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INTRODUCTION: Because of the gap between the high need for cornea tissue for transplantation and the current lower numbers of donors, there is high pressure to succeed in telephone calls to relatives of deceased for obtaining consent. Besides the attempt to increase the rate of donors, these telephone calls should be gentle for relatives and not overstrain professionals. This study assesses the need and expectations of physicians towards communication training in preparation for their task. METHODS: In September-October 2009 a needs assessment for communication training among physicians who currently work or have worked for the eye bank took place. Personal data were recorded and their estimate of the perceived burden as well as needs and potential benefits from a specific communication training for obtaining consent for cornea donation by telephone were assessed using numeric rating scales between 1 (minimum) and 10 (maximum). Results were expressed as mean and range. RESULTS: The participants felt only moderately prepared for the task regarding information content, their emotions and personal convictions. They considered a specific training to be highly meaningful, necessary and helpful. Their highest burden during telephone calls previously experienced was documented as 8.4 and the average burden as 6.3. Accordingly, their willingness to continue working at an eye bank after their rotation was moderate. DISCUSSION: Physicians involved in obtaining consent for cornea donation by telephone feel highly stressed and only moderately prepared for fulfilling the task both for the information content and on the emotional level. Accordingly, specific communication training is perceived as highly meaningful, necessary and helpful. An educational program was developed to meet the needs and expectations of physicians.

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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.

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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.

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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.

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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 microliters to 25 microliters 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.

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PURPOSE: 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. METHODS: Thirty-seven human corneas were stored for < or = 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. RESULTS: 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 periods >6 weeks led to total endothelial necrosis. Biochemical studies showed improving values during the first 4 weeks and acceptable conditions for < or = 6 weeks. CONCLUSION: 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.

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UNLABELLED: Recent studies have shown that the human cornea reflects the metabolic status of the donor. Nevertheless, it has not yet been shown whether this leads to a different tolerance of organ culture and whether these changes are reversible or not. MATERIALS AND METHODS: Eighty-five organ-cultured human corneas were investigated. Endothelial cell density and the concentrations of glucose, lactate, ATP and ADP were determined in each cornea by enzymatic-optical methods. Obduction protocols were examined and five groups of donors could be determined: (1) donors who had died suddenly; (2) donors who had died of malignant processes; (3) donors with septicaemia; (4) renal-insufficient donors; and (5) diabetic donors. RESULTS: Endothelial cell density decreased significantly after 28 days of organ culture. Deswelling for 1 day in a medium containing dextran 500 caused additional cell loss. On the basis of biochemical parameters, the corneas showed recovery after organ culture: especially ATP and ADP concentrations increased. Glucose concentrations decreased and lactate concentration increased in the closed organ culture system. Best recovery was observed in corneas from septic donors and from those who had died of carcinoma. Overall, an equalization of all corneas after the culture period was observed. In contrast, the deswelling period causes deterioration of metabolic status; ATP concentration decreased dramatically, and ADP increase was higher than expected. CONCLUSION: From these data we conclude that there is no donor disease that could lead to exclusion of the cornea from organ culture. In fact, organ culture leads to a recovery of the metabolic status of the human cornea.

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PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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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.

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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 was withdrawn and the fornices were prevented from cicatrization by mechanical treatment 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 therapy. Furthermore it could be demonstrated that the artificial epithelium is an effective tool providing good prognosis for subsequently planned keratoplasty. To enable necessary postoperative inspection blepharoplasty should be avoided.

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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 decreased 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 (chi 2-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.

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BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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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-1 beta (IL-1 beta) 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-1 beta and IL-6 were determined using a specific immunosorbent test (ELISA). RESULTS: It was found that IL-1 beta 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.

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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.

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BACKGROUND: Long-lasting inflammation is a major problem in treatment after severe eye burns and may find expression in an altered elemental composition of the conjunctiva. Particulate contamination of biological tissue induces such inflammatory processes. In the anterior eye segment, trauma or subsequent therapy may give rise to such contamination. Scanning electron microscopy and energy-dispersive X-ray analysis are able to detect traumatic residues of submicron size and changes of the elemental composition. METHODS: Conjunctival specimens from first-time peridectomy of three healthy and nine severely burnt-eyes were examined with scanning electron microscopy and energy-dispersive X-ray analysis. The samples were prepared as cryo- or paraffin sections, mounted on carbon blocks and coated with evaporated elemental carbon. RESULTS: The samples of healthy conjunctiva showed higher concentrations of Na, P and CI. These elements showed lower concentrations in conjunctival stroma of burnt eyes excised before the 20th day after trauma than in material obtained subsequently. In two burnt conjunctival specimens there was severe traumatic contamination with Ca in Ca(OH)2 and CaO burns, and in one case the traumatic substance was Si, in a peroxide plus silicone spray burn. In the remaining six cases, particulate contamination with Fe, Al, Ni, Zn, Cu, Ti and other substances was present in the burnt conjunctivas, while no contamination was detected in the specimens of healthy conjunctivas. CONCLUSIONS: The origin of the contaminant particles is assumed to be the trauma itself and the subsequent therapy. These investigations stress the importance, for clinical purposes, or early peridectomy and contamination-free therapy.

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BACKGROUND: In most clinical centers donor material can not be obtained in necessary 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.

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PURPOSE: Little is known about the metabolic status of human corneas before transplantation. The authors attempted to determine the influence of both cause of death and age on the corneal metabolism. METHODS: Adenosine-triphosphate (ATP), adenosine-diphosphate (ADP), glucose, and lactate were measured in the stroma and endothelium of 30 human corneas. The corneas were divided into four groups according to cause of death and four groups according to age. Corneas from donors with diabetes were excluded. RESULTS: Corneas from patients who died suddenly--because of cardiac infarction, for example--have good metabolic status even 24 hours after death. In corneas of patients with cancer or sepsis, the metabolism has run down. In comparison, corneas from patients with cancer are in better metabolic condition than those from donors with sepsis because they have adapted to catabolism. Corneas donated from patients with renal insufficiency show results somewhere in between. Statistical evaluation revealed significant differences in ATP concentrations for corneas from donors who died suddenly and from patients with renal insufficiency compared to corneas from patients with sepsis. It could be shown that glucose concentrations as a resource for metabolism increase with age. The best ATP-ADP ratios were found in the group of 40- to 59-year-old donors. Nevertheless, differences between the age groups were not statistically significant. CONCLUSION: From our results it can be concluded that the cause of death and systemic metabolism have an influence on corneal metabolism. Results concerning donor age reflect the well-known fact that donor age has no influence on the quality of keratoplasty material.

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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.

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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.

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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.

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