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We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm2) compared to 2%FBS (2113 ± 331 cells/mm2; p = 0.03), and endothelial cell loss was lower (ECL HPL = −0.7% vs. FBS = −3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression.

Cornea transplants are tissue transplants and, as such, must be distinguished from organ transplants (e.g. heart or kidney transplants). However, tissue transplants can only be performed if there are enough donors available to attend to patients in need. Unfortunately, there are too few organ and tissue donors in Germany. All steps involved in processing donor tissues must be performed in accordance with the highest quality standards. All tasks and measures are aimed at improving patient care in the surgical units that are to be supplied. Cornea banks are subject to complex requirements, whose implementation is essential in terms of both infrastructure and personnel. The analysis and identification of essential topics reveal central fields of action that are decisive for implementing the challenges facing cornea banks. Questions of employee qualification, strategic questions due to new transplantation techniques, and changes in the societal perception of organ and tissue donation require the development of strategies that should have a holistic and sustainable effect.Keratoplastiken sind Gewebetransplantationen und als solche von Organtransplantationen (z. B. Herz- oder Nierentransplantationen) zu unterscheiden. Doch auch Gewebetransplantationen können nur durchgeführt werden, wenn Spender in ausreichender Zahl zur Verfügung stehen, um den Bedarf von erkrankten Patienten zu decken. Bedauerlicherweise gibt es in Deutschland zu wenig Organ- und Gewebespender. Für die Bearbeitung der Spendergewebe gelten grundsätzlich hohe Ansprüche an die Qualität und die sachgerechte Durchführung aller Arbeitsschritte. Alle Aktivitäten und Maßnahmen zielen darauf ab, die Patientenversorgung in den zu versorgenden operativen Zentren zu verbessern. Eine Hornhautbank unterliegt dabei gesetzlichen Anforderungen, deren Umsetzung sowohl infrastrukturell als auch personell bedeutsam sind. Fragen der Mitarbeiterqualifikation, strategische Fragen aufgrund von veränderten Transplantationstechniken, Veränderungen in der gesellschaftlichen Wahrnehmung von Organspenden und Gewebespenden fordern die Entwicklung von Planungen, die ganzheitlich und nachhaltig wirken sollten.

Technological progress and societal change are transforming medicine, and cornea banks are no exception. New infectiological factors, statutory requirements, management concepts, globalisation and digitalisation are also influencing how such facilities will operate in the future. The goal of providing high quality material to patients with corneal disease remains unaltered. The present article seeks to shed light on the type of material this will involve and under what circumstances it is to be obtained.Technologischer Fortschritt und gesellschaftliche Entwicklungen verändern die Medizin und machen auch vor Hornhautbanken nicht Halt. Neue infektiologische Aspekte, gesetzliche Vorgaben, Managementkonzepte, Globalisierung und Digitalisierung beeinflussen zusätzlich die zukünftigen Arbeitsinhalte dieser Einrichtungen. Das Ziel einer Bereitstellung von hochwertigem Material zur Versorgung von Patienten mit Hornhauterkrankungen bleibt bestehen. Welche Art von Material dies sein kann und unter welchen Rahmenbedingungen es entsteht soll der vorliegende Beitrag beleuchten.

Journal of Clinical Medicine 11(4), 1109 (2022). doi:10.3390/jcm11041109 special issue: "Special Issue "Corneal Disease & Transplantation" / Special Issue Editors: Vito Romeo; Giulia Coco", Published by MDPI, Basel

Journal of cellular and molecular medicine 25(20), 9647-9659 (2021). doi:10.1111/jcmm.16912, Published by Wiley-Blackwell, Hoboken, NJ

Introduction Since the beginning of the COVID-19 pandemic there has been some debate regarding the risk of transmission through tissue transplantation and tissue banking processes. Aim of the study To analyze the changes that SARS-CoV-2 has caused regarding the harvesting of corneal donor tissue and eye bank activities in Germany. Methods A questionnaire was provided to 26 eye banks in Germany, consisting of questions about adaptations made in the screening of potential donors and the harvesting of corneal tissue following the pandemic spread of SARS-CoV-2. Results Eighteen eye banks actively reduced recruitment of donors and two banks ceased all activity. Additional diagnostic screening was performed in eight banks, using conjunctival swabs and/or nasopharyngeal swabs. In six eye banks, additional protective measures, such as FFP2 masks and/or facial shields, were implemented. Overall, a mean reduction in the number of obtained donor tissues of 17% was observed. Discussion Conjunctival and/or nasopharyngeal swabs of donors have been implemented by a minority. Reasons for not performing additional tests may be moderate sensitivity and lack of validation for postmortem use of RT-PCR testing. Also, the hazard of SARS-CoV-2 entering the corneal donor pool with subsequent transmission might be perceived as theoretical. Face shields provide a sufficient barrier against splash and splatter contamination but may be insufficient against aerosols. Additional face masks would provide support against aerosols, but it remains debatable if corneal harvesting can be considered an aerosol-producing procedure. In the future we expect to see changes in current guidelines because of a surge in scientific activities to improve our understanding of the risks involved with cornea donation in the COVID-19 pandemic, and because current practice may reduce the availability of donor corneas due to new exclusion criteria while the demand remains unchanged.

To assess corneal endothelial cell (CEC) quantity and quality in eye-bank prepared lamellar grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) from organ cultured donor corneas that have not undergone de-swelling by media supplementation with dextran. Prior to graft preparation, corneas that had not undergone de-swelling (n = 30) were placed into fresh storage medium without dextran (KMI). Corneas in the control group (n = 30) were placed in dehydration medium containing 5% dextran (KMII). Subtotal stripping of Descemet’s membrane (DM) was performed manually. Following graft preparation, 10 corneas of each group were cultured further in their respective medium for 24 h, 72 h, or 120 h, respectively. Before and after DM stripping, as well as at the end of culture, CEC numbers were obtained. At the end of culture, CEC morphology was graded using a scoring system and CEC metabolism was assessed by detection of adenosine triphosphate. At 24 h after DM stripping, mean CEC counts (in cells/mm2) were 2204 in corneas stored in KMII, and 2391 in corneas stored in KMI (p = 0.003). This corresponds to a mean relative CEC loss of 12.4% with dextran versus 9.7% without dextran (p = 0.04). At 72 h, CEC counts were 1946 in KMII, and 2289 in KMI (p = 0.004). This corresponds to CEC loss of 23% with dextran versus 14% without dextran (p = 0.009). At 120 h, CEC counts were 2047 in KMII, and 2230 in KMI (p = 0.14). This corresponds to CEC loss of 22.7% with dextran versus 17.2% without dextran (p = 0.14). Also, at 120 h after DM stripping, 6/10 corneas fell below a threshold of 2000 cells/mm2 if stored in medium containing dextran, versus 1/9 corneas if stored without dextran (p = 0.003). Morphological assessment of CEC quality revealed equal scores for cell polymorphism (median = 1), granulation (median = 0) and segmentation (median = 1) in all groups. Lower ATP/protein ratios were observed in corneas stored in medium without dextran at 24 h (p

Purpose: To measure corneal endothelial cell (EC) quality and quantity following Descemet membrane (DM) stripping of human donor corneas and continued storage in organ culture medium containing dextran.Methods: DM stripping was performed in 30 organ cultured, corneoscleral discs. Corneas were divided into 3 groups of 10 corneas each. Baseline mean EC density (cells/mm2) was 2,372 (SD ± 259) in group 1, 2,540 (SD ± 266) in group 2, and 2,665 (SD ± 263) in group 3. Following subtotal DM stripping, culture was continued at 31°C for 24 hours (group 1), 72 hours (group 2), and 120 hours (group 3), respectively. EC density was measured before stripping and at the end of culture. At the end of culture, corneal EC morphology was graded using a scoring system and EC viability was measured by detection of adenosine triphosphate.Results: At the end of culture, mean EC density was 2,159 (SD ± 293) in group 1, 1,946 (SD ± 182) in group 2, and 2,047 (SD ± 225) in group 3. This constitutes an EC loss of 9,1% (SD...

Background: The HCV RNA testing of potential cornea donors frequently relies on blood samples stored pre mortem. The recommended storage time of maximum 72 h frequently excludes a significant fraction of donors. Methods: The influence of storage time of EDTA plasma samples at 4 °C on the viral load measured with the Roche HCV Quantitative Test vs. 2.0 was evaluated for 43 samples from HCV-positive individuals. Results: The mean reduction of the viral load after 4 °C storage for 6-8 days was 0.46 log10 IU/ml (range +0.17 to -1.66 log10 IU/ml). After 1-3 days a mean loss of 0.19 log10 IU/ml (range +0.30 to -1.41 log10 IU/ml) and after 3-5 days of 0.32 log10 IU/ml (range +0.36 to -1.81 log10 IU/ml) was observed. In 23.3% of samples, a viral load reduction ≥ 1 log10 IU/ml (1.0-1.81 log10 IU/ml) was found after prolonged storage (5-8 days). In none of the samples did the HCV load fall below the detection limit. Conclusion: Plasma storage for up to 8 days can quantitatively reduce the HCV RNA load, yet has no influence on the reliability of a qualitative HCV RNA detection by this ultrasensitive test to determine the HCV status of serologically negative cornea donors.

Purpose In this study, we investigated the associations between conjunctival (co) and intraocular (io) swabs and their implications for the contamination rates of organ-cultured corneas. Methods A total of 4177 swabs from 1054 corneas of 527 donors were acquired from the conjunctiva, after disinfection with 5% polyvinylpyrrolidone–iodine solution, and also from the anterior chamber after corneoscleral trepanation (io). Samples were incubated at 22.5 ± 2.5°C and 32.5 ± 2.5°C in thioglycollate broth for 14 days. Donor corneas were cultured in a closed system at 31°C. Microbial differentiation was performed for positive cultures. Results A higher temperature (32.5°C) and the intraocular swab retrieving localization led to significantly higher swab positive rates (32.5°C versus 22.5°C, odds 1.65, p

Amniotic membrane (AM) is an essential tool in ocular surface reconstruction. In this study, we analyzed the differential effects of glycerol and straight storage at − 80 °C for up to 6 months on the structural, biological, and mechanical properties of amniotic membrane (AM). Human placentae of 11 different subjects were analyzed. AMs were stored at − 80 °C, either with a 1:1 mixture of Dulbecco’s modified Eagle medium and glycerol (glycerol) or without any medium or additives (straight). Histological image analysis, tensile strength, cell viability, and basic fibroblast growth factor (bFGF) secretion were evaluated at 0.5, 1, 3, and 6 months. Histologically, neither glycerol nor straight storage significantly altered the epithelial or stromal structure of the AM. However, the cell number of the stroma was significantly reduced during the freezing process, independently of the storage method (p = 0.05–0.001). Tensile strength and Young’s modulus were not influenced by the storage method, but longer storage periods significantly increased the tensile strength of the AMs (p = 0.028). Cell viability was higher in glycerol rather than straight AM samples for up to 3 months of storage (p = 0.047–0.03). Secretion of bFGF at 3 months of storage was significantly higher in glycerol versus straight frozen AM samples (p = 0.04). Glycerol led to higher cell viability and higher bFGF secretion for up to 3 months of AM storage. However, no significant differences between the two methods were observed at 6 months of storage at − 80 °C.

Before corneal transplant surgery, a deswelling process of organ-cultured corneas is required. This study compares the deswelling kinetics of corneas with an intact endothelial cell layer and disrupted or removed endothelium by measuring central corneal thickness (CCT) over time using anterior segment spectral domain optical coherence tomography.Ten donor pairs were cultured in organ culture. The right and left corneas were alternately assigned to one of 2 deswelling groups. Deswelling in the first group [endothelial group (EG)] was induced using a medium with dextran 5%. Corneas of the second group [nonendothelial group (NEG)] were deprived of their endothelial cell layer by trypsinization and were then placed in the same deswelling medium. CCT (mean ± SD) was measured by anterior segment spectral domain optical coherence tomography before deswelling (0 hours) and after 1, 2, 3, 6, 12, 24, 48, 72, and 144 hours. Deswelling kinetics was analyzed through the nonlinear platform in SAS/JMP11 Pro.Before deswelling, CCT was 1071.0 μm (±129.6 μm) and 1133.8 μm (±124.3 μm) in the EG and NEG, respectively. Minimum corneal thickness was obtained after 24 hours in the EG (531.9 ± 47.5 μm) and 6 hours in the NEG (645 ± 81.2 μm). CCT was significantly (P0.01) higher in the NEG than EG after more than 6 hours.Corneal dehydration after organ culture seems to be a multifactorial process, which not only depends on osmotic effects of the deswelling compound but also requires the presence of an intact endothelial cell layer.

Durch den hohen Bedarf und der deutlich geringeren Verfugbarkeit von Hornhauten des Auges besteht eine grose Hoffnung auf das Gelingen arztlicher Akquisegesprache mit Angehorigen zum Einverstandnis in eine Gewebespende. Neben der Steigerung des Erfolgs in der Spendergewinnung sollen Akquisegesprache schutzend fur Angehorige verlaufen und die Arbeitsbelastung des Fachpersonals gering gehalten werden. Diese Arbeit erfasst den Bedarf und Erwartungen an ein Kommunikationstraining zur Vorbereitung auf die Akquisetatigkeit. Im September und Oktober 2009 wurde unter allen Akquisearzten, die in der Hornhautbank arbeiten bzw. gearbeitet haben, eine Bedarfanalyse fur ein Kommunikationstraining vorgenommen. Auf 10-stufigen numerischen Rangskalen wurde das Ausmas der fachlichen, emotionalen und personlichen Vorbereitung, das Ausmas der Einschatzung eines Kommunikationstrainings als sinnvoll, notwendig und hilfreich sowie die durchschnittliche und hochste Belastung und Bereitschaft zur Ausubung der Akquise erfragt. Die Studienteilnehmer fuhlten sich fachlich, emotional und in ihrer personlichen Uberzeugung nur mittelmasig auf die Tatigkeit vorbereitet und erachteten ein Kommunikationstraining fur in hohem Mas sinnvoll, notwendig und hilfreich. Bei mittlerer durchschnittlicher Arbeitsbelastung mit kurzfristigen Hochbelastungsphasen bestand nur eine moderate Bereitschaft zur weiteren Ausfuhrung dieser Tatigkeit. Durch klare Hinweise auf eine nicht als ausreichend empfundene Vorbereitung auf die Akquisetatigkeit und als hoch empfundene Arbeitsbelastung wurde ein Kommunikationsseminar mit einem spezifischen Training der Akquisetelefonate entwickelt. Dieses Training entspricht bedarfsgemas sowohl den Wunschen und Erwartungen des arztlichen Personals als auch den am ehesten beeinflussbaren Variablen im Hinblick auf die Erfolgsquote bei der Spendenakquise.

We report the case of a now 6-year-old girl who was born with different abnormalities in each eye. The right eye showed a total sclerocornea. At the age of 4 years we performed a perforating keratoplasty. On the left eye a large staphyloma developed. The staphyloma was excised and a cornea with a scleral rim was fixed in. This transplant became cloudy under a conjunctival flap and blepharoplasty. In addition to the clinical follow-up, histological and immun-histochemical examinations of the corneas were carried out.

Purpose Descemet membrane (DM) endothelial keratoplasty has improved outcomes of corneal transplantation in patients with corneal endothelial disease. However, the procedure has been criticized for jeopardizing donor tissue during graft preparation. Standardization of this procedure may provide a way toward minimizing tissue loss. For this purpose, we propose the use of a novel tool. Methods Computerized numerical control milling was used to create a blunt instrument, which was used to remove endothelial cells within a defined area in the periphery of donor corneas. Trypan blue was used to stain denuded DM. Graft preparation was continued as per our standard protocol. Transmission electron microscopy was performed on the treated area, and endothelial cell counts were obtained. Results Use of the modified procedure resulted in delineation of a peripheral band of denuded DM, which readily stained with trypan blue. This provided increased visibility of DM during subsequent steps. Transmission electron microscopy confirmed that no structural deficits of DM were induced. Mean endothelial cell loss (±SD) at 24 hours after preparation was 63 (±130) cells per square millimeter in the group prepared with the use of the new instrument (n = 7), versus 116 (±107) cells per square millimeter in the group prepared without the new instrument (n = 7; P = 0.45). Conclusions The device presented here enhances visualization of DM during creation of the peripheral margin for subsequent lifting of the margin and stripping of the graft. This may increase success rates and shorten preparation times and learning periods for DM preparation. DM ultrastructure and endothelial cells were not negatively affected.

Purpose We compared the SLc Original (SLc) and One-Use Plus (OUP) microkeratomes for ultrathin Descemet's stripping automated endothelial keratoplasty (DSAEK) lamella preparation and storage, vis-a-vis accuracy, endothelial cell loss (ECL) and lamellar surface roughness (LSR). Methods Twenty-five human corneas were dissected with single-use heads of different sizes aiming for a posterior lamella (PL) thickness of 85 μm, after which they were incubated for 6 days in a 5% dextran medium. Before preparation (0 hr) and 1, 24, and 144 hr after dissection, ECL and corneal thickness (CCT) were measured by ultrasound pachymetry (USP) and optical coherence tomography (OCT). Lamellar surface roughness (LSR) was assessed by scanning electron microscopy (SEM) and evaluated by two masked observers. Results Prior to cutting, CCTs did not differ between OCT and USP measurements, with a high correlation between the two modalities (r2 = 0.8; p

Purpose Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Methods Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. Result I-ECD was 2812 ± 360/mm2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Conclusion Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve graft quality.

We compared the hand-guided Moria Carriazo-Barraquer (CBm) microkeratome with the fully automatic SLc microkeratome for Descemet's stripping automated endothelial keratoplasty (DSAEK)-lamella preparation and storage, vis-à-vis accuracy, endothelial cell loss (ECL), and lamellar surface roughness (LSR).A total of 18 human corneas were dissected with both the 300 μm CBm multi-use (n = 9) and the 300 µm SLc (n = 9) single-use heads, after which they were incubated for 6 d in a 5% dextran medium. Before preparation (0 h) and 1, 24, and 144 h after dissection, ECL and corneal thickness (CT) were measured by ultrasound pachymetry (USP) and optical coherence tomography (OCT). LSR was assessed by scanning electron microscopy (SEM) and evaluated by three masked observers.Prior to cutting, CTs did not differ significantly between OCT or USP measurements, with a high correlation between the two modalities (r(2)= 0.94, p 0.0001). One hour after preparation the anterior lamella showed a significantly higher dissection depth with the CBm (429.4 ± 21.8 µm) than the SLc (311.7 ± 54.8 µm, p = 0.0006), with the variance of the SLc system showing a trend towards higher values (p = 0.07). Anterior and posterior lamellae swelled significantly in the subsequent culture period. Both groups showed a significant ECL 1 h after preparation (p 0.0001) with no significant difference between the systems (1 h: p = 0.44; CBm: - 9.4%, SLc: -11.7%), which stabilized over 144 h (144 h CBm: -13.9%, 144 h SLc: -10.3%). LSR did not differ significantly between both systems (p = 0.60).The SLc system agrees more with the designated cutting depth than the CBm. The dissection produced a comparable LSR and a ∼10% ECL independently of the system. Further incubation of the prepared lamellae led to a swelling, but no further ECL.

Purpose To study metabolic changes of the human cornea during organ-culture. Morphological changes have been extensively studied, whereas changes in human corneal metabolism have not been investigated yet. Material and methods 106 human corneas were stored for 1, 7, 15, 18, 21 and 28 days in a closed-system under standard eyebank conditions. After storage, glucose, lactate, ATP, ADP and AMP concentrations were determined in each cornea. Results Glucose concentration decreased during the first two weeks with a minimum on day 15. ATP and ADP concentrations increased during the same period of time, but had their minimum later, on day 18. Lactate increased during the culture period up to day 21 and decreased thereafter. Conclusion From these data we conclude that the human cornea recovers during organ-culture, especially during the first two weeks. The changes occurring after a fortnight might be related to the artificial culture conditions. Nevertheless, the metabolic status is better than in post-mortem corneas. The changes may be partly avoided by changing the medium after at least two weeks of organ-culture.

BACKGROUND Metabolic changes of the human donor cornea during organ-culture are not at all reflected by the endothelium. Therefore metabolic investigations have become of increasing interest. It was the aim of this study to determine the correlation between glucose and lactate in storage medium and within the cornea itself and to find thereby an additional parameter for glucose metabolism during organ-culture. METHODS Glucose and lactate were examined in 166 organ-culture medium samples as well as in 106 human corneas by enzymatical optical methods. Investigations were carried out after 1, 7, 15, 21 and 28 days of organ-culture. RESULTS Glucose consumption was highest during the first two weeks of organ-culture. Glucose concentrations showed a good linear correlation between medium samples and the cornea (r = 0.923). The correlation coefficient for lactate was worse (r = 0.733). CONCLUSION Glucose and lactate levels in the organ-culture medium can be used as a marker for glucose metabolism in the cornea.

Background There are two well known systems to culture human corneas prior to transplantation. First, corneal storage at 4 degrees C especially in Optisol medium. Second, organ-culture at physiological temperatures in a modified minimal essential medium (MEM). In the cold storage system the number of endothelial cells after storage might be overestimated because the damaged cells are not able to leave the monolayer. It has been supposed that the lack in energy recruitment is the main reason for that, but has not been proven yet. It was the purpose of this study to describe the energy status of the human cornea after storage in both systems. Materials and methods 32 human corneas were investigated. They were stored for 7 days in Optisol, and for 7 days in MEM plus 1 day in MEM supplemented with 5% dextran 500 and 12 days in modified MEM plus 1 day in MEM supplemented with 5% dextran 500. The endothelial cell density (ECD) as well as the hydration were determined. Glucose, lactate, ATP, ADP and AMP were measured to reflect the energy status. Results Hydration was comparable in all three groups. ECD was slightly higher in Optisol stored corneas, although the amount of damaged cells was much higher. Optisol stored corneas showed a severe anaerobic situation, especially lacate concentrations were increased. In contrast ATP and ADP concentrations were twice as high in MEM than in Optisol stored corneas. Discussion The severe anaerobic situation in Optisol stored corneas leads to a lack in energy recruitment. This reduces the ability of cell function (mitosis) and the function of the monolayer (migration, elimination). Whether these changes are reversible after transplantation has to be determined in future.

• Background: Our study investigated the presence, type and quantity of silicon in the human cornea. We report the results of silicon measurements in the corneas of silicotic individuals, bricklayers and apparently normal human individuals and offer a hypothesis for the mechanism of silicon deposition in the human cornea. • Methods: We examined corneas from 13 deceased subjects who suffered from silicosis, 2 bricklayers and 6 apparently healthy subjects. Cornea samples were examined by energy-dispersive x-ray analysis (EDXA) under calibrated conditions in a scanning electron microscope (SEM). The EDXA detector was a silicon-free germanium crystal. Five distinct layers (epithelium, Bowman's membrane, central stroma, Descemet's membrane and endothelium) were analyzed in each cornea. The method allows simultaneous semiquantitative analysis of, among other elements, silicon, calcium and oxygen. We measured amorphous silicon and visible particles of silicon. • Results: We found amorphous silicon in low concentrations in 38% of the silicotic corneas and in very low concentrations in 29% of the healthy corneas. Bricklayers showed high concentrations of amorphous silicon. These accumulations of silicon were predominantly located in Descemet's membrane. Silicotic corneas showed significantly more silicon-containing particles than corneas of healthy controls (χ2-test,P< 0.01). • Conclusion: Normal corneas contain very low amounts of silicon. Longterm exposure to inhalative silicon dusts results in only very slightly increased levels of amorphous silicon in the cornea. However, silicon-containing particles accumulate in the cornea of silicotic individuals. Bricklayers incorporate more amorphous silicon into the cornea.

Quantification of adenosine phosphates in human corneal extracts has been performed using spectrophotometry. We employed the bioluminescence technique to obtain a more sensitive assay for adenosine phosphates and to reduce the volume of the test sample.The bioluminescence assay for ATP, already known from sterility control, was modified and expanded. Standard curves were established using a standard solution with equimolar concentrations of ATP, ADP and AMP. To monitor the method, adenosine phosphates were measured in 35 human corneal extracts using both spectrophometry and bioluminescence.Linear standard curves ranging from 1 to 45 pmol were established. The two methods yielded comparable results despite the use of a basic dilution of 1:100 for the new technique.Bioluminescence provides a highly sensitive quantification of adenosine phosphates in the human cornea and facilitates an extremely detailed evaluation of the metabolic status of the cornea.

Background Initial surgical treatment of severe eye burns is important for the long lasting healing period. To look closer at the efficacy of the early surgical treatment and the possible consequences for woundhealing, the surgical interventions were examined in an animal experiment. Materials and Methods The right eye of 20 rabbits each was burned with 1 n NaOH for 30 seconds followed by a surgical treatment 48 hours later. In the first study necrosectomy, tenonplasty, application of an artificial epithelium and blepharoplasty were performed. In a comparable study blepharoplasty was withdrawn and the fornices were prevented from cicatrization by mechanical treatment with blunt spatula. Antiinflammatory depotinjection of Volon A 40 was applicated additionally. After an interval of 4-18 weeks the eyes were examined histologically. Results A higher risk for infections could be observed in eyes which underwent blepharorhaphy. Furthermore proliferative processes and corneal ulcerations were predominant. In contrast to these findings the latter complications could be prevented due to the clinical inspection of the artificial epithelium and described mechanical irritation of the fornices in the second study. Discussion It could be shown that proliferative processes are major complications after severe burns of the eye. Nevertheless the intensity could be minimized by the consequent use of blunt spatula in combination with antiinflammatory therapy. Furthermore it could be demonstrated that the artificial epithelium is an effective tool providing good prognosis for subsequenty planned keratoplasty. To enable necessary postoperativ inspection blepharoplasty should be avoided.

Purpose Varying states of hydration during organ culture and subsequent dehydration of corneas with Dextran prior to transplantation cause endothelial cell damage. Hydroxyethyl starch 130 (HES) has been suggested as a permanent dehydrating agent in serum-free cornea culture. This study compares corneas stored in a synthetic medium containing HES with corneas stored and dehydrated under standard conditions. Methods Twenty pairs of human donor corneas were cultivated in MEM with antibiotics at 31°C for 16 days (groups A and B) or 28 days (groups C and D, n=10). Media contained 2%FCS in group A and C, and 7.5 % HES 130 plus 1mU/l human insulin in groups B and D. On day 15 or 27, corneas from group A and C were placed in dehydration medium (MEM + antibiotics + 2%FCS + 5% dextran 500). Endothelial cell density and morphology were investigated at the beginning and end of culture. Corneal thickness was assessed by pachymetry. Keratocyte viability was assessed by TUNEL and Annexin V staining. Results Endothelial cell loss was higher in group A (278.5+/-184.2) vs. B (156.6+/-154; p=0.0293), and in group C (490+/-156.3) vs. D (301.8+/-189.6; p=0.0195), with similar morphology. Final corneal thickness was 882+119µm and 914+119µm in groups A and B, and 889+99µm and 957+87µm in groups C and D, resp. (n.s.). No significant differences were found in TUNEL and Annexin staining. Conclusion The synthetic medium supplemented with HES 130 and insulin improves endothelial cell survival during cornea culture, without adverse effects ion keratocyte viability. It is thus feasible to store donor corneas in fully synthetic medium without the chemically undefined FCS and without the need for dehydration, resulting in increased safety and faster transplant availability.

The authors investigated 32 human corneal buttons of patients suffering from severe eye burns. The keratoplasties were performed in different periods after the burn and collected within five years. Grafts were followed-up until they failed because of ulceration or until rehabilitating keratoplasties were performed. Early (up to six months after the burn) and late (more than 12 months after the burn) obtained corneas showed different cellular reactions in the corneal stroma. Inflammatory cells such as granulocytes and lymphocytes decreased with increasing periods after the burn. More than 12 months after the burn, minor inflammatory cellular reactions but increasing scar formation could be found. Four out of 19 early-, two out of eight intermediate- (between six and 12 months after the burn) and one of the late performed keratoplasties failed and had to be replaced by new corneal grafts. Due to the conditions in the graft beds the explanted corneal grafts showed a cellular reaction in the stroma comparable to the previously explanted burnt corneae. Further complications occurred because of problems concerning the ocular surface. After preliminary therapeutical keratoplasties rehabilitating keratoplasties could be successfully performed in four cases between 19 and 39 months.

We investigated the kind of reactions that occur after injection of a PMMA lens powder into the back skin of rabbit. The lens powder was suspended in NaCl and incomplete Freund's adjuvant to reinforce the immunological reaction. The ELISA test was carried out to detect antibodies against the lens material as a whole and against the UV absorber Tinuvin in particular. We also performed histological and immunohistochemical examinations of the back skin. We did not detect antibodies against either the lens material or against Tinuvin. Histological examination showed a foreign-body or delayed allergic reaction against the lens material. Lymphocytes surrounding the PMMA were found to be mainly of the T-type, which supports the results of the ELISA test.

Following Crede's prophylaxis with silver-nitrate, the cornea of a newborn presented greyish-brown, lime-like plaques on the nasal part of the right eye. A paracentral ulcerating stromal opacification undermined these appositions, when the patient was admitted to the eye-clinic at Aachen. In the material taken in a lamellar keratectomy scanning electron microscopical examination was able to prove the existence of granules, previously described in light-microscopy. These granules measured 100 to 300 nm in diameter and were placed up to 110 microns deep into the corneal stroma of the specimen. An earlier chemical analysis of necrotic material showed no silver specific reaction. By means of EDX-Analysis these granules could be identified as silver-containing. This was once reassured by a newly developed modification of van-Kossa's-staining-method. The fact that the granular deposits contained mainly silver proves that the onset of a sodium-chloride-irrigation did not promote an intended therapeutic silver-chloride-precipitation and therefore had no effects on the silver-nitrate's penetration abilities. Injuries by silver-nitrate-solutions used for Crede's prophylaxis are seldom but still reported. The mechanism of injury in this case of a child, born by sectio remains unknown. Neither the use of an unusual silvernitrate solution, that was taken from a disposable ampoule (Mova-Nitrat) was reported, nor any corneal injury during sectio mentioned.(ABSTRACT TRUNCATED AT 250 WORDS)

Background In most clinical centers donor material can not be obtained in neccessary amounts. Therefore corneas from long-suffering donors have to be accepted, too. It was the aim of this study to demonstrate the influence of the cause of death on the endothelial cell density of fresh human corneas. Material and Methods The endothelial cell count of 81 donor corneas was determined after preparation of the corneo-scleral disc. For all donors the premortal history was evaluated concerning the cause of death and therapy. In this manner donors were divided into five groups: sudden death, carcinoma, septicemia, renal insufficiency and diabetes mellitus. Results There were no significant differences in endothelial cell count between these five groups. Even chemotherapy and radiatio of the donor had no influence on the cell density. Conclusion It may be concluded, that clear corneas have a sufficient endothelial cell density and that the cause of death has no influence on this parameter. Therefore also corneas from donors with long-standing diseases may be accepted for transplantation

• Background: Quantification of glucose and lactate concentrations in human corneal extracts has been performed using spectrophotometry. We employed a bioluminescence technique to obtain a more sensitive assay for glucose and lactate and to reduce the volume of the test sample. • Materials and methods: The NAD(P)H bioluminescence assay (Boehringer Mannheim, Germany) was modified for glucose and lactate. Standard curves were established using a standard solution with 0.004 mM and 0.01 mM concentrations of glucose and lactate, respectively. • Results: Linear standard curves ranging from 0 to 200 pmol for glucose and from 0 to 250 pmol for lactate were established. The sample volume was reduced from 100 μl to 25 μl compared with spectrophotometry. • Discussion: The modified bioluminescence technique provides a highly sensitive quantification of glucose and lactate in the human cornea and thus reveals more details of the overall metabolic status of the tissue.

Corneas are usually stored for a maximum of approximately 30 days in European cornea banks. Although attempts are being made to prolong culture periods, data on their success are extremely limited to date. The following study was carried out to describe the capacities and limits of the established system.Thirty-seven human corneas were stored foror = 12 weeks under standard eye bank conditions [modified minimal essential medium (MEM), 31 degrees C, closed system]. Twenty-one fresh human corneas served as control. Both the adenylate nucleotides and the glucose and lactate concentrations were measured in the tissue (all cellular layers) by using the bioluminescence technique. The endothelial-cell densities also were determined.Endothelial-cell densities decreased from 2,963.4 +/- 58.7 cells/mm2 (fresh) to 2,649 cell/mm2 after 4 weeks and to 2,087 cells/mm2 after 6 weeks. Storage for periods6 weeks led to total endothelial necrosis. Biochemical studies showed improving values during the first 4 weeks and acceptable conditions foror = 6 weeks.From these data, we conclude that long-term organ culture in a closed system is limited to approximately 6 weeks and thereby confirm the clinical results of Früh and Böhnke.

The aim of this histochemical study was to demonstrate the absorption of dextran 500 and its distribution in the cornea after storage under standard eye bank conditions. Furthermore, an attempt was made to distinguish between the soluble and insoluble parts of dextran 500 absorbed by the cornea, in order to see how much dextran remains in the cornea after transplantation.Forty-nine fresh and 65 organ-cultured human corneas were investigated. The corneas were cultured for 28 days in a dextran-free medium, followed by a period of 1-14 days in a medium containing 5% dextran 500. Cryosections were stained by aqueous PAS and a modified alcoholic PAS to determine the amount of dextran.Dextran was not found in the epithelium, stroma or endothelium of fresh human corneas. By contrast, extra- and intracellular positive staining reactions were detected in corneas following storage in a medium containing dextran. Dextran 500 absorption was relatively diffuse in the epithelium after storage in a dextran medium. Initial accumulations were found in the stroma near Bowman's and Descemet's membranes and also in the central part of the cornea, as the period of culture in the medium containing dextran lengthened. We also observed interaction between the stroma and endothelium: decreasing amounts in the endothelium were followed by an increase of same in the stroma. Intracellular deposits of dextran were detected after only one day. A much greater part of the extracellular dextran than previously described was found to be insoluble.As the amount of dextran in the cornea increases over a longer storage time, we conclude that the period of storage in a medium containing dextran should be limited to four days. The fact that the cornea is saturated with dextran after seven days has been shown in further studies to interfere with mitochondrial function and may also cause severe post-operative swelling of the transplant, hence leading to a longer recovery period for the patient.

• Background: The presence of interleukins has been demonstrated in the cornea and other ocular tissues. Although pathogenic mechanisms are unknown, interleukins seem to be involved in inflammatory disorders of the cornea. The present study was undertaken to analyse concentrations of interleukin- Iβ (IL-1β) and interleukin-6 (IL-6) in human corneas with various clinical diagnoses. • Methods: Immediately after keratoplasty 127 explanted human corneas with various corneal diseases were snap frozen and cryosections were prepared for histological examination. Furthermore, the protein content was measured according to the method of Bradford and the concentration of IL-1β and IL-6 were determined using a specific immunosorbent test (ELISA). • Results: It was found that IL-1β and IL-6 level were clearly higher in corneas with ulcerations and distinct inflammatory signs. Lower levels of both interleukins were found in corneas with a weak expression of inflammatory signs. • Conclusions: Keratitis, keratoconus with inflammatory signs, and ulcerating processes showed higher interleukin levels than corneas with non-inflammatory disorders like scar formation, corneal dystrophy and keratoconus. The results could show that, depending on the clinical diagnosis, the inflammatory status of the cornea may be evaluated by the interleukin levels determined in the corneal tissue.

The purpose of this study was to obtain further information on the viability of organ-cultured human cornea. We thus used a specific staining method for succinate dehydrogenase (SDH), which is located in the membrane system of vital mitochondria. We examined fresh and long-term-cultured human corneas. After an initial incubation period in dextran-free culture medium, corneas were stored in a medium containing dextran. With respect to different appearances of the SDH staining, minimal essential medium without dextran seems to have a positive effect on the condition of epithelial cells. After renewal of the medium, keratocytes showed a brief improvement followed by a delayed deterioration, while the endothelial cells were severely damaged. However, best results for all three cell types were observed on the fourth day in a medium containing dextran. We therefore conclude that these corneas were best suited for transplantation.

PURPOSE Tenon plasty has been used to reconstruct the conjunctival surface in severe burns in which ischemic sclera was exposed or undergoing ulceration. A modified impression cytology procedure was applied to investigate the conjunctival epithelium. The quality of the regenerated epithelium on the advanced Tenon sheets was assessed. METHODS The 63 conjunctival samples of eye-burn patients were investigated. Among these, 41 patients had very severe bums. Conjunctival samples were collected from 6 weeks after surgery to 5 years after the accident. They were compared with conjunctival epithelia obtained from 53 normal eyes of healthy volunteers. A 25-mm2 Biopore membrane (Millipore Catalogue PICM 01250) was placed on the bulbar conjunctiva surface in the lower temporal quadrant, at a distance of 3-5 mm from the limbus, till it was soaked with fluid. The ablated cell sheets were stained with periodic acid-Schiff (PAS). RESULTS In all cases, an intact conjunctival epithelium was observed. In healthy eyes, 2,338 epithelial cells/mm2 and 155 goblet cells/ mm2 were found. Eyes after a surgical reconstruction with Tenon plasty resulted only in 1,575 epithelial cells/mm2 and 72 goblet cells/mm2. The differences were highly significant. The ratio of epithelial to goblet cell counts revealed an increase of goblet cells during the postoperative period. CONCLUSION Conjunctival epithelium as well as goblet cell densities were reduced after heat, lime, alkali, and acid burns. However, after concrete burns, cell densities were increased. Tenon plasty provided the regeneration of the fully intact conjunctival epithelium. Goblet cells were present from 6 weeks after the surgery on; their number increased gradually. The stimulation of the goblet cell mucous secretion is discussed.