BASIC INVESTIGATION
What Happens to the Corneal Transplant Endothelium After Penetrating Keratoplasty? Luiz F. Regis-Pacheco, MD,*† and Perry S. Binder, MS, MD†‡
Purpose: The aim of this study was to examine the human corneal endothelium of the transplant donor, wound, and adjacent host to determine the fate of the endothelial cells after penetrating keratoplasty.
Methods: We performed dissecting microscopic overviews and light and scanning electron microscopy on clear corneal transplant specimens obtained 1 month to 47 years after transplantation. The indications for the primary keratoplasty were keratoconus (11), Fuchs endothelial dystrophy (7), bullous keratopathy (6), others (5), and 8 cases without clinical data.
Results: We were able to visualize the wound and perform relative endothelial cell counts in 17 of 37 specimens. The wounds were of 4 shapes: smooth, anterior and/or posterior gaping, and anterior or posterior overriding. Any combination could be seen in the same specimen. Cells migrated from the center of the donor across the donor–host wound toward the host, but in all cases, the cells spread out, enlarged, and were ultimately lost. One case of Fuchs endothelial dystrophy may have had cell migration from the host across the wound to the donor.
Conclusions: We confirmed that donor cells migrate from higher density to lower density across the transplant wound over time. Wound configuration, donor cell health, recipient endothelial health, and probable cell-to-cell contact inhibition are involved in this process. Key Words: corneal transplant, endothelium, wound healing, SEM, morphology (Cornea 2014;33:587–596)
S
pecular microscopic studies have documented that normal human corneas lose a small percent of their endothelium every decade throughout life.1,2 Although controversy exists as to the ability of the endothelium to repair itself,3,4 most agree that the cell loss is irreplaceable.
Received for publication January 23, 2014; revision received February 20, 2014; accepted March 1, 2014. Published online ahead of print April 23, 2014. From the *Department of Ophthalmology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; †National Vision Research Laboratory, San Diego, CA; and ‡Gavin Herbert Eye Institute, University of California, Irvine, CA. Supported by National Vision Research Laboratory. The authors have no conflicts of interest to disclose. Reprints: Perry S. Binder, 2500 6th Avenue, Unit 307, San Diego, CA 92103 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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The success of penetrating corneal transplants is assumed to be related to the quality and quantity of the endothelial cells from the donor; the younger the donor, the greater the endothelial cell density is theorized to account for long-term transplant success in the absence of immunologic rejection or other operative and postoperative complications.5,6 Specular microscopic studies of penetrating keratoplasty cases have documented a progressive loss of endothelial cells; after 10 to 20 years, many corneas need to be retransplanted because of the cell loss. One theory to account for the chronic cell loss assumes trauma of corneal preservation and the surgical technique7; once the initial procedure-related trauma and surgical inflammation is completed, and the graft endothelium replaces the lost cells near the wound, thereafter there is only normal cell loss due to aging.8–10 Another theory supports some impact of the host endothelium migrating onto the donor from the host11–14; the success of the transplant therefore depends on the quality of the host endothelium. The secondary late migration of donor cells to replace diseased or absent host cells along with other proposed mechanisms may account for the documented progressive cell loss.14–17 As endothelial cell density decreases because of migration or apoptosis,8 the remaining donor endothelium is assumed to spread to enlarge and cover the missing areas so that cell density decreases over time as the cell size and cell shape change. These conclusions were reached based on indirect evidence; a few studies have directly examined the human transplant graft–host junction using light or transmission electron microscopy,18–21 but only 1 study has used scanning electron microscopy (SEM).18 We have had the unique opportunity to study the posterior corneal surface in clear penetrating keratoplasty cases. The purpose of the study was to examine these specimens to assess the endothelial cell morphology on both sides of the donor–recipient junction using SEM.
METHODS The study was performed at the Ophthalmology Research Laboratory of the National Vision Research Institute (now closed). Between May 1978 and June 1994, our laboratory received corneal tissue obtained at the time of penetrating keratoplasty from clear corneal transplants, which were optically unacceptable because of high and/or irregular astigmatism that was not correctable, as well as from donor eyes collected through eye banks from those who had died of natural causes months to years after penetrating keratoplasty. www.corneajrnl.com |
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In the case of a regraft, an attempt was made to encompass the donor–recipient junction of the previous transplant in one or more quadrants. Specimens were (1) received in a moist chamber as whole globes, (2) received as a corneal scleral rim in corneal preservation medium, and (3) conveyed by other means, such as a refrigerated balanced salt solution. Specimens obtained from outside our laboratory were refrigerated at +4°C and were shipped to our laboratory. An attempt was made to obtain clinical histories in all cases. On arrival, we performed overview photographs through a dissecting microscope and compared them with the available clinical slit-lamp photographs (Figs. 1A–C). We also created drawings of the original transplant and the current transplant to assist in locating the donor– host junction. According to our laboratory protocol, corneal sections were immediately placed in 2% glutaraldehyde after examination as described above. Specimens designated for SEM were processed as previously described.18,22,23 We performed light microscopy on thick sections designated for transmission electron microscopy in a few specimens to maximize the tissue areas available for SEM study,24 but we report only the SEM here.
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Each SEM specimen was scanned at low power (·9–·20) to obtain specimen orientation compared with any available clinical drawings. Because specimens were obtained over many months, the tissues were not coprocessed, and therefore, variations in tissue shrinkage would prevent a direct comparison of the cell density from 1 donor button with another.23 A minimum of 4 scanning electron micrographs, enlarged up to ·200, were made of each area examined. For a better definition of the cell borders, it was necessary to obtain electron micrographs enlarged from ·500 to ·700. It is known that the inclination or tilt of the SEM specimen may exaggerate the shapes of the cells that could be detrimental to obtaining comparative size, shape, and density estimates. Therefore, inclination or tilt of the tissue was adjusted throughout the SEM procedures to maintain visualization in the best possible position perpendicular to the area being examined. The images of electron micrographs were preferably taken in the flattest possible positions and between any folds in Descemet membrane. Table 1 lists the specimen studied, the original recipient diagnosis, the time from the original transplant to the repeat transplant, and any other clinical information (when this information was available to us) (Table 2).
FIGURE 1. A, Case 14. Clinical photograph before repeat transplant for high astigmatism. B, Dissecting microscopic view of case 14. Note the 2 sutures seen in the clinical photograph above. The entire transplant wound is encompassed by the larger trephine diameter. C, Continuous (left, dissecting microscopic photograph, case 35) or interrupted (right, dissecting microscopic photograph, case 37) sutures marked the site of the transplant wounds.
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Corneal Transplant Endothelium
TABLE 1. Specimen Clinical Information
Tissue
Drawing and/or Dissecting Photo
Donor Age/ Gender
PKP No.
OS OD OS OS OD NA OD OD OS OD OS OD OS OS OD OD OD OD OS OD OS OS OS
CS CS CS CS B B B CS CS B CS CS B B B B S S S B Eye Eye B
No Yes No No Yes Yes No No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes
13 6 wk NA NA 20 yr NA 37 yr NA NA 27/? NA 46 66/? 55/? 60/? 57/F 19/? NA NA NA NA NA 53/M
1 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 2
OD OD OS OS OD OD OD OD NA OD OD OD OS NA
½B CS Eye B B B B ½B B B B Eye Eye ½B
No Yes No Yes Yes No No No Yes Yes Yes No No No
53/M NA NA 41/? NA 28/M 72/M 66/M NA 49/M NA NA NA NA
2 1 ? 2 1 2 1 1 1 1 2 NA NA 1
Case
Age/ Sex
Pretransplant Diagnosis
POPKP, mo
Eye
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
81/F 78/F 73/M 65/M 47/F 54/F 77/F 73/F 73/F 62/F 71/M 68/M 85/F 65/M 42/F 35/M 42/M 76/M 76/M 98/F 98/F 82/F 39/M
ABK PBK Fuchs Fuchs HSV NA Fuchs ABK ABK Interstitial Keratitis NA Leukoma Interstitial keratitis Fuchs KC KC Interstitial keratitis NA NA NA NA NA KC
24 12 54 31 204 NA 32 65 81 140 NA 84 48 36 156 180 240 NA NA NA NA NA 31
24 25 26 27 28 29 30 31 32 33 34 35 36 37
81/F 66/M 59/F 47/F ?/M 80/F 71/M 53/F 69/F 43/F 58/F 86/F 72/F 63/F
Fuchs PBK PBK KC KC KC KC KC KC KC KC Fuchs? NA Fuchs
54 64 NA 156 216 564 444 298 492 240 NA NA NA NA
Diagnosis at Latest PKP Immune rejection Steep pediatric cornea Early PBK Corneal abscess Partial failure Clear PKP Steep cornea Clear PKP Clear PKP Corneal thinning NA Clear PKP Clear failed autograft Clear PKP Steep cornea Steep cornea Irregular cornea NA NA NA NA NA Recurrent KC; nonhealing epi-defect NA NA NA NA Recurrent KC Irregular square graft Recurrent KC; square graft Recurrent KC; hydrops NA Irregular astigmatism High astigmatism NA NA Irregular astigmatism
Data from eye banks, donating surgeons, and chart reviews. ABK, aphakic bullous keratopathy; B, corneal button; CS, corneal scleral rim; EpiDefect, chronic nonhealing epithelial defect; Eye, whole eye bank eye; Fuchs, Fuchs endothelial dystrophy; HSV, herpes simplex keratitis; KC, keratoconus; M/F, male/female; NA, data not available; PBK, pseudophakic bullous keratopathy; PKP, penetrating keratoplasty; POPKP, postoperative time from last transplant to excision; ?, NA; ½ B, half corneal button; 19 PKP, original transplant; 29 PKP, repeat transplant number 2.
Cell Counting We attempted to count a minimum of 50 contiguous endothelial cells in each electron micrograph: in the center of the donor, mid-way between the donor and wound, close to the wound, over the wound, and across the wound on to the host. The fewest cells counted in a given image were 50, the maximum were 150, and all but 3 images had more than 80 cells counted. This procedure was based on the study by Berry et al,25 which demonstrated that counting of as few as 10 cells is adequate for determining the endothelial cell density, assuming that these cells are uniformly distributed. 2014 Lippincott Williams & Wilkins
In cases where the cell borders were difficult to visualize, the SEM negatives were scanned under high resolution (300–1200 dpi; V60 scanner, Epson, Inc) and converted to a positive image for cell counting and morphological analysis using Adobe Photoshop CS 5 (Adobe, Inc).
Morphological Analysis Changes in cell shape, which might suggest cell migration, were clinically analyzed. An attempt was made to document the presence of bare Descemet membrane to www.corneajrnl.com |
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TABLE 2. Endothelial Cells and Wound Description From Scanning Electron Microscopy Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Visualize Wound? Yes Yes Yes No No Yes Yes No No Yes May be Yes Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes No No
Donor Cell Morphology Small, regular Small, hexagonal Large, 5–6 sides Small, distorted, guttata Large, postmortem artifact Distorted, small, overlapping Small, hexagonal Postmortem artifact Large, distorted Large, 5–6 sides Distorted, guttata Postmortem artifact ? Large, 5–6 sides Small, 5 and 6 sides, some ruptured Small, hexagonal Small, postmortem artifact Small, postmortem artifact Large, 5–6 sides Postmortem artifact None seen Large, postmortem artifact Few large cells Guttata, distorted, postmortem artifact None seen Large, 5 and 6 sides Small, stressed Large, overlapping, spreading None seen Small, hexagonal Very large, 4–6 sides Large, variable None seen None seen None seen None seen
Cell Migration
Host Cells Description
Donor to host Same size up to wound Donor to host ? No Similar both sides ? ? ? Donor to host ? ? ? Donor to host ? ?
Enlarge, then disappear None seen Guttata None seen None seen Similar both sides None seen None seen None seen Guttata Similar both sides None seen Yes Large None seen Yes None seen No Large, distorted None seen None seen None seen None seen None seen No Yes Same as donor ? None seen None seen None seen Yes Yes None seen None seen None seen None seen
? Donor to host ? ? ? No ? Donor to host ? Yes May be ? ? ? No ? ? ? ? ?
Morphological description of donor, host, and donor–host junction. Maybe, some evidence of migration (see explanation in Results); None seen, less than 20 cells present (unable to make a determination); ?, Unable to make a determination.
investigate the individual characteristics of endothelial cells and their morphological changes, which might suggest possible cell migration. In our analysis, all cases were divided in accordance with the preoperative diagnosis, their primary indication for corneal transplant: keratoconus (11), Fuchs endothelial dystrophy (7), corneal edema (aphakic and pseudophakic bullous keratopathy) (6), and other indications (5) (Table 1).
RESULTS Clinical Specimens We obtained documentation to study 37 specimens that had sufficient tissue to examine the donor–host junction
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(Table 1). Additional clear corneal transplant specimens had either insufficient clinical information, were only processed for light microscopy, or were not processed because of various technical reasons. Ten cases had clinical slit-lamp photographs obtained before the repeat transplant, and 14 cases had dissecting microscopic overview photographs of the excised specimens (Fig. 1B). The original indications for the primary keratoplasty are listed in Table 1. Fuchs endothelial dystrophy (cases 3, 4, 7, 14, 24, 29, and 35), keratoconus (cases 15, 16, 23, and 27–34), and bullous keratopathy (cases 1, 2, 8, 9, 25, and 26) were the most common diagnoses. Eight cases had no clinical diagnosis available (cases 6, 11, 18, 19, 20, 21, 22, and 36), and a few scattered diagnoses made up the remaining specimens (cases 5, 10, 12, 13, and 17). The indications for the regrafts included recurrent 2014 Lippincott Williams & Wilkins
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keratoconus, steep corneas associated with a pediatric donor, and/or irregular/high astigmatism unresponsive to relaxing or wedge resections. Nine specimens represented a second transplant and 2 had no information about the transplant number; the remainder were obtained after an initial transplant that had been performed 1 to 47 years earlier (Table 1). Figure 1A displays a clinical slit-lamp photograph, and Figure 1B its counterpart dissecting microscopic overview photograph. Figure 2 shows a case of a square graft obtained 47 years after the primary transplant operation by Ramon Castroviejo, MD. The diameters of the excised tissue, where known, varied from 7.5 to 10 mm with the most common diameter being 8 mm. The second transplant trephine diameters were mostly 0.5 mm greater than the previous transplant diameters.
Cell Density We were able to perform relative cell densities from the SEM photographs in 17 of the specimens. In an attempt to locate more cells in poor images, we subsequently digitized each SEM negative and enhanced each negative; this permitted us to visualize more cells in 10 additional specimens, but the cell borders were either blurry or not present in sufficient quantity or quality to permit an accurate count. In 6 specimens, we were unable to see cells anywhere in the specimen despite the material being described as a clear graft. Because all of our specimens were prepared over several years by different electron microscopy technicians using different equipment, we assume the inability to see cells in
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clear grafts was due to inadequate tissue processing, prolonged death to fixation, inadequate sputter coating, and/or other technical issues. Unfortunately, all of the 37 specimens were discarded when our laboratory closed in 1997, therefore we were unable to reprocess the tissues.
Morphology of the Donor
For most specimens, the most common finding was the ability to visualize endothelial cells in the donor. We found various central donor cell morphologies from different specimens, which shows how much variability in the center of a clear donor can be present with various host diagnoses and posttransplant time. Stated differently, our examination of different areas within the same clear corneal transplant donor tissue showed great variation in size, shape, and distribution of the cells. The decrease in the cell density and the increase in the cell pleomorphism over a distance suggest cell migration in the direction of the lower cell density, and cells with increased size and pleomorphism. Because our cell counting technique was relative for each specimen and the calculation was based on the assumed tissue shrinkage percentages,23,26 we were not able to make any conclusions regarding cell density versus postoperative time, cell density versus host diagnosis at the time of tissue excision, donor age, or cell density versus diameter of the excision. We were able to analyze changes in cell dimensions (shape and size) in many specimens within the same donor tissue.
Host–Donor Junction Sutures marked the wounds in some cases (Figs. 1A–C). We found 4 general shapes of the donor–host junction. The wound could be tight, that is, well apposed; in these specimens, the transition from donor to host was smooth (Fig. 2). In other specimens, a wound gaped open anterior to posterior toward the anterior chamber apparently making it difficult for cells to transition across the gape. The donor could be retracted into the host cornea away from the anterior chamber below the level of the host forcing endothelial cells to migrate “down the hill” to the level of the host. Alternatively, the host could be deeper in the anterior chamber versus the donor so that cells migrating from the donor to the host would have to move “uphill.” Any combination of these shapes might be present in the same specimen. Figures 3 and 4A to F show examples of different wound configurations. Figure 1C is a view of dissecting microscopy of cases 35 and 37 obtained from eye bank specimens showing a continuous 11-0 suture (left) and interrupted 10-0 sutures (right) across the transplant wounds. Some specimens included 360 degrees of the wound, whereas others were created by decentering the trephine to encompass some of the previous wound so that only a portion of the wound might be present. We were able to easily see endothelial cells on the donor side in most specimens, over the wound in 16 and into the host in 13 (Figs. 4A–F, 5A, B).
FIGURE 2. Case 29. Square graft for Fuchs endothelial dystrophy. The lines mark the outer border of the square graft performed by Ramon Castroviejo 47 years ago. SEM ·20. 2014 Lippincott Williams & Wilkins
Morphology of the Host Surfaces and Cells We assessed these cells the same way as we assessed the donor cells. In addition, we looked at the host surface peripheral www.corneajrnl.com |
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with a similar appearance to the donor cells (Fig. 4E). Case 32 (also a second penetrating keratoplasty) also had a similar appearing endothelium on the host and donor, whereas case 33 (original transplant) had host cells that were 5 and 6 sided with areas of bare Descemet membrane being apparent. Figure 4B is an example of a bare, rough host consisting of fibrocellular material commonly seen in failed grafts— a retrocorneal membrane.27 The donor cells appear to be on the surface of the donor side of the wound, but there is no evidence of these cells on the host side.
Cell Migration Across the Transplant Wound
FIGURE 3. Example of SEM morphology in case 6 from an 8-mm button. The numbers refer to each of the subsequent figures in sequence. Case 6. The clinical history is not available. Overview SEM of one half of the excised cornea showing 3 areas of the donor from the center to the periphery up to the circular donor–host wound that is easily seen. Area 4 represents the host. SEM ·10.
to the wound for cell presence and quality. Figures 5A and B show various host stroma and cell appearances. In all cases where we could detect endothelial cells on the host side of the wound, the cells appeared to be enlarging, spreading, and/ or assuming a more pleomorphic appearance. Further away from the wound, the cell density would decrease and cell processes would spread to cover more of the host. Eventually, the cells were not able to cover the host and bare areas of Descemet membrane so that the fibrocellular surface of the host would be exposed (Fig. 5B). In cases of Fuchs endothelial dystrophy, the guttate excrescences on the surface of the host Descemet membrane would be exposed. There were 7 cases of a primary transplant in a recipient with keratoconus (cases 15, 16, 23, 27, 28, 30, 32, and 33); we were able to obtain cell counts centrally in 6 cases, but we could only see cells in 4 keratoconus host cases (cases 16, 27, 32, and 33). A large area of host cells was seen in case 16, but for some reason, the electron microscopy technician did not scan that area, which appeared to have a high density of cells similar to that of the donor when viewed at ·40. The host cells on case 16 were enlarged and contained 4-, 5-, and 6-sided cells whose surfaces were rough and appeared of different electron reflectivity compared with the donor cells. Case 27 (a second penetrating keratoplasty) had cells of 4 to 6 sides
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If the cell density decreased from the center to the periphery of the donor, we assumed that the cells were being “lost” by a spreading and enlarging process. In the circumstance where the central morphology consisted of small, hexagonal-shaped, uniform density cells, as we looked toward the donor periphery, we saw increasing pleomorphism and/or increasing cell size, we assumed cell movement toward the periphery. Change in appearance of the cell surface (smooth to rough and/or electron reflectivity) from central to periphery was similarly considered as a sign of cell movement. At the donor–host junction, we looked for appearance of the cells across the wound (Figs. 4A–F, 5A, B). If the cells increased in size, changed shape from a regular 6-sided hexagon to cells with fewer sides, demonstrated a decrease in density, and/or appeared to spread out over the host, we assumed that there was centrifugal movement, that is, movement toward the host. We expected to see a high density of host cells in our keratoconus cases because keratoconus hosts have normal cell densities.6,28 In only 4 keratoconus cases where we had donor and host cells present, we could see similar cell morphology in the donor and host in 2 of the specimens. We therefore could not confirm a host-to-donor (centripetal) cell migration or contact inhibition between donor and host cells when a host cell population is healthy, although we suspect this is the case clinically because of the high success rate of corneal transplants for keratoconus. In case 3 (Fig. 4F), obtained 54 months after a transplant for a host diagnosis of Fuchs endothelial dystrophy, guttae are easily seen on the host side (upper right). The cell appearance and density were very similar on both sides of the ridge-like wound. Guttae were also present on the donor at the edge of the ridge and into the donor. We did not have any information about the donor, but at the time of the regraft, the surgeon thought that the cornea was slightly edematous. The findings strongly suggest cell migration from the host to the donor, but one cannot eliminate the possibility of the donor having subclinical Fuchs endothelial dystrophy. But if this were the case, one would expect to see many more guttae in the donor. We had 7 primary grafts for keratoconus and 3 primary grafts for Fuchs endothelial dystrophy. All of these 10 eyes had evidence of cell migration from the donor to the recipient.
DISCUSSION Evidence supports an acute loss of donor endothelium at the time of surgery that is due to a combination of surgical 2014 Lippincott Williams & Wilkins
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FIGURE 4. Examples of various donor–host wounds. A, Overview of case 20 showing a depression between the donor and host (“gully”) on the top and a very even wound that is completely closed on the right. Specimen of a 98-year-old donor who died after keratoplasty, obtained from eye bank. Clinical data unavailable. SEM ·20. B, Case not reported herein, but previously published. There is a slight ridge at the donor–host junction (arrowheads). The endothelium of the donor ([D], left) appears to slide over the wound onto a rough recipient (R) stroma and then disappears. SEM ·20. Reproduced with permission from CV Mosby, Figures 1–16E; Transactions of the New Orleans Academy of Ophthalmology (page 23).18 Clinical data not available. C, Case 10 (interstitial keratitis, transplant 2). Donor (left) protruding into anterior chamber so that endothelium has to migrate “uphill” onto host (right). As cells cross the wound, their morphology changes to larger cells that attempt to spread out over the host stroma and are eventually lost. SEM ·200. D, Case 14 (Fuchs dystrophy, first transplant). Ridge (arrows) at the donor (bottom)–host (top) junction (dashed lines). Cells enlarge and spread “uphill” onto the host surface where they make a sheet 10 to 15 cells wide before being unable to surface the bare host stroma. SEM ·200. E, Case 27 (keratoconus, second transplant). Sharply defined donor (lower, left)–host (upper, right) wound with a smooth border (dashed line). Endothelium appears to have successfully migrated onto the host as the cell shapes are similar on both sides of the wound immediately adjacent to the wound, but the cells enlarge in the periphery of the host with loss of some cell junctions (upper right). SEM ·200. F, Case 3 obtained 54 months after a transplant for a host diagnosis of Fuchs endothelial dystrophy. Guttae are easily seen on the host side (upper right). The cell appearance and density was very similar on both sides of the ridge-like wound (D). Guttae were also present on the donor at the edge of the ridge and into the donor. We did not have any information about the donor, but at the time of the regraft, the surgeon thought that the cornea was slightly edematous. The findings strongly suggest cell migration from the host to the donor, but one cannot eliminate the possibility of the donor having subclinical Fuchs endothelial dystrophy. But if this was the case, one would expect to see many more guttata in the donor.
trauma, corneal harvest trauma, and preservation damage.7,29 After keratoplasty, there is a greater rate of cell loss compared with the normal aging process.8,15,29–31 Some of this loss can be attributed to postkeratoplasty complications such as immuno 2014 Lippincott Williams & Wilkins
logic rejection, glaucoma, secondary surgical insults (inflammation, intraocular lens damage, vitreous touch, etc), but many grafts do not have any of these issues and yet they suffer progressive cell loss and ultimate failure from apoptosis.8 www.corneajrnl.com |
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FIGURE 5. Examples of endothelial cell morphology on the host side of the transplant wound. A, Case 14 (Fuchs dystrophy, first transplant). The donor center is down to the right away from this image. Cells seem to be spreading away from the donor onto a surface of Descemet membrane that appears to contain diffuse guttata. Bare areas of Descemet membrane are further away from the wound (above) where the endothelial cells can no longer cover the surface. SEM ·500. B, Host side of wound from case 16 (keratoconus, transplant one). The center of the donor is above this image. The host appears to have a rough surface away from the wound. The cells appear to enlarge further away from the wound. SEM ·400.
There is uncertainty as to the mechanism(s) of a traumatic, progressive postkeratoplasty endothelial cell loss that is greater than the normal aging process. There is scant evidence that the human endothelium is capable of mitosis,3,4,13,32 so it is assumed that donor cells move to areas that are devoid of cells or areas of lesser cell density, and therefore, the central donor cell density has to decrease over time unless replaced by host cells.30,32–37 Studies of graft success rates have suggested that healthy host endothelium (eg, keratoconus) is related to better longitudinal outcomes6 compared with hosts with diseased or absent endothelium (eg, pseudophakic bullous keratopathy).16,38 Basic to this assumption is that host cells can migrate across the transplant wound to repopulate the donor or at least prevent or slow down central cell migration to the host. In vitro animal and human organ culture experiments that used whole corneas have demonstrated centripetal and centrifugal movements of cells after keratoplasty37 or wounding.39 One study that assessed corneal transplants in rabbits has demonstrated cell movement across the wound from the donor to the recipient40; and most recently, there is additional evidence that endothelial cells can migrate from the donor to the host.17,41,42 Special techniques have been used to image the host endothelium using conventional specular microscopy,23,43 but because of the optics of these systems, the transplant wounds themselves could not be directly visualized. One study found donor cell survival to be highly variable and that cell migration was not fully explained by spreading of cells. They were convinced that cells migrated across the transplant wound with the recipient cells replacing the donor cells in 10 of 36 specimens.14 Although there have been several pathological studies of failed corneal grafts, only 1 study imaged the clear corneal transplant wound using SEM but did not discuss the findings. In the review of corneal anatomy by Binder et al,18 2 scanning electron micrographs of the donor–host junction were shown, but there was no discussion regarding cell migration
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(Fig. 4B). Our present article is the first to demonstrate the donor–host junction using SEM in an analysis of clear corneal transplants in which we directly document cell migration across the wound onto the host. As the cells moved across the wound, they tended to enlarge and spread to cover the host in areas devoid of endothelium either over bare Descemet membrane or over fibrocellular tissue presumably a residual of previous endothelial cell loss and replacement wound healing27,44 (Figs. 4, 5). If the host diagnosis was Fuchs endothelial dystrophy, we could detect guttae in the host, and in some cases in the donor at the edge of the wound (Fig. 4F). Although we had several host diagnoses of keratoconus in primary grafts, we were not able to document cell movement toward the donor either because we could not detect host cells (cases 6, 15, 23, and 28) or donor cells (cases 28 and 30) in these specimens. We ascribed the inability to see cells despite a pregraft diagnosis of a clear corneal transplant to prolonged postmortem preservation time or inadequate SEM tissue preparation or examination. Other studies have also documented the absence of cells in cases of clear corneal transplants even when pregraft specular microscopy found the presence of cells.14,23 We demonstrated the highly variable cell sizes and shapes within the same donor cornea described as being “clear” which has been previously described14,16,23,25,29,30,43,45–47 (Figs. 3–5). This variable size and shape of donor cells after keratoplasty suggests unstable, stressed, or enlarging cells related to peripheral wound-healing events.29 Focal areas of cell loss were also seen within the donor, which has been previously documented.29 A small area of examination (eg, conventional specular microscopy) in such specimens would lead to an erroneous cell density in the case of nonuniform size, shape, and density of cells. Our study confirms the biexponential model of cell loss after keratoplasty15,16 and documents that one reason for continued cell loss over time in addition to apoptosis is consistent with progressive donor cell migration centrifugally across the wound to replace cells in a host with a poor or missing 2014 Lippincott Williams & Wilkins
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endothelium. Because studies of keratoconus transplants (normal, healthy endothelium) show consistently higher survival rates than grafts into hosts with poor or no endothelium, for example, bullous keratopathy,16 it is easy to conclude that in keratoconus transplants either the donor cells are contactinhibited as they meet the host cells and/or the host cells migrate centripetally across the wound to replace the weak, healthy, or diseased donor cells.14,17 Cases of borderline host endothelium (Fuchs endothelial dystrophy) may be able to repopulate the donor depending on cell variables we currently cannot measure (Fig. 4F). For example, Price et al48 found equivalent graft survival rates between keratoconus and Fuchs dystrophy. Instances of late clearing of corneal grafts in hosts with unhealthy endothelium11 or in cases of attempted Descemet stripping endothelial keratoplasty41,49,50 have been reported, which suggest centripetal cell movement. We found 4 different wound configurations in our specimens. It would seem that the smoother the wound, the easier the cell migration (Figs. 2–5), but that if there is a large disparity in height and/or a wound gape, it would be more difficult for endothelial cells to migrate across compared with migration across a smooth, well-apposed wound. If a host has no cells and or the wound is not conducive to cell migration, one would anticipate a chronic loss of cells associated with a continuous centrifugal cell movement. Based on our morphology studies herein, in the case of Descemet stripping endothelial keratoplasty or Descemet’s stripping automated endothelial keratoplasty, the large height disparity between the host and donor would lend itself to chronic cell loss. If we can create better posterior wound coadaptation through better technology,51 we may be able to increase the survival rate of our transplants. Variation in surgeon surgical expertise undoubtedly plays a role; square grafts created by one expert corneal surgeon have survived for many decades (cases 29 and 30) (Fig. 2). Our study has several weaknesses. We were only able to study 37 specimens. Based on our analyses, we had some cases of poor tissue preparation, lack of some clinical information, tissue shrinkage artifacts,23,26 and lack of complete assessment of host areas. This was evident from cases with cells seen on specular microscopy before tissue excision, but absence of cells with SEM. Although we had several specimens where the endothelium clearly moved across the wounds from the donor to the host, we had other specimens where we could not identify such cell migration. We were especially disappointed by not documenting cell migration from the host to the donor in our cases of keratoconus. To correct these errors, in addition to the obvious technical issues, we suggest obtaining larger diameter excisions, immunohistochemistry,17 radio labeling techniques, and the use of wide-field specular microscopy.52
ACKNOWLEDGMENTS The authors thank those organizations and surgeons who kindly provided tissue for this study (the San Diego Eye Bank, Lions Eye Bank Portland, and Arizona Lions Eye Bank), Frank Price, Jr, MD, Jeffrey L. Katz, MD, Lee T. Nordan, MD, and 2014 Lippincott Williams & Wilkins
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Edward L. Shaw, MD. We thank Max Moore for microscopy expertise. The authors dedicate this article to the memory of Frank M. Polack, MD, who kindly provided the initial experimental pathologic and scanning electron microscopy experience for one of the (P.S.B.) authors. REFERENCES 1. Abib FC, Barreto J Jr. Behavior of corneal endothelial density over a lifetime. J Cataract Refract Surg. 2001;27:1574–1578. 2. Cheng H, Jacobs PM, McPherson K, et al. Precision of cell density estimates and endothelial cell loss with age. Arch Ophthalmol. 1985; 103:1478–1481. 3. Laing RA, Neubauer L, Oak SS, et al. Evidence for mitosis in the adult corneal endothelium. Ophthalmology. 1984;91:1129–1134. 4. Treffers WF. Human corneal endothelial wound repair. In vitro and in vivo. Ophthalmology. 1982;89:605–613. 5. Lass JH, Beck RW, Benetz BA, et al. Baseline factors related to endothelial cell loss following penetrating keratoplasty. Arch Ophthalmol. 2011;129:1149–1154. 6. Gordon MO, Steger-May K, Szczotka-Flynn L, et al. Baseline factors predictive of incident penetrating keratoplasty in keratoconus. Am J Ophthalmol. 2006;142:923–930. 7. Alqudah AA, Terry MA, Straiko MD, et al. Immediate endothelial cell loss after penetrating keratoplasty. Cornea. 2013;32:1587–1590. 8. Albon J, Tullo AB, Aktar S, et al. Apoptosis in the endothelium of human corneas for transplantation. Invest Ophthalmol Vis Sci. 2000;41: 2887–2893. 9. Bourne WM. Penetrating keratoplasty with fresh and cryopreserved corneas. Donor endothelial cell survival in primates. Arch Ophthalmol. 1978;96:1073–1074. 10. Bourne WM, O’Fallon WM. Endothelial cell loss during penetrating keratoplasty. Am J Ophthalmol. 1978;85:760–766. 11. Baum JL, Late spontaneous clearing of corneal grafts. Arch Ophthalmol. 1977;95:1538–1539. 12. Chi HH, Teng CC, Katzin HM, et al. The fate of endothelial cells in corneal homografts. Am J Ophthalmol. 1965;59:186–191. 13. Wollensak G, Green WR. Analysis of sex-mismatched human corneal transplants by fluorescence in situ hybridization of the sex-chromosomes. Exp Eye Res. 1999;68:341–346. 14. Lagali N, Stenevi U, Claesson M, et al. Donor and recipient endothelial cell population of the transplanted human cornea: a two-dimensional imaging study. Invest Ophthalmol Vis Sci. 2010;51:1898–1904. 15. Armitage WJ, Dick AD, Bourne WM. Predicting endothelial cell loss and long-term corneal graft survival. Invest Ophthalmol Vis Sci. 2003;44: 3326–3331. 16. Böhringer D, Böhringer S, Poxleitner K, et al. Long-term graft survival in penetrating keratoplasty: the biexponential model of chronic endothelial cell loss revisited. Cornea. 2010;29:1113–1117. 17. Merjava S, Malinova E, Liskova P, et al. Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium. Histochem Cell Biol. 2011;136:93–101. 18. Binder PS, Wickham MG, Zavala EY, et al. Corneal anatomy and wound healing. In: Symposium on Medical and Surgical Diseases of the Cornea. Transactions of the New Orleans Academy of Ophthalmology 1980. St Louis, MO: CV Mosby; 1–35. 19. Hatanaka H, Koizumi N, Okumura N, et al. A study of host corneal endothelial cells after non-Descemet stripping automated endothelial keratoplasty. Cornea. 2013;32:76–80. 20. Obata H, Tsuru T. Corneal wound healing from the perspective of keratoplasty specimens with special reference to the function of the Bowman layer and Descemet membrane. Cornea. 2007;26(9 suppl l):S82–S89. 21. Stewart RM, Kaye SB, Batterbury M, et al. Histopathological features associated with endothelial keratoplasty failure. Invest Ophthalmol Vis Sci. 2009;50: E-Abstract 2215. 22. Binder PS. Barraquer lecture. What we have learned about corneal wound healing from refractive surgery. Refract Corneal Surg. 1989;5: 98–120. 23. Binder PS, Akers P, Zavala EY. Endothelial cell density determined by specular microscopy and scanning electron microscopy. Ophthalmology. 86;1979:1831–1847.
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24. Binder PS, Rock ME, Schmidt KC, et al. High-voltage electron microscopy of normal human cornea. Invest Ophthalmol Vis Sci. 1991;32:2234–2243. 25. Berry CC, Binder PS, Kahn M, Distribution of cells areas in normal and transplanted corneas. Exp Eye Res. 1980;31:623–635. 26. Virtanen J, Uusitalo H, Palkama A, et al. The effect of fixation on corneal endothelial cell dimensions and morphology in scanning electron microscopy. Acta Ophthalmol (Copenh). 1984;62:577–585. 27. Lang GK, Green WR, Maumenee AE. Clinicopathologic studies of keratoplasty eyes obtained post mortem. Am J Ophthalmol. 1986;101:28–40. 28. Pramanik S, Musch DC, Sutphin JE, et al. Extended long-term outcomes of penetrating keratoplasty for keratoconus. Ophthalmology. 2006;113: 1633–1638. 29. Bell KD, Campbell RJ, Bourne WM. Pathology of late endothelial failure: late endothelial failure of penetrating keratoplasty: study with light and electron microscopy. Cornea. 2000;19:40–46. 30. Bourne WM, Hodge DO, Nelson LR. Corneal endothelium five years after transplantation. Am J Ophthalmol. 1994;118:185–196. 31. Ing JJ, Ing HH, Nelson LR, et al. Ten-year postoperative results of penetrating keratoplasty. Ophthalmology. 1998;105:1855–1865. 32. He Z, Campolmi N, Gain P, et al. Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans. Stem Cells. 2012;30:2523–2534. 33. Bourne WM, Shearer DR. Effects of long-term rigid contact lens wear on the endothelium of corneal transplants for keratoconus 10 years after penetrating keratoplasty. CLAO J. 1995;21:265–267. 34. Matsuda M, Bourne WM. Long-term morphologic changes in the endothelium of transplanted corneas. Arch Ophthalmol. 1985;103:1343–1346. 35. Olsen EG, Davanger M. The healing of human corneal endothelium. An in vitro study. Acta Ophthalmol (Copenh). 1984;62:885–892. 36. Waring GO III, Bourne WM, Edelhauser HF, et al. The corneal endothelium. Normal and pathologic structure and function. Ophthalmology. 1982;89:531–590. 37. Matsuda M, Sawa M, Edelhauser HF, et al. Cellular migration and morphology in corneal endothelial wound repair. Invest Ophthalmol Vis Sci. 1985;26:443–449. 38. Sugar A, Meyer RF, Heidemann D, et al. Specular microscopic follow-up of corneal grafts for pseudophakic bullous keratopathy. Ophthalmology. 1985;92:325–330.
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39. Van Horn DL, Sendele DD, Seideman S, et al. Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci. 1977;16:597–613. 40. Olson RJ, Levenson JE. Migration of donor endothelium in keratoplasty. Am J Ophthalmol. 1977;84:711–714. 41. Hos D, Heindl LM, Bucher F, et al. Evidence of donor corneal endothelial cell migration from immune reactions occurring after descemet membrane endothelial keratoplasty. Cornea. 2014;33:331–334. 42. Jacobi C, Zhivov A, Korbmacher J, et al. Evidence of endothelial cell migration after descemet membrane endothelial keratoplasty. Am J Ophthalmol. 2011;152:537–542.e2. 43. Rao GN, Aquavella JV. Peripheral recipient endothelium following corneal transplantation. Ophthalmology. 1981;88:50–55. 44. Polack FM. The endothelium of failed corneal grafts. Am J Ophthalmol. 1975;79:251–261. 45. Rao GN, Shaw EL, Arthur E, et al. Morphological appearance of the healing corneal endothelium. Arch Ophthalmol. 1978;96: 2027–2030. 46. Bourne WM, Kaufman HE. The endothelium of clear corneal transplants. Arch Ophthalmol. 1976;94:1730–1732. 47. Bourne WM. Chronic endothelial cell loss in transplanted corneas. Cornea. 1983;2:289–294. 48. Price FW Jr, Whitson WE, Marks RG. Graft survival in four common groups of patients undergoing penetrating keratoplasty. Ophthalmology. 1991;98:322–328. 49. Shah RD, Randleman JB, Grossniklaus HE. Spontaneous corneal clearing after Descemet’s stripping without endothelial replacement. Ophthalmology. 2012;119:256–260. 50. Ziaei M, Barsam A, Mearza AA. Spontaneous corneal clearance despite graft removal in Descemet stripping endothelial keratoplasty in Fuchs endothelial dystrophy. Cornea. 2013;32:e164–e166. 51. Vetter JM, Butsch C, Faust M, et al. Irregularity of the posterior corneal surface after curved interface femtosecond laser-assisted versus microkeratome-assisted descemet stripping automated endothelial keratoplasty. Cornea. 2013;32:118–124. 52. Borderie VM, Boëlle PY, Touzeau O, et al. Predicted long-term outcome of corneal transplantation. Ophthalmology. 2009;116: 2354–2360.
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What Happens to the Corneal Transplant Endothelium After Penetrating Keratoplasty? Luiz F. Regis-Pacheco, MD,*† and Perry S. Binder, MS, MD†‡
Purpose: The aim of this study was to examine the human corneal endothelium of the transplant donor, wound, and adjacent host to determine the fate of the endothelial cells after penetrating keratoplasty.
Methods: We performed dissecting microscopic overviews and light and scanning electron microscopy on clear corneal transplant specimens obtained 1 month to 47 years after transplantation. The indications for the primary keratoplasty were keratoconus (11), Fuchs endothelial dystrophy (7), bullous keratopathy (6), others (5), and 8 cases without clinical data.
Results: We were able to visualize the wound and perform relative endothelial cell counts in 17 of 37 specimens. The wounds were of 4 shapes: smooth, anterior and/or posterior gaping, and anterior or posterior overriding. Any combination could be seen in the same specimen. Cells migrated from the center of the donor across the donor–host wound toward the host, but in all cases, the cells spread out, enlarged, and were ultimately lost. One case of Fuchs endothelial dystrophy may have had cell migration from the host across the wound to the donor.
Conclusions: We confirmed that donor cells migrate from higher density to lower density across the transplant wound over time. Wound configuration, donor cell health, recipient endothelial health, and probable cell-to-cell contact inhibition are involved in this process. Key Words: corneal transplant, endothelium, wound healing, SEM, morphology (Cornea 2014;33:587–596)
S
pecular microscopic studies have documented that normal human corneas lose a small percent of their endothelium every decade throughout life.1,2 Although controversy exists as to the ability of the endothelium to repair itself,3,4 most agree that the cell loss is irreplaceable.
Received for publication January 23, 2014; revision received February 20, 2014; accepted March 1, 2014. Published online ahead of print April 23, 2014. From the *Department of Ophthalmology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; †National Vision Research Laboratory, San Diego, CA; and ‡Gavin Herbert Eye Institute, University of California, Irvine, CA. Supported by National Vision Research Laboratory. The authors have no conflicts of interest to disclose. Reprints: Perry S. Binder, 2500 6th Avenue, Unit 307, San Diego, CA 92103 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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The success of penetrating corneal transplants is assumed to be related to the quality and quantity of the endothelial cells from the donor; the younger the donor, the greater the endothelial cell density is theorized to account for long-term transplant success in the absence of immunologic rejection or other operative and postoperative complications.5,6 Specular microscopic studies of penetrating keratoplasty cases have documented a progressive loss of endothelial cells; after 10 to 20 years, many corneas need to be retransplanted because of the cell loss. One theory to account for the chronic cell loss assumes trauma of corneal preservation and the surgical technique7; once the initial procedure-related trauma and surgical inflammation is completed, and the graft endothelium replaces the lost cells near the wound, thereafter there is only normal cell loss due to aging.8–10 Another theory supports some impact of the host endothelium migrating onto the donor from the host11–14; the success of the transplant therefore depends on the quality of the host endothelium. The secondary late migration of donor cells to replace diseased or absent host cells along with other proposed mechanisms may account for the documented progressive cell loss.14–17 As endothelial cell density decreases because of migration or apoptosis,8 the remaining donor endothelium is assumed to spread to enlarge and cover the missing areas so that cell density decreases over time as the cell size and cell shape change. These conclusions were reached based on indirect evidence; a few studies have directly examined the human transplant graft–host junction using light or transmission electron microscopy,18–21 but only 1 study has used scanning electron microscopy (SEM).18 We have had the unique opportunity to study the posterior corneal surface in clear penetrating keratoplasty cases. The purpose of the study was to examine these specimens to assess the endothelial cell morphology on both sides of the donor–recipient junction using SEM.
METHODS The study was performed at the Ophthalmology Research Laboratory of the National Vision Research Institute (now closed). Between May 1978 and June 1994, our laboratory received corneal tissue obtained at the time of penetrating keratoplasty from clear corneal transplants, which were optically unacceptable because of high and/or irregular astigmatism that was not correctable, as well as from donor eyes collected through eye banks from those who had died of natural causes months to years after penetrating keratoplasty. www.corneajrnl.com |
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In the case of a regraft, an attempt was made to encompass the donor–recipient junction of the previous transplant in one or more quadrants. Specimens were (1) received in a moist chamber as whole globes, (2) received as a corneal scleral rim in corneal preservation medium, and (3) conveyed by other means, such as a refrigerated balanced salt solution. Specimens obtained from outside our laboratory were refrigerated at +4°C and were shipped to our laboratory. An attempt was made to obtain clinical histories in all cases. On arrival, we performed overview photographs through a dissecting microscope and compared them with the available clinical slit-lamp photographs (Figs. 1A–C). We also created drawings of the original transplant and the current transplant to assist in locating the donor– host junction. According to our laboratory protocol, corneal sections were immediately placed in 2% glutaraldehyde after examination as described above. Specimens designated for SEM were processed as previously described.18,22,23 We performed light microscopy on thick sections designated for transmission electron microscopy in a few specimens to maximize the tissue areas available for SEM study,24 but we report only the SEM here.
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Each SEM specimen was scanned at low power (·9–·20) to obtain specimen orientation compared with any available clinical drawings. Because specimens were obtained over many months, the tissues were not coprocessed, and therefore, variations in tissue shrinkage would prevent a direct comparison of the cell density from 1 donor button with another.23 A minimum of 4 scanning electron micrographs, enlarged up to ·200, were made of each area examined. For a better definition of the cell borders, it was necessary to obtain electron micrographs enlarged from ·500 to ·700. It is known that the inclination or tilt of the SEM specimen may exaggerate the shapes of the cells that could be detrimental to obtaining comparative size, shape, and density estimates. Therefore, inclination or tilt of the tissue was adjusted throughout the SEM procedures to maintain visualization in the best possible position perpendicular to the area being examined. The images of electron micrographs were preferably taken in the flattest possible positions and between any folds in Descemet membrane. Table 1 lists the specimen studied, the original recipient diagnosis, the time from the original transplant to the repeat transplant, and any other clinical information (when this information was available to us) (Table 2).
FIGURE 1. A, Case 14. Clinical photograph before repeat transplant for high astigmatism. B, Dissecting microscopic view of case 14. Note the 2 sutures seen in the clinical photograph above. The entire transplant wound is encompassed by the larger trephine diameter. C, Continuous (left, dissecting microscopic photograph, case 35) or interrupted (right, dissecting microscopic photograph, case 37) sutures marked the site of the transplant wounds.
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Corneal Transplant Endothelium
TABLE 1. Specimen Clinical Information
Tissue
Drawing and/or Dissecting Photo
Donor Age/ Gender
PKP No.
OS OD OS OS OD NA OD OD OS OD OS OD OS OS OD OD OD OD OS OD OS OS OS
CS CS CS CS B B B CS CS B CS CS B B B B S S S B Eye Eye B
No Yes No No Yes Yes No No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes
13 6 wk NA NA 20 yr NA 37 yr NA NA 27/? NA 46 66/? 55/? 60/? 57/F 19/? NA NA NA NA NA 53/M
1 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 2
OD OD OS OS OD OD OD OD NA OD OD OD OS NA
½B CS Eye B B B B ½B B B B Eye Eye ½B
No Yes No Yes Yes No No No Yes Yes Yes No No No
53/M NA NA 41/? NA 28/M 72/M 66/M NA 49/M NA NA NA NA
2 1 ? 2 1 2 1 1 1 1 2 NA NA 1
Case
Age/ Sex
Pretransplant Diagnosis
POPKP, mo
Eye
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
81/F 78/F 73/M 65/M 47/F 54/F 77/F 73/F 73/F 62/F 71/M 68/M 85/F 65/M 42/F 35/M 42/M 76/M 76/M 98/F 98/F 82/F 39/M
ABK PBK Fuchs Fuchs HSV NA Fuchs ABK ABK Interstitial Keratitis NA Leukoma Interstitial keratitis Fuchs KC KC Interstitial keratitis NA NA NA NA NA KC
24 12 54 31 204 NA 32 65 81 140 NA 84 48 36 156 180 240 NA NA NA NA NA 31
24 25 26 27 28 29 30 31 32 33 34 35 36 37
81/F 66/M 59/F 47/F ?/M 80/F 71/M 53/F 69/F 43/F 58/F 86/F 72/F 63/F
Fuchs PBK PBK KC KC KC KC KC KC KC KC Fuchs? NA Fuchs
54 64 NA 156 216 564 444 298 492 240 NA NA NA NA
Diagnosis at Latest PKP Immune rejection Steep pediatric cornea Early PBK Corneal abscess Partial failure Clear PKP Steep cornea Clear PKP Clear PKP Corneal thinning NA Clear PKP Clear failed autograft Clear PKP Steep cornea Steep cornea Irregular cornea NA NA NA NA NA Recurrent KC; nonhealing epi-defect NA NA NA NA Recurrent KC Irregular square graft Recurrent KC; square graft Recurrent KC; hydrops NA Irregular astigmatism High astigmatism NA NA Irregular astigmatism
Data from eye banks, donating surgeons, and chart reviews. ABK, aphakic bullous keratopathy; B, corneal button; CS, corneal scleral rim; EpiDefect, chronic nonhealing epithelial defect; Eye, whole eye bank eye; Fuchs, Fuchs endothelial dystrophy; HSV, herpes simplex keratitis; KC, keratoconus; M/F, male/female; NA, data not available; PBK, pseudophakic bullous keratopathy; PKP, penetrating keratoplasty; POPKP, postoperative time from last transplant to excision; ?, NA; ½ B, half corneal button; 19 PKP, original transplant; 29 PKP, repeat transplant number 2.
Cell Counting We attempted to count a minimum of 50 contiguous endothelial cells in each electron micrograph: in the center of the donor, mid-way between the donor and wound, close to the wound, over the wound, and across the wound on to the host. The fewest cells counted in a given image were 50, the maximum were 150, and all but 3 images had more than 80 cells counted. This procedure was based on the study by Berry et al,25 which demonstrated that counting of as few as 10 cells is adequate for determining the endothelial cell density, assuming that these cells are uniformly distributed. 2014 Lippincott Williams & Wilkins
In cases where the cell borders were difficult to visualize, the SEM negatives were scanned under high resolution (300–1200 dpi; V60 scanner, Epson, Inc) and converted to a positive image for cell counting and morphological analysis using Adobe Photoshop CS 5 (Adobe, Inc).
Morphological Analysis Changes in cell shape, which might suggest cell migration, were clinically analyzed. An attempt was made to document the presence of bare Descemet membrane to www.corneajrnl.com |
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TABLE 2. Endothelial Cells and Wound Description From Scanning Electron Microscopy Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Visualize Wound? Yes Yes Yes No No Yes Yes No No Yes May be Yes Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes No No
Donor Cell Morphology Small, regular Small, hexagonal Large, 5–6 sides Small, distorted, guttata Large, postmortem artifact Distorted, small, overlapping Small, hexagonal Postmortem artifact Large, distorted Large, 5–6 sides Distorted, guttata Postmortem artifact ? Large, 5–6 sides Small, 5 and 6 sides, some ruptured Small, hexagonal Small, postmortem artifact Small, postmortem artifact Large, 5–6 sides Postmortem artifact None seen Large, postmortem artifact Few large cells Guttata, distorted, postmortem artifact None seen Large, 5 and 6 sides Small, stressed Large, overlapping, spreading None seen Small, hexagonal Very large, 4–6 sides Large, variable None seen None seen None seen None seen
Cell Migration
Host Cells Description
Donor to host Same size up to wound Donor to host ? No Similar both sides ? ? ? Donor to host ? ? ? Donor to host ? ?
Enlarge, then disappear None seen Guttata None seen None seen Similar both sides None seen None seen None seen Guttata Similar both sides None seen Yes Large None seen Yes None seen No Large, distorted None seen None seen None seen None seen None seen No Yes Same as donor ? None seen None seen None seen Yes Yes None seen None seen None seen None seen
? Donor to host ? ? ? No ? Donor to host ? Yes May be ? ? ? No ? ? ? ? ?
Morphological description of donor, host, and donor–host junction. Maybe, some evidence of migration (see explanation in Results); None seen, less than 20 cells present (unable to make a determination); ?, Unable to make a determination.
investigate the individual characteristics of endothelial cells and their morphological changes, which might suggest possible cell migration. In our analysis, all cases were divided in accordance with the preoperative diagnosis, their primary indication for corneal transplant: keratoconus (11), Fuchs endothelial dystrophy (7), corneal edema (aphakic and pseudophakic bullous keratopathy) (6), and other indications (5) (Table 1).
RESULTS Clinical Specimens We obtained documentation to study 37 specimens that had sufficient tissue to examine the donor–host junction
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(Table 1). Additional clear corneal transplant specimens had either insufficient clinical information, were only processed for light microscopy, or were not processed because of various technical reasons. Ten cases had clinical slit-lamp photographs obtained before the repeat transplant, and 14 cases had dissecting microscopic overview photographs of the excised specimens (Fig. 1B). The original indications for the primary keratoplasty are listed in Table 1. Fuchs endothelial dystrophy (cases 3, 4, 7, 14, 24, 29, and 35), keratoconus (cases 15, 16, 23, and 27–34), and bullous keratopathy (cases 1, 2, 8, 9, 25, and 26) were the most common diagnoses. Eight cases had no clinical diagnosis available (cases 6, 11, 18, 19, 20, 21, 22, and 36), and a few scattered diagnoses made up the remaining specimens (cases 5, 10, 12, 13, and 17). The indications for the regrafts included recurrent 2014 Lippincott Williams & Wilkins
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keratoconus, steep corneas associated with a pediatric donor, and/or irregular/high astigmatism unresponsive to relaxing or wedge resections. Nine specimens represented a second transplant and 2 had no information about the transplant number; the remainder were obtained after an initial transplant that had been performed 1 to 47 years earlier (Table 1). Figure 1A displays a clinical slit-lamp photograph, and Figure 1B its counterpart dissecting microscopic overview photograph. Figure 2 shows a case of a square graft obtained 47 years after the primary transplant operation by Ramon Castroviejo, MD. The diameters of the excised tissue, where known, varied from 7.5 to 10 mm with the most common diameter being 8 mm. The second transplant trephine diameters were mostly 0.5 mm greater than the previous transplant diameters.
Cell Density We were able to perform relative cell densities from the SEM photographs in 17 of the specimens. In an attempt to locate more cells in poor images, we subsequently digitized each SEM negative and enhanced each negative; this permitted us to visualize more cells in 10 additional specimens, but the cell borders were either blurry or not present in sufficient quantity or quality to permit an accurate count. In 6 specimens, we were unable to see cells anywhere in the specimen despite the material being described as a clear graft. Because all of our specimens were prepared over several years by different electron microscopy technicians using different equipment, we assume the inability to see cells in
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clear grafts was due to inadequate tissue processing, prolonged death to fixation, inadequate sputter coating, and/or other technical issues. Unfortunately, all of the 37 specimens were discarded when our laboratory closed in 1997, therefore we were unable to reprocess the tissues.
Morphology of the Donor
For most specimens, the most common finding was the ability to visualize endothelial cells in the donor. We found various central donor cell morphologies from different specimens, which shows how much variability in the center of a clear donor can be present with various host diagnoses and posttransplant time. Stated differently, our examination of different areas within the same clear corneal transplant donor tissue showed great variation in size, shape, and distribution of the cells. The decrease in the cell density and the increase in the cell pleomorphism over a distance suggest cell migration in the direction of the lower cell density, and cells with increased size and pleomorphism. Because our cell counting technique was relative for each specimen and the calculation was based on the assumed tissue shrinkage percentages,23,26 we were not able to make any conclusions regarding cell density versus postoperative time, cell density versus host diagnosis at the time of tissue excision, donor age, or cell density versus diameter of the excision. We were able to analyze changes in cell dimensions (shape and size) in many specimens within the same donor tissue.
Host–Donor Junction Sutures marked the wounds in some cases (Figs. 1A–C). We found 4 general shapes of the donor–host junction. The wound could be tight, that is, well apposed; in these specimens, the transition from donor to host was smooth (Fig. 2). In other specimens, a wound gaped open anterior to posterior toward the anterior chamber apparently making it difficult for cells to transition across the gape. The donor could be retracted into the host cornea away from the anterior chamber below the level of the host forcing endothelial cells to migrate “down the hill” to the level of the host. Alternatively, the host could be deeper in the anterior chamber versus the donor so that cells migrating from the donor to the host would have to move “uphill.” Any combination of these shapes might be present in the same specimen. Figures 3 and 4A to F show examples of different wound configurations. Figure 1C is a view of dissecting microscopy of cases 35 and 37 obtained from eye bank specimens showing a continuous 11-0 suture (left) and interrupted 10-0 sutures (right) across the transplant wounds. Some specimens included 360 degrees of the wound, whereas others were created by decentering the trephine to encompass some of the previous wound so that only a portion of the wound might be present. We were able to easily see endothelial cells on the donor side in most specimens, over the wound in 16 and into the host in 13 (Figs. 4A–F, 5A, B).
FIGURE 2. Case 29. Square graft for Fuchs endothelial dystrophy. The lines mark the outer border of the square graft performed by Ramon Castroviejo 47 years ago. SEM ·20. 2014 Lippincott Williams & Wilkins
Morphology of the Host Surfaces and Cells We assessed these cells the same way as we assessed the donor cells. In addition, we looked at the host surface peripheral www.corneajrnl.com |
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with a similar appearance to the donor cells (Fig. 4E). Case 32 (also a second penetrating keratoplasty) also had a similar appearing endothelium on the host and donor, whereas case 33 (original transplant) had host cells that were 5 and 6 sided with areas of bare Descemet membrane being apparent. Figure 4B is an example of a bare, rough host consisting of fibrocellular material commonly seen in failed grafts— a retrocorneal membrane.27 The donor cells appear to be on the surface of the donor side of the wound, but there is no evidence of these cells on the host side.
Cell Migration Across the Transplant Wound
FIGURE 3. Example of SEM morphology in case 6 from an 8-mm button. The numbers refer to each of the subsequent figures in sequence. Case 6. The clinical history is not available. Overview SEM of one half of the excised cornea showing 3 areas of the donor from the center to the periphery up to the circular donor–host wound that is easily seen. Area 4 represents the host. SEM ·10.
to the wound for cell presence and quality. Figures 5A and B show various host stroma and cell appearances. In all cases where we could detect endothelial cells on the host side of the wound, the cells appeared to be enlarging, spreading, and/ or assuming a more pleomorphic appearance. Further away from the wound, the cell density would decrease and cell processes would spread to cover more of the host. Eventually, the cells were not able to cover the host and bare areas of Descemet membrane so that the fibrocellular surface of the host would be exposed (Fig. 5B). In cases of Fuchs endothelial dystrophy, the guttate excrescences on the surface of the host Descemet membrane would be exposed. There were 7 cases of a primary transplant in a recipient with keratoconus (cases 15, 16, 23, 27, 28, 30, 32, and 33); we were able to obtain cell counts centrally in 6 cases, but we could only see cells in 4 keratoconus host cases (cases 16, 27, 32, and 33). A large area of host cells was seen in case 16, but for some reason, the electron microscopy technician did not scan that area, which appeared to have a high density of cells similar to that of the donor when viewed at ·40. The host cells on case 16 were enlarged and contained 4-, 5-, and 6-sided cells whose surfaces were rough and appeared of different electron reflectivity compared with the donor cells. Case 27 (a second penetrating keratoplasty) had cells of 4 to 6 sides
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If the cell density decreased from the center to the periphery of the donor, we assumed that the cells were being “lost” by a spreading and enlarging process. In the circumstance where the central morphology consisted of small, hexagonal-shaped, uniform density cells, as we looked toward the donor periphery, we saw increasing pleomorphism and/or increasing cell size, we assumed cell movement toward the periphery. Change in appearance of the cell surface (smooth to rough and/or electron reflectivity) from central to periphery was similarly considered as a sign of cell movement. At the donor–host junction, we looked for appearance of the cells across the wound (Figs. 4A–F, 5A, B). If the cells increased in size, changed shape from a regular 6-sided hexagon to cells with fewer sides, demonstrated a decrease in density, and/or appeared to spread out over the host, we assumed that there was centrifugal movement, that is, movement toward the host. We expected to see a high density of host cells in our keratoconus cases because keratoconus hosts have normal cell densities.6,28 In only 4 keratoconus cases where we had donor and host cells present, we could see similar cell morphology in the donor and host in 2 of the specimens. We therefore could not confirm a host-to-donor (centripetal) cell migration or contact inhibition between donor and host cells when a host cell population is healthy, although we suspect this is the case clinically because of the high success rate of corneal transplants for keratoconus. In case 3 (Fig. 4F), obtained 54 months after a transplant for a host diagnosis of Fuchs endothelial dystrophy, guttae are easily seen on the host side (upper right). The cell appearance and density were very similar on both sides of the ridge-like wound. Guttae were also present on the donor at the edge of the ridge and into the donor. We did not have any information about the donor, but at the time of the regraft, the surgeon thought that the cornea was slightly edematous. The findings strongly suggest cell migration from the host to the donor, but one cannot eliminate the possibility of the donor having subclinical Fuchs endothelial dystrophy. But if this were the case, one would expect to see many more guttae in the donor. We had 7 primary grafts for keratoconus and 3 primary grafts for Fuchs endothelial dystrophy. All of these 10 eyes had evidence of cell migration from the donor to the recipient.
DISCUSSION Evidence supports an acute loss of donor endothelium at the time of surgery that is due to a combination of surgical 2014 Lippincott Williams & Wilkins
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FIGURE 4. Examples of various donor–host wounds. A, Overview of case 20 showing a depression between the donor and host (“gully”) on the top and a very even wound that is completely closed on the right. Specimen of a 98-year-old donor who died after keratoplasty, obtained from eye bank. Clinical data unavailable. SEM ·20. B, Case not reported herein, but previously published. There is a slight ridge at the donor–host junction (arrowheads). The endothelium of the donor ([D], left) appears to slide over the wound onto a rough recipient (R) stroma and then disappears. SEM ·20. Reproduced with permission from CV Mosby, Figures 1–16E; Transactions of the New Orleans Academy of Ophthalmology (page 23).18 Clinical data not available. C, Case 10 (interstitial keratitis, transplant 2). Donor (left) protruding into anterior chamber so that endothelium has to migrate “uphill” onto host (right). As cells cross the wound, their morphology changes to larger cells that attempt to spread out over the host stroma and are eventually lost. SEM ·200. D, Case 14 (Fuchs dystrophy, first transplant). Ridge (arrows) at the donor (bottom)–host (top) junction (dashed lines). Cells enlarge and spread “uphill” onto the host surface where they make a sheet 10 to 15 cells wide before being unable to surface the bare host stroma. SEM ·200. E, Case 27 (keratoconus, second transplant). Sharply defined donor (lower, left)–host (upper, right) wound with a smooth border (dashed line). Endothelium appears to have successfully migrated onto the host as the cell shapes are similar on both sides of the wound immediately adjacent to the wound, but the cells enlarge in the periphery of the host with loss of some cell junctions (upper right). SEM ·200. F, Case 3 obtained 54 months after a transplant for a host diagnosis of Fuchs endothelial dystrophy. Guttae are easily seen on the host side (upper right). The cell appearance and density was very similar on both sides of the ridge-like wound (D). Guttae were also present on the donor at the edge of the ridge and into the donor. We did not have any information about the donor, but at the time of the regraft, the surgeon thought that the cornea was slightly edematous. The findings strongly suggest cell migration from the host to the donor, but one cannot eliminate the possibility of the donor having subclinical Fuchs endothelial dystrophy. But if this was the case, one would expect to see many more guttata in the donor.
trauma, corneal harvest trauma, and preservation damage.7,29 After keratoplasty, there is a greater rate of cell loss compared with the normal aging process.8,15,29–31 Some of this loss can be attributed to postkeratoplasty complications such as immuno 2014 Lippincott Williams & Wilkins
logic rejection, glaucoma, secondary surgical insults (inflammation, intraocular lens damage, vitreous touch, etc), but many grafts do not have any of these issues and yet they suffer progressive cell loss and ultimate failure from apoptosis.8 www.corneajrnl.com |
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FIGURE 5. Examples of endothelial cell morphology on the host side of the transplant wound. A, Case 14 (Fuchs dystrophy, first transplant). The donor center is down to the right away from this image. Cells seem to be spreading away from the donor onto a surface of Descemet membrane that appears to contain diffuse guttata. Bare areas of Descemet membrane are further away from the wound (above) where the endothelial cells can no longer cover the surface. SEM ·500. B, Host side of wound from case 16 (keratoconus, transplant one). The center of the donor is above this image. The host appears to have a rough surface away from the wound. The cells appear to enlarge further away from the wound. SEM ·400.
There is uncertainty as to the mechanism(s) of a traumatic, progressive postkeratoplasty endothelial cell loss that is greater than the normal aging process. There is scant evidence that the human endothelium is capable of mitosis,3,4,13,32 so it is assumed that donor cells move to areas that are devoid of cells or areas of lesser cell density, and therefore, the central donor cell density has to decrease over time unless replaced by host cells.30,32–37 Studies of graft success rates have suggested that healthy host endothelium (eg, keratoconus) is related to better longitudinal outcomes6 compared with hosts with diseased or absent endothelium (eg, pseudophakic bullous keratopathy).16,38 Basic to this assumption is that host cells can migrate across the transplant wound to repopulate the donor or at least prevent or slow down central cell migration to the host. In vitro animal and human organ culture experiments that used whole corneas have demonstrated centripetal and centrifugal movements of cells after keratoplasty37 or wounding.39 One study that assessed corneal transplants in rabbits has demonstrated cell movement across the wound from the donor to the recipient40; and most recently, there is additional evidence that endothelial cells can migrate from the donor to the host.17,41,42 Special techniques have been used to image the host endothelium using conventional specular microscopy,23,43 but because of the optics of these systems, the transplant wounds themselves could not be directly visualized. One study found donor cell survival to be highly variable and that cell migration was not fully explained by spreading of cells. They were convinced that cells migrated across the transplant wound with the recipient cells replacing the donor cells in 10 of 36 specimens.14 Although there have been several pathological studies of failed corneal grafts, only 1 study imaged the clear corneal transplant wound using SEM but did not discuss the findings. In the review of corneal anatomy by Binder et al,18 2 scanning electron micrographs of the donor–host junction were shown, but there was no discussion regarding cell migration
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(Fig. 4B). Our present article is the first to demonstrate the donor–host junction using SEM in an analysis of clear corneal transplants in which we directly document cell migration across the wound onto the host. As the cells moved across the wound, they tended to enlarge and spread to cover the host in areas devoid of endothelium either over bare Descemet membrane or over fibrocellular tissue presumably a residual of previous endothelial cell loss and replacement wound healing27,44 (Figs. 4, 5). If the host diagnosis was Fuchs endothelial dystrophy, we could detect guttae in the host, and in some cases in the donor at the edge of the wound (Fig. 4F). Although we had several host diagnoses of keratoconus in primary grafts, we were not able to document cell movement toward the donor either because we could not detect host cells (cases 6, 15, 23, and 28) or donor cells (cases 28 and 30) in these specimens. We ascribed the inability to see cells despite a pregraft diagnosis of a clear corneal transplant to prolonged postmortem preservation time or inadequate SEM tissue preparation or examination. Other studies have also documented the absence of cells in cases of clear corneal transplants even when pregraft specular microscopy found the presence of cells.14,23 We demonstrated the highly variable cell sizes and shapes within the same donor cornea described as being “clear” which has been previously described14,16,23,25,29,30,43,45–47 (Figs. 3–5). This variable size and shape of donor cells after keratoplasty suggests unstable, stressed, or enlarging cells related to peripheral wound-healing events.29 Focal areas of cell loss were also seen within the donor, which has been previously documented.29 A small area of examination (eg, conventional specular microscopy) in such specimens would lead to an erroneous cell density in the case of nonuniform size, shape, and density of cells. Our study confirms the biexponential model of cell loss after keratoplasty15,16 and documents that one reason for continued cell loss over time in addition to apoptosis is consistent with progressive donor cell migration centrifugally across the wound to replace cells in a host with a poor or missing 2014 Lippincott Williams & Wilkins
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endothelium. Because studies of keratoconus transplants (normal, healthy endothelium) show consistently higher survival rates than grafts into hosts with poor or no endothelium, for example, bullous keratopathy,16 it is easy to conclude that in keratoconus transplants either the donor cells are contactinhibited as they meet the host cells and/or the host cells migrate centripetally across the wound to replace the weak, healthy, or diseased donor cells.14,17 Cases of borderline host endothelium (Fuchs endothelial dystrophy) may be able to repopulate the donor depending on cell variables we currently cannot measure (Fig. 4F). For example, Price et al48 found equivalent graft survival rates between keratoconus and Fuchs dystrophy. Instances of late clearing of corneal grafts in hosts with unhealthy endothelium11 or in cases of attempted Descemet stripping endothelial keratoplasty41,49,50 have been reported, which suggest centripetal cell movement. We found 4 different wound configurations in our specimens. It would seem that the smoother the wound, the easier the cell migration (Figs. 2–5), but that if there is a large disparity in height and/or a wound gape, it would be more difficult for endothelial cells to migrate across compared with migration across a smooth, well-apposed wound. If a host has no cells and or the wound is not conducive to cell migration, one would anticipate a chronic loss of cells associated with a continuous centrifugal cell movement. Based on our morphology studies herein, in the case of Descemet stripping endothelial keratoplasty or Descemet’s stripping automated endothelial keratoplasty, the large height disparity between the host and donor would lend itself to chronic cell loss. If we can create better posterior wound coadaptation through better technology,51 we may be able to increase the survival rate of our transplants. Variation in surgeon surgical expertise undoubtedly plays a role; square grafts created by one expert corneal surgeon have survived for many decades (cases 29 and 30) (Fig. 2). Our study has several weaknesses. We were only able to study 37 specimens. Based on our analyses, we had some cases of poor tissue preparation, lack of some clinical information, tissue shrinkage artifacts,23,26 and lack of complete assessment of host areas. This was evident from cases with cells seen on specular microscopy before tissue excision, but absence of cells with SEM. Although we had several specimens where the endothelium clearly moved across the wounds from the donor to the host, we had other specimens where we could not identify such cell migration. We were especially disappointed by not documenting cell migration from the host to the donor in our cases of keratoconus. To correct these errors, in addition to the obvious technical issues, we suggest obtaining larger diameter excisions, immunohistochemistry,17 radio labeling techniques, and the use of wide-field specular microscopy.52
ACKNOWLEDGMENTS The authors thank those organizations and surgeons who kindly provided tissue for this study (the San Diego Eye Bank, Lions Eye Bank Portland, and Arizona Lions Eye Bank), Frank Price, Jr, MD, Jeffrey L. Katz, MD, Lee T. Nordan, MD, and 2014 Lippincott Williams & Wilkins
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Edward L. Shaw, MD. We thank Max Moore for microscopy expertise. The authors dedicate this article to the memory of Frank M. Polack, MD, who kindly provided the initial experimental pathologic and scanning electron microscopy experience for one of the (P.S.B.) authors. REFERENCES 1. Abib FC, Barreto J Jr. Behavior of corneal endothelial density over a lifetime. J Cataract Refract Surg. 2001;27:1574–1578. 2. Cheng H, Jacobs PM, McPherson K, et al. Precision of cell density estimates and endothelial cell loss with age. Arch Ophthalmol. 1985; 103:1478–1481. 3. Laing RA, Neubauer L, Oak SS, et al. Evidence for mitosis in the adult corneal endothelium. Ophthalmology. 1984;91:1129–1134. 4. Treffers WF. Human corneal endothelial wound repair. In vitro and in vivo. Ophthalmology. 1982;89:605–613. 5. Lass JH, Beck RW, Benetz BA, et al. Baseline factors related to endothelial cell loss following penetrating keratoplasty. Arch Ophthalmol. 2011;129:1149–1154. 6. Gordon MO, Steger-May K, Szczotka-Flynn L, et al. Baseline factors predictive of incident penetrating keratoplasty in keratoconus. Am J Ophthalmol. 2006;142:923–930. 7. Alqudah AA, Terry MA, Straiko MD, et al. Immediate endothelial cell loss after penetrating keratoplasty. Cornea. 2013;32:1587–1590. 8. Albon J, Tullo AB, Aktar S, et al. Apoptosis in the endothelium of human corneas for transplantation. Invest Ophthalmol Vis Sci. 2000;41: 2887–2893. 9. Bourne WM. Penetrating keratoplasty with fresh and cryopreserved corneas. Donor endothelial cell survival in primates. Arch Ophthalmol. 1978;96:1073–1074. 10. Bourne WM, O’Fallon WM. Endothelial cell loss during penetrating keratoplasty. Am J Ophthalmol. 1978;85:760–766. 11. Baum JL, Late spontaneous clearing of corneal grafts. Arch Ophthalmol. 1977;95:1538–1539. 12. Chi HH, Teng CC, Katzin HM, et al. The fate of endothelial cells in corneal homografts. Am J Ophthalmol. 1965;59:186–191. 13. Wollensak G, Green WR. Analysis of sex-mismatched human corneal transplants by fluorescence in situ hybridization of the sex-chromosomes. Exp Eye Res. 1999;68:341–346. 14. Lagali N, Stenevi U, Claesson M, et al. Donor and recipient endothelial cell population of the transplanted human cornea: a two-dimensional imaging study. Invest Ophthalmol Vis Sci. 2010;51:1898–1904. 15. Armitage WJ, Dick AD, Bourne WM. Predicting endothelial cell loss and long-term corneal graft survival. Invest Ophthalmol Vis Sci. 2003;44: 3326–3331. 16. Böhringer D, Böhringer S, Poxleitner K, et al. Long-term graft survival in penetrating keratoplasty: the biexponential model of chronic endothelial cell loss revisited. Cornea. 2010;29:1113–1117. 17. Merjava S, Malinova E, Liskova P, et al. Recurrence of posterior polymorphous corneal dystrophy is caused by the overgrowth of the original diseased host endothelium. Histochem Cell Biol. 2011;136:93–101. 18. Binder PS, Wickham MG, Zavala EY, et al. Corneal anatomy and wound healing. In: Symposium on Medical and Surgical Diseases of the Cornea. Transactions of the New Orleans Academy of Ophthalmology 1980. St Louis, MO: CV Mosby; 1–35. 19. Hatanaka H, Koizumi N, Okumura N, et al. A study of host corneal endothelial cells after non-Descemet stripping automated endothelial keratoplasty. Cornea. 2013;32:76–80. 20. Obata H, Tsuru T. Corneal wound healing from the perspective of keratoplasty specimens with special reference to the function of the Bowman layer and Descemet membrane. Cornea. 2007;26(9 suppl l):S82–S89. 21. Stewart RM, Kaye SB, Batterbury M, et al. Histopathological features associated with endothelial keratoplasty failure. Invest Ophthalmol Vis Sci. 2009;50: E-Abstract 2215. 22. Binder PS. Barraquer lecture. What we have learned about corneal wound healing from refractive surgery. Refract Corneal Surg. 1989;5: 98–120. 23. Binder PS, Akers P, Zavala EY. Endothelial cell density determined by specular microscopy and scanning electron microscopy. Ophthalmology. 86;1979:1831–1847.
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24. Binder PS, Rock ME, Schmidt KC, et al. High-voltage electron microscopy of normal human cornea. Invest Ophthalmol Vis Sci. 1991;32:2234–2243. 25. Berry CC, Binder PS, Kahn M, Distribution of cells areas in normal and transplanted corneas. Exp Eye Res. 1980;31:623–635. 26. Virtanen J, Uusitalo H, Palkama A, et al. The effect of fixation on corneal endothelial cell dimensions and morphology in scanning electron microscopy. Acta Ophthalmol (Copenh). 1984;62:577–585. 27. Lang GK, Green WR, Maumenee AE. Clinicopathologic studies of keratoplasty eyes obtained post mortem. Am J Ophthalmol. 1986;101:28–40. 28. Pramanik S, Musch DC, Sutphin JE, et al. Extended long-term outcomes of penetrating keratoplasty for keratoconus. Ophthalmology. 2006;113: 1633–1638. 29. Bell KD, Campbell RJ, Bourne WM. Pathology of late endothelial failure: late endothelial failure of penetrating keratoplasty: study with light and electron microscopy. Cornea. 2000;19:40–46. 30. Bourne WM, Hodge DO, Nelson LR. Corneal endothelium five years after transplantation. Am J Ophthalmol. 1994;118:185–196. 31. Ing JJ, Ing HH, Nelson LR, et al. Ten-year postoperative results of penetrating keratoplasty. Ophthalmology. 1998;105:1855–1865. 32. He Z, Campolmi N, Gain P, et al. Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans. Stem Cells. 2012;30:2523–2534. 33. Bourne WM, Shearer DR. Effects of long-term rigid contact lens wear on the endothelium of corneal transplants for keratoconus 10 years after penetrating keratoplasty. CLAO J. 1995;21:265–267. 34. Matsuda M, Bourne WM. Long-term morphologic changes in the endothelium of transplanted corneas. Arch Ophthalmol. 1985;103:1343–1346. 35. Olsen EG, Davanger M. The healing of human corneal endothelium. An in vitro study. Acta Ophthalmol (Copenh). 1984;62:885–892. 36. Waring GO III, Bourne WM, Edelhauser HF, et al. The corneal endothelium. Normal and pathologic structure and function. Ophthalmology. 1982;89:531–590. 37. Matsuda M, Sawa M, Edelhauser HF, et al. Cellular migration and morphology in corneal endothelial wound repair. Invest Ophthalmol Vis Sci. 1985;26:443–449. 38. Sugar A, Meyer RF, Heidemann D, et al. Specular microscopic follow-up of corneal grafts for pseudophakic bullous keratopathy. Ophthalmology. 1985;92:325–330.
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39. Van Horn DL, Sendele DD, Seideman S, et al. Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci. 1977;16:597–613. 40. Olson RJ, Levenson JE. Migration of donor endothelium in keratoplasty. Am J Ophthalmol. 1977;84:711–714. 41. Hos D, Heindl LM, Bucher F, et al. Evidence of donor corneal endothelial cell migration from immune reactions occurring after descemet membrane endothelial keratoplasty. Cornea. 2014;33:331–334. 42. Jacobi C, Zhivov A, Korbmacher J, et al. Evidence of endothelial cell migration after descemet membrane endothelial keratoplasty. Am J Ophthalmol. 2011;152:537–542.e2. 43. Rao GN, Aquavella JV. Peripheral recipient endothelium following corneal transplantation. Ophthalmology. 1981;88:50–55. 44. Polack FM. The endothelium of failed corneal grafts. Am J Ophthalmol. 1975;79:251–261. 45. Rao GN, Shaw EL, Arthur E, et al. Morphological appearance of the healing corneal endothelium. Arch Ophthalmol. 1978;96: 2027–2030. 46. Bourne WM, Kaufman HE. The endothelium of clear corneal transplants. Arch Ophthalmol. 1976;94:1730–1732. 47. Bourne WM. Chronic endothelial cell loss in transplanted corneas. Cornea. 1983;2:289–294. 48. Price FW Jr, Whitson WE, Marks RG. Graft survival in four common groups of patients undergoing penetrating keratoplasty. Ophthalmology. 1991;98:322–328. 49. Shah RD, Randleman JB, Grossniklaus HE. Spontaneous corneal clearing after Descemet’s stripping without endothelial replacement. Ophthalmology. 2012;119:256–260. 50. Ziaei M, Barsam A, Mearza AA. Spontaneous corneal clearance despite graft removal in Descemet stripping endothelial keratoplasty in Fuchs endothelial dystrophy. Cornea. 2013;32:e164–e166. 51. Vetter JM, Butsch C, Faust M, et al. Irregularity of the posterior corneal surface after curved interface femtosecond laser-assisted versus microkeratome-assisted descemet stripping automated endothelial keratoplasty. Cornea. 2013;32:118–124. 52. Borderie VM, Boëlle PY, Touzeau O, et al. Predicted long-term outcome of corneal transplantation. Ophthalmology. 2009;116: 2354–2360.
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