Routine evaluation of endothelium in human donor corneas

Summary Specular microscopy allows a direct evaluation of the corneal endothelium in intact donor eyes. It is therefore superior to previous methods for assessing donor material for penetrating keratoplasties. In a study of 278 eyes (139 donors) 226 could be examined by specular microscopy without any preparatory major manipulations. Twenty-four pairs and 12 single eyes (27%) of the 226 corneas were considered not suitable for grafts according to their morphologic endothelial changes. The discarded eyes were found all normal at conventional slit-lamp examination. Specular microscopy of the endothelium was not possible in 52 eyes as the corneal swelling was already too advanced.

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Zusammenfassung Die Auflichtmikroskopie erlaubt eine direkte Untersuchung und Beurteilung des Hornhautendothels am intakten Spenderauge. Sie ist damit bisherigen Untersuchungsmethoden an Genauigkeit überlegen und ermöglicht eine kritischere Auslese des Spendermaterials für perforierende Keratoplastiken. Unter Augenbank-bedingungen wurde die routinemäßige Anwendbarkeit an einer Serie von 278 Augen (139 Spender) geprüft. 226 Augen konnten ohne einschneidende Vorbereitungen mit dem Auflichtmikroskop untersucht werden. Darunter waren 24 Augenpaare und 12 einzelne Partneraugen (27%) mit morphologischen Endothelveränderungen, die eine weitere Verwendung als Spendermaterial ausschlossen. Bei 52 Augen war das Endothel wegen bereits fortgeschrittener Hornhautquellung mit dem Auflichtmikroskop nicht zu untersuchen. Bei der vorgängigen Spaltlampenuntersuchung waren bei allen Augen der untersuchten Serie keine pathologischen Befunde zu erheben.

     

Integrity of epithelium and endothelium in organ-cultured human corneas.

PURPOSE. To examine components of the junctional complex and the actin cytoskeleton and the incidence of apoptosis in epithelium and endothelium of organ-cultured human corneas. METHODS. Human corneas, either organ-cultured for 1 to >28 days or excised directly from eyes stored in moist chambers, were stained with antibodies to ZO-1, vinculin, and caspase 3 coupled to FITC-conjugated secondary antibody. These markers were combined with rhodamine-phalloidin staining for F-actin and DAPI labeling for DNA. The corneas were examined by confocal microscopy. RESULTS. The depth of the epithelium was reduced during organ culture, but no changes were observed in the distribution of ZO-1 or vinculin, or in the F-actin cytoskeleton. The appearance of apoptotic epithelial cells positive for caspase 3 or with condensed DNA increased with time after 14 days in organ culture, but there was no correlation with donor age. ZO-1 and F-actin staining patterns in endothelium were similarly undisturbed by organ culture, but apoptotic endothelial cells were only rarely seen and then only after >28 days in organ culture. CONCLUSIONS. Organ culture maintained the integrity of tight junctions and the actin cytoskeleton in epithelial and endothelial cell layers. Apoptosis was evident in epithelium but was observed rarely in the endothelium and then only after extended periods in organ culture.     

Long-term survival of endothelium following transplantation of corneas stored by organ culture.

This study reports corneal graft survival, endothelial cell changes, and visual outcome in 20 patients who received some of the first corneas stored by organ culture in the Corneal Transplant Service Eye Bank in Bristol. Mean donor age was 48 years (SD 15, n = 20) and corneas were stored for an average of 21 days (SD 7, n = 20). Preoperative endothelial cell density was 2334 cells/mm2 (SD 235, n = 18) and this fell by 8% (SD 12) to 2158 cells/mm2 (SD 372) within the first 2 months following transplantation. In 13 patients, endothelial cell density thereafter declined exponentially with a half-life of 41 months (SD 17, n = 12; one patient excluded as an outlier). Corneas that suffered rejection episodes showed the highest rates of loss of endothelial cells. Endothelial cell loss 4 years after transplantation was 46% (SD 16, n = 12), which was similar to the postoperative decline in cell density reported for corneas stored for far shorter periods in McCarey-Kaufman medium at 4 degrees C.     

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time

PURPOSE: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. METHODS: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. RESULTS: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. CONCLUSIONS: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.     

深低温冻存角膜复温后内皮细胞活性的变化

目的 探讨深低温冻存角膜复温后内皮细胞活性变化的规律。方法 冻存牛角膜复温后,分别行器官培养（Ａ组）及ｏｐｔｉｓｏｌ保存（Ｂ组）,检测皮内细胞密度、存活率（ＥＳＲ）及形态。结果 Ａ组培养６ｈ密度下降了７１．２％,２４ｈＥＳＲ明显下降（２７．２％）;Ｂ组保存７２ｈ密度下降了７３．６％（３７．２％）。结论 深低温冻存可造成内此细胞的潜伏性损伤,判定其活性在复温后组织培养６ｈ后进行。复温后Ｏｐｔｉｓｏｌ保存能改善细胞的潜在性损伤。     

Heterologous transplantation versus enhancement of human corneal endothelium

The ability to successfully transplant human corneal endothelium would offer a significant advance in the treatment of many corneal diseases. To investigate the feasibility of this, we established cultures of endothelial cells derived from neonatal human corneas. Eye bank donor corneas were either enhanced with a suspension of cultured endothelial cells or underwent endothelial cell removal and subsequent replacement with cultured endothelium. Following a 48-h incubation, the corneas were transplanted into the eyes of nonhuman primates. Over a 12-month period, 67% of the corneas with complete endothelial cell replacement thinned and remained clear, with a mean corneal thickness of 0.57 mm. Enhanced corneal buttons demonstrated a significantly lower success rate (35%), with opacified and thickened corneas. Control eyes in which the native endothelium was removed demonstrated advanced corneal edema and vascularization, with a mean corneal thickness in excess of 1 mm. By utilizing established tissue-culture techniques, we have demonstrated that human corneal endothelium, when cultured and subsequently transplanted, retains its in vivo pump function. Although further studies are warranted, these results indicate that transplanted human corneal endothelial cells can function normally and suggest the possibility of endothelial cell replacement for therapeutic purposes.     

Experiences with the transplantation of tissue-cultured corneas

Between October 1982 and May 1984, 21 cultivated donor corneas were successfully grafted in 20 patients at Innsbruck Eye Clinic. The average duration of cultivation was 10.9 days; the average number of endothelial cells prior to cultivation was 3,050 per mm2, and after cultivation 2,850 per mm2.     

Evaluation of the endothelium of human donor corneas by induced dilation of intercellular spaces and trypan blue

The endothelium of 30 pairs of human cadaver corneas was stained by trypan blue and the intercellular spaces were visualized by induced dilation prior to corneal culture. Trypan blue staining and induced dilation of intercellular spaces by 0.9% and 0.45% NaCl were found to be atraumatic. Only a fraction of damaged cells were stained by trypan blue. Endothelial cell losses in culture did not correlate with the number of trypan-blue stained cells, the post-mortem time, or donor age.     

Expression of angiogenesis-related factors in human corneas after cultivated oral mucosal epithelial transplantation

Purpose. We analyzed the expression of angiogenesis-related factors in corneal tissues that had undergone previously autologous cultivated oral mucosal epithelial transplantation (COMET). Methods. Six eyes from four chemically- and two thermally-injured patients with limbal stem cell deficiency who received COMET to promote wound healing were studied retrospectively. Immunoconfocal microscopy was performed on corneal specimens from the patients after COMET, as well on normal corneas, conjunctiva, and oral mucosa for keratin 8, fibroblast growth factor-2 (FGF-2), VEGF, collagen XVIII (endostatin), pigment epithelium-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), tissue inhibitor of metalloproteinase-3 (TIMP-3), thrombospondin-1 (TSP-1), and interleukin-1 receptor antagonist (IL-1ra). Results. FGF-2, VEGF, endostatin, PEDF, and IL-1ra were detected in all the samples, with signals for FGF-2, VEGF, and IL-1ra localized to the full-thickness epithelial layer, as signals for endostatin limited to the basement membrane. Expression of PEDF varied in tissues, with a preferential expression in the suprabasal epithelial layer. FGF-2 and IL-1ra were abundantly expressed in the basal epithelial layer in specimens with increased stratification. Signals for sFlt-1, TIMP-3, and TSP-1 were detected in normal corneal epithelium, and in a specimen containing corneal epithelium, but were negative in all other specimens. Conclusions. Expression of FGF-2, VEGF, PEDF, endostatin, and IL-1ra was similar in normal corneas, conjunctiva, oral mucosa, and corneas after COMET. Expression of sFlt-1, TIMP-3, and TSP-1 was limited to normal corneas and negative for other tissues. A lack of the aforementioned antiangiogenic factors may contribute to the peripheral corneal neovascularization seen after COMET.     

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.