Sabine Salla

Explorative study of interleukin levels in the human cornea

Abstract• 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.

CHANGES IN HUMAN DONOR CORNEAS PRESERVED FOR LONGER THAN 4 WEEKS

Purpose Corneas are usually stored for a maximum of ∼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 less than equal to12 weeks under standard eye bank conditions [modified minimal essential medium (MEM), 31°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 less than equal to6 weeks. Conclusion From these data, we conclude that long-term organ culture in a closed system is limited to ∼6 weeks and thereby confirm the clinical results of Früh and Böhnke.

Remarks on the vitality of the human cornea after organ culture.

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.

Conjunctival tissue examination in severe eye burns: a study with scanning electron microscopy and energy-dispersive X-ray analysis

Abstract•Background: Longlasting 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.

Employment of bioluminescence for the quantification of adenosine phosphates in the human cornea

Abstract• 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.

Bitte um Einverständnis in eine Hornhautspende

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.ZusammenfassungHintergrundDurch den hohen Bedarf und der deutlich geringeren Verfügbarkeit von Hornhäuten des Auges besteht eine große Hoffnung auf das Gelingen ärztlicher Akquisegespräche mit Angehörigen zum Einverständnis in eine Gewebespende. Neben der Steigerung des Erfolgs in der Spendergewinnung sollen Akquisegespräche schützend für Angehörige verlaufen und die Arbeitsbelastung des Fachpersonals gering gehalten werden. Diese Arbeit erfasst den Bedarf und Erwartungen an ein Kommunikationstraining zur Vorbereitung auf die Akquisetätigkeit.MethodeIm September und Oktober 2009 wurde unter allen Akquiseärzten, die in der Hornhautbank arbeiten bzw. gearbeitet haben, eine Bedarfanalyse für ein Kommunikationstraining vorgenommen. Auf 10-stufigen numerischen Rangskalen wurde das Ausmaß der fachlichen, emotionalen und persönlichen Vorbereitung, das Ausmaß der Einschätzung eines Kommunikationstrainings als sinnvoll, notwendig und hilfreich sowie die durchschnittliche und höchste Belastung und Bereitschaft zur Ausübung der Akquise erfragt.ErgebnisseDie Studienteilnehmer fühlten sich fachlich, emotional und in ihrer persönlichen Überzeugung nur mittelmäßig auf die Tätigkeit vorbereitet und erachteten ein Kommunikationstraining für in hohem Maß sinnvoll, notwendig und hilfreich. Bei mittlerer durchschnittlicher Arbeitsbelastung mit kurzfristigen Hochbelastungsphasen bestand nur eine moderate Bereitschaft zur weiteren Ausführung dieser Tätigkeit.DiskussionDurch klare Hinweise auf eine nicht als ausreichend empfundene Vorbereitung auf die Akquisetätigkeit und als hoch empfundene Arbeitsbelastung wurde ein Kommunikationsseminar mit einem spezifischen Training der Akquisetelefonate entwickelt. Dieses Training entspricht bedarfsgemäß sowohl den Wünschen und Erwartungen des ärztlichen Personals als auch den am ehesten beeinflussbaren Variablen im Hinblick auf die Erfolgsquote bei der Spendenakquise.AbstractIntroductionBecause 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.MethodsIn 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.ResultsThe 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.DiscussionPhysicians 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.

A sensitive assay for the quantification of glucose and lactate in the human cornea using a modified bioluminescence technique

Abstract• 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.

Assessment of conjunctival epithelium after severe burns and surgical reconstruction with Tenon plasty by means of a modified impression cytology procedure.

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.

Gewebezustand humaner Hornhäute vor und nach Organkultur Einfluß der Todesursache des Spenders

Frühere Untersuchungen haben gezeigt, daß die humane Hornhaut den Stoffwechsel des Gesamtorganismus widerspiegelt. Bislang ist nicht geklärt, welchen Einfluß die Todesursache auf das Verhalten der Hornhaut in der Organkultur hat.Material und Methode: Es wurden 85 Organ-kultivierte humane Hornhäute untersucht. Die Endothelzellzahl sowie die biochemischen Parameter Glukose, Laktat, ATP und ADP wurden bestimmt. Die Sektionsprotokolle der Spender wurden ausgewertet und 5 Spendergruppen definiert: 1. plötzlich verstorbene Spender; 2. Spender nach Karzinom; 3. septische Spender; 4. niereninsuffiziente und 5. diabtetische Spender.Ergebnisse: In allen Spendergruppen nahm die Endothelzellzahl nach 28tägiger Lagerung deutlich ab. Die Entquellung für nur 1 Tag führte zu einem etwa gleich großen Zellverlust. Die biochemischen Parameter verbesserten sich in allen Gruppen. Es kam zu einer weitgehenden Angleichung, so daß Hornhäute von Spendern mit Sepsis oder Karzinom am stärksten von der Organkultur profitierten. Die Entquellung hatte hingegen einen durchweg negativen Effekt auf den Stoffwechsel.Schlußfolgerungen: Die Ergebnisse zeigen, daß es keine Erkrankung gibt, die primär die Lagerung einer Hornhaut in der Organkultur ausschließt; vielmehr kommt es zu einer Angleichung des Gewebes während der Kultivierung.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.

Instrument to Enhance Visualization of Descemet Membrane During Graft Preparation for DMEK Surgery.

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.