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Influence of Ultrastructural Corneal Graft Abnormalities on the Outcome of Descemet Membrane Endothelial Keratoplasty
Influence of Ultrastructural Corneal Graft Abnormalities on the Outcome of Descemet Membrane Endothelial Keratoplasty ¨ TZER-SCHREHARDT, THEOFILOS TOURTAS, AND FRIEDRICH E. KRUSE JULIA M. WELLER, URSULA SCHLO PURPOSE:
To investigate if ultrastructural alterations in the Descemet membrane (DM) are correlated with the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Retrospective cohort study. METHODS: SETTING: Institutional, single-center. STUDY POPULATION: One hundred and twelve residual DM specimens obtained after DM stripping. MAIN OUTCOME MEASURES: Incidence of ultrastructural abnormalities in transmission electron microscopy, graft detachment rate, graft failure rate, best-corrected visual acuity (BCVA), endothelial cell density (ECD), and central corneal thickness (CCT). Examination dates were on the day before DMEK and 1, 3, 6, and 12 months after surgery. RESULTS: Abnormalities in the ultrastructure of DM were found in 16 of 112 specimens (14%) (abnormal DM group), comprising deposits of long-spacing collagen, fine filaments (proteoglycans), a posterior collagenous layer, pseudoexfoliative material, and guttae. The secondary graft failure rate was significantly higher in the abnormal DM group compared with the normal DM group (P [ .001). There was a trend for an increased graft detachment rate in the abnormal DM group (11/16) compared with the normal DM group (42/96) (P [ .103). There was no significant difference in mean CCT and ECD after surgery. Mean CCT in the eyes with graft failure in the abnormal DM group at the last follow-up before regrafting was 850 mm, indicating endothelial failure with stromal edema. CONCLUSION: This study reveals a correlation between ultrastructural alterations of DM in donor corneas and the graft failure rate after DMEK. Thus, graft failure after DMEK not only is determined by surgical trauma and postoperative events but may also be influenced by intrinsic, graft-specific features. (Am J Ophthalmol 2016;169:58–67. Ó 2016 Elsevier Inc. All rights reserved.)
Supplemental Material available at AJO.com. Accepted for publication Jun 6, 2016. From the Department of Ophthalmology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany. Inquiries to Julia M. Weller, Department of Ophthalmology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany; e-mail: [email protected]
58
©
2016
D
ESCEMET
MEMBRANE
ENDOTHELIAL
KERATO-
plasty (DMEK), a technique for endothelial grafting in which the graft consists of Descemet membrane (DM) and endothelium only, has gained increasing importance during the last 10 years.1,2 In contrast to earlier techniques for corneal transplantation, the demands on DMEK are not only to replace diseased tissue but also to gain near-normal visual function. This is enabled by the fact that corneal structure after DMEK closely resembles its physiological constitution.3 Outcomes after DMEK are determined by different factors: (1) Intraoperative complications: Intraoperative factors influencing DMEK outcomes are the experience of the surgeon,4 the surgical technique (for example, no-touch technique),2,5 and ocular comorbidities (for example, an unstable iris-lensdiaphragm).6 (2) Postoperative complications: The most common complication of DMEK is graft detachment requiring repeated intracameral air injections. Because of the use of an undersized (compared to the descemetorrhexis size) and well-centered graft,7,8 sulfur hexafluoride gas,9 and a standardized surgical technique,10 the graft detachment rate has been decreased from up to 82% in the beginnings of DMEK11 to values below 20%, nowadays requiring additional air injections in only 5%–10%.10 Endothelial immune reactions occurring in less than 1% play only a minor role in the postoperative course after DMEK.12 (3) Preoperative characteristics of the graft: Graft quality is determined by the origin and storage conditions of the donor tissue. Beneficial factors for DMEK include donor age above 50–60 years,13,14 absence of diabetes mellitus in the donor,15 and organ culture conditions.16 The individual structural features of Descemet membrane, for example its thickness, may also be a contributing factor (unpublished data). DM is the specialized, 10- to 15-mm-thick basement membrane of the corneal endothelium consisting of collagens type IV, VIIII, and XVIII; glycoproteins; and proteoglycans.17–20 It is composed of a fetal anterior banded
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layer (ABL), about 3 mm in thickness, and a postnatal posterior nonbanded layer (PNBL), which gradually thickens with age, about 1 mm per decade, by continuous secretion by the endothelial cells.17,19 Owing to this lifelong appositional growth, the PNBL provides a historical record of endothelial function, and ultrastructural abnormalities in this layer reflect intrinsic disturbances in endothelial function.17 In our previous studies investigating structural characteristics of DM, DM-stroma interface, and posterior corneal stroma, we have provided evidence of interindividual morphologic and biochemical variations of DM influencing graft preparation.18,21 We further showed that in a series of primary DMEK failures, the majority (8/14) of failed grafts revealed ultrastructural abnormalities indicative of a subclinical preoperative corneal endothelial dysfunction, which may have contributed to DMEK failure.22 Based on these observations, we hypothesize that DMEK grafts may display interindividual ultrastructural variations, and that abnormalities in ultrastructure indicating a preexisting endothelial dysfunction may affect the postoperative clinical outcome. Therefore, in a retrospective, single-center, nonrandomized consecutive series, 112 residual DM specimens obtained after DM stripping were examined by transmission electron microscopy, and the ultrastructural findings were correlated with the postoperative clinical parameters, for example, postoperative visual acuity, central corneal thickness, endothelial cell density, and graft detachment (rebubbling) rates.
METHODS THE STUDY COMPLIED WITH THE TENETS OF THE DECLARA-
tion of Helsinki and adhered to all state laws of the country. The Institutional Review Board of the University of Erlangen-Nu¨rnberg, Germany, waived the need for approval. Informed consent was obtained from the patients. TISSUE
SPECIMENS
AND
SURGICAL
PROCEDURE:
Corneal donor tissue (n ¼ 112) was obtained from various eye banks in Europe and the United States. European donor corneas (86%) were organ-cultured for 385 6 18 hours at 34 C in Dulbecco’s modified Eagle’s medium containing penicillin and streptomycin (Biochrom, Berlin, Germany) and fetal calf serum (Linaris, Bettingen am Main, Germany). The donor corneas obtained from US eye banks (14%) were short-term cultured at 4 C in Optisol-GS (Bausch & Lomb, Irvine, California, USA) for 216 6 87 hours. Mean age of all donors was 72 6 10 years, mean storage time was 363 6 128 hours, and mean preoperative endothelial cell density was 2527 6 216 cells/mm2. DMEK procedures were performed in 112 eyes of 105 consecutive patients suffering from Fuchs endothelial VOL. 169
corneal dystrophy (n ¼ 92), primary DMEK failure (n ¼ 7), primary failure after Descemet stripping automated endothelial keratoplasty (n ¼ 2), pseudophakic bullous keratopathy (n ¼ 2), failed penetrating keratoplasty (n ¼ 1), and keratopathy in pseudoexfoliation syndrome (n ¼ 1) between April 7, 2010 and September 29, 2011. Mean age of patients was 69 6 9 years, 48% of patients were male and 52% female, and there were 57% right eyes and 43% left eyes. Immediately prior to transplantation, the endothelial cell–DM complex (EDM) was stripped from the donor corneal stroma as described previously in detail.2 Briefly, the corneoscleral buttons were mounted on a suction block (Hanna trephination system; Moria Instruments, Antony, France) and the endothelium was marked by gentle touch with an 8.0 mm trephine and stained with 0.06% trypan blue (Vision Blue; D.O.R.C. Deutschland GmbH, Berlin Germany) for 60 seconds. The submerged EDM peripheral to the mark was incised with a razor blade. The central edge of the EDM was lifted with a round knife (UltraSharp MVR knife; Alcon Grieshaber, Schaffhausen, Switzerland). The central EDM was stripped using 2 forceps and transferred into the patient’s eye. The remaining peripheral EDMs adjacent to the 8-mm graft edge were processed for transmission electron microscopy according to standard protocols. Graft transplantation was performed as previously described in detail.2 Briefly, the recipient’s DM was removed from the stroma using an inverted hook (Price endothelial keratoplasty hook; Moria SA) in a central 9mm area. The graft roll was injected into the anterior chamber and positioned centrally, and a small air bubble was injected into the lumen of the roll. The graft was spread out on the surface of the iris by increasing the air bubble and centered by variation of the air/fluid content. Then, the air bubble was removed and an air bubble injected between the graft and the iris to attach the graft at the stromal backside. Finally, the anterior chamber was filled with an air bubble. After 60 minutes, the air bubble was reduced to 50% of the anterior chamber volume. DMEK was combined with phacoemulsification and implantation of an intraocular lens (triple-DMEK) in 55 of 112 eyes (49%). CLINICAL EVALUATION:
Clinical outcome parameters of patients analyzed in this study were best-corrected visual acuity (BCVA), central corneal thickness (CCT; Pentacam; Oculus, Wetzlar, Germany), and endothelial cell density (ECD; SeaEagle; Rhine-Tec GmbH, Krefeld, Germany). A previous analysis revealed no differences between measurements of the CCT with Pentacam Scheimpflug technique and ultrasound pachymetry (data not shown). The Pentacam data only are presented because these measurements are more reproducible. The occurrence of graft detachments was assessed at the slit lamp and by the use of slit-lamp optical coherence tomography (Heidelberg Engineering, Heidelberg, Germany). Significant graft detachment requiring a repeated
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59
air injection was defined as detachment of more than 1 quadrant of the graft with a gap of more than 1 corneal thickness. Primary graft failure was defined as persisting corneal edema after surgery without initial clearance of the cornea within the first 3 months after surgery. Secondary graft failure was defined as corneal edema after initial clearance of the stroma. Examination dates were on the day before DMEK and 1, 3, 6, and 12 months after surgery. TRANSMISSION ELECTRON MICROSCOPY:
The residual peripheral EDM complexes (n ¼ 112) were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 48 hours, postfixed in 2% buffered osmium tetroxide for 2 hours, dehydrated in graded alcohol concentrations, and embedded in epoxy resin according to standard protocols. Then, 1-mm semithin sections for orientation were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (EM 906E; Carl Zeiss AG, Oberkochen, Germany). The specimens were examined and assessed independently by 2 examiners experienced in transmission electron microscopy (U.S.S., J.W.) regarding thickness and ultrastructural features (abnormal fibrillary inclusions, abnormal collagen deposits, etc) of DM. Three different levels of sectioning were examined per specimen. In order to demonstrate the reliability of the approach investigating the corneoscleral rims, we provided some examples showing comparable ultrastructural alterations in both peripheral rims and central DM specimens obtained from patients after re-DMEK (Figure 1). Care was taken to avoid outer regions containing Hassall-Henle bodies at the periphery of the cornea (Figure 2); these wart-like excrescences of the posterior surface of DM contain collagenous and fibrillary materials together with numerous fissures filled by endothelial cell processes, and are believed to reflect physiological aging processes of the cornea. According to the assessment of both examiners, the EDMs were classified as ultrastructurally normal or abnormal.
STATISTICAL ANALYSIS:
IBM SPSS software version 20.0 (IBM, Armonk, New York, USA) was used for statistical analysis. Differences of samples between groups were assessed by Mann-Whitney U test. Categorical data were analyzed with x2 test and Fisher exact test, if the expected value of each group was less than 5. The significance level was set at P ¼ .05.
RESULTS ULTRASTRUCTURAL FINDINGS:
Transmission electron microscopy was performed on 112 residual EDM specimens at 3 different levels of sectioning. Most of the EDM
60
specimens analyzed (96/112; 86%) revealed a regular thickness of DM and a normal structure comprising a narrow interfacial matrix zone (w1 mm), an anterior banded layer (w2–3 mm), and an amorphous posterior nonbanded layer (9–11 mm) without any fibrillary inclusions (Figure 3). The endothelial cell layer was frequently discontinuous owing to forceps manipulation of the peripheral EDM margins. Sixteen of 112 specimens (14%) revealed abnormalities in the ultrastructure of the DM. These comprised abnormal inclusions of collagen fibers representing long-spacing collagen (n ¼ 11, 69%) and/or fine filaments, probably representing proteoglycans (n ¼ 2, 13%), within the posterior nonbanded layer of DM, either in a lamellar ribbonlike or a rather diffuse pattern (Figure 3). Another 2 of the 16 specimens (13%) revealed a fibrillar collagenous layer deposited posteriorly onto the nonbanded layer of the DM proper (Figure 3). One specimen showed retrocorneal deposits of typical fibrillar pseudoexfoliative material indicative of pseudoexfoliation syndrome of the donor, and 1 specimen showed typical guttae formations indicative of Fuchs endothelial corneal dystrophy of the donor (Figure 3). Mean thickness of all DM specimens was 15.8 6 5.5 mm; there were no significant differences in DM thickness between the structurally normal (15.7 6 5.5 mm) and the structurally abnormal (16.6 6 5.5 mm) specimens (P ¼ .455). ThedonorcorneaswithnormalorabnormalDMdidnotshow anysignificantdifferencesregardingtypeoftissueculture(shortterm/long-term culture: normal DM group, 14%/86%; abnormal DM group,33%/66%, P ¼ .164), mean age of donor (normal DM group, 73 6 10 years; abnormal DM group, 73 6 12 years, P ¼ .916), preoperative endothelial cell density (normal DM group, 2529 6 220/mm2; abnormal DM group, 25146201/mm2,P¼.781),orthemeanstoragetime(normal DM group, 368 6 125 hours; abnormal DM group, 339 6 141 hours,P¼.859). CORRELATION OF CLINICAL OUTCOMES AFTER DESCEMET MEMBRANE ENDOTHELIAL KERATOPLASTY WITH ULTRASTRUCTURAL FINDINGS: Clinicalparameters
were compared between eyes having received a structurally normalEDMgraft(n¼96,normalDMgroup)orastructurally abnormalEDMgraft(n¼16,abnormalDMgroup).Therewas no significant difference in the patients’ mean age at surgery (normalDMgroup,6969years;abnormalDMgroup,7266years, P¼.407).Fiftyof96eyesofthenormalDMgroupand5of16eyesin theabnormalDMgroupunderwenttriple-DMEK(P¼.177). Graft detachment requiring an additional air injection into the anterior chamber occurred in 42 of 96 eyes (44%) in the normal DM group and in 11 of 16 eyes (69%) in the abnormal DM group; this difference did not reach statistical significance (P ¼ .103). Graft failure was noticed in 13 of 112 eyes and revealed a statistically significant difference between both groups: in the abnormal DM group graft failures occurred in 6 of 16 eyes (38%, secondary failure in 4 of 6 eyes), and in the
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FIGURE 1. Corresponding ultrastructural abnormalities at the center and the periphery of the Descemet membrane. Transmission electron micrographs showing ultrastructural abnormalities within the posterior nonbanded layer (PNBL) in failed Descemet membrane endothelial keratoplasty (DMEK) grafts (Left column) and in corresponding peripheral donor endothelial-Descemet membrane (EDM) rims (Right column). Deposits of long-spacing collagens and proteoglycans are seen in the failed DMEK grafts (central) as well as in the peripheral rims.
normal DM group in 7 of 96 eyes (7%, secondary failure in 1 of 7 eyes) (P ¼ .003). Respecting only secondary graft failures, the difference was highly significant (P ¼ .001). In the group of normal DM eyes, the (only) secondary graft failure occurred after 9 months. In the group of abnormal DM eyes, the secondary graft failures occurred after 6, 9, 36, and 53 months. Applying the Bonferroni correction for multiple statistical tests, the significance level for this study has to be set at VOL. 169
.002 (29 single tests). At this level, the comparison of overall graft failure rates between the 2 groups is not statistically significant (P ¼ .003), but the comparison of secondary graft failures is still statistically significant (P ¼ .001). AnalyzingthelastCCTvaluebeforeregrafting,themeanCCT intheabnormalDMgroupwas850mm(range591–1267mm), indicatingendothelialfailurewithstromaledema. Postoperative clinical results (BCVA, CCT, ECD) are shown in Table 1 for all eyes, eyes with abnormal DMs,
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61
FIGURE 2. Hassall-Henle bodies at the periphery of the cornea. Transmission electron micrographs showing HassallHenle bodies at the periphery of the cornea, which were avoided for the analysis of ultrastructural abnormalities. These wart-like excrescences of the posterior surface of Descemet membrane contain collagenous and fibrillary materials together with numerous fissures filled by endothelial cell processes, and are believed to reflect physiological aging processes of the cornea.
and eyes with normal DMs. Regarding BCVA, a significant difference between both groups was found after 3 and 6 months postoperatively. However, there was a significant difference in BCVA already before DMEK surgery. Therefore, the change of BCVA was analyzed and a significant difference between both groups was found between the preoperative BCVA and BCVA 1 month after DMEK. In the abnormal DM group (starting with a worse BCVA), the increase of BCVA was higher (0.72 6 0.61) than in the normal DM group (0.24 6 0.53, P ¼ .019). No significant differences were found between groups regarding CCT and ECD before DMEK and 1, 3, 6, and 12 months after DMEK, respectively. Subgroup analyses for the organ-cultured grafts and for eyes with Fuchs endothelial corneal dystrophy are shown in Tables 2 and 3, respectively.
DISCUSSION IN THIS RETROSPECTIVE CONSECUTIVE SERIES, WE EXAM-
ined 112 residual DM specimens obtained during DMEK surgery by transmission electron microscopy and correlated the ultrastructural findings with the clinical outcomes after DMEK. Based on our previous study by Cirkovic and associates,22 who described ultrastructural abnormalities of DM in the majority of failed primary DMEK grafts, we hypothesized that similar ultrastructural abnormalities may also occur in efficient DMEK grafts but may have an influence on the postoperative clinical outcome. Ultrastructural abnormalities in the DM specimens analyzed comprised abnormal inclusions of collagen 62
(long-spacing collagen or fibrillary collagen) or proteoglycans within PNBL of DM and abnormal depositions of a posterior collagenous layer or pseudoexfoliation fibrils on the posterior surface of DM. Deposits of long-spacing collagen have been described in DM specimens of diabetic rats and humans, increasing with age, as well as in Fuchs endothelial corneal dystrophy and iridocornealendothelial syndrome.23–26 Whether the DM specimens revealing long-spacing collagen were derived from diabetic donors is not known, because this information is not provided by European eye banks. However, it is conceivable that ultrastructural abnormalities in DM account for the difficulties in DMEK graft preparation observed in corneas from diabetic donors.15 Further studies are necessary to analyze the ultrastructural features of DM from diabetic donors. Abnormal inclusions of proteoglycans in DM have not been reported previously. In contrast, a fibrous posterior collagenous layer has been shown to be deposited on the posterior surface of DM in various pathologic conditions, including pseudophakic bullous keratopathy and Fuchs endothelial corneal dystrophy. This layer is thought to be produced by impaired and attenuated endothelial cells, which start to transdifferentiate into myofibroblasts as a common final pathway following endothelial dysfunction and damage.27 One specimen of our series revealed typical guttae formations, and another specimen showed retrocorneal deposits of typical pseudoexfoliation fibrils pointing to the presence of Fuchs endothelial corneal dystrophy or pseudoexfoliation syndrome in the donors. The question arises why the pseudoexfoliation deposits and guttae were overlooked by the eye bank, which approved the corneas for transplantation. Probabaly, the delicate pseudoexfoliation deposits and guttae were simply too small to be detected by specular microscopy. The main criterion for the approval of corneal buttons is the endothelial cell density. Since the endothelial cell density was normal in these corneal buttons, the deposits were probably subclinical (ie, the respective endothelial dysfunctions were only beginning). However, the endothelial cells of pseudoexfoliation corneas continue to produce pseudoexfoliation material after transplantation and might thereby have led to a secondary graft failure. This study confirmed our hypothesis that ultrastructural abnormalities might be a risk factor for graft failure after DMEK. There was a significant correlation between ultrastructural findings and the secondary graft failure rate. Although the overall graft failure rate between the 2 groups is not significant after Bonferroni correction, there is a clinically relevant difference between the 2 groups (failure rate 7% vs 38%). We did not find a significant difference in CCT and ECD after DMEK in eyes that have received ultrastructurally abnormal grafts compared with those that have received structurally normal grafts, although we found a significant difference in the graft failure rate between both groups.
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FIGURE 3. Transmission electron micrographs showing ultrastructural abnormalities within the posterior nonbanded layer (PNBL) and on the endothelial side of Descemet membrane (DM). (First row, left) Normal DM with anterior banded layer (ABL, top), PNBL (middle), and endothelial cells (bottom). (First row, right) Fibrillary depositions within the PNBL (arrow). (Second row, left) Lamellar ribbon-like distributed long-spacing collagens (arrow) and diffusely distributed fibrillary depositions within the PNBL (arrowhead). (Second row, right) Ribbon-like distributed long-spacing collagens (arrow; insert: magnification of long-spacing collagens). (Third row, left) Layer with proteoglycans between the ABL and PNBL (arrow; insert: magnification of proteoglycans). (Third row, right) Posterior collagenous layer on the endothelial side of Descemet membrane (arrow). (Fourth row, left) Pseudoexfoliation deposits (arrow). (Fourth row, right) Guttae formations indicative of Fuchs endothelial corneal dystrophy of the donor (arrow).
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TABLE 1. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preoperative BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Change of BCVA preoperative – 1 month Change of BCVA 1 to 3 months Change of BCVA 3 to 6 months Change of BCVA 6 to 12 months
All Patients (n ¼ 112)
Normal DM Group (n ¼ 96)
Abnormal DM Group (n ¼ 16)
P Valuea
689 6 129 mm 559 6 109 mm 554 6 134 mm 548 6 92 mm 542 6 85 mm 2526 6 216/mm2 1544 6 309/mm2 1514 6 315/mm2 1532 6 321/mm2 1480 6 292/mm2 0.73 6 0.48 0.40 6 0.46 0.37 6 0.43 0.28 6 0.35 0.21 6 0.30 0.32 6 0.56
681 6 118 mm 556 6 111 mm 548 6 111 mm 540 6 64 mm 546 6 90 mm 2529 6 220/mm2 1550 6 319/mm2 1531 6 331/mm2 1532 6 332/mm2 1479 6 292/mm2 0.65 6 0.42 0.37 6 0.47 0.32 6 0.38 1.21 6 0.20 0.21 6 0.31 0.24 6 0.53
723 6 173 mm 572 6 102 mm 585 6 219 mm 602 6 201 mm 517 6 33 mm 2514 6 201 1516 6 259 1431 6 208 1534 6 239 1484 6 308 1.2 6 0.65 0.54 6 0.44 0.64 6 0.60 0.73 6 0.69 0.26 6 0.17 0.72 6 0.61
.530 .509 .836 .865 .266 .781 .652 .246 .792 .491 .002* .085 .012* .007* .136 .019*
0.02 6 0.35
0.05 6 0.36
0.14 6 0.3
.182
0.01 6 0.15
0.02 6 0.1
0.08 6 0.31
.782
0.02 6 0.2
0.01 6 0.14
0.15 6 0.47
.778
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
This discrepancy can be explained by the fact that the graft failure occurred at different intervals after DMEK, ranging from 3 to 53 months. In case of graft failure, the eyes were regrafted within 3 months and excluded from further analysis. The last CCT value before regrafting was 850 mm on average. Since these values were measured at different follow-up visits, they did not influence the statistical analysis of mean CCT in a significant way. The high graft detachment rate in the present study is attributed to the surgical technique, which was not standardized at the time of surgery (2010–2011) compared to nowadays. It might be objected that the graft failures were also caused by the surgical technique and not by the ultrastructural abnormalities of the DM. Summarizing the graft failure rates and rebubbling rates of the 2 groups, 44% of eyes in the normal DM group needed rebubblings, but only 7% failed (ie, 17% of eyes requiring rebubbling failed), whereas in the abnormal DM group 38% of eyes had graft failure, which is 55% compared to eyes requiring rebubblings (69%). This discrepancy (17% vs 55%) cannot be explained by the surgical technique because the technique was similar in all eyes. Therefore, we attribute the higher graft failure rate in the abnormal DM 64
group to the ultrastructural abnormalities and not to the surgical technique. No endothelial graft rejection occurred in the eyes included in this study. The graft failures occurred without obvious reason (for example, graft rejection, herpes endotheliitis) but may be partially ascribed to preexisting endothelial damage, as reflected by the ultrastructural abnormalities of DM specimens. Since there is a discrepancy in the rate of abnormal grafts between organ-cultured and Optisol-cultured grafts (shortterm/long-term culture: normal DM group, 14%/86%; abnormal DM group, 33%/66%, P ¼ .164), we performed a subanalysis of the organ-cultured grafts only (Table 2). The ultrastructural abnormalities described in this investigation were mostly observed within deeper zones of the posterior nonbanded layer of the Descemet membrane. Owing to the lifelong growth of Descemet membrane, it can be assumed that those buried depositions reflect disturbances of endothelial function during the lifetime rather than postmortem influences of culture conditions during the storage period. Therefore, the authors believe that the storage conditions cannot have caused the ultrastructural abnormalities observed.
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TABLE 2. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty in the Subgroup of Organ-Cultured Donor Corneas Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preoperative BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Rebubbling (n) Graft failure (n)
All Patients (n ¼ 97)
701 6 137 mm 553 6 109 mm 554 6 141 mm 552 6 98 mm 532 6 46 mm 2507 6 204/mm2 1562 6 323/mm2 1532 6 327/mm2 1543 6 316/mm2 1468 6 262/mm2 0.76 6 0.51 0.34 6 0.31 0.37 6 0.44 1. 6 0.32 0.18 6 0.14 44/97 9/97
Normal DM Group (n ¼ 85)
Abnormal DM Group (n ¼ 12)
693 6 124 mm 549 6 107 mm 544 6 113 mm 536 6 53 mm 533 6 46 mm 2501 6 201/mm2 1563 6 334/mm2 1534 6 342/mm2 1535 6 331/mm2 1456 6 269/mm2 0.69 6 0.44 0.30 6 0.26 0.32 6 0.40 0.20 6 0.12 0.17 6 0.14 36/85 5/85
745 6 200 mm 579 6 119 mm 620 6 266 mm 657 6 223 mm 515 6 44 mm 2539 6 227/mm2 1552 6 270/mm2 1520 6 204/mm2 1600 6 178/mm2 1599 6 130/mm2 1.36 6 0.70 0.57 6 0.46 0.65 6 0.62 0.73 6 0.75 0.26 6 0.10 8/12 4/12
P Valuea
.705 .562 .572 .236 .631 .644 .798 .790 .388 .221 .005* .039* .012* .09 .126 .101 .013*
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
TABLE 3. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty in the Subgroup of Patients With Fuchs Endothelial Corneal Dystrophy Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preop BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Rebubbling (n) Graft failure (n)
All Patients (n ¼ 92)
673 6 111 mm 555 6 89 mm 541 6 91 mm 539 6 65 mm 542 6 89 mm 2533 6 218/mm2 1559 6 296/mm2 1519 6 328/mm2 1527 6 301/mm2 1494 6 297/mm2 0.68 6 0.39 0.40 6 0.45 0.31 6 0.31 0.27 6 0.25 0.23 6 0.29 45/92 7/92
Normal DM Group (n ¼ 81)
Abnormal DM Group (n ¼ 11)
676 6 114 mm 551 6 84 mm 544 6 97 mm 540 6 66 mm 546 6 94 mm 2540 6 228/mm2 1576 6 312/mm2 1536 6 348/mm2 1522 6 310/mm2 1496 6 299/mm2 0.66 6 0.37 0.40 6 0.47 0.29 6 0.30 0.24 6 0.23 0.23 6 0.31 37/81 4/81
656 6 91 mm 583 6 122 mm 528 6 44 mm 534 6 61 mm 517 6 33 mm 2592 6 136/mm2 1457 6 153/mm2 1431 6 179/mm2 1572 6 237/mm2 1485 6 308/mm2 0.88 6 0.47 0.38 6 0.24 0.46 6 0.37 0.39 6 0.33 0.24 6 0.14 8/11 3/11
P Valuea
.720 .505 .966 .718 .282 .523 .340 .264 .582 .656 .014* .544 .070 .065 .148 .086 .056
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
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Apart from the donor tissue storage, the underlying corneal pathology might be another possible bias of this study. Corneal endothelial cells of patients with Fuchs endothelial corneal dystrophy have been shown to be able to repopulate after DMEK.28 Therefore, a subanalysis of the eyes with the diagnosis of Fuchs endothelial corneal dystrophy was performed (Table 3). However, the number of graft failures in this subgroup was only 7. A comparison with the eyes of this study, in which DMEK was performed for another indication (n ¼ 20), is limited by the different cohort sizes and small subgroups. Therefore, it is difficult to draw any conclusions from this subgroup. Analysis of BCVA showed significant differences between both groups already preoperatively. Eyes having received structurally abnormal grafts had worse preoperative BCVA values than those receiving normal grafts. An explanation for this finding might be bias of preoperative BCVA data by the fact that some eyes had cataracts of different degrees at the time of surgery and others were already pseudophakic. The value of our study is limited by the fact that only the residual DM remnants obtained from the periphery of the donor corneas were evaluated, since ultrastructural alter-
ations in the periphery may not necessarily adversely affect the quality of central grafts. Therefore we had performed an ultrastructural comparison of peripheral rims and central DM specimens (Figure 1), which shows a comparable ultrastructure. Other technologies that have been established to visualize corneal structure are inferior to electron microcopy owing to a lower resolution. For example, laser scanning confocal microscopy has a resolution of only 1 mm, which is 3 orders of magnitude lower than the resolution of electron microscopy. Because the deposits of longspacing collagen found in this study measure only 70– 150 nm, electron microscopy is the only method to detect them. Furthermore, the relatively small number of abnormal DMs decreases the value of the statistical analysis. In conclusion, this study reveals a correlation between ultrastructural alterations of DM in donor corneas and the graft failure rate after DMEK. Thus, graft failure after DMEK is determined not only by surgical and postoperative factors but also by intrinsic, graft-specific features. Retrospective ultrastructural analysis of residual DM remnants might be important for clinicians to assess the reasons for graft failure.
¨ TZER-SCHREHARDT. THE STUDY FUNDING/SUPPORT: GRANTS FROM DFG (GERMAN RESEARCH FOUNDATION) FOR U. SCHLO data and the interpretation of the data are independent of this funding. Financial disclosures: The following authors have no financial disclosures: Julia M. Weller, Ursula Schlo¨tzer-Schrehardt, Theofilos Tourtas, and Friedrich E. Kruse. All authors attest that they meet the current ICMJE criteria for authorship. We thank Elke Meyer (technical assistant, Department of Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany) for the excellent preparation of electron microscopy specimens.
REFERENCES 1. Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 2006; 25(8):987–990. 2. Kruse FE, Schrehardt US, Tourtas T. Optimizing outcomes with Descemet’s membrane endothelial keratoplasty. Curr Opin Ophthalmol 2014;25(4):325–334. 3. Livny E, Parker JS, van der Kaaij M, et al. Postmortem ultrastructural analysis of a cornea transplanted with Descemet membrane endothelial keratoplasty. Cornea 2014;33(8): 790–794. 4. Dapena I, Ham L, Droutsas K, van Dijk K, Moutsouris K, Melles GR. Learning curve in descemet’s membrane endothelial keratoplasty: first series of 135 consecutive cases. Ophthalmology 2011;118(11):2147–2154. 5. Satue´ M, Rodrı´guez-Calvo-de-Mora M, Naveiras M, Cabrerizo J, Dapena I, Melles GR. Standardization of the Descemet membrane endothelial keratoplasty technique: outcomes of the first 450 consecutive cases. Arch Soc Esp Oftalmol 2015;90(8):356–364. 6. Weller JM, Tourtas T, Kruse FE. Feasibility and outcome of Descemet membrane endothelial keratoplasty in complex anterior segment and vitreous disease. Cornea 2015;34(11): 1351–1357.
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7. Tourtas T, Schlomberg J, Wessel JM, Bachmann BO, Schlo¨tzer-Schrehardt U, Kruse FE. Graft adhesion in descemet membrane endothelial keratoplasty dependent on size of removal of host’s descemet membrane. JAMA Ophthalmol 2014;132(2):155–161. 8. Ro¨ck T, Bramkamp M, Bartz-Schmidt KU, Ro¨ck D, Yo¨ru¨k E. Causes that influence the detachment rate after Descemet membrane endothelial keratoplasty. Graefes Arch Clin Exp Ophthalmol 2015;253(12):2217–2222. 9. Terry MA, Straiko MD, Veldman PB, et al. Standardized DMEK technique: reducing complications using prestripped tissue, novel glass injector, and sulfur hexafluoride (SF6) gas. Cornea 2015;34(8):845–852. 10. Rodrı´guez-Calvo-de-Mora M, Quilendrino R, Ham L, et al. Clinical outcome of 500 consecutive cases undergoing Descemet’s membrane endothelial keratoplasty. Ophthalmology 2015;122(3):464–470. 11. Tourtas T, Laaser K, Bachmann BO, Cursiefen C, Kruse FE. Descemet membrane endothelial keratoplasty versus descemet stripping automated endothelial keratoplasty. Am J Ophthalmol 2012;153(6):1082–1090. 12. Price MO, Price FW Jr, Kruse FE, Bachmann BO, Tourtas T. Randomized comparison of topical prednisolone acetate 1% versus fluorometholone 0.1% in the first year after descemet membrane endothelial keratoplasty. Cornea 2014;33(9):880–886.
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13. Heinzelmann S, Hu¨ther S, Bo¨hringer D, Eberwein P, Reinhard T, Maier P. Influence of donor characteristics on descemet membrane endothelial keratoplasty. Cornea 2014; 33(6):644–648. 14. Gorovoy IR, Cui QN, Gorovoy MS. Donor tissue characteristics in preparation of DMEK grafts. Cornea 2014;33(7): 683–685. 15. Greiner MA, Rixen JJ, Wagoner MD, et al. Diabetes mellitus increases risk of unsuccessful graft preparation in Descemet membrane endothelial keratoplasty: a multicenter study. Cornea 2014;33(11):1129–1133. 16. Laaser K, Bachmann BO, Horn FK, Schlo¨tzer-Schrehardt U, Cursiefen C, Kruse FE. Donor tissue culture conditions and outcome after descemet membrane endothelial keratoplasty. Am J Ophthalmol 2011;151(6):1007–1018. 17. Johnson DH, Bourne WM, Campbell RJ. The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch Ophthalmol 1982;100(12):1942–1947. 18. Schlo¨tzer-Schrehardt U, Bachmann BO, Laaser K, Cursiefen C, Kruse FE. Characterization of the cleavage plane in Descemet’s membrane endothelial keratoplasty. Ophthalmology 2011;118(10):1950–1957. 19. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci 1984;25(12):1402–1415. 20. Kabosova A, Azar DT, Bannikov GA, et al. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci 2007;48: 4989–4999.
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21. Schlo¨tzer-Schrehardt U, Bachmann BO, Tourtas T, et al. Reproducibility of graft preparations in Descemet’s membrane endothelial keratoplasty. Ophthalmology 2013;120(9):1769–1777. 22. Cirkovi c A, Schlo¨tzer-Schrehardt U, Weller JM, Kruse FE, Tourtas T. Clinical and ultrastructural characteristics of graft failure in DMEK: 1-year results after repeat DMEK. Cornea 2015;34(1):11–17. 23. Akimoto Y, Sawada H, Ohara-Imaizumi M, Nagamatsu S, Kawakami H. Change in long-spacing collagen in Descemet’s membrane of diabetic Goto-Kakizaki rats and its suppression by antidiabetic agents. Exp Diabetes Res 2008;2008:818341. 24. Rehany U, Ishii Y, Lahav M, Rumelt S. Ultrastructural changes in corneas of diabetic patients: an electronmicroscopy study. Cornea 2000;19(4):534–538. 25. Levy SG, Moss J, Sawada H, Dopping-Hepenstal PJ, McCartney AC. The composition of wide-spaced collagen in normal and diseased Descemet’s membrane. Curr Eye Res 1996;15(1):45–52. 26. Levy SG, McCartney AC, Sawada H, Dopping-Hepenstal PJ, Alexander RA, Moss J. Descemet’s membrane in the iridocorneal-endothelial syndrome: morphology and composition. Exp Eye Res 1995;61(3):323–333. 27. Waring GO 3rd. Posterior collagenous layer of the cornea. Ultrastructural classification of abnormal collagenous tissue posterior to Descemet’s membrane in 30 cases. Arch Ophthalmol 1982;100(1):122–134. 28. Jacobi C, Zhivov A, Korbmacher J, et al. Evidence of endothelial cell migration after descemet membrane endothelial keratoplasty. Am J Ophthalmol 2011;152(4):537–542.
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To investigate if ultrastructural alterations in the Descemet membrane (DM) are correlated with the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK). DESIGN: Retrospective cohort study. METHODS: SETTING: Institutional, single-center. STUDY POPULATION: One hundred and twelve residual DM specimens obtained after DM stripping. MAIN OUTCOME MEASURES: Incidence of ultrastructural abnormalities in transmission electron microscopy, graft detachment rate, graft failure rate, best-corrected visual acuity (BCVA), endothelial cell density (ECD), and central corneal thickness (CCT). Examination dates were on the day before DMEK and 1, 3, 6, and 12 months after surgery. RESULTS: Abnormalities in the ultrastructure of DM were found in 16 of 112 specimens (14%) (abnormal DM group), comprising deposits of long-spacing collagen, fine filaments (proteoglycans), a posterior collagenous layer, pseudoexfoliative material, and guttae. The secondary graft failure rate was significantly higher in the abnormal DM group compared with the normal DM group (P [ .001). There was a trend for an increased graft detachment rate in the abnormal DM group (11/16) compared with the normal DM group (42/96) (P [ .103). There was no significant difference in mean CCT and ECD after surgery. Mean CCT in the eyes with graft failure in the abnormal DM group at the last follow-up before regrafting was 850 mm, indicating endothelial failure with stromal edema. CONCLUSION: This study reveals a correlation between ultrastructural alterations of DM in donor corneas and the graft failure rate after DMEK. Thus, graft failure after DMEK not only is determined by surgical trauma and postoperative events but may also be influenced by intrinsic, graft-specific features. (Am J Ophthalmol 2016;169:58–67. Ó 2016 Elsevier Inc. All rights reserved.)
Supplemental Material available at AJO.com. Accepted for publication Jun 6, 2016. From the Department of Ophthalmology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany. Inquiries to Julia M. Weller, Department of Ophthalmology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany; e-mail: [email protected]
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©
2016
D
ESCEMET
MEMBRANE
ENDOTHELIAL
KERATO-
plasty (DMEK), a technique for endothelial grafting in which the graft consists of Descemet membrane (DM) and endothelium only, has gained increasing importance during the last 10 years.1,2 In contrast to earlier techniques for corneal transplantation, the demands on DMEK are not only to replace diseased tissue but also to gain near-normal visual function. This is enabled by the fact that corneal structure after DMEK closely resembles its physiological constitution.3 Outcomes after DMEK are determined by different factors: (1) Intraoperative complications: Intraoperative factors influencing DMEK outcomes are the experience of the surgeon,4 the surgical technique (for example, no-touch technique),2,5 and ocular comorbidities (for example, an unstable iris-lensdiaphragm).6 (2) Postoperative complications: The most common complication of DMEK is graft detachment requiring repeated intracameral air injections. Because of the use of an undersized (compared to the descemetorrhexis size) and well-centered graft,7,8 sulfur hexafluoride gas,9 and a standardized surgical technique,10 the graft detachment rate has been decreased from up to 82% in the beginnings of DMEK11 to values below 20%, nowadays requiring additional air injections in only 5%–10%.10 Endothelial immune reactions occurring in less than 1% play only a minor role in the postoperative course after DMEK.12 (3) Preoperative characteristics of the graft: Graft quality is determined by the origin and storage conditions of the donor tissue. Beneficial factors for DMEK include donor age above 50–60 years,13,14 absence of diabetes mellitus in the donor,15 and organ culture conditions.16 The individual structural features of Descemet membrane, for example its thickness, may also be a contributing factor (unpublished data). DM is the specialized, 10- to 15-mm-thick basement membrane of the corneal endothelium consisting of collagens type IV, VIIII, and XVIII; glycoproteins; and proteoglycans.17–20 It is composed of a fetal anterior banded
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layer (ABL), about 3 mm in thickness, and a postnatal posterior nonbanded layer (PNBL), which gradually thickens with age, about 1 mm per decade, by continuous secretion by the endothelial cells.17,19 Owing to this lifelong appositional growth, the PNBL provides a historical record of endothelial function, and ultrastructural abnormalities in this layer reflect intrinsic disturbances in endothelial function.17 In our previous studies investigating structural characteristics of DM, DM-stroma interface, and posterior corneal stroma, we have provided evidence of interindividual morphologic and biochemical variations of DM influencing graft preparation.18,21 We further showed that in a series of primary DMEK failures, the majority (8/14) of failed grafts revealed ultrastructural abnormalities indicative of a subclinical preoperative corneal endothelial dysfunction, which may have contributed to DMEK failure.22 Based on these observations, we hypothesize that DMEK grafts may display interindividual ultrastructural variations, and that abnormalities in ultrastructure indicating a preexisting endothelial dysfunction may affect the postoperative clinical outcome. Therefore, in a retrospective, single-center, nonrandomized consecutive series, 112 residual DM specimens obtained after DM stripping were examined by transmission electron microscopy, and the ultrastructural findings were correlated with the postoperative clinical parameters, for example, postoperative visual acuity, central corneal thickness, endothelial cell density, and graft detachment (rebubbling) rates.
METHODS THE STUDY COMPLIED WITH THE TENETS OF THE DECLARA-
tion of Helsinki and adhered to all state laws of the country. The Institutional Review Board of the University of Erlangen-Nu¨rnberg, Germany, waived the need for approval. Informed consent was obtained from the patients. TISSUE
SPECIMENS
AND
SURGICAL
PROCEDURE:
Corneal donor tissue (n ¼ 112) was obtained from various eye banks in Europe and the United States. European donor corneas (86%) were organ-cultured for 385 6 18 hours at 34 C in Dulbecco’s modified Eagle’s medium containing penicillin and streptomycin (Biochrom, Berlin, Germany) and fetal calf serum (Linaris, Bettingen am Main, Germany). The donor corneas obtained from US eye banks (14%) were short-term cultured at 4 C in Optisol-GS (Bausch & Lomb, Irvine, California, USA) for 216 6 87 hours. Mean age of all donors was 72 6 10 years, mean storage time was 363 6 128 hours, and mean preoperative endothelial cell density was 2527 6 216 cells/mm2. DMEK procedures were performed in 112 eyes of 105 consecutive patients suffering from Fuchs endothelial VOL. 169
corneal dystrophy (n ¼ 92), primary DMEK failure (n ¼ 7), primary failure after Descemet stripping automated endothelial keratoplasty (n ¼ 2), pseudophakic bullous keratopathy (n ¼ 2), failed penetrating keratoplasty (n ¼ 1), and keratopathy in pseudoexfoliation syndrome (n ¼ 1) between April 7, 2010 and September 29, 2011. Mean age of patients was 69 6 9 years, 48% of patients were male and 52% female, and there were 57% right eyes and 43% left eyes. Immediately prior to transplantation, the endothelial cell–DM complex (EDM) was stripped from the donor corneal stroma as described previously in detail.2 Briefly, the corneoscleral buttons were mounted on a suction block (Hanna trephination system; Moria Instruments, Antony, France) and the endothelium was marked by gentle touch with an 8.0 mm trephine and stained with 0.06% trypan blue (Vision Blue; D.O.R.C. Deutschland GmbH, Berlin Germany) for 60 seconds. The submerged EDM peripheral to the mark was incised with a razor blade. The central edge of the EDM was lifted with a round knife (UltraSharp MVR knife; Alcon Grieshaber, Schaffhausen, Switzerland). The central EDM was stripped using 2 forceps and transferred into the patient’s eye. The remaining peripheral EDMs adjacent to the 8-mm graft edge were processed for transmission electron microscopy according to standard protocols. Graft transplantation was performed as previously described in detail.2 Briefly, the recipient’s DM was removed from the stroma using an inverted hook (Price endothelial keratoplasty hook; Moria SA) in a central 9mm area. The graft roll was injected into the anterior chamber and positioned centrally, and a small air bubble was injected into the lumen of the roll. The graft was spread out on the surface of the iris by increasing the air bubble and centered by variation of the air/fluid content. Then, the air bubble was removed and an air bubble injected between the graft and the iris to attach the graft at the stromal backside. Finally, the anterior chamber was filled with an air bubble. After 60 minutes, the air bubble was reduced to 50% of the anterior chamber volume. DMEK was combined with phacoemulsification and implantation of an intraocular lens (triple-DMEK) in 55 of 112 eyes (49%). CLINICAL EVALUATION:
Clinical outcome parameters of patients analyzed in this study were best-corrected visual acuity (BCVA), central corneal thickness (CCT; Pentacam; Oculus, Wetzlar, Germany), and endothelial cell density (ECD; SeaEagle; Rhine-Tec GmbH, Krefeld, Germany). A previous analysis revealed no differences between measurements of the CCT with Pentacam Scheimpflug technique and ultrasound pachymetry (data not shown). The Pentacam data only are presented because these measurements are more reproducible. The occurrence of graft detachments was assessed at the slit lamp and by the use of slit-lamp optical coherence tomography (Heidelberg Engineering, Heidelberg, Germany). Significant graft detachment requiring a repeated
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air injection was defined as detachment of more than 1 quadrant of the graft with a gap of more than 1 corneal thickness. Primary graft failure was defined as persisting corneal edema after surgery without initial clearance of the cornea within the first 3 months after surgery. Secondary graft failure was defined as corneal edema after initial clearance of the stroma. Examination dates were on the day before DMEK and 1, 3, 6, and 12 months after surgery. TRANSMISSION ELECTRON MICROSCOPY:
The residual peripheral EDM complexes (n ¼ 112) were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 48 hours, postfixed in 2% buffered osmium tetroxide for 2 hours, dehydrated in graded alcohol concentrations, and embedded in epoxy resin according to standard protocols. Then, 1-mm semithin sections for orientation were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (EM 906E; Carl Zeiss AG, Oberkochen, Germany). The specimens were examined and assessed independently by 2 examiners experienced in transmission electron microscopy (U.S.S., J.W.) regarding thickness and ultrastructural features (abnormal fibrillary inclusions, abnormal collagen deposits, etc) of DM. Three different levels of sectioning were examined per specimen. In order to demonstrate the reliability of the approach investigating the corneoscleral rims, we provided some examples showing comparable ultrastructural alterations in both peripheral rims and central DM specimens obtained from patients after re-DMEK (Figure 1). Care was taken to avoid outer regions containing Hassall-Henle bodies at the periphery of the cornea (Figure 2); these wart-like excrescences of the posterior surface of DM contain collagenous and fibrillary materials together with numerous fissures filled by endothelial cell processes, and are believed to reflect physiological aging processes of the cornea. According to the assessment of both examiners, the EDMs were classified as ultrastructurally normal or abnormal.
STATISTICAL ANALYSIS:
IBM SPSS software version 20.0 (IBM, Armonk, New York, USA) was used for statistical analysis. Differences of samples between groups were assessed by Mann-Whitney U test. Categorical data were analyzed with x2 test and Fisher exact test, if the expected value of each group was less than 5. The significance level was set at P ¼ .05.
RESULTS ULTRASTRUCTURAL FINDINGS:
Transmission electron microscopy was performed on 112 residual EDM specimens at 3 different levels of sectioning. Most of the EDM
60
specimens analyzed (96/112; 86%) revealed a regular thickness of DM and a normal structure comprising a narrow interfacial matrix zone (w1 mm), an anterior banded layer (w2–3 mm), and an amorphous posterior nonbanded layer (9–11 mm) without any fibrillary inclusions (Figure 3). The endothelial cell layer was frequently discontinuous owing to forceps manipulation of the peripheral EDM margins. Sixteen of 112 specimens (14%) revealed abnormalities in the ultrastructure of the DM. These comprised abnormal inclusions of collagen fibers representing long-spacing collagen (n ¼ 11, 69%) and/or fine filaments, probably representing proteoglycans (n ¼ 2, 13%), within the posterior nonbanded layer of DM, either in a lamellar ribbonlike or a rather diffuse pattern (Figure 3). Another 2 of the 16 specimens (13%) revealed a fibrillar collagenous layer deposited posteriorly onto the nonbanded layer of the DM proper (Figure 3). One specimen showed retrocorneal deposits of typical fibrillar pseudoexfoliative material indicative of pseudoexfoliation syndrome of the donor, and 1 specimen showed typical guttae formations indicative of Fuchs endothelial corneal dystrophy of the donor (Figure 3). Mean thickness of all DM specimens was 15.8 6 5.5 mm; there were no significant differences in DM thickness between the structurally normal (15.7 6 5.5 mm) and the structurally abnormal (16.6 6 5.5 mm) specimens (P ¼ .455). ThedonorcorneaswithnormalorabnormalDMdidnotshow anysignificantdifferencesregardingtypeoftissueculture(shortterm/long-term culture: normal DM group, 14%/86%; abnormal DM group,33%/66%, P ¼ .164), mean age of donor (normal DM group, 73 6 10 years; abnormal DM group, 73 6 12 years, P ¼ .916), preoperative endothelial cell density (normal DM group, 2529 6 220/mm2; abnormal DM group, 25146201/mm2,P¼.781),orthemeanstoragetime(normal DM group, 368 6 125 hours; abnormal DM group, 339 6 141 hours,P¼.859). CORRELATION OF CLINICAL OUTCOMES AFTER DESCEMET MEMBRANE ENDOTHELIAL KERATOPLASTY WITH ULTRASTRUCTURAL FINDINGS: Clinicalparameters
were compared between eyes having received a structurally normalEDMgraft(n¼96,normalDMgroup)orastructurally abnormalEDMgraft(n¼16,abnormalDMgroup).Therewas no significant difference in the patients’ mean age at surgery (normalDMgroup,6969years;abnormalDMgroup,7266years, P¼.407).Fiftyof96eyesofthenormalDMgroupand5of16eyesin theabnormalDMgroupunderwenttriple-DMEK(P¼.177). Graft detachment requiring an additional air injection into the anterior chamber occurred in 42 of 96 eyes (44%) in the normal DM group and in 11 of 16 eyes (69%) in the abnormal DM group; this difference did not reach statistical significance (P ¼ .103). Graft failure was noticed in 13 of 112 eyes and revealed a statistically significant difference between both groups: in the abnormal DM group graft failures occurred in 6 of 16 eyes (38%, secondary failure in 4 of 6 eyes), and in the
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FIGURE 1. Corresponding ultrastructural abnormalities at the center and the periphery of the Descemet membrane. Transmission electron micrographs showing ultrastructural abnormalities within the posterior nonbanded layer (PNBL) in failed Descemet membrane endothelial keratoplasty (DMEK) grafts (Left column) and in corresponding peripheral donor endothelial-Descemet membrane (EDM) rims (Right column). Deposits of long-spacing collagens and proteoglycans are seen in the failed DMEK grafts (central) as well as in the peripheral rims.
normal DM group in 7 of 96 eyes (7%, secondary failure in 1 of 7 eyes) (P ¼ .003). Respecting only secondary graft failures, the difference was highly significant (P ¼ .001). In the group of normal DM eyes, the (only) secondary graft failure occurred after 9 months. In the group of abnormal DM eyes, the secondary graft failures occurred after 6, 9, 36, and 53 months. Applying the Bonferroni correction for multiple statistical tests, the significance level for this study has to be set at VOL. 169
.002 (29 single tests). At this level, the comparison of overall graft failure rates between the 2 groups is not statistically significant (P ¼ .003), but the comparison of secondary graft failures is still statistically significant (P ¼ .001). AnalyzingthelastCCTvaluebeforeregrafting,themeanCCT intheabnormalDMgroupwas850mm(range591–1267mm), indicatingendothelialfailurewithstromaledema. Postoperative clinical results (BCVA, CCT, ECD) are shown in Table 1 for all eyes, eyes with abnormal DMs,
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FIGURE 2. Hassall-Henle bodies at the periphery of the cornea. Transmission electron micrographs showing HassallHenle bodies at the periphery of the cornea, which were avoided for the analysis of ultrastructural abnormalities. These wart-like excrescences of the posterior surface of Descemet membrane contain collagenous and fibrillary materials together with numerous fissures filled by endothelial cell processes, and are believed to reflect physiological aging processes of the cornea.
and eyes with normal DMs. Regarding BCVA, a significant difference between both groups was found after 3 and 6 months postoperatively. However, there was a significant difference in BCVA already before DMEK surgery. Therefore, the change of BCVA was analyzed and a significant difference between both groups was found between the preoperative BCVA and BCVA 1 month after DMEK. In the abnormal DM group (starting with a worse BCVA), the increase of BCVA was higher (0.72 6 0.61) than in the normal DM group (0.24 6 0.53, P ¼ .019). No significant differences were found between groups regarding CCT and ECD before DMEK and 1, 3, 6, and 12 months after DMEK, respectively. Subgroup analyses for the organ-cultured grafts and for eyes with Fuchs endothelial corneal dystrophy are shown in Tables 2 and 3, respectively.
DISCUSSION IN THIS RETROSPECTIVE CONSECUTIVE SERIES, WE EXAM-
ined 112 residual DM specimens obtained during DMEK surgery by transmission electron microscopy and correlated the ultrastructural findings with the clinical outcomes after DMEK. Based on our previous study by Cirkovic and associates,22 who described ultrastructural abnormalities of DM in the majority of failed primary DMEK grafts, we hypothesized that similar ultrastructural abnormalities may also occur in efficient DMEK grafts but may have an influence on the postoperative clinical outcome. Ultrastructural abnormalities in the DM specimens analyzed comprised abnormal inclusions of collagen 62
(long-spacing collagen or fibrillary collagen) or proteoglycans within PNBL of DM and abnormal depositions of a posterior collagenous layer or pseudoexfoliation fibrils on the posterior surface of DM. Deposits of long-spacing collagen have been described in DM specimens of diabetic rats and humans, increasing with age, as well as in Fuchs endothelial corneal dystrophy and iridocornealendothelial syndrome.23–26 Whether the DM specimens revealing long-spacing collagen were derived from diabetic donors is not known, because this information is not provided by European eye banks. However, it is conceivable that ultrastructural abnormalities in DM account for the difficulties in DMEK graft preparation observed in corneas from diabetic donors.15 Further studies are necessary to analyze the ultrastructural features of DM from diabetic donors. Abnormal inclusions of proteoglycans in DM have not been reported previously. In contrast, a fibrous posterior collagenous layer has been shown to be deposited on the posterior surface of DM in various pathologic conditions, including pseudophakic bullous keratopathy and Fuchs endothelial corneal dystrophy. This layer is thought to be produced by impaired and attenuated endothelial cells, which start to transdifferentiate into myofibroblasts as a common final pathway following endothelial dysfunction and damage.27 One specimen of our series revealed typical guttae formations, and another specimen showed retrocorneal deposits of typical pseudoexfoliation fibrils pointing to the presence of Fuchs endothelial corneal dystrophy or pseudoexfoliation syndrome in the donors. The question arises why the pseudoexfoliation deposits and guttae were overlooked by the eye bank, which approved the corneas for transplantation. Probabaly, the delicate pseudoexfoliation deposits and guttae were simply too small to be detected by specular microscopy. The main criterion for the approval of corneal buttons is the endothelial cell density. Since the endothelial cell density was normal in these corneal buttons, the deposits were probably subclinical (ie, the respective endothelial dysfunctions were only beginning). However, the endothelial cells of pseudoexfoliation corneas continue to produce pseudoexfoliation material after transplantation and might thereby have led to a secondary graft failure. This study confirmed our hypothesis that ultrastructural abnormalities might be a risk factor for graft failure after DMEK. There was a significant correlation between ultrastructural findings and the secondary graft failure rate. Although the overall graft failure rate between the 2 groups is not significant after Bonferroni correction, there is a clinically relevant difference between the 2 groups (failure rate 7% vs 38%). We did not find a significant difference in CCT and ECD after DMEK in eyes that have received ultrastructurally abnormal grafts compared with those that have received structurally normal grafts, although we found a significant difference in the graft failure rate between both groups.
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FIGURE 3. Transmission electron micrographs showing ultrastructural abnormalities within the posterior nonbanded layer (PNBL) and on the endothelial side of Descemet membrane (DM). (First row, left) Normal DM with anterior banded layer (ABL, top), PNBL (middle), and endothelial cells (bottom). (First row, right) Fibrillary depositions within the PNBL (arrow). (Second row, left) Lamellar ribbon-like distributed long-spacing collagens (arrow) and diffusely distributed fibrillary depositions within the PNBL (arrowhead). (Second row, right) Ribbon-like distributed long-spacing collagens (arrow; insert: magnification of long-spacing collagens). (Third row, left) Layer with proteoglycans between the ABL and PNBL (arrow; insert: magnification of proteoglycans). (Third row, right) Posterior collagenous layer on the endothelial side of Descemet membrane (arrow). (Fourth row, left) Pseudoexfoliation deposits (arrow). (Fourth row, right) Guttae formations indicative of Fuchs endothelial corneal dystrophy of the donor (arrow).
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TABLE 1. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preoperative BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Change of BCVA preoperative – 1 month Change of BCVA 1 to 3 months Change of BCVA 3 to 6 months Change of BCVA 6 to 12 months
All Patients (n ¼ 112)
Normal DM Group (n ¼ 96)
Abnormal DM Group (n ¼ 16)
P Valuea
689 6 129 mm 559 6 109 mm 554 6 134 mm 548 6 92 mm 542 6 85 mm 2526 6 216/mm2 1544 6 309/mm2 1514 6 315/mm2 1532 6 321/mm2 1480 6 292/mm2 0.73 6 0.48 0.40 6 0.46 0.37 6 0.43 0.28 6 0.35 0.21 6 0.30 0.32 6 0.56
681 6 118 mm 556 6 111 mm 548 6 111 mm 540 6 64 mm 546 6 90 mm 2529 6 220/mm2 1550 6 319/mm2 1531 6 331/mm2 1532 6 332/mm2 1479 6 292/mm2 0.65 6 0.42 0.37 6 0.47 0.32 6 0.38 1.21 6 0.20 0.21 6 0.31 0.24 6 0.53
723 6 173 mm 572 6 102 mm 585 6 219 mm 602 6 201 mm 517 6 33 mm 2514 6 201 1516 6 259 1431 6 208 1534 6 239 1484 6 308 1.2 6 0.65 0.54 6 0.44 0.64 6 0.60 0.73 6 0.69 0.26 6 0.17 0.72 6 0.61
.530 .509 .836 .865 .266 .781 .652 .246 .792 .491 .002* .085 .012* .007* .136 .019*
0.02 6 0.35
0.05 6 0.36
0.14 6 0.3
.182
0.01 6 0.15
0.02 6 0.1
0.08 6 0.31
.782
0.02 6 0.2
0.01 6 0.14
0.15 6 0.47
.778
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
This discrepancy can be explained by the fact that the graft failure occurred at different intervals after DMEK, ranging from 3 to 53 months. In case of graft failure, the eyes were regrafted within 3 months and excluded from further analysis. The last CCT value before regrafting was 850 mm on average. Since these values were measured at different follow-up visits, they did not influence the statistical analysis of mean CCT in a significant way. The high graft detachment rate in the present study is attributed to the surgical technique, which was not standardized at the time of surgery (2010–2011) compared to nowadays. It might be objected that the graft failures were also caused by the surgical technique and not by the ultrastructural abnormalities of the DM. Summarizing the graft failure rates and rebubbling rates of the 2 groups, 44% of eyes in the normal DM group needed rebubblings, but only 7% failed (ie, 17% of eyes requiring rebubbling failed), whereas in the abnormal DM group 38% of eyes had graft failure, which is 55% compared to eyes requiring rebubblings (69%). This discrepancy (17% vs 55%) cannot be explained by the surgical technique because the technique was similar in all eyes. Therefore, we attribute the higher graft failure rate in the abnormal DM 64
group to the ultrastructural abnormalities and not to the surgical technique. No endothelial graft rejection occurred in the eyes included in this study. The graft failures occurred without obvious reason (for example, graft rejection, herpes endotheliitis) but may be partially ascribed to preexisting endothelial damage, as reflected by the ultrastructural abnormalities of DM specimens. Since there is a discrepancy in the rate of abnormal grafts between organ-cultured and Optisol-cultured grafts (shortterm/long-term culture: normal DM group, 14%/86%; abnormal DM group, 33%/66%, P ¼ .164), we performed a subanalysis of the organ-cultured grafts only (Table 2). The ultrastructural abnormalities described in this investigation were mostly observed within deeper zones of the posterior nonbanded layer of the Descemet membrane. Owing to the lifelong growth of Descemet membrane, it can be assumed that those buried depositions reflect disturbances of endothelial function during the lifetime rather than postmortem influences of culture conditions during the storage period. Therefore, the authors believe that the storage conditions cannot have caused the ultrastructural abnormalities observed.
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TABLE 2. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty in the Subgroup of Organ-Cultured Donor Corneas Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preoperative BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Rebubbling (n) Graft failure (n)
All Patients (n ¼ 97)
701 6 137 mm 553 6 109 mm 554 6 141 mm 552 6 98 mm 532 6 46 mm 2507 6 204/mm2 1562 6 323/mm2 1532 6 327/mm2 1543 6 316/mm2 1468 6 262/mm2 0.76 6 0.51 0.34 6 0.31 0.37 6 0.44 1. 6 0.32 0.18 6 0.14 44/97 9/97
Normal DM Group (n ¼ 85)
Abnormal DM Group (n ¼ 12)
693 6 124 mm 549 6 107 mm 544 6 113 mm 536 6 53 mm 533 6 46 mm 2501 6 201/mm2 1563 6 334/mm2 1534 6 342/mm2 1535 6 331/mm2 1456 6 269/mm2 0.69 6 0.44 0.30 6 0.26 0.32 6 0.40 0.20 6 0.12 0.17 6 0.14 36/85 5/85
745 6 200 mm 579 6 119 mm 620 6 266 mm 657 6 223 mm 515 6 44 mm 2539 6 227/mm2 1552 6 270/mm2 1520 6 204/mm2 1600 6 178/mm2 1599 6 130/mm2 1.36 6 0.70 0.57 6 0.46 0.65 6 0.62 0.73 6 0.75 0.26 6 0.10 8/12 4/12
P Valuea
.705 .562 .572 .236 .631 .644 .798 .790 .388 .221 .005* .039* .012* .09 .126 .101 .013*
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
TABLE 3. Comparison of the Central Corneal Thickness, Endothelial Cell Density, and Best-Corrected Visual Acuity (LogMAR) in All Eyes, Eyes With Normal Descemet Membrane, and Eyes With Abnormal Descemet Membrane Before and 1, 3, 6, and 12 Months After Descemet Membrane Endothelial Keratoplasty in the Subgroup of Patients With Fuchs Endothelial Corneal Dystrophy Group
CCT preoperative CCT 1 month CCT 3 months CCT 6 months CCT 12 months ECD preoperative ECD 1 month ECD 3 months ECD 6 months ECD 12 months BCVA preop BCVA 1 month BCVA 3 months BCVA 6 months BCVA 12 months Rebubbling (n) Graft failure (n)
All Patients (n ¼ 92)
673 6 111 mm 555 6 89 mm 541 6 91 mm 539 6 65 mm 542 6 89 mm 2533 6 218/mm2 1559 6 296/mm2 1519 6 328/mm2 1527 6 301/mm2 1494 6 297/mm2 0.68 6 0.39 0.40 6 0.45 0.31 6 0.31 0.27 6 0.25 0.23 6 0.29 45/92 7/92
Normal DM Group (n ¼ 81)
Abnormal DM Group (n ¼ 11)
676 6 114 mm 551 6 84 mm 544 6 97 mm 540 6 66 mm 546 6 94 mm 2540 6 228/mm2 1576 6 312/mm2 1536 6 348/mm2 1522 6 310/mm2 1496 6 299/mm2 0.66 6 0.37 0.40 6 0.47 0.29 6 0.30 0.24 6 0.23 0.23 6 0.31 37/81 4/81
656 6 91 mm 583 6 122 mm 528 6 44 mm 534 6 61 mm 517 6 33 mm 2592 6 136/mm2 1457 6 153/mm2 1431 6 179/mm2 1572 6 237/mm2 1485 6 308/mm2 0.88 6 0.47 0.38 6 0.24 0.46 6 0.37 0.39 6 0.33 0.24 6 0.14 8/11 3/11
P Valuea
.720 .505 .966 .718 .282 .523 .340 .264 .582 .656 .014* .544 .070 .065 .148 .086 .056
BCVA ¼ best-corrected visual acuity; CCT ¼ central corneal thickness; DM ¼ Descemet membrane; ECD ¼ endothelial cell density. For analysis of the BCVA only patients without ocular comorbidities are included. a Significant differences are indicated with an asterisk.
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Apart from the donor tissue storage, the underlying corneal pathology might be another possible bias of this study. Corneal endothelial cells of patients with Fuchs endothelial corneal dystrophy have been shown to be able to repopulate after DMEK.28 Therefore, a subanalysis of the eyes with the diagnosis of Fuchs endothelial corneal dystrophy was performed (Table 3). However, the number of graft failures in this subgroup was only 7. A comparison with the eyes of this study, in which DMEK was performed for another indication (n ¼ 20), is limited by the different cohort sizes and small subgroups. Therefore, it is difficult to draw any conclusions from this subgroup. Analysis of BCVA showed significant differences between both groups already preoperatively. Eyes having received structurally abnormal grafts had worse preoperative BCVA values than those receiving normal grafts. An explanation for this finding might be bias of preoperative BCVA data by the fact that some eyes had cataracts of different degrees at the time of surgery and others were already pseudophakic. The value of our study is limited by the fact that only the residual DM remnants obtained from the periphery of the donor corneas were evaluated, since ultrastructural alter-
ations in the periphery may not necessarily adversely affect the quality of central grafts. Therefore we had performed an ultrastructural comparison of peripheral rims and central DM specimens (Figure 1), which shows a comparable ultrastructure. Other technologies that have been established to visualize corneal structure are inferior to electron microcopy owing to a lower resolution. For example, laser scanning confocal microscopy has a resolution of only 1 mm, which is 3 orders of magnitude lower than the resolution of electron microscopy. Because the deposits of longspacing collagen found in this study measure only 70– 150 nm, electron microscopy is the only method to detect them. Furthermore, the relatively small number of abnormal DMs decreases the value of the statistical analysis. In conclusion, this study reveals a correlation between ultrastructural alterations of DM in donor corneas and the graft failure rate after DMEK. Thus, graft failure after DMEK is determined not only by surgical and postoperative factors but also by intrinsic, graft-specific features. Retrospective ultrastructural analysis of residual DM remnants might be important for clinicians to assess the reasons for graft failure.
¨ TZER-SCHREHARDT. THE STUDY FUNDING/SUPPORT: GRANTS FROM DFG (GERMAN RESEARCH FOUNDATION) FOR U. SCHLO data and the interpretation of the data are independent of this funding. Financial disclosures: The following authors have no financial disclosures: Julia M. Weller, Ursula Schlo¨tzer-Schrehardt, Theofilos Tourtas, and Friedrich E. Kruse. All authors attest that they meet the current ICMJE criteria for authorship. We thank Elke Meyer (technical assistant, Department of Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany) for the excellent preparation of electron microscopy specimens.
REFERENCES 1. Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea 2006; 25(8):987–990. 2. Kruse FE, Schrehardt US, Tourtas T. Optimizing outcomes with Descemet’s membrane endothelial keratoplasty. Curr Opin Ophthalmol 2014;25(4):325–334. 3. Livny E, Parker JS, van der Kaaij M, et al. Postmortem ultrastructural analysis of a cornea transplanted with Descemet membrane endothelial keratoplasty. Cornea 2014;33(8): 790–794. 4. Dapena I, Ham L, Droutsas K, van Dijk K, Moutsouris K, Melles GR. Learning curve in descemet’s membrane endothelial keratoplasty: first series of 135 consecutive cases. Ophthalmology 2011;118(11):2147–2154. 5. Satue´ M, Rodrı´guez-Calvo-de-Mora M, Naveiras M, Cabrerizo J, Dapena I, Melles GR. Standardization of the Descemet membrane endothelial keratoplasty technique: outcomes of the first 450 consecutive cases. Arch Soc Esp Oftalmol 2015;90(8):356–364. 6. Weller JM, Tourtas T, Kruse FE. Feasibility and outcome of Descemet membrane endothelial keratoplasty in complex anterior segment and vitreous disease. Cornea 2015;34(11): 1351–1357.
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7. Tourtas T, Schlomberg J, Wessel JM, Bachmann BO, Schlo¨tzer-Schrehardt U, Kruse FE. Graft adhesion in descemet membrane endothelial keratoplasty dependent on size of removal of host’s descemet membrane. JAMA Ophthalmol 2014;132(2):155–161. 8. Ro¨ck T, Bramkamp M, Bartz-Schmidt KU, Ro¨ck D, Yo¨ru¨k E. Causes that influence the detachment rate after Descemet membrane endothelial keratoplasty. Graefes Arch Clin Exp Ophthalmol 2015;253(12):2217–2222. 9. Terry MA, Straiko MD, Veldman PB, et al. Standardized DMEK technique: reducing complications using prestripped tissue, novel glass injector, and sulfur hexafluoride (SF6) gas. Cornea 2015;34(8):845–852. 10. Rodrı´guez-Calvo-de-Mora M, Quilendrino R, Ham L, et al. Clinical outcome of 500 consecutive cases undergoing Descemet’s membrane endothelial keratoplasty. Ophthalmology 2015;122(3):464–470. 11. Tourtas T, Laaser K, Bachmann BO, Cursiefen C, Kruse FE. Descemet membrane endothelial keratoplasty versus descemet stripping automated endothelial keratoplasty. Am J Ophthalmol 2012;153(6):1082–1090. 12. Price MO, Price FW Jr, Kruse FE, Bachmann BO, Tourtas T. Randomized comparison of topical prednisolone acetate 1% versus fluorometholone 0.1% in the first year after descemet membrane endothelial keratoplasty. Cornea 2014;33(9):880–886.
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SEPTEMBER 2016
13. Heinzelmann S, Hu¨ther S, Bo¨hringer D, Eberwein P, Reinhard T, Maier P. Influence of donor characteristics on descemet membrane endothelial keratoplasty. Cornea 2014; 33(6):644–648. 14. Gorovoy IR, Cui QN, Gorovoy MS. Donor tissue characteristics in preparation of DMEK grafts. Cornea 2014;33(7): 683–685. 15. Greiner MA, Rixen JJ, Wagoner MD, et al. Diabetes mellitus increases risk of unsuccessful graft preparation in Descemet membrane endothelial keratoplasty: a multicenter study. Cornea 2014;33(11):1129–1133. 16. Laaser K, Bachmann BO, Horn FK, Schlo¨tzer-Schrehardt U, Cursiefen C, Kruse FE. Donor tissue culture conditions and outcome after descemet membrane endothelial keratoplasty. Am J Ophthalmol 2011;151(6):1007–1018. 17. Johnson DH, Bourne WM, Campbell RJ. The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch Ophthalmol 1982;100(12):1942–1947. 18. Schlo¨tzer-Schrehardt U, Bachmann BO, Laaser K, Cursiefen C, Kruse FE. Characterization of the cleavage plane in Descemet’s membrane endothelial keratoplasty. Ophthalmology 2011;118(10):1950–1957. 19. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci 1984;25(12):1402–1415. 20. Kabosova A, Azar DT, Bannikov GA, et al. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci 2007;48: 4989–4999.
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21. Schlo¨tzer-Schrehardt U, Bachmann BO, Tourtas T, et al. Reproducibility of graft preparations in Descemet’s membrane endothelial keratoplasty. Ophthalmology 2013;120(9):1769–1777. 22. Cirkovi c A, Schlo¨tzer-Schrehardt U, Weller JM, Kruse FE, Tourtas T. Clinical and ultrastructural characteristics of graft failure in DMEK: 1-year results after repeat DMEK. Cornea 2015;34(1):11–17. 23. Akimoto Y, Sawada H, Ohara-Imaizumi M, Nagamatsu S, Kawakami H. Change in long-spacing collagen in Descemet’s membrane of diabetic Goto-Kakizaki rats and its suppression by antidiabetic agents. Exp Diabetes Res 2008;2008:818341. 24. Rehany U, Ishii Y, Lahav M, Rumelt S. Ultrastructural changes in corneas of diabetic patients: an electronmicroscopy study. Cornea 2000;19(4):534–538. 25. Levy SG, Moss J, Sawada H, Dopping-Hepenstal PJ, McCartney AC. The composition of wide-spaced collagen in normal and diseased Descemet’s membrane. Curr Eye Res 1996;15(1):45–52. 26. Levy SG, McCartney AC, Sawada H, Dopping-Hepenstal PJ, Alexander RA, Moss J. Descemet’s membrane in the iridocorneal-endothelial syndrome: morphology and composition. Exp Eye Res 1995;61(3):323–333. 27. Waring GO 3rd. Posterior collagenous layer of the cornea. Ultrastructural classification of abnormal collagenous tissue posterior to Descemet’s membrane in 30 cases. Arch Ophthalmol 1982;100(1):122–134. 28. Jacobi C, Zhivov A, Korbmacher J, et al. Evidence of endothelial cell migration after descemet membrane endothelial keratoplasty. Am J Ophthalmol 2011;152(4):537–542.
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