 2 or 3 or neurological disease, major head wounds or days and were allowed to stabilize for 7 days prior to drowning were excluded. These studies use human use, except as indicated. tissue designated "exempt" under category number five by the Public Health Service (PHS). The protocol Microscopy/Morphology/Vitality has been accepted by both the PHS and the Oregon Health Sciences University Human Subjects Com- Small wedges, 2 mm at the circumference, were cut mittee. L-glutamine, 100X Antibiotic-antimycotic from corneoscleral explants with a double-edged mixture, Dulbecco's modified Eagle medium with razor blade and fixed for 4 hr in 2.5% glutaraldehyde, 2% paraformaldehyde, 5 mM calcium chloride, 130 added L-glutamine and glucose, Dulbecco's phos- mM sucrose and 100 mM sodium cacodylate (pH phate buffered saline and 0.4% Trypan blue stain in 7.4). The wedges were then washed three times in this normal saline were obtained from GIBCO (Santa buffer without aldehydes, post-fixed for 1 hr in 1% Clara, CA). GAG degrading enzymes (Miles Labora- osmium tetroxide in the same buffer, washed in the tories, Elkhart, IN), cellulose acetate electrophoresis 3 same buffer, dehydrated through graded alcohols to supplies and standards were as described earlier. Ra- propylene oxide, embedded in Epon and sectioned dioactive precursors: D-[6-3H(N)]-glucosamine hy- on an LKB (Bromma, Sweden) III microtome with a drochloride (~20 Ci/mmol) and carrier-free 35S-sul- glass or diamond knife for light or transmission elec- fate (~43 Ci/mmol) and Atomlite liquid scintillation tron microscopy. Sections were viewed and photo- solution were purchased from New England Nuclear graphed on a Zeiss (Oberkochen, West Germany) (Boston, MA). Epon 812 and cacodylic acid (sodium Photomicroscope or a Siemens (Hamburg, West Ger- salt) were purchased from Ernest F. Fullam (Schenec- many) Elmiskop-101 TEM. Parallel wedges were tady, NY); EM grade glutaraldehyde was purchased vital stained with 0.4% trypan blue in saline for 30 in 10 ml sealed vials from Stevens Metallurgical min at room temperature and processed for light mi- Corp. (New York, NY); paraformaldehyde was pur- croscopy. Several eyes were received within 1 to 4 hr chased from Mallinckrodt (St. Louis, MO); osmium postmortem (designated as "fresh" in the text) and tetroxide was purchased from Polysciences (Warring- immediately fixed for electron microscopy. ton, PA); Alcian Blue 8GX was purchased from Al- drich (Milwaukee, WI); papain type IV and cysteine Radiolabel Incorporation hydrochloride were purchased from Sigma Chemical (St. Louis, MO); Bio-Gel P-6DG desalting gel and Explants, stabilized for 1 week in culture except as columns were purchased from Bio-Rad Labs (Rich- indicated, were changed to fresh media containing mond, CA). All other chemicals were the best Re- 100 /LtCi of 35S-sulfate and 75 /xCi of 3H-glucosamine agent Grade available. per dish and incubated under standard culture con- ditions for the times indicated. Media and label were changed every 48 hr. A standard labeling time of 48 Explant Organ Culture hr was used where no time is indicated. At the desig- Explants were removed by a scleral incision 2 mm nated times, explants were removed from culture and the TM, sclera and cornea separated under a dissect- posterior to the limbus and lifted with a gentle teas- 3 ing-away of the iris and ciliary body. Associated iris ing scope as described previously. root was removed with tweezers without disruption of the TM or scleral spur. This complete, undivided GAG Isolation and Analysis corneoscleral explant was used for culture. Using The methods for isolation, partial purification, cel- aseptic technique, the explants were rinsed in 10 ml lulose acetate electrophoresis (CAE) and sequential of Dulbecco's phosphate buffered saline and incu- enzymatic degradation of the GAGs have been de- bated, concave side up, for 30 min at room tempera- scribed in detail previously.3 Briefly, tissue lipids were ture in 10 ml of Dulbecco's modified Eagle medium extracted with chloroform/methanol; explant pro- (DMEM) with IX added Antibiotic-antimycotic teins were digested with papain; proteins were precip- mixture (final incubation concentrations: 100 units/ itated with trichloroacetic acid (TCA); small mole- ml Penicillin G, 100 Mg/ml Streptomycin sulfate, 0.25 cules, TCA and free radiolabeled were removed by Downloaded from iovs.arvojournals.org on 10/01/2021 92 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Jonuory 1988 Vol.
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