Pathogenesis of Chandler's Syndrome, Essential Iris Atrophy and the Cogan-Reese Syndrome

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1 June 1986 Vol. 27/6 Investigative Ophthalmology & Visual Science A Journal of Dosic and Clinical Research Articles Pathogenesis of Syndrome, Essential Iris Atrophy and the Cogan-Reese Syndrome /. Alterations of the Cornea/ Endorhelium Jorge A. Alvarado, Collin G. Murphy, Maria Maglio, and John Hefheringron Eight keratoplasty and 14 trabeculectomy specimens from syndrome, Essential Iris Atrophy, and the Cogan-Reese syndrome were studied by electron microscopic and morphometric methods. The corneal endothelium in these conditions undergoes the most varied and complex alterations of any of the endotheliopathies so far studied. The size, shape, and density are altered, and the apical surface shows a myriad of abrmalities including alterations of the intercellular borders and junctions, and formation of numerous microvilli, filopodia, and "blebs." Whereas many cells have features indicative of metabolic activity, and others may have undergone division, still others appear to have been injured as they are disrupted and necrotic. There is also evidence for the presence of a low-grade, long-standing chronic inflammation and an associated loss of contact inhibition with formation of multiple endothelial layers. These changes do t encompass the entire endothelium, as some regions remain relatively unaffected, and each specimen presents a unique morphology. The endothelium is most affected in cases of Essential Iris Atrophy. Some changes may be related to such processes as cell migration and reparative activities. However, the presence of cell necrosis (apoptosis) and chronic inflammation (endotheliitis) may be more specifically related to the ICE syndrome endotheliopathy. The slit lamp and specular microscopy findings characteristic of this disease are correlated with the described histologic abrmalities. Invest Ophthalmol Vis Sci 27: , 1986 syndrome, Essential Iris Atrophy, and the Cogan-Reese syndrome are three clinical manifestations of a disease entity of unkwn etiology conveniently referred to as the irido-corneal-endothelial or ICE syndrome. 1 As described by Campbell, 2 the endothelial cells in these diseases are primarily affected and have the ability to migrate into the surrounding tissues. Migration into the trabecular meshwork may by itself cause some trabecular dysfunction and even glaucoma. Endothelial colonization of the anterior iris surface and deposition of an ectopic Descemet's membrane may lead to development of peripheral anterior synechiae, with probable glaucoma, ectropion uveae, From the Department of Ophthalmology, University of California Medical Center, San Francisco, California. Supported by NEI Grants EY 02068, EY 02162, by Research to Prevent Blindness, Inc., and by That Man May See, Inc. Submitted for publication: June 5, Reprint requests: Jorge Alvarado, MD, Department of Ophthalmology, U-490, University of California School of Medicine, San Francisco, CA pupillary displacement, and iris hole formation in some cases. The nature of the basic endothelial abrmality in the ICE syndrome is still t understood. There have been limited opportunities to study the endothelium because most of the corneal specimens previously examined had remaining endothelial cells. Seven corneal buttons from typical ICE syndrome cases have been examined by three groups of investigators, 3 " 5 but only two specimens of Patel et al 5 have a substantial number of endothelial cells available for study. Not only do these specimens have a remarkable paucity of endothelial cells, but degenerating cells are also present. In contrast, two atypical cases 67 show that the endothelium may have epithelial-like characteristics similar to those described for posterior polymorphous dystrophy. 8 " 10 Thus, while the former studies suggest that the endothelial disorder is degenerative in nature, the latter point to a transformation or metaplasia to epitheliallike cells. The present study is a comprehensive ultrastructural survey based on new quantitative-morphometric 853

2 854 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1986 Vol. 27 Table 1. Ice syndrome specimens: keratoplasty syndrome specimens Accession number D.v. Age/sex Contributor Disease severity /M Bourne, WM Bullous keratopathy. Glaucoma controlled surgically. 151 and Cogan- Reese* 62/F Boruchoff, SA Shaffer, RN Intermittent edema. Glaucoma requiring filtration surgery X /F Fine, M Hetherington, J Moderate chronic edema. Glaucoma controlled medically /M Kramer, S Intermittent edema. Recurrent erosions. Mild glaucoma /F Alvarado, J Advanced chronic edema. Glaucoma controlled with filtration surgery. Essential iris atrophy specimens 219 Essential iris atrophy 38/F Shaw, EL Hoskins, DH Moderately severe chronic edema with controlled glaucoma. 351 Essential iris atrophy 63/F Ostler, B Severe chronic edema with controlled glaucoma. 703 Essential iris atrophy 55/F Alvarado, J Severe/chronic edema. No glaucoma. Seen by Dr. Chandler. Table 2. Ice syndrome specimens: trabeculectomy Accession number* syndrome' specimens DA. & Cogan-Reese Essential iris atrophy' specimens Essential iris atrophy & Cogan-Reese Essential iris atrophy Essential iris atrophy Essential iris atrophy Essential iris atrophy Essential iris atrophy Age/sex 28/F 30/F 37/M 33/F 35/F 44/F 61/F 32/M 46/F 47/F 50/F 30/M 48/M 39/F Contributor Hetherington, J Hetherington, J Hetherington, J Hetherington, J Hetherington, J Hetherington, J Alvarado, J Hoskins, DH Hetherington, J Hoskins, DH Hoskins, DH Hoskins, DH Hetherington, J Hetherington, J * Accession numbers 74 and 246, 99 and 229, 103 and 359 are pairs of specimens obtained from the same patient. methods and direct comparisons of the endothelium in the ICE syndrome and rmals." Our collection of 22 ICE syndrome specimens is the largest so far studied. Many endothelial cells were present in our specimens, allowing us to measure changes in cell shape, size, and density as well as to describe new and striking alterations in their appearance. There is a specific type of cell necrosis and a low-grade chronic inflammation that may contribute to the endothelial cell loss characteristic of these diseases. Epithelial-like cells were t observed in our specimens. Materials and Methods Specimen Collection and Preparation Eight of the 22 specimens were obtained at keratoplasty (Table 1), and 14 specimens containing mostly the peripheral cornea were obtained at trabeculectomy (Table 2). Forty-three rmal postnatal corneas ranging from birth to 98 years of age served as controls. Findings from the examination of these rmal specimens have been previously published."" 13 Our standard methods were used to fix, embed, section, and examine tissues by light (LM), transmission (TEM), and scanning (SEM) electron microscopy."" 15 Informed consent was obtained from patients or relatives before the study, following the usual procedures.

3 No. 6 CORNEAL ENDOTHEUUM IN THE ICE SYNDROME / Alvorodo er ol. 855 Table 3. ICE syndrome keratoplasty specimens: scanning electron microscopy Accession number Prominent zipper-like margins* Microvilli* Filopodia Bleb formation (grouped blebs) Endothelial discontinuities or gaps Syndrome Specimens many rare sparse many areas of denudation seen Essential iris atrophy \, at edge many * +1 = scarce; +2 = small patches of cells; +3 = more extensive region of cells; +4 = large areas affected. Microscopic Examination Alternate quadrants of the corneal buttons were examined by SEM or jointly by TEM and LM. The cells from the two ncontiguous quadrants examined by SEM were scored for such surface features as cell margins, microvilli, filopodia, blebs, and areas without an endothelial lining and exposure or "baring" of Descemet's membrane (Table 3). The remaining quadrants were examined by TEM to study other ultrastructural features of the endothelial cells. These same two quadrants had ten step-serial, 1-yum thick sections taken at 30-yum intervals. To measure the cellularity or endothelial cell density, we counted the number of nuclei/ 100 fxm of endothelial length on montages made from light micrographs of these sections. The peripheral corneal endothelium in 14 trabeculectomy specimens was also examined by SEM (Table 4) and TEM. Morphometric and Statistical Studies We used a series of verlapping scanning electron micrographs to study changes in endothelial cell shape. The cell perimeter and area were measured for each cell using a Talos digitizing pad interfaced with a DEC PDP 11/70 computer. We used the ratio of the perimeter to the square root of the area to define a "shape-ratio". 16 These measurements could be performed in seven corneal buttons and in 17 rmal corneas of similar ages. The degree of distortion or skewness of endothelial shape can be determined by comparing it to that of an ideal shape such as the circle. The shape-ratio is 3.55 for a circle and 3.77 for a hexagon. All computed shape-ratios derived from measured cells are rmalized to that of a circle, which is assigned a value of 1.00, so that a hexagon would have a corresponding value of Table 4. ICE syndrome trabeculectomy specimens: scanning electron microscopy of peripheral cornea Accession number Prominent zipper-like margins* Microvilli* Filopodia Bleb formation (grouped blebs) Endothelial discontinuities or gaps syndrome specimens Essential iris atrophy specimens = scarce; +2 = small patches of cells.

4 856 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1986 Vol. 27 The cell density or cellularity of the corneal endothelium was measured in five of the corneal buttons. The other specimens did t have sufficient remaining tissue to perform this study. Cell densities in the ICE corneas were compared with those previously established for rmal corneas." We used t-tests to compare "shape-ratios" in rmal and ICE corneas and to compare variability in measured "shape-ratios" and areas between the two groups. For the cell density, we used rmal tolerance intervals derived from our previous study" to compare the cell density from each specimen with rmals. In all comparisons, the data were weighted according to the number of observations (cells measured or tissue sections scored). Results We follow an anatomical outline in presenting our findings so that changes involving entire cells are described first, followed by those affecting a portion of the cell. Tables 3 and 4 show the distribution of five abrmalities among the eight corneal buttons and 14 trabeculectomy specimens studied. Cell Size, Shape, and Density The corneal endothelium of patients with the ICE syndrome has a marked alteration of cell size and shape. Although it is possible to find cells with a rmal hexagonal shape and an otherwise healthy appearance, especially early in the disease process (Fig. 1), by the time keratoplasty is necessary, many of the endothelial cells have an abrmal configuration as shown in Figure 2. The degree of pleomorphism can be quite marked, with some cells exhibiting triangular, quadrangular, or other unusual shapes. Along with these changes in cell shape, one also finds a highly irregular cell size with an intermingling of large and small cells (Fig. 2). To measure alterations of the cell size and shape, we computed a "shape-ratio" (see Methods), and ted a departure from the rmal shape-ratios in five of seven corneal specimens (Fig. 3). In general, the endothelial shape-ratio is higher than rmal (P < ; see Table 5) for the ICE specimens. However, the shape-ratios in two specimens, from patients who were judged to be clinically less affected (numbers 151 and 275), fall within the rmal control range. The standard deviations of the shape-ratios and area measurements (Table 6) are also higher than rmal for the ICE corneas (P < and P < , respectively). The cell areas are more variable and greater (2.3X) than rmals (Fig. 4 and Table 6). As shown in Figure 5, the ICE corneas have a lower density of endothelial cells than rmals. Three of the five specimens with cell density measurements have a clearly decreased concentration of endothelial cells; one specimen (number 151) has a borderline rmal density and ather (number 275) has a rmal density. The latter two specimens are the same shown above to have rmal shape-ratios and they were obtained from patients considered to be least affected clinically. These abrmalities in endothelial cell shape, size, and density are accompanied by scattered and isolated necrotic cells as shown in Figures 6A and 7A. Cell necrosis occurs in all of the corneal buttons examined. These necrotic cells often occur as single cells encircled by irregularly shaped neighboring cells (Figs. 6A and 7A), suggesting that necrosis occurred prior to keratoplasty and fixation of these tissues. In other cases, one can see that the decrease in endothelial cell density can eventually produce a loss in continuity of the endothelial cell lining with baring of Descemet's membrane as shown in Figure 6B. This cell "dropout" disrupts the rmal mosaic pattern of repeating hexagons, and the endothelium in the ICE syndrome often acquires an irregular, "crazy-quilt" pattern (Figs. 2, 6A, and 7A). These alterations in endothelial cell shape, size, and density may be related to earlier necrotic events and to migration of these cells toward neighboring tissues. Abrmalities of the Apical Endothelial Surface Examination of the endothelial apical surface by SEM demonstrates a variety of abrmalities. While some cells in the least affected areas appear rmal (Fig. 1), in other areas the cells display abrmal intercellular borders and junctions, as well as numerous microvilli, filopodia, and "blebs". These changes, for the most part, are indicative of metabolically active cells as mentioned below. Intercellular borders and junctions: The rmal apical intercellular border is rather narrow (less than 1 nm) and formed by flat, undulating, and overlapping cell margins." In the ICE syndrome, however, the cell borders are often prominent due to the formation of numerous, finger-like, interlocking processes. In six of the eight corneal buttons the intercellular borders are approximately as wide as in the rmals (about 1 fim), but they have numerous processes that give them a prominent appearance as shown in Figures 2A, C, and D. In two of the eight corneal buttons from patients in relatively early stages of the ICE syndrome (numbers 151 and 275), almost all of the endothelial cells viewed by SEM had the widest, most abrmally complex cell borders we observed. In these two cases, one of which is illustrated in Figure 7, the finger-like processes are more numerous and much longer than those shown in Figure 2, measuring about 2 ^.m, so that the width

5 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er ol. 857 Fig. 1. Normal-appearing corneal endothelium in the ICE syndrome. A, Cells with a rmal hexagonal shape and rmal cell margins. Each cell contains a large oval nucleus (specimen number 74, SEM, 2400X). B, Higher magnification of the same specimen. Note narrow, undulating cell margins (SEM, 4800X). C, This cell has numerous mitochondria, scattered glycogen deposits (arrows), and a microvilli at the apical surface. Dm = Descemet's membrane. (Specimen number 151, TEM. 12,500X). D, This cell has abundant mitochondria, rough-surfaced endoplasmic rcticulum, and cytoplasmic micronlaments (specimen number 277, TEM, I2,5OOX).

6 F: 858 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1986 Vol. 27 Mg. z. Alierea corneai enaomenum in me iv^n synurume iacivij. iiiu^imuuiib m t\, a, mu \^uic lt^^tll ai mt wm iiia&iiuii_aiiwii iu muumu, the differences in cell size and shape. A, Large cells with prominent margins, irregular size and shape. Some cells contain many microvilli (specimen number 2494, I000X). B, Smaller, irregularly shaped cells with rmal margins (specimen number 359, 1000X). C, Cells with irregular shapes and sizes and prominent margins. Some cells have densely packed microvilli, whereas adjacent cells have (specimen number 277, I000X). D, High magnification of irregularly shaped cells with prominent margins (specimen number 2l 9, 2200X).

7 No. 6 CORNEAL ENDOTHEUUM IN THE ICE SYNDROME / Alvorodo er ol o "219* A O 275 O Early ICE n = 2 Late ICE n = 5 A Normals n =17 Table 6. Comparison of variability in corneal endothelial cell shape and area in ICE syndrome (n = 7) and rmals (n = 17) Normals ICE F P* Mean standard deviations Shape-ratio < Area < Fig. 3. Shape-ratios computed for endothelial cells from scanning electron micrographs of rmal and ICE syndrome corneas. of the intercellular border is about 4 ^im, compared to the rmal width of I ^m. Furthermore, the fingerlike processes are intimately interlocked with one ather, forming striking "zipper-like" arrangements, as seen in Figures 7C and D. The eight keratoplasty specimens are arranged according to the severity of the condition based on clinical criteria, as shown in Tables I and 3. The least severely affected individual, with a diagsis of syndrome, is at the top, while the most severely affected, with a diagsis of Essential Iris Atrophy (El A), appears at the bottom. Individuals with less affected corneas, or who were operated earlier in the disease course, seem to have the most prominent endothelial cell borders. This alteration occurs more commonly at earlier stages of the disease when the endothelial cells appear to be metabolically active and less frequently in advanced cases. By TEM, one can compare the different types of intercellular junctions found in the ICE syndrome. Figure 8A shows an example of a rmal-looking junction, where there is a simple overlapping of the apical margin of one cell over its neighbor, the cells are joined by a tight junction, and there is a short path along the intercellular space from the anterior chamber to Descemet's membrane. However, in ICE specimens from the early stages, these corneal endothelial cell borders are associated with more complex intercellular junctions, which differ from those in rmals in at least Age * T-test for the null hypothesis that the difference between rmal and ICE syndrome equals zero. two respects. First, the path from the anterior chamber towards Descemet's membrane along the intercellular space can be rather long and tortuous as shown in Figure 8B. Secondly, in one case from a patient at an early stage in the development of the disease process, the intercellular space is occluded by an increased number of junctions (Figs. 8B and C). This arrangement represents a departure from the rmal or from cases of advanced ICE syndrome with chronic corneal edema. Later in the disease, the junctions may disappear (Fig. 8D) and gaps form between the cells. Thus, the endothelial barrier is effectively disrupted and chronic corneal edema ensues. Microvilli: Although 10 to 20 microvilli are rmally found on the apical surface of endothelial cells," in the ICE corneas 100 or more microvilli may be seen in some cells (Figs. 2, 6, and 7), particularly in cases of syndrome (see Table 3). These structures can be observed by TEM as short and thin (approxi- O Early ICE n-2 Late ICE rt.s A Normal n-17 Table 5. Comparison of corneal endothelial cell shape in ICE syndrome (n = 7) and rmal corneas (n = 17) Normals ICE Mean shape-ratio SD < * T-test for the null hypothesis that the difference between the mean shape ratios equals zero Age-Normals SO Age-ICE Fig. 4. Areas of endothelial cells measured on scanning electron micrographs in rmal and ICE syndrome corneas. Triangles and circles = mean areas; lines represent standard deviations.

8 860 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1986 Vol > J ft Age 0 Early ICE Late ICE # 301 #* Fig. 5. Cellularity of endothelium in rmal and ICE corneas. Dashed line = regression line determined for 56 rmal prenatal and postnatal specimens, ages 12 wk gestation to 98 years of age. Hatched area = tolerance intervals computed from rmal specimens such thai 80% of all future rmal populations have a 0.95 probability of falling within these boundaries. mately 100 X 500 nm) projections from the apical surface (Fig. IC). Cells with many microvilli occur sporadically, so that a cell with abundant microvilli may lie adjacent to one with only a of them (Fig. 2C). The same two specimens which have cells with prominent "zipper-like" borders (numbers 151 and 275) also have cells with the greatest number of microvilli (Fig. 7). In other specimens, only a microvilli can be found in most cells (see Figs. 1, 2A and B). Microvilli have been observed in five of seven corneas examined by previous investigators 3 " 5 and in all of our corneal buttons. Fitopodia: Filopodia are thin processes similar to microvilli except that they are longer, measuring up to 14 jum in length in our specimens (Fig. 9). They commonly originate near the cell borders and probably extend into the anterior chamber in vivo; however, in our fixed specimens they are visible as fine lines extending across the cell surfaces as shown in Figures 9A and B. True filopodia were observed by SEM in four of our corneal button specimens; their presence appears to be inversely related to disease severity (Table 3). Previously, only the one corneal button examined by Richardson 4 showed such filopodia. Rodrigues et al 17 found such structures in half of their trabeculectomy specimens, as did we (Table 4). Blebs: In specimens with the ICE syndrome, some cells have large and bizarre apical irregularities resembling grouped blebs and folds (Figs. 9-11; Tables 3 and 4). Typically, regions with blebbed cells lie adjacent to necrotic cells. These structures were commonly found rig. «. IXCI'IUM^ anu uaiuig ui L/CM-CIIICI S iiiciiiuialit. JL^IVI. n, ngvium. un ^anuwy, wim-n UUJ iu«hj pitwmu...^...,,.^..v-, ^^^...g,..., nucleus. Note surrounding intact but irregularly shaped cells (specimen number 151, 860X). B, Cell borders have opened up (arrows) to reveal the surface of Desccmet's membrane (specimen number 275, 2400X).

9 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er ol. 861 Fig. 7. Prominent intercellular cell borders in specimen number 151, SEM. A, Low power view. Note irregular shapes, scattered patches of necrosis (arrows) and wide and prominent cell margins (260X). B, Higher power view of same region. Cells have abundant microvilli as well as fine filopodia (arrows) extending from the cell margins (1000X). C, Higher power view of the zipper-like margins (3000X). D, High power view of the complex interlocking projections which constitute the wide and zipper-like cell margins (10,000x).

10 862 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1986 Vol. 27 Fig. 8. Intercellular borders, TEM, A, Normal appearing tight junction (arrow) joins neighboring endothelial cells at the apical surface in this specimen. This junction (arrow) is intimately associated with the terminal web at apical surface (small arrowheads). A typical process extends for a short distance over the adjacent cell (large arrowheads). The intercellular border follows a short path to its basal termination at Descemet's membrane (Dm) (specimen 301, I5.77OX). B, Complex intercellular junction (specimen 99). Apically, there is a very long process extending over the adjacent cell (arrows). Below the surface there is a highly convoluted intercellular border (19.000X). C, At higher magnification of B, multiple punctate occlusions can be seen to obstruct the space between these cells (38,000X). D, Convoluted intercellular border is apparently devoid of junctions. Dm = Descemel's membrane (specimen 151, 49,400x). Electrondense deposits in cytoplasm are glycogen (arrow). in the cells which have zipper-like intercellular borders, microvilli, and filopodia as shown in Figure 9. The blebs assume several forms: one type, as shown in Figures 9C and D, consists of raised, angular ridges of folded plasma membrane near the cell borders, is associated with filopodia, and several may be present in one cell; two other types of protuberances were found in the centers of cells with or filopodia, microvilli, r prominent cell borders, as shown in Figure 10. One of these two subtypes (Figs. I0A and B) is circular and composed of many microblebs; the other is formed from large smooth sheets of plasma membrane, which assume a ring- or crater-like configuration in the center of each cell (Figs. IOC and D). Typically, one such central protrusion exists, although we observed some cells with two symmetrical structures (Figs. 10A-C). Grouped blebs were observed in four of the eight corneal buttons (Table 3) and they occur in both syndrome and EIA specimens. Although "blebbing" has been previously observed in the ICE syndrome endothelium, 4 ' 17 these grouped blebs have t been described or illustrated in previous studies, r are we aware that these structures have been observed in other diseases of the corneal endothelium,

11 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er ol. 860 Fig. 9. Filopodia and blebs in altered corneal endothelium (specimen 151, SEM), A, Many thin filopodia (arrows) extend from the cell margin. Raised and folded blebs arise from the cell surface near margins (2600X). B, Higher magnifications of filopodia, some of which extend more than 14 ^m. Two filopodia arise at the center of the cell (arrow) (4400X). C, Higher magnification of folded surface blebs (6000X). D, High magnification of raised blebs associated with the zipper-like cell margins (I l,000x).

12 864 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / June 1986 Vol <- Fig. 10. "Grouped blebs" in ICE syndrome endothelium (SEM). (A) Convoluted protrusions in centers of endothelial cells. Note cell with two "grouped blebs" (arrow) (specimen 219, 2000X). (B) Higher magnification of cells in A. Note complexity of blebs (4000X). (C) Smoothsurfaced "grouped blebs" in cell centers. One cell has two of the large protrusions (specimen 351, 2000X). An adjacent cell has two small blebs similar to those in Figure 9. (D) Crater-shaped "grouped blebs" in ather ICE specimen (specimen 326, 2200X). except in one specimen of ours from a patient with an acute herpes simplex keratouveitis and a secondary glaucoma. By TEM, one can see that the grouped blebs represent a protrusion of the cytoplasm overlying or adjacent to the nucleus (Figs. 11A and B). The cytoplasm of the bleb is altered in that the cytoplasmic microfilaments that form the "terminal web" just below the apical surface end abruptly where the bleb begins (Fig. 11B). Cells containing these blebs are frequently found adjacent to necrotic cells having a pale cytoplasm (see below). In one case, adjoining cells, each having grouped blebs, were observed by TEM to overlie the necrotic remains of a third endothelial cell (Fig. 11 A). In some blebs the cytoplasm contains numerous elements of the rough-surfaced endoplasmic reticulum and Golgi as shown in Figures 11A and B. Other blebs resemble large packets of specialized organelles con-

13 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er ol. 865 Kig. 11. "Grouped blebs" in ICE syndrome endothelium cornea (TEM). A, Adjoining endothelial cells each have a "grouped bleb" protruding from the apical surface (arrowheads). These two cells overlie a necrolic cell (arrow). Note occluding junction (double arrows). Dm = Descemet's membrane (specimen 275, 4300X), B, Higher magnification of "grouped bleb" in A. Protruding portion of cell contains many organelles. Note that terminal web (arrow) does t extend into the "grouped bleb" (25,000x). C, "Grouped bleb" containing many polyribosomes. Dm = Descemet's membrane (specimen 74, 16.5OOX).

14 866 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1986 Vol. 27 Fig. 12. A, Large cluster of binucleale cells (specimen 219, SEM, 1000X). B, Transmission electron micrograph of a cell with two nuclei (arrows) (specimen 275, 9000X). C, The elongated cell in this scanning electron micrograph may be undergoing mitosis (specimen 219, 1400X). taining both free and polyribosomes and elements of the smooth surfaced endoplasmic reticulum (Fig. 11C). Nuclear and Cytoplasmic Abrmalities In two corneas from patients with early stages of the ICE syndrome, we have seen several cells with two nuclei (Fig. 12). Presumably, these binucleated cells have undergone nuclear division in the absence of cytoplasmic division, a process kwn as endomitosis. 18 We have been able to correlate this SEM finding directly with TEM where we also observed single corneal endothelial cells containing two nuclei as shown in Figure 12B. We have ted a binucleate cell in a rmal cornea"; however, such cells are very rare and are usually found only in individuals of an advanced age. In contrast, in some specimens with the ICE syndrome, we observed some large clusters of binucleate cells in young individuals. Mitotic figures are difficult to find even in rmal prenatal corneal endothelial cells that are kwn to be actively dividing." However, one specimen with the ICE syndrome (Fig. 12C) contained a configuration that resembles a putative mitotic figure illustrated in a specular microscopy study in a case of graft rejection reported by Laing et al. 19 Thus, it appears that mitosis may occur in the corneal endothelium of patients with the ICE syndrome. We examined many cells from these specimens, looking for keratin filaments and desmosomes. Only one cell with a filaments was found in specimen number 151, and desmosomal junctions were t observed. Thus, we could t find evidence to support

15 No. 6 CORNEAL ENDOTHEUUM IN THE ICE SYNDROME / Alvorodo er ol. 867 the findings of Hirst et al 6 and Quigley et al, 7 who observed large numbers of epithelial-like cells containing keratin filaments and desmosomal junctions. By SEM, three main types of cells are detected: healthy cells, cells with surface modifications, and frankly necrotic cells. By TEM, one common finding is the occurrence of "rarefied" or abrmally electronlucent cells. Our impression is that there is a reduction in microfilaments, rough-surfaced endoplasmic reticulum, mitochondria and other cytoplasmic organelles, as well as a change in the electron density of the cytosol itself in these cells, resulting in an electron-lucent appearance. These alterations probably indicate an imminent necrosis that may t always involve surface changes detectable by SEM. In other cells, TEM shows that the endothelial cells have an extraordinary number of cytoplasmic organelles and these cells appear to be rather vigorous and healthy (Fig. 1). The overall picture observed by TEM is cell activation mixed with evidence of altered, damaged, and even necrotic cells. The lightly staining cells are often situated next to cells with rmal cytoplasm (Fig. 11A) as well as activated cells (Fig. 1C). Inflammatory Cells and Other Endothelial Abrmalities In six of eight corneal buttons, we observed lymphocytes either on the surface of the endothelium (Fig. 13A) or at an open intercellular border (Fig. 13B). The morphology of these cells by SEM is the same previously reported for peripheral lymphocytes. 20 As seen by TEM, these lymphocytes are sometimes completely wedged between or beneath corneal endothelial cells (Figs. 13Cand D). In several small regions of one specimen (number 275), the endothelial cells form multiple layers (Fig. 14). This growth of one layer of corneal endothelial cells over ather has t been previously reported. The region shown in Figures 14A and B consists of three to four cell layers with a necrotic cell on the surface. By SEM, one can see (Fig. 14C) layers of overlapping cells, where long filopodia of one cell extend over the surface of a neighboring cell. It is likely that these cells are longer contact-inhibited and thus can grow over each other. 21 ' 22 Discussion Our finding of a substantial number of endothelial cells in our specimens represents the major difference between our study and that of previous investigators who observed a remarkable paucity of such cells. 3 " 5 The endothelial cell density measurements in two of our five specimens were rmal. Bourne 23 has recently demonstrated by specular microscopy that rmal or higher than rmal cell densities occur in some ICE syndrome patients. Corneal edema had been previously explained by a sparseness of endothelial cells, but our findings show that the edema is related to the many cells with multiple abrmalities of the endothelial fluid barrier. These amalies include abrmal intercellular borders, gaps in the endothelial lining, nfunctioning necrotic cells, and a decreased endothelial cell population (P < ). Bourne and Brubaker 24 have shown that before chronic edema develops, the endothelial barrier is actually more impermeable than in rmals. This observation correlates very well with our discovery that early in the disease course there is a narrowing of the intercellular path by an increased number of junctions, as well as a lengthening of this passageway due to the formation of long and tortuous paths. The constellation of described findings taken together probably occurs only in the ICE syndrome endothelium. Posterior polymorphous dystrophy (PPD) is difficult to distinguish from the ICE syndrome both histologically and clinically, 8 " 10 although its hereditary nature, bilaterality, and presence of epithelial-like cells are table differences. We will analyze the described endothelial alterations in relation to certain processes and conditions present in these diseases. In this manner we may discover which alterations are specifically linked to the ICE syndrome endotheliopathy. One process may be related to reparative activities induced by the injury and loss of endothelial cells. The formation of microvilli, filopodia, and zipper-like junctions has been ted during the reparative process following wounding of the endothelium in animal experiments, 2526 in humans whose endothelial cells have been inadvertently "burned" during Argon-laser trabeculoplasty, 27 and in human corneal endothelium treated with Nd:YAG-laser prior to enucleation or keratoplasty. 26 Thus, the presence of these structures in the ICE syndrome diseases may simply reflect the fact that this endothelium is also engaged in reparative activities. Ather process is related to the migration of the endothelial cells to neighboring tissues, which would be expected to involve cell activation, locomotion, and division. We observed many activated endothelial cells containing large concentrations of mitochondria and other cytoplasmic organelles. Filopodia, microvilli, and blebs also represent "forms of cellular motility" 17 and/ or cellular activation associated with cell spreading, 28 mitosis, 2930 and transformed cells 31 in other systems. Zipper-like intercellular borders have been observed when the entire endothelial layer shifts or moves towards a wound. 25 ' 26 These junctions, with their fingerlike, interlocking processes, may hold the moving cells together and maintain the partial integrity of the migrating endothelial layer.

16 868 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1986 Vol. 27 Fig. 13. Inflammatory cells. A, Cell on endothelial surface of ICE cornea (specimen 151. SEM, 6000X). B, Cell (arrow) between cndothelial cell margins (specimen 151. SEM, 6000X). C, Inflammatory cells (arrows) beneath endothelium (specimen 275, 5O5OX). Dm - Descemet's membrane. D, Inflammatory cell adjacent to Descemet's membrane (Dm) (specimen 301, TEM, 865OX). Cell division may be necessary to provide a sufficient number of cells to cover the surfaces of the cornea, trabecular, and iris tissues. Endomitosis, or nuclear division without cytokinesis, leads to the formation of enlarged binucleate cells to cover denuded areas. It is thought that by acquiring multiple sets of chromosomes cells can enlarge, 3233 as occurs in megakaryocytes and giant cells. We saw direct evidence for true mitosis except, perhaps, in one cell shown in Figure 12B. However, the appearance of central blebs on the apical

17 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er at. 869 B Dm Fig. 14. Multiple cell layers in specimen 275. A, Light micrograph. Two or three rows of nuclei are visible in this region. Darkly stained cells at surface (arrow) are necrolic (I300X). Dm = Desccmct's membrane. B,TEM. Three to four cell layers arc visible. Mitochondria (arrows) are of rmal endothelial morphology. The surface cell is necrotic. Dm = Descemefs membrane (TEM, 825OX). C, Two overlapping layers (arrows) of cells can be seen. Note prominent cell borders and long filopodia (SEM. 3000X). Y surface of many cells may represent indirect evidence of mitosis. The compound blebs we observed resemble the mitotic blebs seen by SEM in cultured ovarian cells. 29 " 31 We believe that the "black dots" and "bright bodies" observed by Bourne 23 and others 19 by specular microscopy correspond to the grouped blebs as discussed below. Bourne's comment that in the ICE syn- drome these structures occur in areas where "perhaps the cells are proliferating abrmally" is particularly pertinent. In these regions, the endothelium has abrmally small cells with unusually high cell densities. 19 We have previously postulated that such blebs may represent specialized areas for synthesis of nuclear and plasma membranes in dividing cells. 34

18 870 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1986 Vol. 27 Ather important consideration is whether the lowgrade inflammation and cell necrosis are secondary to certain conditions present in these ICE syndrome e or even to the surgical source of our specimens. These conditions include the presence of an elevated intraocular pressure (IOP), glaucoma, glaucoma therapy and chronic corneal edema. Two corneal buttons come from patients who did t have a pressure elevation or glaucoma. The other six buttons come from patients whose intraocular pressure had been rmal for some time prior to the keratoplasty. No systematic differences were observed between these two types of cornea specimens regarding any of the described endothelial alterations. The 14 trabeculectomy specimens came from patients with an elevated IOP who were using medications for glaucoma. We actually found less prounced endothelial alterations in these specimens than in the corneal buttons. We have attributed this difference to the fact that these specimens came from earlier stages of the disease process. Does an elevated IOP, as observed in primary open angle glaucoma (POAG), produce some of these endothelial changes? We have examined the central corneal endothelium from e of patients with POAG, using multiple sections as employed in this study, and found a slightly decreased endothelial cellularity. However, inflammatory cells, cell necrosis, r the striking endothelial alterations seen in the ICE syndrome were ted. In chronic corneal edema some studies have shown a endothelial cells with microvilli and filopodia but evidence of chronic inflammation. 35 We have examined 100 specimens of Fuchs' dystrophy in the same meticulous manner. In these cases of chronic edema, we found evidence for an inflammatory response confined to the endothelial layer as observed in the ICE syndrome. Similarly, Waring 35 found evidence of endotheliitis or pervasive cell necrosis in cases of Fuchs' endothelial dystrophy. We have studied other cases of chronic endothelial edema, such as in congenital hereditary endothelial dystrophy and PPD and observed that, while an occasional inflammatory cell can be found, these are t as frequent or localized exclusively to the endothelial layer as in the ICE syndrome. We are aware that cell injury and/or necrosis by itself, as may occur during endothelial trauma, is a strong stimulant for an inflammatory response. However, these are transient responses and in the case of the ICE syndrome, we are concerned about long-standing, lowgrade inflammation. The specimen source may have influenced our findings. The corneal specimens included in this study are obtained at keratoplasty. Three e obtained at enucleation are also part of our collection, and we have observed similar findings in both types of specimens. Scheie and Yaff 36 have remarked about the presence of inflammation in the iris and vitreous body in one eye, and Eagle et al 1 mentioned that in 10 out of 16 e examined they found evidence for iridocyclitis. Thus, it appears that extensive evidence of inflammation has been observed by investigators who have examined whole e. Although the possibility of an inflammatory process was raised as early as 1903 by Harms, 37 most electron-microscopy reports have t provided evidence for the presence of inflammatory cells on or within the endothelium, other than an occasional macrophage. 5 Consequently, Shields has remarked that an inflammatory reaction is t an important histopathologic aspect of these diseases. 38 The evidence observed in our specimens by LM, TEM, and SEM is clear-cut and convincing, since these inflammatory cells were sharply confined to the endothelial cell layer in six of eight corneal buttons. We have insufficient information to determine whether this inflammation represents a reaction specifically to the corneal endothelium, as in an autoimmune response, a response to the presence of a viral or other antigen, or a nspecific response due to the presence of injured and necrotic cells. The pervasive cell necrosis observed in all of our specimens also may be fundamentally related to the ICE syndrome endotheliopathy. The damaged cells are t only those enlarged, pleomorphic cells, but also the small, hexagonal, healthy-appearing cells seen by SEM. We have referred to this cell loss as due to "necrosis"; however, we are intrigued by the fact that the injured cells have many of the associated findings characteristic of a specific type of cell necrosis kwn as apoptosis. 39 "Apoptosis appears to be an active, energy-dependent process that occurs under physiologic as well as pathologic conditions, affects individual cells scattered throughout a tissue, and does t evoke inflammation." 39 Furthermore, in addition to the usual rapid nuclear and cytoplasmic condensation, apoptotic death is associated with an "exuberant protrusion of the cell surface" 39 such as shown in our cases. The major difference between apoptosis and necrosis is that the latter is a degenerative process that affects groups of contiguous cells or tracts of tissue and is accompanied by inflammation and scar formation. Apoptotic cell death may also be cell-mediated and in this regard, our observation of lymphocytes within the endothelial layer is particularly relevant. Further studies to classify these lymphocytes and to characterize fully this process of cell necrosis are certainly indicated. Finally, we want to comment on some of the important clinical signs of the ICE syndrome observed by slit-lamp and specular microscopy. At the slit lamp, one of the most useful diagstic features is the ease with which one can observe the individual corneal endothelial cells. Our studies suggest that this is due to the presence of widened cell borders (4X) and larger than rmal cells (2.3X). The endothelium in Chan-

19 No. 6 CORNEAL ENDOTHELIUM IN THE ICE SYNDROME / Alvorodo er ol. 871 dler's syndrome has the most complex borders and is less affected than in EIA, having a rmal cell density in some cases (numbers 151 and 275) and areas containing essentially rmal-looking cells. Bourne 23 has recently described cases of syndrome containing a relatively rmal endothelium. By specular microscopy two signs of endothelial alterations are recognized in the ICE syndrome. One is said to be pathogmonic and is related to the presence of large oval bodies within the cell boundaries. 40 These bodies are sometimes dark surrounded by a light background or light surrounded by a dark background They occupy a surface area which averages 19.9% (SD = 7.3%; n = 34) as measured on the micrographs published by Hirst et al. 40 This proportion is remarkably similar to that occupied by the nucleus of endothelial cells (21.1%; SD = 5.7%; n = 54). The oval shape of these structures is also similar to that of the nucleus of endothelial cells as observed by SEM. In addition, one cell in the Hirst study (see their Fig. 2), contains two of these oval structures, occupying 32.1% of the cell surface, which is also comparable to the 28.8% (SD = 4.6%; n = 14) occupied by the paired nuclei in our binucleate cells. Thus, we believe that these oval bodies observed by specular microscopy are the nuclei of endothelial cells. Why are the nuclei so easily visualized in the ICE syndrome while they are usually t observed in specular microscopy of the rmal corneal endothelium? We believe that this clarity is related to the progressive cytoplasmic alterations observed in the ICE endothelium, beginning with "rarefication" of the cytoplasm with alterations in the content of cytoplasmic organelles and continuing with the disruption of the apical plasma membrane as shown by SEM. The initial visualization of the nucleus may be related to this cytoplasmic rarefication and the "reversal" of the pattern of light and dark may be related to the onset of actual cell necrosis with rupture of the cell membrane. An alternative explanation has been offered by Hirst et al 4043 who have proposed that these specular microscopy images are related to the presence of microvilli in the endothelial cells. It is difficult for us to see how the microvilli can produce images by specular microscopy that resemble the nucleus in shape, size, and location, when these structures are t found distributed precisely in a supranuclear location. Furthermore, this latter explanation does t seem to account well for the pattern reversal. If microvilli are indeed responsible for these patterns, then cases of epithelial downgrowth, where microvilli are really abundant, would be expected to have these specular microscopy images as well. Yet such images are said to be pathogmonic for the ICE syndrome. 40 Lastly, Eagle and Shields, 44 in a report which appeared after this paper had been prepared, find evidence to support the idea that increased density of microvilli contribute to the characteristic specular microscopic picture seen in the ICE syndrome. Specular microscopy also shows smaller round structures with either a bright or dark appearance located near cell centers ' 45 Bourne 23 has stated that the histopathologic counterpart of such images is unkwn, and Laing et al 45 have proposed that they may represent a peculiar cytoplasmic organelle. Our studies show that the size, location, shape, and general appearance of grouped blebs correspond rather well to those of these specular microscopy images. The proportion of cell surface occupied by these round structures averages 8.4% (SD = 5.0%; n = 18) in published specular micrographs 21 and 10.6% (SD = 5.8%; n = 47) in our scanning micrographs. They are located in the same supranuclear portion of the apical surface as seen in specular micrographs. Thus these specular microscopy images probably represent a "blebbing" of the apical cell membrane rather than a cytoplasmic organelle. In conclusion, our studies have given us a new understanding of the cellular alterations occurring in the ICE syndrome endothelium. We find evidence that these cells actively migrate to colonize surrounding tissues and that this migration may be promoted by a loss of contact-inhibition and restricted motility. This activation process seems to occur concomitantly with cell damage, necrosis, and a continued decrease in cell density. Our newly discovered evidence for an inflammatory process helps explain the cell necrosis and possible transformation of these endothelial cells. In the accompanying paper 46 we present evidence that this may be an acquired disease, possibly of viral etiology. Our findings also provide a histopathologic basis for the most important signs of the ICE syndrome endothelium observed clinically by slit-lamp and specular microscopy examination. Key words: human corneal endothelium, ICE syndrome, syndrome, Essential Iris Atrophy, Cogan-Reese syndrome, electron microscopy, morphometry, glaucoma Ackwledgments The authors thank Dr. R. Shatter, Dr. M. Fine, Dr. S. A. Boruchoff, Dr. W. M. Bourne, Dr. S. Kramer, Dr. B. Ostler, Dr. E. L. Shaw, and Dr. D. H. Hoskins for kindly providing us with so many valuable specimens. We also thank Richard P. Juster, PhD, of the Scientific Computing Services, UCSF, who conducted the statistical analyses. We also appreciate valuable comments on the manuscript made by Dr. E. L. Howes, Jr. Dr. Howes also brought to our attention the similarities between apoptotic cell death and the type of endothelial cell necrosis observed in these diseases. Michelle Murphy's editorial assistance is gratefully ackwledged. References 1. Eagle R, Font R, YafFM, and Fine B: The iris naevus (Cogan- Reese) syndrome: light and electron microscopic observations. Br J Ophthalmol 64:446, 1980.

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