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The Balance between Corneal Transparency and EdemaThe Proctor Lecture

Henry F. Edelhauser

The corneas of all animal species remain transparent be-cause of the epithelial barrier, the stromal latticelike ar-

rangement of the collagen fibrils, and the endothelial cells.Depending on the species, the corneal epithelium, the cornealendothelium, or both have a major role in regulating cornealhydration. The ultimate goal is for the cornea to remain trans-parent and to provide the major refractive function of the eye.The one exception is that of the aquatic fishes, since theirlenses serve in the major refractive role.

The comparative optical characteristics of the vertebrateeye have been eloquently summarized in Gordon Walls’ text,The Vertebrate Eye and Its Adaptive Radiations.1 The pur-pose of this review is to discuss the various factors that regu-late corneal hydration and transparency and to illustrate someclinical situations in which the function of the corneal endo-thelium becomes compromised.

CORNEAL STRUCTURAL ADAPTATIONS

The animal cornea consists of a basic structure of two coveringlayers—the epithelium and endothelium—and a central colla-gen stroma (Fig. 1). Variations in the thickness of the cornealepithelium are a structural adaptation that occurs in fishes andallows them to adapt to a wide variety of environmental con-ditions. The fish cornea, as well as that of all other animals,remains transparent, because it transmits greater than 90% ofthe incident visible light. Besides structural adaptations, cor-neal transparency is preserved by cellular active transportmechanisms, which keep the hydrophilic corneal stroma lesshydrated than it can become. The ideal physiological cornealhydration is approximately 78% in humans.2 An increase ordecrease from this ideal level can result in corneal opacity.Several studies have established that the limiting layers of thecornea are the epithelium and endothelium, which are sites ofactive ion transport that regulate the hydration of the hydro-philic stroma.3–6 Further studies have shown in mammals thata reduction of corneal metabolism (i.e., anaerobiosis, or low-ered temperature) adversely affects the barrier properties andtransport functions of both the epithelium and endothelium,resulting in an increase in corneal thickness and a loss oftransparency.4,7 The elasmobranch (sharks, skates, and rays)cornea resists swelling because of unique structural adapta-tions in the stroma (i.e., sutural fibers). Smelser7 has elegantlyshown that the shark cornea does not swell or become edem-atous from a loss of epithelial barrier function. The corneas ofmore advanced teleosts (salt and freshwater bony fish) swell

and become edematous and opaque when the epithelial barrieris compromised.

Various structural and metabolic adaptations develop in thecornea that enable aquatic animals to maintain transparency.8

For example, elasmobranchs have a relatively thin cornea (160�m) of which the epithelium makes up approximately onethird of the thickness.9–11 The corneal stroma of elasmo-branchs resists swelling when placed in deionized water orwhen held at 4°C for 12 hours.7,9 This is also a characteristic ofthe spectacle of the sea lamprey, which is the most primitivefish in the evolutionary tree (Fig. 1).12,13 Stromal swelling islimited in these animals, because sutural fiber complexes serveto hold the collagen lamellae together by interconnecting ad-jacent collagen bundles (Fig. 1). The sutural fibers have beenfound to be composed of type I collagen fibrils.14 The imper-meable nature of the elasmobranch corneal epithelium, as inmost other fishes studied, is another major factor that contrib-utes to the maintenance of corneal transparency.8 In contrast,the corneal endothelium of the elasmobranch is primitive andhas a minor physiological role in corneal hydration. It can bestbe observed histologically or with specular microscopy.9,10,12

The more advanced marine teleosts (e.g., sculpin) differfrom the elasmobranchs in that the teleost cornea is composedof two distinct layers. It can be mechanically or histologicallyseparated into an outer stroma and an inner stroma (Fig. 1).This was first described by Fisher and Zadunaisky,9 who visu-alized five morphologically distinct zones with the specularmicroscope. The outer and inner stroma are not substantiallyattached to one another, but appear to be slightly adherent toone another through a mucoid layer (similar to that foundbetween the primary spectacle and the cornea of the sealamprey).15 In contrast to the elasmobranch corneal epithe-lium, the epithelium of salt water teleosts comprises only 14%of the total corneal thickness. It has been shown to be imper-meable to water8; however, the stroma becomes edematousand opaque when kept at 2°C for up to 22 hours.9 Evaluationof swelling characteristics have shown that the outer stromaexhibits more swelling than the inner stroma.7 This greaterswelling ability is related to the different anatomic arrange-ments of the collagen fibrils in the two layers. The collagenfibrils of the inner layer are compact and highly interwoven,whereas the collagen fibers of the outer layer are spread apartand loose (Fig. 1). With the existence of a large osmoticgradient across the cornea, the loose stroma of the outer layermay easily become overhydrated. Thus, an additional barriercontrolling corneal hydration in the salt water teleost is foundin the endothelium and possibly anterior to Descemet’s mem-brane. These fish possess a thicker Descemet’s membrane anda more well-developed endothelium than the elasmobranchs.Ultrastructure studies have shown an extensive iridescent la-mellar structure anterior to Descemet’s membrane that mayassist in removing corneal water and conserving intraocularfluid. This structure has also been speculated to function as arefraction gradient for absorbing various light rays.

From the Emory Eye Center, Department of Ophthalmology,Emory University School of Medicine, Atlanta, Georgia.

Supported in part by National Eye Institute Grants EY00933 andP30-EY06360, an unrestricted grant from Research to Prevent Blind-ness, and a grant from the Alcon Research Institute.

Disclosure: H.F. Edelhauser, Alcon Research Institute (F)Corresponding author: Henry F. Edelhauser, Emory Eye Center,

1365-B Clifton Road, N.E., Atlanta, GA 30322; [email protected].

DOI:10.1167/iovs.05–1139Investigative Ophthalmology & Visual Science, May 2006, Vol. 47, No. 5Copyright © Association for Research in Vision and Ophthalmology

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The freshwater teleost (e.g., rainbow trout) illustrates thenext evolutionary step in the development of the cornea. Itmaintains ideal hydration and transparency by possessing athick corneal epithelium that comprises 40% of the total cor-neal thickness16,17 (Fig. 1). This has been described bySmelser7 for the carp cornea, one species of freshwater teleost.Therefore, both the elasmobranch and the teleost maintaintheir ideal hydration levels and transparency, primarily by vir-tue of their epithelia. The freshwater teleost cornea has onecontinuous stroma that consists of bands of collagen fibrilsinterspersed with keratocytes. On removal of the corneal epi-thelium, marked stromal swelling occurs in fresh water.13

The central corneal thickness of most mammalian corneasvaries from 0.200 mm in the guinea pig to 0.809 mm in thebovine cornea; human corneas typically average �0.540 mm inthickness (Fig. 1). In mammals, corneal transparency is primar-ily maintained by the corneal endothelium.11 Similar to less-advanced animals, corneal edema and stromal swelling result inopacification. Both the epithelium and endothelium of themammalian cornea prevent corneal swelling by functioning asdiffusion barriers to the fluid (tears or aqueous humor) and byacting as sites of active ion transport, to induce the osmoticmovement of the water out of the stroma.6,18–21 Metabolicenergy is essential for maintaining the barrier function andionic pumps of mammalian corneal boundary layers, particu-larly the corneal endothelium. Although the epithelium of themammalian cornea is a highly resistant barrier to the diffusionof tears, it also has ionic pump sites for the active transport ofsodium18 and chloride ions.21

A high epithelial mitotic index ensures rapid wound repairof the human corneal epithelium and, thus, maintenance ofbarrier function. By comparison, the human corneal endothe-lium (composed of 350,000–500,000 cells; 2000–5000 cells/mm2) has a limited capacity for mitosis and serves as a limiteddiffusion barrier to aqueous humor, yet possesses numerousmetabolic pump sites for the active transport of ions and fluidmovement. The corneal endothelium serves the major role of

maintaining corneal hydration in terrestrial mammals. It is alsothe primary cellular area of concern in human disease states orwhen endothelial cells become damaged. When a large num-ber of cells are damaged due to disease, the endothelium losesboth its barrier and pump functions, resulting in corneal swell-ing and opacity.

Clinically, when one evaluates the mammalian cornea witha slit lamp, it appears as one homogenous transparent tissue.However, from a histologic, ultrastructural, and biochemicalpoint of view, there is a difference between the anterior thirdand the posterior two thirds of the human cornea. The ana-tomic separation of the corneal stroma into an anterior andposterior region in salt water teleost fishes may help explainfrom a teleologic viewpoint why this structural and biochem-ical difference developed in humans.

In the mammalian cornea, the anterior stroma has less waterthan the posterior stroma (3.04 g H2O/g dry weight for theanterior and 3.85 g H2O/gm dry weight for the posterior).22

This difference may be due to atmospheric drying through thecorneal epithelium and distribution of the two proteoglycansof the corneal stroma. Previous biochemical studies haveshown that the anterior corneal stroma has a higher ratio ofdermatan sulfate to keratan sulfate.23 Similarly, the anteriorstroma has less glucose (3.89 �M/g H2O) than does the poste-rior stroma (4.93 �M/g H2O).22 This latter finding is not unex-pected, since corneal glucose is obtained from the aqueoushumor and the corneal epithelium utilizes most of the freeglucose.

The clinical importance of the proteoglycan ratios in themammalian corneal stroma relates to stromal hydration andwater distribution. Dermatan sulfate has less water-sorptivecapacity, but greater water-retentive capacity; whereas, thekeratan sulfate possesses a greater water-sorptive capacity, buta meager ability to retain the stromal water.23,24 Therefore,when corneal edema typically occurs, stromal swelling is pre-dominant in the posterior stroma. In addition, because theposterior stroma has a lower water-retention capacity, due to

FIGURE 1. Comparative anatomy of corneas for the sea lamprey, elasmobranchs, salt water, and freshwater teleosts and mammals.

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the higher ratio of keratan sulfate to dermatan sulfate, it isrelatively easy for the corneal endothelium to remove thewater once the barrier and metabolic pump have becomere-established.

CORNEAL ENDOTHELIAL DEVELOPMENT AND

CELL DENSITY

The anatomic and physiological development of the rabbitcorneal endothelium has been studied by Stiemke et al.,25 whocorrelated the morphology, hydration, and Na/K ATPase pumpsite density from birth to young adult age. They found that thebody weight and the diameter and surface area of the corneaincrease in the rabbit from birth to 3 months of life. As thecornea enlarges from birth to adulthood, there is a markeddecrease in corneal endothelial cell density (ECD; from 10,577to 4,077 cells/mm2; Fig. 2). In addition to the decrease in ECD,the endothelial cells develop tight junctions, the endothelialpermeability decreases, the number of endothelial cell pumpsites and density increase, and the total corneal hydrationdecrease (Fig. 3), resulting in a transparent cornea. Althoughnot as well studied, a similar developmental process presum-ably occurs in the human cornea, since the central ECD de-creases from birth when there are 5000–6000 cells/mm2 toadulthood when there are 2500–3000 cells/mm2.26–33

Recently, Amann et al.34 measured the corneal ECD in thecentral, paracentral, and peripheral zones of normal humancorneas. Their results showed that the human cornea has anincreased ECD more peripherally: peripheral � paracentral �central (Fig. 4). The superior peripheral region of the corneal

endothelium consistently had the highest ECD. The increase inthe peripheral ECD suggests that a reserve storage populationof cells resides near Schwalbe’s line, to spread and remodel, if

FIGURE 2. Alizarin red photomicrographs of the central rabbit cornealendothelium: (A) newborn (10,575 cells/mm2), (B) 7 day, (C) 13 day(6752 cells/mm2), (D) 20 day (5001 cells/mm2), (E) 3 month (4077cell/mm2). [Bar, 20 �m]. The right panel shows the correspondingtransmission electron micrographs of the endothelium illustrating theapparent progressive thinning of the endothelial cells along with anincreasing regularity of cell height and thickening of Descemet’s mem-brane. DM � Descemet’s membrane, Bar, 2 �m. Copyright 1991 fromStiemke MM, Edelhauser HF, Geroski DH. The developing cornealendothelium: correlation of morphology, hydration and Na/K ATPasepump site density. Curr Eye Res. 1991;10:145–156. Reproduced bypermission of Taylor and Francis Group, LLC., http://www.taylorand-francis.com.

FIGURE 3. Corneal endothelial permeability of the New Zealand Whiterabbit as a function of age with and without the corneal endothelium (A).Na/K ATPase pump site density per endothelial cell as a function of age(B). Corneal hydration as a function of age (C). (A) Adapted with permis-sion from Stiemke MM, McCartney MD, Cantu-Crouch D, Edelhauser HF.Maturation of the corneal endothelial tight junction. Invest OphthalmolVis Sci. 1991;32:2757–2765. © ARVO. (B, C) Adapted from Stiemke MM,Edelhauser HF, Geroski DH. The developing corneal endothelium: corre-lation of morphology, hydration and Na/K ATPase pump site density. CurrEye Res. 1991;10:145–156. Reproduced by permission of Taylor andFrancis Group, LLC., http://www.taylorandfrancis.com. Copyright 1991.

IOVS, May 2006, Vol. 47, No. 5 The Proctor Lecture 1757


corneal endothelial damage occurs. Figure 5 shows the age-dependent human ECD within these three different cornealregions from the second through the seventh decades of life.The ECDs were determined from 75 human eye bank corneasthat were stained with alizarin red. Figure 5 shows that theECD decreases at an annual rate of 0.59% centrally, 0.45%paracentrally, and 0.40% peripherally. Data from this study andothers34–37 have shown that the ECD adjacent to Schwalbe’sline is approximately 20% to 30% higher than in the centralcornea. It has therefore been suspected that the endothelialcells around Schwalbe’s line may serve as a regenerative zonethat is able to provide a slow continuous population of cells tothe central cornea. Unfortunately, in the human cornea, thisregenerative capacity is quite limited, as opposed to that of therabbit. It has been documented that a loss of central endothe-lial cells occurs at a rate of 0.5% to 0.6% per year38 in humancorneas, similar to the data shown in Figure 5. This is a smalldecline and amounts to a loss of only approximately 100 to 500cells per year from the central corneal endothelium. Theknowledge about the increased peripheral ECD in humans,particularly superiorly, is most important for the clinician tounderstand, since this area can be damaged by common in-traocular surgeries, particularly cataract or refractive surgeryinvolving anterior chamber intraocular lenses (IOLs) or phakic

IOLs. Increased peripheral ECD appears to be unique to hu-mans, as similar observations have not been made in othermammals (Table 1).

It is interesting to note that, in a comparison of the centralECD from a Japanese population to that in an age-matched U.S.population, the mean central ECD in the Japanese was signif-icantly higher than that of the U.S. cohort.39 The increasedcentral ECD appears to provide Japanese individuals with agreater functional reserve for future endothelial cell damage.

Human corneal endothelial cells have been shown to havea limited ability to divide both in vivo and in tissue culture. Thisfact has been well-documented for a number of years. Becauseof progressive corneal endothelial cell loss (i.e., cell death) inhumans, coverage of the posterior corneal surface by endothe-lium with increasing age relies on cellular migration andspreading (i.e., cells sliding from the periphery and enlargingto cover the surface areas of the defect) for repair. Attempts tounderstand the limited division potential of human centralendothelial cells have been limited due to our current knowl-edge of corneal endothelial cell biology. Joyce et al.40 havesuggested that TGF-�2 may keep human corneal endothelialcells (HCECs) in a nonreplicative state. Paull and Whikehart41

have shown that both p53 and Tap63 are elevated in thenormal central human corneal endothelium, which suggests

FIGURE 4. Diagram illustrating a 5% increase in corneal endothelialcell density 2 mm from the center of the cornea center and the 10%increase in endothelial cell density 4 mm from the center of the cornea.Reprinted with permission from Edelhauser HF. The resiliency of thecorneal endothelium to refractive and intraocular surgery. Cornea.2000;19:263–273.

FIGURE 5. Human corneal endothe-lial cell density in the central, para-central, and limbal (far peripheral ad-jacent to Schwalbe’s line) betweenthe second and seventh decades. (n),the number of corneas counted.

TABLE 1. Endothelial Cell Densities Determined from AlizarinRed–Stained Cornea

Central Paracentral Peripheral

Mouse (28 g; n � 10) 2845 � 209 2866 � 376 2610 � 320Rat (370 g; n � 10) 2601 � 280 2506 � 231 2146 � 390Rabbit (4–5 lb; n � 5) 3926 � 382 3733 � 504 3612 � 365Rabbit 2.4 y (9.6 lb; n � 3) 2619 � 169 2593 � 428 2308 � 215Primate

Rhesus 3597 — 4049Baboon 4082 — 3861Macaque

7 y 3747 — 254610 y 3540 — 249016 y 2703 — 1995

Data are expressed as cells per square millimeter � SD.



that these proteins may also inhibit the endothelial cell cycleby keeping them in the G1 phase. Our current understanding isthat HCECs are held in the G1 phase of the cell cycle andcannot readily pass into the S phase, even when stimulated byinjury.42 Because of the recently reported increased peripheralhuman ECD and studies by Bednarz et al.43 that showed a zoneof endothelial cell regeneration in the peripheral region of thehuman cornea near Schwalbe’s line, it has been hypothesizedthat the peripheral endothelial cells may act as an adult stemcell reservoir. Recently, a study by Whikehart et al.44 sup-ported this view when they found that central human cornealendothelial cells lack telomerase activity, but endothelial cellsadjacent to Schwalbe’s line have this enzyme activity. Similarly,these peripheral endothelial cells stained positive with BrdU,which is a marker for dividing cells. Thus, adult endothelialstem cells may exist near Schwalbe’s line to supply new cellsfor both the corneal endothelium and the trabecular mesh-work.45,46

CORNEAL ENDOTHELIAL BARRIER AND PUMP

The corneal endothelium forms an anatomic and physiologicalbarrier between the nutrient-rich aqueous humor and the cor-neal stroma. Tight junctions are an integral component of theendothelial barrier, connecting cells at the apical-most part ofthe lateral membrane. Tight junctions of the corneal endothe-lium are focally present around endothelial cells and serve torestrict selectively the extracellular diffusion of some ions andmacromolecules. Because the tight junctions of the cornealendothelium are known to be “leaky, ” the enhanced perme-ability is advantageous, because it permits diffusion of mostnutrients into the stroma from the aqueous humor.47 Metabolicpump sites (Na,K-ATPase) are also found on the lateral mem-branes of corneal endothelial cells, which serve to transportNa� ions from the corneal stroma to the aqueous humor.6,48,49

Overall, this dynamic pump–leak system maintains cornealdeturgescence and permits sufficient nutrient delivery into thestroma and epithelium. Disruption of the corneal endothelialcells typically results in corneal edema, swelling, and opacity.For the pump–leak system to function properly, the tightjunctions must be intact and continually function as an endo-thelial barrier. The ultrastructure of the corneal endothelialtight junctions has been studied extensively.50–54 The specificmolecules that comprise the tight junctions are F-actin, ZO-1,and �- and �-cadherin.52,54 We have recently shown55 that thecorneal endothelium also contains the junctional adhesion mol-ecules JAM-1 and AF6 (Fig. 6). We have shown that blockingthe JAM-1 protein with anti-JAM-1 antibody when the tightjunctions are disrupted with a Ca2�-free irrigating solutionprevents tight junction formation when Ca2� is added back tothe solution. Overall, these studies confirm that the ZO-1,JAM-1, and AF6 junctional proteins are both effective in main-taining the endothelial tight junctions complex and in main-taining the endothelial barrier.

FIGURE 6. Transmission electron micrograph of a human corneal en-dothelial tight junctional complex (A). Localization of ZO-1 and JAM-1in rabbit corneal endothelial tight junctions. (Rabbit corneas were

fixed with 10% acetic acid in ethanol and blocked with 1% BSA).Corneas were immunolabeled with a monoclonal antibody againstZO-1 (B, green) or tight junctional marker JAM-1 (C, green). Nuclei areshown in blue. As shown in the figure JAM-1 localizes in tight junctionsof corneal endothelial cells with a staining pattern identical with thatof tight junctional marker ZO-1. Confocal cross-sections of cornealendothelium (D) confirms that JAM-1 localizes in the apical part of theendothelial junction. Reprinted with permission from Mandell KJ,Holley GP, Parkos CA, Edelhauser HF. Antibody blockade of junctionaladhesion molecule-A in rabbit corneal endothelial tight junctions pro-duces corneal swelling. Invest Ophthalmol Vis Sci. 2006;47:in press. ©ARVO.



The importance of tight junctions in controlling endothelialbarrier function was confirmed by measuring permeability inrabbit and human corneas after in vitro corneal endothelialperfusion with 5-(6)-carboxy fluorescein as a permeabilitytracer. Removal of the endothelium in the rabbit cornea in-creased permeability from 3.19 � 10�4 to 31.21 � 10�4 cmmin�1. In the human donor cornea, the permeability increasedfrom 2.26 � 10�4 to 12.85 � 10�4 cm min�1.56 In the healthynormal human cornea, there was no correlation between en-dothelial permeability, donor age, or ECD. There is, however,an increase in corneal endothelial permeability in human sub-jects with acute or severe endothelial damage or disease. Forexample, patients with Fuchs’ endothelial dystrophy have adefect in the corneal endothelial barrier function and an in-crease in endothelial permeability.57

The corneal endothelial pump site density has been quan-titated with tritiated ouabain (one Na,K-ATPase site binds onemolecule of 3H-ouabain) in human and rabbit corneas. Therabbit corneal endothelium was found to have 3.0 � 106

sites/cell,58,59 and the human corneal endothelium had 2.1 �106 sites/cell. It is interesting to note that in healthy, normalhuman corneas—where there is a 0.6% decrease per year in

central ECD with age—the endothelial pump site density re-mains constant with age.60 In contrast, patients with cornealendothelial cell damage or disease have increased endothelialpump site densities as endothelial permeability increases.57–60

Hence, the increased metabolic pump sites are presumably anadaptation to compensate for the increased corneal endothelialpermeability to a certain degree.

Corneal endothelial wound repair after damage usually fol-lows a three-stage process. The first stage is characterized bythe establishment of an initial coverage of the wound bymigration of adjacent endothelial cells, which forms a tempo-rary incomplete barrier with minimal pump sites and incom-pletely formed tight junctions. In the second stage, the barrier(i.e., tight junctions) and subsequently the pump functionsreturn to normal levels, the endothelial cells form irregularpolygons, the corneal thickness typically returns to normal,and transparency is restored. The third stage involves remod-eling of the endothelial cells to form more regular hexagons.58

Based on several human studies measuring central ECD byspecular microscopy, it has been substantiated that the normaldecrease in human ECD is �0.6% per year from age 15 to 85years. In comparison, after a penetrating keratoplasty (PKP),the central endothelial cell loss of the donor button over thefirst postoperative year averages 34.1%.61

Human corneas before PKP are typically stored in Opti-sol-GS Solution (Chiron Vision, Irvine, CA) for up to 10 to 15days before surgery. We have shown that human corneasstored less than 5 days in Optisol-GS have 1.1 � 0.1 apoptoticcells/mm2 and corneas stored over 22 days have 4.9 � 0.2apoptotic cells/mm2, and storage beyond 22 days increases thenumber of apoptotic cells exponentially (Figs. 7, 8). Komuro etal.62 have also reported slight endothelial apoptosis in Optisol-GS–stored corneas. They also reported that endothelial apopto-sis correlates best with TUNEL-positive keratogenic corneas,but least with storage time. Thus, human Optisol-GS storage(which is the preferred storage media in the United States)causes minimal apoptosis and the marked decrease in ECDafter penetrating keratoplasty is apparently due to surgicaltrauma.

In summary, the healthy normal human corneal endothe-lium maintains corneal transparency because of increased pe-ripheral ECD, tight junctions that form a leaky barrier to diffu-sion, and a large number of Na,K-ATPase pump sites. Finally,

FIGURE 7. Montage of images a 10-day Optisol-GS–stored human cor-nea. The endothelium was strippedfrom Descemet’s membrane andstained with TUNEL stain for apopto-tic cells. Right: normal corneal endo-thelial cell nuclei stained red withpropidium iodide and Apoptotic cellsstained green.

FIGURE 8. Number of apoptotic cells per square millimeter in thehuman corneal endothelium versus days of Optisol-GS storage (mean �SE).



these studies also have shown that there is minimal apoptosisof healthy normal endothelial cells with age, enabling themonolayer of corneal endothelial cells to remain intact over anormal lifespan (Fig. 9).

FACTORS LEADING TO CORNEAL EDEMA

Mishima63 first described the substantial physiological reserveof the human corneal endothelium. He also showed thatchronic corneal endothelial cell decompensation typically oc-curs when the central ECD declines to 700–400 cells/mm2.There is therefore an enormous reserve of extra endothelialcells to withstand intraocular surgery, trauma, and disease.Figure 10 compares the ultrastructure of the normal humancorneal endothelium to that of the corneal endothelium withpseudophakic bullous keratopathy (PBK). The latter corneatypically reveals through specular microscopy a decreased cen-tral ECD, increased polymegethism (larger than normal endo-thelial cells), and indistinct endothelial cell details, which cor-relates to the ultrastructure of a Descemet’s membrane that issparsely covered by endothelial cells and a thin, newly se-creted, diffuse banded posterior collagenous layer.64 In compari-son, Fuchs’ endothelial dystrophy has a specular microscopicappearance similar to chronic PBK, with the additional findingof having many focal dark spots due to the guttae, which arefocal thick deposits of the banded posterior collagenous layer(Fig. 11). Of note, transmission electron microscopy of corneasaffected by Fuchs’ dystrophy also shows degenerative-appear-ing corneal endothelial cells covering the guttae.

During the early development of phacoemulsification(1970s), marked corneal edema was frequently noted aftersurgery. One of the main causes of this postoperative edema

FIGURE 9. Summary diagram of normal corneal endothelial functionfor maintaining corneal transparency.

FIGURE 10. Scanning and transmis-sion electron micrographs of a nor-mal human corneal endothelium. (A,B) The endothelial cells are hexago-nal, have tight junctions, and containnumerous mitochondria. Acute pseu-dophakic bullous keratopathy (C, D).There is loss of endothelial cells andtight junctions (C). The stressed en-dothelial cells have laid down a dif-fuse, banded posterior collagenouslayer (D).

FIGURE 11. Specular microscopy of a corneal endothelium withFuchs’ endothelial dystrophy (A). Transmission electron microscopy ofa cornea with Fuchs’ dystrophy (B, C). Note that the degenerative-appearing endothelial cells cover the guttae. The black areas on thespecular photograph show up because the refractive index of the thinareas of endothelial cells covering the guttae are similar to aqueoushumor.



was the use at that time of an “incomplete” intraocular irrigat-ing solution (e.g., PlasmaLyte 148; Baxter Healthcare, Deer-field, IL).65,66 This incomplete intraocular irrigating solutionwhen perfused onto the corneal endothelium caused a break-down of the corneal endothelial tight junctions (Fig. 12) result-ing in corneal edema.66 Watsky and Edelhauser67 have shownthat corneal endothelial permeability is substantially increasedwhen perfused with a Ca2�-free medium. Similarly, we re-ported that when corneal endothelial intracellular reducedglutathione was oxidized by thiol-oxidation with diamide, theendothelial tight junctions also became disrupted (Fig. 13) andbarrier function was lost.68 Transmission electron microscopyfurther revealed that the endothelial cells separated at theapical junctions and that microfilaments in the apical endothe-lial cytoplasm formed dense contractile bands. It was con-cluded from this study that the ratio of reduced to oxidizedglutathione in the endothelial cells has a role in the mainte-nance of the endothelial barrier function by protecting thetight junctions from separating. Ultimately, this led to thedevelopment of the first commercially available “complete”intraocular irrigating solution, BSS Plus (Alcon Laboratories,Fort Worth, TX) for cataract and retina intraocular surgery. BSSPlus contains five essential ions (Na�, K�, Ca2� Mg�, and Cl�),bicarbonate as a buffer, dextrose as an energy source, andglutathione as an antioxidant.

PH AND OSMOTIC TOLERANCE OF THE

CORNEAL ENDOTHELIUM

With all the ophthalmic solutions, medications, and otheragents currently used during routine phacoemulsification cat-aract surgery, it is important to know the pH and osmotictolerance range of the human corneal endothelium. Studieshave shown that the corneal endothelium has a pH tolerancebetween 6.8 and 8.2, which is similar to that of the naturalaqueous humor bicarbonate buffer system.69 Moreover, duringcataract surgery, the osmolality of the anterior chamber caneasily vary because of the variety of drugs and solutions used inirrigation or injection of the eye. This variation can cause theendothelial cells to become swollen, degenerated, apoptotic,or even necrotic. If all the essential ions are present, cornealendothelial cells have been shown to tolerate a wide range ofosmolalities from 250 to 350 mOsmoles.70 Therefore, both the

FIGURE 12. Scanning electron micrograph of rabbit corneal endothe-lium perfused for 1.5 hours with Ca2�-free intraocular irrigating solu-tion. The cells are separating at the tight junctions (A). Transmissionelectron micrograph of the same cornea showing loss of the apicaltight junctional complexes (B).

FIGURE 13. Scanning electron micrograph (SEM) of rabbit cornealendothelium after perfusion with 4 � 10�4 M diamide for 15 minutes(A). The endothelial monolayer is disrupted at the tight junctions. After30 minute perfusion with 4 � 10�4 M diamide, the endothelial cellsform clusters of flat islands and larger areas of Descemet’s membraneare exposed (B). (A) Reproduced from The Journal of Cell Biology,1976, Vol. 68, 567–578 by copyright permission of the RockefellerUniversity Press.



pH and osmolality of the intraocular solution are critical inmaintaining the corneal endothelium.

TOXIC ENDOTHELIAL CELL DESTRUCTION AND TOXIC

ANTERIOR SEGMENT SYNDROME

Toxic endothelial cell destruction (TECD) is a syndrome ofcorneal endothelial cell decompensation caused by toxicagents that are introduced into the eye, most commonly duringcataract surgery. TECD was first described by Breebaart et al.71

and Nuyts et al.72 in 1990 as severe postoperative cornealedema—star-shaped Descemet’s folds, a two-fold increase incorneal thickness, and visual acuity at count fingers within 1 to3 days after cataract surgery. The mechanism of corneal edemais related to the breakdown of the corneal endothelial barrierfunction and endothelial cell death. This syndrome has com-monly been linked to a detergent solution used in ultrasonicbath cleaners for disinfecting ophthalmic surgical instruments.The main accompanying factor causing this syndrome is thatreusable cannulas, which are used to re-form the anteriorchamber, often retain residual viscoelastics. During the ultra-sonic cleaning procedure, the viscoelastic residue, or some-times the reusable cannula itself, accumulates toxic ultrasonicdetergent residue. When the anterior chamber is re-formedwith the toxic residue present, the result is TECD. TECD hasalso been reported from other contaminants such as the heavymetal ions Cu2� and Zn2� (which coat the hubs of surgicalinstruments and cannulas).73,74

The cases of toxic anterior segment syndrome (TASS) ap-pear similar to TECD, but with additional damage to the iris,trabecular meshwork, and occasionally the lens. TASS presentsclinically as acute postoperative corneal edema with hypopyonand fibrin in the anterior chamber. One of the main causes ofTASS is retained residues of viscoelastics and detergents on orin reusable ophthalmic instruments used during phacoemulsi-fication. Ophthalmic instruments in the United States are com-monly cleaned with an enzymatic detergent containing subtili-sin, a proteolytic enzyme produced from the fermentation ofBacillus licheniformis. It appears that residual viscoelastics oninstruments and/or cannulas can absorb this enzyme, and, evenwhen autoclaved to 120°C, the enzyme does not becomedeactivated, because the enzyme requires higher temperatures

(�140°C) for deactivation.75 In laboratory experiments, whensubtilisin is perfused directly onto the rabbit corneal endothe-lium, corneal swelling occurs, increasing in severity with in-creasing enzyme dose (Fig. 14), along with structural damageto corneal endothelial cells (Fig. 15). When the same commer-

FIGURE 14. Mean swelling of rabbitcorneas after endothelial perfusionwith subtilisin A. In all cases, cornealswelling of the endothelium perfer-fused with the subtilisin A com-pound was significantly increasedover the control (BSS plus; AlconLaboratories, Fort Worth, TX). Notethat the increased corneal swellingoccurs with the higher dose.

FIGURE 15. Scanning and transmission electron microscopy of thecorneal endothelium of rabbit cornea perfused with subtilisin A. Thecontrol corneal endothelium was perfused for 4 hours with BSS plus(A, B). The subtilisin-perfused corneal endothelium was perfused with100 �g/mL for 30 minutes (C, D) and 90 minutes (E, F). After 30minutes, the endothelial cells were swollen, with loss of microvilli.After 90 minutes of subtilisin A perfusion, there was marked endothe-lial cell edema, loss intracellular junctional complexes, and some evi-dence of endothelial cell death.



cial enzymatic detergent was further evaluated, it was found tohave a marked effect on endothelial junctional proteins andendothelial plasma membrane components (Fig. 16) as cornealendothelial permeability increased 3.6-fold (6.25 � 10�4 to22.74 � 10�4 cm/min).75,76 It should be noted that when theanterior chamber is re-formed after cataract surgery, agentsplaced in the anterior chamber generally remain for at least 4.5hours. Therefore, since the detergent–enzyme contacts theendothelium up to 4.5 hours, there is ample time for serioustoxicity to occur.

CORNEAL SWELLING AND EDEMA

The predominant osmotic gradient across the corneal endothe-lium is related to sodium ion activity in the aqueous humor.77

Normally, the sodium concentration gradient in the anteriorchamber results in a net hydrostatic force to draw water os-motically out of the stroma. This effect is somewhat blunted byother ionic gradients, which reduce the net hydrostatic forceacross the endothelium to 30.4 mm Hg. When the cornealendothelium is damaged, there is loss of both the cornealendothelial barrier and pump function followed by a loss ofionic gradients, ultimately resulting in corneal edema andswelling. Histopathology of a human cornea with marked cor-neal edema typically shows a loss of artifactual clefting in thestroma, a marked increase in corneal thickness, and intercellu-lar and extracellular edema of the basal epithelial cell layer ofthe epithelium. Ultrastructural studies of edematous humancorneas in the anterior, middle, and posterior stroma (Fig. 17)show that the collagen fibrils are not equally distant from eachother after edema occurs, which may partially explain the hazecaused by corneal edema.78 In addition, corneal edema is

FIGURE 16. Scanning electron micrograph (A) of rabbit corneal endo-thelium perfused with 1% Medline Enzymatic Detergent solution,which contains subtilisin, shows swollen intracellular regions anddisrupted intracellular junctions. Transmission electron micrographs(B, C) shows increased intracellular vacuolization (arrows), abnormalsubcellular organelles, and irregular plasma membrane (original mag-nification, �4350). Reprinted with permission from Parikh C, SippyBD, Martin DF, Edelhauser HF. Effects of enzymatic sterilization deter-gents on the corneal endothelium. Arch Ophthalmol. 2002:120:165–172. © 2002, American Medical Association. All rights reserved.

FIGURE 17. Transmission electron microscopy of the edematous cor-neal stroma showing the collagen fibrils in the anterior (A), middle (B),and posterior (C) regions of the stroma. There is a diffuse separation ofthe fibrils in each region of the stroma, with the largest changes in themiddle and posterior regions. Original magnification, �95,400.



associated with loss of stromal proteoglycans (keratan sulfateand dermatan sulfate)79 and hydropic degeneration or cell lysisof keratocytes. Figure 18 summarizes the causes of cornealedema that have been described in this review. If a compro-mised endothelium already exists, all of these factors can havea more rapid onset.

EFFECT OF CORNEAL ENDOTHELIAL

DECOMPENSATION IN LASIK CORNEAS

In human corneas that have been treated by laser in situkeratomileusis (LASIK), the central and paracentral lamellarinterface wound between the flap and stromal bed has limitedwound strength that averages around 2.4% of normal cornealstroma as measured with a tensiometer.80 The LASIK scarvaries in thickness from 0.4 to 11.4 �m thick (mean thickness,4.5 �m). It has been found to be hypocellular and is composedpredominantly of abnormally large, non–fibril-bound proteo-glycans.81,82 In cases of endothelial decompensation, the post-LASIK cornea preferentially becomes edematous posteriorlyand in the interface wound. This sometimes results in thedevelopment of a fluid pocket between the flap and bed at thehypocellular primitive stromal scar (Fig. 19). Recent laboratorystudies conducted on post-LASIK human eye bank corneashave shown that a lamellar fluid pocket can develop at theLASIK interface wound within 3 hours, if there is a loss of thecorneal endothelial barrier pump function (Fig. 20).83 Thispost-LASIK complication emphasizes the importance of havinga good functioning endothelium with a high ECD (i.e., goodbarrier and pump function).

This review describes the teleologic development of thecornea and shows the importance of the corneal epitheliumand endothelium in maintaining corneal transparency. In mam-malian species, the balance between corneal transparency andedema is controlled predominantly by the corneal endothelialbarrier and metabolic pump function. Loss of either the meta-bolic pump or the barrier function results in edema and loss oftransparency.

Acknowledgments

My very sincere and deeply appreciative thanks to ARVO, the AwardsSelection Committee, The Board of Trustees, and colleagues and mem-bers of ARVO for bestowing this great honor on me. It is quiteimpossible to express fully my appreciation for being selected toreceive the Proctor Medal. In accepting an award of this sort no personstands alone; with the awardee are all of the teachers and all of thegraduate students, fellows, and coworkers who have contributed tothe awardee’s work. I consider The Proctor Medal to be the highestkudo any ophthalmic researcher can receive, because it is awarded bymen and woman whom I respect beyond all others. It will be myproudest possession!A special thanks goes to my wife Barbara and to my family, who havebeen so supportive over the past years. I would also like to thankGeorge Smelser, PhD, the 1961 Proctor Medal awardee who suggestedthat I join ARVO, V. Everett Kinsey, PhD (the 1952 Proctor awardee),and Venkat Reddy, PhD (the 1979 Friedenwald awardee), who helpedme publish my first paper in IOVS in 1965. Over the past 39 years, Ihave had the support of two great Ophthalmology Department Chairs,Richard O. Schultz, MD, MS, at the Medical College of Wisconsin, andThomas M. Aaberg, Sr, MD, MSPH, at Emory University, both of whomhave recognized the importance of basic scientific and translationalresearch. I thank them for their unending support!I would like to thank and give a special tribute to the many graduatestudents, postdoctoral fellows, administrative assistants, and col-leagues who have conducted the research and aided my laboratoryover the years and who have made this award possible. A specialthanks to Glenn Holley, BS, my long-time laboratory assistant, NancyL’Hernault, BS, for her skill with electron microscopy, Patrick DeLeon,BA, for his medical illustrations, and Daniel Dawson, MD, for help inthe laboratory and manuscript review.

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FIGURE 19. Histopathology of a human corneal button after a compli-cated LASIK procedure. There was severe loss of corneal endothelialcells, posterior stromal edema, and a fluid pocket between the flap andbed at the central and paracellular interface scar.

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