Human Trabecular Mesh Work Organ Culture: Morphology and Glycosaminoglycan Synthesis

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 enzymatic 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

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 conditions 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. 29

molecular sieve chromatography on Bio-Gel P-6DG in Figure 1. At this level of resolution, it is difficult to columns; and the GAGs were collected by precipita- detect significant changes until at least 4 weeks. By 7 tion from an ethanol/potassium acetate solution. weeks (Fig. lc), deterioration is quite apparent. For electrophoretic studies, these semi-purified The transmission electron micrographs shown in GAGs were dissolved in 5 fil of water and 0.25 /A Figure 2 are of the TM of fresh eyes that were ob- aliquots were electrophoresed on cellulose acetate in tained and placed in fixative within 2 hr postmortem. a Beckman (Fullerton, CA) Microzone apparatus for Figure 3 is an electron micrograph of the TM of an 2 hr at 4 mamps using 0.3 M cadmium acetate buffer eye that was enucleated 3 hr and fixed 43 hr post- (pH 4.1). The cellulose acetate membranes were mortem. The trabecular morphology in this micro- stained with Alcian Blue for visualization of the graph is typical of the worst eyes that we accept for GAGs. Each lane of the membrane was cut into 20 culture. Compared to fresh eyes or 7-day cultured slices (1 mm wide); the slices and both ends of each explants, the TMs of these eyes have a large number lane were counted in Atomlite in a Packard Tri-carb of electron-lucent vesicles (arrow) throughout the cy- (4000 Series, Downers Grove, IL) liquid scintillation toplasm and exhibit occasional loss of beam integrity counter. The amounts of each isotope were deter- near the cellular surface (*). Figure 4a and b shows mined using a program that corrects to disintegra- two electron micrographs of typical TMs after 1 week tions per minute (DPM) in external standard mode in culture. The morphology is quite similar to that of with automatic gain compensation; corrections are fresh specimens (Fig. 2). based upon dual-label quench curves using Packard Figure 4c and d contains electron micrographs of 3H-toluene and 14C-toluene quench standards or the TM of explants which had been in culture for 14 35S-sulfate quenched with chloroform. Isotope days (c) and 21 days (d). Occasionally, a few cells (less counting separations allowed less than 5% uncor- than 5%) can be found at 21 days which have begun rected cross-channel spill. DPM values for 35S-sul- to show the beginnings of possible cytoplasmic fate were corrected for radioactive decay. changes (see Fig. 5 for more extreme examples). By For sequential enzymatic degradation studies, the 28 days (Fig. 5a, b), however, appreciable beam dis- enzyme sequence to degrade each respective GAG integration (*) is observed and more cells can be (given below in parenthesis after the enzyme name) found which exhibit nuclear changes (arrows) or loss was as detailed previously,3 ie Streptomyces hyal- of cytoplasmic integrity (just to left of arrow in a). uronidase (HA), chondroitinase AC (CS), chondroi- The proportion of damaged cells is still low at 28 days tinase ABC (DS), keratanase (KS) and heparatinase (between 5% and 20%). By 49 days in culture (Fig. (HS). The remaining unidentified material which was 5c), cellular degradation is very advanced. The cells resistant to all enzymatic degradation is termed have released most of their cytoplasmic contents, the "REM." Instead of applying the respective degraded membranes are compromised (arrow) and the beams GAG samples to CAE as previously reported, the su- are disorganized (*). Vesicles are quite apparent by pernatant from each alcohol precipitation step was this time as is an increased amount of amorphous dried in a scintillation vial, dissolved in 100 /A of material in the spaces between the beams. The nu- water and then counted in 10 ml of Atomlite. Culture clear changes beginning to appear at 28 days (arrows medium from some experiments was digested with 5a, b) are becoming pronounced (double arrows 5c). papain and then treated identically to the tissue samples. The number of eyes used in individual experi- Cellulose Acetate Electrophoresis of GAGs ments is indicated in the text or figure captions. In Figure 6, a comparison of the electrophoretic Results profiles of radiolabel incorporation into trabecular, corneal and scleral GAGs, after a 48 hr incubation Morphology of Explant Organ Cultures with label under standard culture conditions, is presented. Figure 6a shows the CAE profile of 3H-gluco- Tissue wedges were evaluated for trypan blue ex- samine and 35S-sulfate incorporation into trabecular clusion and light microscopic morphology after 0, 1, GAGs. Figure 6b shows the 35S-sulfate incorporation 3, 5, 7, 10, 14, 21 and 28 days in culture. Tissue into the corneal and scleral GAGs. These profiles are morphology and vital staining, as judged by light mi- the averages of three separate experiments, and are croscopy, indicate that the explants regain apparently typical of many others that we have conducted. In normal morphology and essentially 100% viability these experiments, the GAGs from the three tissues of within 3 to 5 days in culture and maintain this for at one explant were run in parallel lanes on the same least 1 month (data, not shown). Light micrographs of acetate strips and therefore reflect identical electro- explants after 7, 28 and 49 days in culture are shown phoretic conditions.

Downloaded from iovs.arvojournals.org on 10/01/2021 Fig. 1. Light micrographs of corneoscleral explants. After culture for 7 (a), 28 (b) and 49 (c) days, thick sections were cut from fixed and embedded wedges, stained with 0.1%> toluidine blue in 0.1% sodium borate and photographed. Schlemm's canal is seen near the center with the trabecular meshwork in the upper right and the sclera in the lower left of each photomicrograph. Magnifica- tions: (a) X400, (b) X250. (c) X210. A*

Fig. 2. Transmission electron micrographs of fresh human TM. Micrograph (a) °*"a "fresh" human eye. fixed within 3 hr postmortem, showing trabecular cells and adjacent beam from the deep corneoscleral region near the juxtacanalicular area at a magnification of XI2.667. Note pigment granules (P), the extracellular matrix beams (*), the plasma membrane integrity (arrow), the close association of the beams with lamina lucida and lamina densa of the basement membrane at the cell surface and the lack of open beams without cells between them and the open trabecular spaces (S). Lower magnification (X6933) micrograph (b) showing beams {*), villous cytoplasmic projections into the spaces (S) between trabecular cells and beams and the typical trabecular nucleus with the nuclear membrane and closely-associated electron dense heterochromatin (arrow).

Downloaded from iovs.arvojournals.org on 10/01/2021 94 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988 Vol. 29

Fig. 3. Electron micrograph of juxtacanalicular meshwork of an eye showing an example of the maximum deterioration due to storage prior to culture that we observed in acceptable eyes. This eye was enucleated 3 hr postmortem and had been stored at 5°C for 43 hr. Note the increased number of electron-lucent vacuoles (arrow) apparent in the cytoplasm, random scattering of ribosomes throughout the cytoplasm and the lack of close beam-membrane association (*) which may reflect reduced rates of extracellular matrix biosynthesis or increased release of matrix degradative enzymes. These areas of minor extracellular matrix disruption are frequently seen near cytoplasmic regions containing increased numbers of electron-lucent vacuoles. SC marks Schlemm's canal. The magnification is XI 1,733.

Incorporation Time Courses amount for each GAG) were observed when the raw DPMs were averaged. Most of this variability is due In Figure 1, the results from time courses for the to differences in the total incorporation of label, total incorporation of 3H-glucosamine into the rather than from the biochemical analysis. When rep- GAGs of the TM, cornea and sclera are presented. licate analyses of pooled samples are conducted, the Similar curves are obtained for 35S-sulfate incorpora- variations are quite small. tion (data not shown). In the TM, the incorporation These GAG profiles are distinctive for each radio- of both labels is at steady state within 2 days. This is label and for each tissue. The relative incorporation not the case for either the cornea or the sclera, both of of 3H-glucosamine into the trabecular GAGs is: which show increasing incorporation for at least 7 22.3% HA, 27.9% CS, 21.3% DS, 5.9% KS, 17.7% HS days. The CAE profiles for the time courses of 35S- and 4.9% unidentified material. The relative incorpo- sulfate and 3H-glucosamine incorporation into the ration of 35S-sulfate into trabecular GAGs is: 0% TM show that the relative distribution into the re- HA, 32.9% CS, 34.8% DS, 7.7% KS, 13.8% HS and spective peaks is constant over the times studied (data 11.1% unidentified material. not shown). Effects of In Vitro Incubation Times Upon GAG Sequential Enzymatic Analysis of the GACs of Profiles theTM Based upon the morphological studies (Figs. 1-5), Sequential enzymatic degradation of the GAGs it appears that the TMs of explants that have been in from the TMs, corneas and scleras of explants re- culture for between 7 and 21 days resemble closely sulted in the relative incorporation profiles presented the TMs of fresh eyes. To determine a range of in in Figures 8 and 9, respectively. For comparison pur- vitro incubation times that produces a profile of tra- poses, we have included in Figure 8 (solid bars) the becular GAGs resembling that determined earlier for relative amounts of Alcian blue staining determined noncultured eyes,3 explants were cultured for several in our previous studies with uncultured human tra- time intervals prior to the initiation of the standard becular GAGs.3 The explants were preequilibrated in 48 hr incubation with radiolabels; GAGs were then culture for 7 days and incubated for 48 hr with 35S- analyzed as before. This study used standard explant sulfate and 3H-glucosamine. The GAGs were ex- conditions and eyes that were obtained within 48 hr tracted and analyzed enzymatically as outlined in the and were enucleated within 3 hr postmortem. The Methods section. To check for incomplete degrada- exception to this was the group of eyes which are tion in the individual steps, several pools of three to designated "fresh." In this group, labeling in culture five TMs were analyzed without increasing the en- was begun within 4 hr postmortem. The results for zyme incubation times or enzyme concentrations; no 3H-glucosamine incorporation into the respective significant differences were observed. The means of GAGs are presented in Figure 10. When the GAG the percentages of incorporation into each GAG have profiles which result from the different preequilibra- relatively small standard errors (SEM). Somewhat tion times are analyzed statistically, four obvious larger SEM values (approaching 10% of the total groups emerge. These groups are: (1) Explants from

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 1 HUMAN TRABECULAR GLYCO5AMINOGLYCAN5 / Acorr er ol. 95

Fig. 4. Electron micrographs of explants after 7 to 21 days in culture. After explants were in culture for 7 days, they were processed for TEM and typical micrographs are presented showing (a) trabecular celts and beams from the juxtacanalicular region and at lower magnification (b) cells and beams of the TM from the corneoscleral region. Micrographs are also presented of the cells and beams in the deep corneoscleral region of an explant that had been in culture for 14 days (c) and in the central corneoscleral region of an explant that had been in culture for 21 days (d). The magnifications are X9600, X7867, X8000 and XI 1,333, respectively. Note the prominent Golgi (arrow in a), phagocytic and/or secretory vesicles (arrow in d), mitochondria (arrow in c), rough endoplasmic reticulum (arrow in b), "curly" collagen (• in a) and the close association of the plasma membranes with each other and with the adjacent beams.

fresh eyes; (2) explants from 0 to 5 days in culture; (3) and the profiles obtained (Fig. 10), statistical analysis explants from 7 to 14 days in culture; and (4) explants is presented as a comparison of groups 2 and 4 above from 21 and 28 days in culture. Because of the resem- to the combined groups 1 and 3 of fresh and 7 to 14 blance between the trabecular GAG profiles that we days in culture. The (*) indicates significance at level previously obtained for pools of eyes prior to culture3 ofP< 0.005.

Downloaded from iovs.arvojournals.org on 10/01/2021 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1988 Vol. 29

Fig. 5. Electron micrographs of explants maintained for 28 and 49 days in culture. Micrographs of two explants are shown after 28 days in culture focusing on: (a) the corneoscleral region and (b) the juxtacanalicular region. These two views are not really "typical" of the whole explant, but were se- lected to emphasize the significant loss of beam integrity (* in b), decreased amounts of electron dense material in the beams near the cell surface (** in b), apparent compromise of the plasma membrane (near the beams in a), the presence of spaces between the nucleus and the nuclear membrane (arrow in b) and the change in appearance of the nucleus itself (arrow in a) that are becoming more apparent after this long in culture. SC marks Schlemm's canal. A lower-magnification micrograph (c) of the corneoscleral meshwork of an explant that had been maintained in culture for 49 days is also presented to show the extreme widespread beam disintegration (*), cellular degeneration with severe loss of membrane integrity and release of cytoplasmic components (single arrows) and the nuclear deterioration (double arrows) that predominates the explants after this long in culture. The original magnifications are x 13,200, x 12,000 and X3600, respectively.

As can be seen in Figure 10, several of the GAGs Other Observations and Controls are produced in significantly different amounts, when To test for an effect of the antibiotics in the media compared to the fresh and 7 to 14 day culture groups. upon the GAG profile, pairs of eyes from the same HA is produced in higher amounts from 0 to 5 days donor were cultured, one with and one without the in vitro, and in lower amounts from 21 to 28 days in addition of the antibiotic/antimycotic solution to the vitro. The other GAGs are different either in the media, for 7 days and then labeled for 48 hr. No early, or in the late groups, but not in both. No simple significant differences in GAG profiles were observed overall pattern for GAG production as a function of (data not shown) within four respective pairs of ex- culture preequilibration time is apparent. The results plants. from the analysis of 35S-sulfate incorporation into Individual variation (standard error of the mean the respective GAGs obtained in this study (data not < 10%) in the total radiolabel incorporated into tra- shown) are similar, although not identical and the becular GAGs in the cultures is much larger than the changes are slightly less pronounced. No general pat- observed differences between paired eyes from the terns are apparent here either. same donor. Retrospective regression analysis indi-

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 1 HUMAN TRABECULAR GLYCO5AMINOGLYCANS / Acorr er ol. 97

5OOO 8OO A TM TM 7OO « 3H . 35S CORNEA 6OO SCLERA JCE) tn 500 2

4-OO (C

3OO -SULfATE 2OO 35S-

1OO

O O CAE SLICE NUMBER 9OOO 01 23456789 10 B DAYS INCUBATED WITH PRECURSORS r 8OOO „ CORNEA Fig. 7. Time courses for the total incorporation of 3H-glucosa- SCLERA mine into the GAGs of the TM, cornea and sclera after various T • periods of continuous radiolabel exposure. Explants were preequil- 01 6OOO ibrated for 7 days prior to initiation of time courses. Media were changed every 48 hr and fresh label added. Values are means in 2 5OOO which three complete explants were used for each time point. I5 4OOO

En 3OOO and by very noticeable increases in media turbidity. c/> in •*» 2OOO Careful attention to the media and microscopic ob- i servation of media aliquots are recommended to safe- 1OOO guard against this infrequent problem. o Analysis of the radiolabeled macromolecules in the CAE SLICE NUMBER culture medium shows that less than 10% of the label incorporation into GAGs is secreted without being Fig. 6. Cellulosie acetate electrophoretic profiles of radiolabel incorporation into GAGs of the TM, cornea and sclera after standard 7-day preequilibration followed by 48 hr of incubation with labels. Panel A shows CAE profiles of 3H-glucosamine (solid line) 40 and 35S-sulfate (dashed line) incorporation into the GAGs of the

TM. Panel B shows the CAE profiles of 35S-sulfate incorporation TO 35 1 WM AB into the cornea (solid lines) and the sclera (dashed lines). Electro- OF 1 • 3H phoresis was in the direction of the arrow (negative-to-positive) and 30 1=1 3SS values are averages of three experiments using separate single ex- CO plants. Individual experiments used the three tissues from the same 1 25 explant and were run on the same CAE strip. 1 | 20 1 1 8 MINOGL o 15 | cates no significant effect of donor age on individual o o GAG production. However, it should be mentioned 10 Illllll ll

that in this analysis, the proportion of donors which IIHIIlj were under 50 years of age was small (9 of 41 total) 5 i w-\ and more data would be required to draw a meaning- ful conclusion on the effect of donor age. HA CS DSil KlS HS REM TYPE OF GLYCOSAMINOGLYCAN As might be expected, reduced culture pH has drastic effects upon GAG profiles and morphology, Fig. 8. Trabecular GAG incorporation profiles obtained by sequential enzymatic analysis. Explants were preequilibrated for 7 therefore media with a colored pH-indicator are ad- days, incubated with radiolabels for 48 hr and then the individual vantageous. Infrequently, yeast infections can occur GAGs were analyzed. Relative incorporation values for 3H-gluco- in cultures, nearly always in pairs of eyes suggesting samine (3H, open boxes) and 35S-sulfate (35S, hatched boxes) are the donor as the source of infection. In these cases the compared with Alcian Blue staining values (AB, solid boxes) re- 3 complete explants are quickly permeated (within 12 ported previously for pools of noncultured eyes. Incorporation values are means with vertical lines showing the SEM for 18 sam- hr) by these microorganisms. Infections are very eas- ples analyzed in five separate experiments; 12 were analyzed indi- ily detectable by media color changes (acidification vidually and the remaining six samples were analyzed as pools of turns the phenol red indicator yellow), a yeasty odor tissue from three explants each.

Downloaded from iovs.arvojournals.org on 10/01/2021 98 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1988 Vol. 29

4O eyes and to that reported previously by other investi- L 20 28 30 3H—GLU gators. ' " Comparison of our ultrastructural re- 35S-S sults with those of "activated" trabecular cells in cul- o ture7'22'26'31 leads us to conclude that our TM cells are to not "activated." Micrographs of TMs from explants 3 25 i >- 1 that have been in culture between 7 and 21 days do O 20 not show the migration from the beams, changes in z ^ 15 the heterochromatin and nuclear structure, increased numbers of mitochondria, overly prominent Golgi 1O complex, increased rough endoplasmic reticulum 5 with enlarged cisterns, or generally enlarged cells that are characteristic of this "activation." Cellular migra- O li nil liHA ICS DS KS HS REM tion is normally observed from meshwork explants which have been exposed to serum or growth factors, to phagocytic loads or to proteinases. In addition, 3H-GLU 35S-S migration of cells from the beams is observed from TMs which have been separated from the cornea and sclera before culture.7-20-22'25'26'29 From our studies, we cannot differentiate between these two potential contributing factors. The trabecular GAG profile that is synthesized by fresh explants or by explants from eyes stored for up to 48 hr and then cultured for 1 week prior to initiation of experiments is very similar to the GAG profile

HA CS DS KS HS REM TYPE OF GLYCOSAMINOGLYCAN Fig. 9. Corneal and scleral GAG incorporation profiles obtained by sequential enzymatic analysis. The incorporation profiles of 3H-glucosamine (solid bars) and 35S-sulfate (hatched bars) into the GAGs of the cornea (A) and sclera (B) in explant organ culture are shown. The actual explants and experimental details are the same as those presented in the caption to Figure 8, omitting the Alcian Blue data.

integrated into the extracellular matrix of the explant. No individual GAG predominates the labeled material released into the media by the explants. HA CS DS KS HS REM TYPE OF GLYCOSAMINOGLYCAN Discussion • P < 0.005 From these studies, we conclude that serum-free Fig. 10. Comparison of trabecular GAG incorporation profiles corneoscleral explant organ culture provides a satis- after various preequilibration times in vitro. Labeling was for 48 hr, begun after the designated number of days in vitro. Only 3H-glu- factory system for studies of the extracellular matrix cosamine data are shown here. Dark bars show the results for of the trabecular meshwork. With very fresh eyes, "fresh" explants (n = 5); horizontal-hatched bars show the results these studies can be initiated immediately. However, for explants labeled after from 0 to 5 days in culture (n = 14); open if the eyes have been stored for more than a few hours bars show the results for explants labeled after from 7 to 14 days in postmortem, then a 1 week preequilibration period in culture (n = 18); cross-hatched bars show results from cultures labeled after from 21 to 28 days in culture (n = 16). All eyes had culture is required prior to initiation of experiments. been stored at 5°C for up to 48 hr prior to initiation of culture, Sometime between 2 and 3 weeks in culture detri- except those designated "fresh," which were obtained within 4 hr mental changes begin to occur and results become postmortem and labeled immediately. Percentages of total 3H-glu- questionable. The supportive evidence for these con- cosamine incorporation values are means with vertical lines show- clusions is summarized below. ing the SEM and the (*) marks those which are significantly different (P < 0.005) from the mean values of all explants from the The ultrastructure of trabecular meshworks, which "fresh" and the 7 to 14 day groups as determined by unpaired have been in explant organ culture for a period of student t-test analysis. All analyses were conducted on individual from 7 to 21 days, is quite similar to that of "fresh" explants.

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 1 HUMAN TRABECULAR, GLYCO5AMINOGLYCANS / Acorr er ol. 99

reported previously for the TMs of noncultured TM as regards its morphology or its GAGs. Clearly, human eyes.3 A strict comparison is not possible since we have not evaluated other aspects of trabecu- here, since Alcian Blue staining is not quantitatively lar behavior, we cannot comment beyond this limited identical to either 3H-glucosamine or 35S-sulfate in- contention. It is also important to mention that care- corporation into the individual GAGs. However, the ful interpretation will be required in evaluations of profiles should be, and are, sufficiently similar for us the effects of drugs or natural modulators upon the to conclude that within the time window from 7 to at TM in these corneoscleral explants, since trabecular least 14 days or when "fresh" eyes are used the cor- changes could be indirect as a consequence of neoscleral explant culture system provides a very changes in the adjacent tissues. good approximation of the in vivo state of the TM. Trabecular cell culture is one step further removed This conclusion has recently been verified by the use from the in vivo state and early studies with dividing of a new microanalytic method to determine the trabecular cells20 found very high HA levels synthe- GAG content of individual human TMs which had 32 34 33 sized and secreted into the media. A GAG profile not been cultured. ' In one of these studies, very similar to ours has recently been reported for human trabecular GAG microanalysis results were 27 3 densely-confluent trabecular cell cultures. This lat- compared directly with our published values and ter study reported the same, very high hyaluronic acid very good agreement was observed. synthesis (75-91% of the total incorporation into The incorporation profile of the trabecular GAGs GAGs) that has been reported for most trabecular cell is also clearly distinct from that of the cornea or the cultures, while the cells were in the proliferative sclera in our culture system. The time courses for phase.7'18"20*22'27 However, it is only after 2-3 weeks in total radiolabel incorporation are also quite different; cell culture, or when dense confluence is reached, that the maximal incorporation of both labels into the the profile of trabecular GAGs changes; the produc- TM, which probably reflects a steady-state balance tion of HA drops to around 10% while the production between GAG synthesis and turnover, occurs long of other GAGs, particularly HS, increases.7-20'22-27 We before the maximum is reached in the other tissues of have recently compared the proteoglycans produced the explant. This probably means that the extracellu- by our explant organ culture with those produced in lar matrix of the TM is more dynamic than that of the trabecular cell culture36"38 and found the similarities adjacent tissues, although other explanations are pos- to be more striking than the differences. This has also sible. Electrophoretic migration profiles of the GAGs recently been verified.39 of these three tissues are also quite different. These We conclude then that either cell culture or cor- three lines of evidence and the additional fact that neoscleral explant organ culture will reproduce the very little labeled macromolecular material is found intact GAG profile faithfully, if treated appropriately, free in the medium argue strongly against the possi- and that both systems have considerable potential, bility of the cornea or sclera contributing to our deter- albeit somewhat different ones, for future studies. mination of the trabecular GAG profile. Key words: aqueous outflow pathway, glycosaminoglycans, One difficulty of all culture studies is establishing human corneoscleral explant organ culture, trabecular ex- the extent to which the behavior of the cells in culture tracellular matrix reflects the in vivo state. The relatively undisturbed state of corneoscleral explants, the relatively avascu- Acknowledgments lar nature of the TM and the exclusion of serum or We are grateful to Emmanuel Tanne, MD, Mike Mar- growth factors favor a close resemblance between quette and Mike Gordon of the Oregon Lions' Eye Banks cultured and in vivo trabecular behavior. Observa- for providing human eyes; Mary Westcott-Hodson for tions similar to ours have been reported for the cor- technical assistance, Anne Truesdale for print and film pro- neal GAGs in a very similar corneoscleral explant cessing, Daniel Carr for assistance with some experiments organ culture system.24-35 We do observe minor cor- and Drs. Jon Polansky, Paul Knepper and Edmund Crean for suggestions and ideas. Brief accounts of portions of this neal opacity and hydration in the explant cultures, work have been presented in abstract form.1-2 suggesting that our conditions may be less than per- fect for some types of long-term studies of the cornea. References We also observe differences between scleral CS levels 3 1. Acott TS, Passo MS, Westcott M, and Van Buskirk EM: Tra- in our culture (Fig. 9b) and our previous analysis. becular meshwork glycosaminoglycans in excised primate eyes We do not assume that the sclera is maintained opti- and in human organ cultures. ARVO Abstracts. Invest Oph- mally in this culture system, since there are no serum thalmol Vis Sci 24(Suppl):86, 1983. or growth factors in the media and the sclera is a 2. Acott TS, Westcott-Hodson M, Samples JR, and Van Buskirk EM: Human trabecular meshwork glycosaminoglycan produc- vascular tissue. We do contend, from our results, that tion in explant organ culture. ARVO Abstracts. Invest Oph- the other tissues in the explant are not affecting the thalmol Vis Sci 25(Suppl):100, 1984.

Downloaded from iovs.arvojournals.org on 10/01/2021 100 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Jonuory 1988 Vol. 29

3. Acott TS, Westcott M, Passo MS, and Van Buskirk EM: Tra- 22. Rohen JW, Schachtschabel DO, Lutjen-Drecoll E, Rohrbach becular meshwork glycosaminoglycans in human and cyno- M, and Berghoff K: Morphological and biochemical studies on molgus monkey eyes. Invest Ophthalmol Vis Sci 26:1320, cell and tissue cultures of human trabecular meshwork. In 1985. Glaucoma Update II, Krieglstein GK and Leydhecker W, edi- 4. Barany EH: In vitro studies of the resistance to flow through tors. Amsterdam, Springer-Verlag, 1983, pp. 39-43. the angle of the anterior chamber. Acta Societatis Medicorum 23. Van Buskirk EM, Pond V, Rosenquist RC, and Acott TS: Upsal 54:260, 1953. Argon laser trabeculoplasty: Studies on mechanism of action. 5. Barany EH and Scotchbrook S: Influence of testicular hyal- Ophthalmology 91:1005, 1984. uronidase on the resistance to flow through the angle of the 24. Funderburgh JL, Stenzel-Johnson PR, and Chandler JW: anterior chamber. Acta Physiol Scand 30:240, 1954. Corneal glycosaminoglycan synthesis in long-term organ cul- 6. Grierson I, Lee WR, and Abraham S: A light microscopic ture. Invest Ophthalmol Vis Sci 24:208, 1983. study of the effects of testicular hyaluronidase on the outflow 25. Polansky JR, Weinreb RN, Baxter JD, and Alvarado J: system of a baboon (Papio cynocephalus). Invest Ophthalmol Human trabecular cells: I. Establishment in tissue culture and Vis Sci 18:356, 1979. growth characteristics. Invest Ophthalmol Vis Sci 18:1043, 7. Rohen JW and Liitjen-Drecoll E: Biology of the trabecular 1979. meshwork. In Basic Aspects of Glaucoma Research, Liitjen- 26. Grierson I, Marshall J, and Robbins E: Human trabecular Drecoll E, editor. Stuttgart, K.F. Schattauer Verlag, 1982, pp. meshwork in primary culture: A morphological and autoradio- 41-66. graphic study. Exp Eye Res 37:349, 1983. 8. Knepper PA, Collins JA, Weinstein HG, and Breen M: 27. Crean EV, Tyson SL, and Richardson TM: Factors influencing Aqueous outflow pathway complex carbohydrate synthesis in glycosaminoglycan synthesis by calf trabecular meshwork cell vitro. Invest Ophthalmol Vis Sci 24:1546, 1984. cultures. Exp Eye Res 43:365, 1986. 9. Knepper PA, Farbman AI, and Telser AG: Aqueous outflow 28. Hogan MJ, Alvarado JA, and Weddell JE: Histology of the pathway glycosaminoglycans. Exp Eye Res 32:265, 1981. Human Eye: An Atlas and Textbook. Philadelphia, W.B. 10. Borcherding MS, Blacik LJ, Sittig RA, Bizzell JW, Breen M, SaundersCo., 1971. and Weinstein HG: Proteoglycans and collagen fibreorganiza - 29. Alvarado JA, Wood I, and Polansky JR: Human trabecular tion in human corneoscleral tissue. Exp Eye Res 21:59, 1975. cells: II. Growth pattern and ultrastructural characteristics. In- 11. Zimmerman LE: Demonstration of hyaluronidase-sensitive vest Ophthalmol Vis Sci 23:464, 1982. acid mucopolysaccharide. Am J Ophthalmol 44:1, 1957. 30. Tripathi RC and Tripathi BJ: Human trabecular endothelium, 12. Richardson TM: Distribution of glycosaminoglycans in the corneal endothelium, keratocytes, and scleral fibroblasts in aqueous outflow system of the cat. Invest Ophthalmol Vis Sci primary culture: A comparative study of growth characteris- 22:319, 1982. tics, morphology, and phagocytic activity by light and scanning 13. Rodrigues MM, Katz SI, Foidart J-M, and Spaeth GL: Colla- electron microscopy. Exp Eye Res 35:611, 1982. gen, factor VIII antigen, and immunoglobins in the human 31. Rohen JW, Schachtschabel DO, and Whermann R: Structural aqueous drainage channels. Am Acad Ophthalmol 87:337, changes of human and monkey trabecular meshwork follow- 1980. ing in vitro cultivation. Graefes Arch Clin Exp Ophthalmol 14. Hotchkiss ML, Brown RH, Newsome DA, and Pollack IP: 218:225, 1982. Harvesting trabecular meshwork for tissue culture and the use 32. Collins JA and Knepper PA: Analysis of human trabecular of type IV collagen and heparin sulfate to identify trabecular meshwork glycosaminoglycans by high performance liquid cells. ARVO Abstracts. Invest Ophthalmol Vis Sci chromatography. ARVO Abstracts. Invest Ophthalmol Vis Sci 26(Suppl):lll, 1985. 26(Suppl):333, 1985. 15. Alvarado J, Polansky J, and Newsome D: Human trabecular 33. Collins JA, Gum GG, Palmberg PF, and Knepper PA: Micro- meshwork ECM: Fluorescence staining of collagen subtypes, scale analysis of glycosaminoglycans from single human tra- laminin, fibronectin and heparan sulfate proteoglycan using becular meshwork. ARVO Abstracts. Invest Ophthalmol Vis monospecific antibodies. ARVO Abstracts. Invest Ophthalmol Sci27(Suppl):162, 1986. VisSci26(Suppl):lll, 1985. 34. Goossens W, Higbee RG, Palmberg PF, and Knepper PA: Histochemical identification of glycosaminoglycans in pri- 16. Knepper PA, Farbman AI, and Telser AG: Exogenous hyal- mary open angle glaucoma. ARVO Abstracts. Invest Ophthal- uronidases and degradation of hyaluronic acid in the rabbit eye. Invest Ophthalmol Vis Sci 25:286, 1984. mol Vis Sci 27(Suppl):162, 1986. 35. Klintworth GK and Smith CF: A comparative study of extra- 17. Van Buskirk EM and Brett J: The canine eye: In vitro dissolu- cellular sulfated glycosaminoglycans synthesized by rabbit tion of the barriers to aqueous outflow. Invest Ophthalmol Vis corneal fibroblasts in organ and confluent cultures. Lab Invest Sci 17:258, 1978. 35:258, 1976. 18. Schachtschabel DO, Bigalke B, and Rohen JW: Production of 36. Acott TS, Truesdale AT, Samples JR, and Van Buskirk EM: glycosaminoglycans by cell cultures of the trabecular mesh- Trabecular extracellular matrix synthesis in human explant work of the primate eye. Exp Eye Res 24:71, 1977. organ culture. ARVO Abstracts. Invest Ophthalmol Vis Sci 19. Schachtschabel DO, Rohen JW, Wever J, and Sames K: Syn- 27(Suppl):211, 1986. thesis and composition of glycosaminoglycans by cultured 37. Nobis C, Polansky J, Acott T, Van Buskirk EM, and Alvarado human trabecular meshwork cells. Graefes Arch Clin Exp J: Evaluations of proteoglycans and glycoproteins from cul- Ophthalmol 218:113, 1982. tured human trabecular cells. ARVO Abstracts. Invest Oph- 20. Polansky JR, Wood IS, Maglio MT, and Alvarado JA: Trabec- thalmol Vis Sci 27(Suppl):164, 1986. ular meshwork cell culture in glaucoma research: Evaluation of 38. Acott TS, Nobis CA, and Van Buskirk EM: Studies of human biological activity and structural properties of human trabecu- trabecular extracellular matrix proteoglycans. 7th Interna- lar cells in vitro. Ophthalmology 91:580, 1984. tional Congress of Eye Res #510, 1986. 21. Hassell JR, Newsome DA, and Ballintine EJ: Synthesis of ex- 39. Elvart JL, Kurosawa A, Yue BYJ, Einer VM, and Tso MOM: tracellular matrix components by trabecular meshwork in Monkey trabecular meshwork cells in culture: Morphologic organ culture. ARVO Abstracts. Invest Ophthalmol Vis Sci and biochemical studies. ARVO Abstracts. Invest Ophthalmol 19(Suppl):273, 1980. VisSci27(Suppl):l65, 1986.

Downloaded from iovs.arvojournals.org on 10/01/2021