moderate fuchs' endothelial dystrophy atpase pump site density

Investigative Ophthalmology & Visual Science, Vol. 30, No. 7, July 1989Copyright © Association for Research in Vision and Ophthalmology

Moderate Fuchs' Endorhelial DystrophyATPase Pump Site Density

Mitchell D. McCartney,*% Thomas O. Wood,f and Barbara J. McLaughlin*

The Na+, K+-ATPase pump site density on corneal endothelial cells from Fuchs' endothelial dys-trophy corneas has been shown to be drastically decreased in end-stage disease (McCartney et al,Invest Ophthalmol Vis Sci 28:1955,1987) and significantly increased in the early stages (Geroski etal, Ophthalmology 92:759,1985) as compared to normal endothelium. In order to provide values forcorneas between these two extremes, eye bank corneas from donors with no evidence of corneal edemabut with guttata across the extent of the cornea were processed for autoradiography as well asimmunohistochemistry. Pump site density was increased compared to end-stage disease but was lessthan values reported for either functional tissue or early stage disease. Similarly, immunohistochem-istry results showed the amount of Na+, K+-ATPase antibody localization to be increased in respect toend-stage disease, but reduced as compared to functional tissue. These results suggest that pump sitedensity on endothelial cells affected with Fuchs' endothelial dystrophy follows a gradual declinetowards end-stage disease values as opposed to a sudden sharp deterioration after an initial increase.Invest Ophthalmol Vis Sci 30:1560-1564,1989

Fuchs' endothelial dystrophy is a primary disorderof the corneal endothelium that accounts for approxi-mately 10% of the penetrating keratoplasty per-formed annually.1 Fuchs' is a progressive disease thatis inherited as an autosomal dominant trait and gen-erally affects women more severely.2 Characteristicguttata form posterior to Descemet's membrane andthe endothelial cells get progressively thinner withirregular borders as the disease advances. Concomi-tant with these morphological changes corneal edemaand thickness increase.

Previous studies on early Fuchs' endothelial dys-trophy have reported increased permeability3"5 andincreased Na+, K+-ATPase pump site density ascompared to normal.6 In end-stage disease, wherethere is edema from central to peripheral regions of

From the *Department of Ophthalmology and Visual Sciences,University of Louisville School of Medicine, Kentucky Lions EyeResearch Institute, Louisville, Kentucky and the tDepartment ofOphthalmology, The College of Medicine, The University of Ten-nessee, Memphis, Tennessee.

i Current address: Alcon Laboratories, Inc., Fort Worth, Texas.Supported by USPHS Grant EY-05673 and Baptist Memorial

Hospital Clinical Innovations Fund, Baptist Memorial Hospital,Memphis, Tennessee.

Submitted for publication: August 12, 1988; accepted January18, 1989.

Reprint requests to: Dr. Barbara J. McLaughlin, Department ofOphthalmology, University of Louisville, Kentucky Lions Eye Re-search Institute, 301 East Muhammad Ali Boulevard, Louisville,KY 40202.

the cornea, the pump site density is significantly re-duced in comparison to functional corneal endothe-lium.7 In order to obtain values between these twoextremes, the current study has used autoradiographyand immunohistochemistry to examine eye bankcorneas with no history of edema, but with guttataacross the width of the cornea.

Materials and Methods

Two eye bank corneas (69-year-old female and 79-year-old female), obtained within 1 hr postmortem,were judged to have "moderate" Fuchs' endothelialdystrophy. Corneal guttata were present across thewidth of the cornea, but no case history of cornealedema was noted. The corneas were excised from theglobes and the corneas placed in McKarey-Kaufmanmedia.8 The corneas were then divided in half withone-half being processed for autoradiography and theother for immunohistochemistry (see below).

Two keratoconus corneas were obtained at thetime of transplant surgery from keratoconus eyes thatwere examined preoperatively to ensure that therewere no endothelial abnormalities or previous historyof hydrops. Corneal buttons were placed inMcCarey-Kaufman media8 immediately after surgi-cal dissection and transported to the laboratory whereprocessing for immunohistochemical localization ofNa+, K+-ATPase began within the same time periodas for the Fuchs' corneas.

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No. 7 MODERATE FUCHS' ATPASE PUMP SITE DEN5ITY / McCorrney er ol 1561

Autoradiography

The half of the cornea designated for autoradiogra-phy was processed according to the methodologypreviously described.7 Briefly, the cornea was cut inhalf with the half serving as a control being preincu-bated in 10~6 M nonradioactive (cold) ouabain (ICNPharmaceutical, Plainview, NY). The preincubationof the tissue with nonradioactive ouabain providedcompetitive inhibition for the noncovalent bindingsteroid nucleus of the radioactive ouabain with thereceptor.9 Both halves were then incubated in 7X 10~7 M 3H-ouabain (New England Nuclear, Bos-ton, MA: specific activity 20 Ci/mMol) for 1 hr. Fol-lowing extensive rinsing in 0.1 M sodium cacodylatebuffer (Polysciences, Warrington, PA), the halveswere frozen in liquid nitrogen, freeze-dried, osmi-cated under vacuum and embedded in Spurr resin.Semithin sections (0.5 nm) were placed on glassslides, covered with Kodak NTB-2 emulsion (East-man Kodak, Rochester, NY), exposed and photo-graphically developed.

In order to calculate the number of silver grains per100 Jim2 of cell, bright and darkfield light micro-graphs were taken of the same section. Cell area mea-surements were calculated from morphometric anal-ysis of the brightfield photographs which corre-sponded to those areas photographed in darkfield forsilver grain counts. Silver grains were counted onlyover those areas shown to contain endothelial cells inbrightfield micrographs. Established formulas10 wereused to determine which silver grains should be con-sidered arising from radiation bound to the tissue.Background silver grain density was determinedusing the alternating grid square method" over areascontaining no tissue. Comparisons were subsequentlymade between the experimental and control tissue foreach cornea. Silver grain development in the basalepithelium served to confirm the viability of the tech-nique for each cornea.

Immunohistochemistry

The half of the Fuchs' corneas designated for im-munohistochemistry as well as the keratoconus sur-gical buttons removed at the time of transplant sur-gery were processed according to the methodologypreviously described.l2 Briefly, the tissue was fixed for1 hr in an aldehyde fixative (3% paraformaldehyde,0.1% glutaraldehyde), rinsed and cryosectioned. In-cubation of the tissue in the primary antisera ob-tained from whole serum goat anti-Na+, K+-ATPase(Accurate Chemical and Scientific Corporation,Westburg, NY) using a modification of the(NH4)2SO4 precipitation technique of Jonak13 and

Fig. 1. Darkfield light micrograph showing the silver grain den-sity over the endothelium from the periphery of a "moderate"Fuchs' endothelial dystrophy cornea. Silver grain (arrows) densityvaried between central (Fig. 2) and peripheral areas. However, bothcentral and peripheral grain densities were decreased in compari-son to functional tissue. S = stroma; bar = 20 ^m.

secondary antisera, swine anti-goat horseradish per-oxidase (HRP) conjugate (Boehringer MannheimBiochemicals, Indianapolis, IN), was for 12 hreach at4°C with three washes with 0.05 M tris buffered sa-line (TBS) of 20 min each before and between anti-sera. Following a final 20 min wash with TBS, thelocation of the HRP-antibody complex was visual-ized using the chromagen 3,3'diaminobenzidine tet-rahydrochloride (DAB) (Sigma, St. Louis, MO).

Control tissue was incubated in either: (1) nonim-mune serum followed by secondary antibody; (2) pri-mary antibody that had been preabsorbed with excessantigen (rabbit kidney Na+, K+-ATPase) followed bysecondary antibody; or (3) secondary antibody alone.The tissue was then processed in the identical mannerto experimental tissue.

Results

AutoradiographyThe endothelium (Fig. I) and the basal epithelium

of the moderate Fuchs' corneas showed silver graindevelopment that was different from the values cal-culated for control tissue (Experimental: 41.35; Con-trol: 5.11 per 100 nm2). Qualitatively, this silver graindevelopment appeared to vary from one region toanother in these corneas (Figs. 1, 2). Silver grain den-sity in the control endothelium was greatly reduced(Fig. 3). When the silver grain density over the endo-thelial cells was quantified on the moderate Fuchs'corneas, there was an average of 41.35 silver grainsper 100 ^m2.


1562 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1989 Vol. 30

Fig. 2. Darkfield light micrograph showing silver grain deposi-tion over the endothelium from the central region of a "moderate"Fuchs1 endothelial dystrophy cornea. The silver grain (arrows)densities varied between central and peripheral (Fig. 1) regions. S= stroma; bar = 20 ftm.

I m munohistochemistry

En face sections of the endothelium from the kera-toconus buttons showed an even distribution of anti-body labeling along the hexagonally arranged lateralmembranes of these functional cells (Fig. 4). Sectionsof the moderate Fuch's endothelial dystrophy corneas

Fig. 3. Darkfield light micrograph showing silver grain develop-ment in the control half of the cornea illustrated in Figure 1. Silvergrain (arrows) density in the endothelium of the control tissue wasdrastically reduced in comparison to experimental tissue. A fold inthe tissue section (open arrows) appears as a white line in thisdarkfield micrograph. S = stroma; bar = 20 fim.

showed a patchy antibody labeling (Fig. 5) along thelateral borders of these pleomorphic cells. No sub-stantial differences could be detected in the amountof antibody labeling between central and peripheralendothelial cells. Control tissue incubated with non-immune serum or preabsorbed primary antibodyshowed little or no antibody labeling.

Discussion

Previous studies on Na+, K+-ATPase pump sitedensity in end-stage Fuchs' endothelial dystrophy, astage characterized by pan-corneal edema, have indi-cated that the pump site density is significantly re-duced in comparison to functional tissue.7 In con-trast, the pump site density on early Fuchs' dystrophyis significantly increased as compared to normal cor-neal endothelium.6 It appears that as the disease pro-gresses there is a transition from increased pumpdensity to decreased pump site density during end-stage disease. It is not known as yet when the transi-tion from increased to decreased pump site densityoccurs and whether this transition is abrupt or grad-ual. The current study has examined corneas with"moderate" Fuchs' endothelial dystrophy using auto-radiography and immunohistochemistry in an effortto provide data for a disease stage between these twoextremes.

Corneas used in this study had guttata across thewidth of the cornea but no reported history of cornealedema. Accordingly, these corneas were judged to beat a stage of disease between early and end-stage.Quantitative assessment of the endothelial pump sitedensity on these moderate Fuchs' corneas showedthat pump site density appeared to be increased incomparison to end-stage disease7 but less than valuesreported for functional corneal endothelium7 andearly Fuchs' dystrophy.6 These data suggest that thetransition from increased levels in early Fuchs' to thelow levels in end-stage disease is a gradual rather thanan abrupt decline. The starting point of this decline orwhat the triggering mechanism is remains to be elu-cidated by obtaining even earlier stages than wereavailable in the current study.

The Na+, K+-ATPase antibody aliquots used inthis study had previously been used to label func-tional and dysfunctional human corneal endothe-lium as well as the positive control tissue, rabbit kid-ney.12 Antibody labeling on the Fuchs' corneas in thisstudy appeared to be increased from the amount oflabeling previously shown in end-stage disease, butstill was qualitatively less than functional tissue. Na+,K+-ATPase has previously been shown to be local-ized to lateral membranes of corneal endothelialcells.14"16 The antibody labeling of the lateral mem-


No. 7 MODERATE FUCHS' ATPASE PUMP SITE DENSITY / McCartney er al 1560

Fig. 4. Light micrographof cryosectioned functionalcorneal endothelium (kera-toconus) incubated in theprimary antibody (goatanti-Na+, K+-ATPase) fol-lowed by incubation in thesecondary antibody (swineanti-goat HRP). This enface section shows denseantibody labeling (arrows)along the lateral borders ofthe endothelial cells. Bar= 20 ^m.

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branes of keratoconus endothelial cells, corneal endo-thelial cells previously shown to be analogous to eyebank corneal endothelium,7'12'17"20 appears to be uni-form, while labeling on end-stage disease endothelialcells is deposited sparsely on the lateral membranes ofthese thinned cells.12 The patchy deposition of theantibody label in the current study, in a morphologi-cal pattern midway between functional and dysfunc-tional corneal endothelium, suggests that the distri-bution of Na+, K+-ATPase as well as the number ofpump sites may change as the disease progresses.

Studies on the early stages of Fuchs' endothelialdystrophy have reported both increased endothelialpermeability3"5 as well as endothelial permeabilitythat is not significantly different from control eyes.21

What initially appears as conflicting evidence may infact be due to the variability of this disease entityand/or a difficulty in staging disease severity. Fuchs'endothelial dystrophy is a progressive disease thatstarts centrally and spreads peripherally.22"25 This

progression has been visualized using a cytochemicalstain, cytochrome oxidase,19 which is an indicator ofthe functional activity of the cells.26 It appears thatthere is a spectrum of disease severity containedwithin one cornea and that this spectrum may not beaccounted for in any of the disease staging paradigms.Krachmer et al27 and Wilson and Bourne28 have sug-gested stages that are a reflection of the severity ofguttata present during slit-lamp examination. Al-though these stages can be used for relative divisionsof the overall severity of the disease, the inherentproblem of variation amongst the cells, which hasbeen noted using invasive techniques,1719 remains.These difficulties were evident in the current studywhere the overall stage of the disease was classified as"moderate" but silver grain densities were increasedin peripheral cells compared to central cells. In stud-ies on Fuchs' endothelial dystrophy the overall dis-ease severity may be classified in broad terms, but apotential problem exists whenever cellular parame-

Fig. 5. Light micrographof a cryosectioned "moder-ate" Fuchs' endothelial dys-trophy cornea incubated ingoat anti-Na+, K+-ATPaseantibody followed by swineanti-goat HRP. The anti-body labeling (arrows) ap-peared patchy along the lat-eral borders of these pleo-morphic cells. Bar = 20 ̂ m.


1564 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1989 Vol. 30

ters from various studies are compared. Future stud-ies may require comparisons based on the overallseverity of the disease as well as the measurement ofindividual parameters in discrete areas of the cornea.

In conclusion, this study has provided data sug-gesting that the endothelial cell pump site density on"moderate" Fuchs' endothelial dystrophy corneas ismidway between functional and end-stage diseasevalues. We suggest that there is a gradual transitionfrom the increase Na+, K+-ATPase pump site valuesin early Fuchs'6 to the decreased end-stage valuesshown for these cells,7 rather than an abrupt deterio-ration after an initial increase.

Key words: ATPase, autoradiography, immunohistochem-istry, moderate Fuchs' endothelial dystrophy, human

References

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15. Leuenberger P and Novikoff A: Localization of transportadensosine triphosphatase in rat cornea. J Cell Biol 60:721,1974.

16. Tervo T and Palkama A: Electron microscopic localization ofadenosine triphosphatase (Na+, K+-ATPase) activity in the ratcornea. Exp Eye Res 21:269, 1975.

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18. McLaughlin BJ, Wood TO, and Caldwell RB: Quantitativeanalysis of freeze-fracture changes in human corneal endothe-lium dysfunction. ARVO Abstracts. Invest Ophthalmol Vis Sci27(Suppl):71, 1986.

19. Tuberville AW, Wood TO, and McLaughlin BJ: Cytochromeoxidase activity of Fuchs' endothelial dystrophy. Curr Eye Res5:939, 1986.

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21. Wilson SE, Bourne WM, and Brubaker RF: Effect of dexa-methasone on corneal endothelial function in Fuchs' dys-trophy. Invest Ophthalmol Vis Sci 29:357, 1988.

22. Waring III GO, Rodrigues MM, and Laibson PR: Cornealdystrophies: II. Endothelial dystrophies. Surv Ophthalmol23:147, 1978.

23. Hogan MJ, Wood I, and Fine M: Fuchs' endothelial dystrophyof the cornea. Am J Ophthalmol 78:363, 1974.

24. Keyes J and Holmberg A: The fine structure of the cornea inFuchs' endothelial dystrophy. Invest Ophthalmol 3:47, 1964.

25. Iwamoto T and Devoe AG: Electron microscopic studies onFuchs' combined dystrophy: I. Posterior portion of the cornea.Invest Ophthalmol 10:9, 1971.

26. Wong-Riley MTT: Changes in the visual system of monocu-larly sutured or enucleated cats demonstrable with cytochromeoxidase histochemistry. Brain Res 171:11, 1979.

27. Krachmer JH, Purcell JJ, Young CW, and Bucher KD: Corneaendothelial dystrophy: A study of 64 families. Arch Ophthal-mol 96:2036, 1978.

28. Wilson SE and Bourne WM: Corneal endothelial function inFuchs' dystrophy. Fifteenth Cornea Research Conference,Boston, Massachusetts, 1987, p. 58.


moderate fuchs' endothelial dystrophy atpase pump site density

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