Katrin Engelmann

DIFFERENT CHARACTERISTICS OF ENDOTHELIAL CELLS FROM CENTRAL AND PERIPHERAL HUMAN CORNEA IN PRIMARY CULTURE AND AFTER SUBCULTURE

SummarySeveral methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.

Physiological features of primary cultures and subcultures of human retinal pigment epithelial cells before and after cryopreservation for cell transplantation

Abstractu2002· Background: One striking disadvantage of in vitro culturing of human retinal pigment epithelial (RPE) cells is the loss of epithelial differentiation and specific cell function during culture. This may be one of the main reasons for the failure of RPE cell transplantation. The aim of this study was to evaluate cell culture conditions ensuring the maintenance of differentiation and function of RPE cells after subcultivation and storage in liquid nitrogen.u2002· Methods: Enzymatically isolated cells were seeded onto coated culture dishes, cultured with a specially formulated improved growth medium until confluence and then cryopreserved in liquid nitrogen for 16–66 months. HLA class I and II typing was performed before cryopreservation and after thawing. Expression of Ca2+ channels in primary, first-passage and cryopreserved RPE cells was studied using the patch-clamp technique.u2002· Results: After cryopreservation no loss of any HLA antigen was detectable in 12 of 14 cell strains studied. Patch-clamp experiments demonstrated that high-threshold L-type Ca2+ channels, which are typical for freshly isolated cells, could be detected in first-passage and cryopreserved RPE cells only when improved culture conditions were employed, not in conventionally cultured cells. The characteristics of these channels showed little change in subcultured cells compared to primary cultures.u2002· Conclusion: This is the first study showing the maintenance of adult human RPE-specific cell differentiation and characteristics in vitro after primary culture and after cryopreservation using improved cell culture methods. The optimization and quality control of cell culture is an important prerequisite for successful cell transplantation.

Endotoxins modulate the autocrine function of organ cultured donor corneas and increase the incidence of endothelial cell death

BACKGROUND/AIMS Bacterial endotoxin is a potent inflammatory stimulator, the local and systemic responses thereby elicited being mediated via the release of cytokines from diverse cell types. Under physiological conditions, the corneal endothelium is protected from these toxins by the epithelial and vascular barriers, but in organ culture these safeguards are no longer operative, and such substances will therefore have ready access to this cell layer. The consequences of such exposure may take the form of overt damage to the endothelium and/or a more discreet influence on the cornea’s immunological status, the effects of which may be realised only after transplantation, by its poor performance. The media bathing organ cultured donor corneas were monitored for the presence of various cytokine mediators of the inflammatory response before and after incubation with endotoxin, and these data compared with those pertaining to endothelial cell morphology and numerical density. METHODS Six pairs of fellow donor corneas were cultured for an initial equilibration period of 10 days and then transferred to fresh medium; thereafter, one of each pair was incubated in the absence, and the other in the presence, of endotoxin (50 μg/ml ≡ 25u2009000 units/ml), and culturing continued for a further 10 days. Samples of medium were withdrawn at regular intervals throughout the 20 days and screened for the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, and TNF by ELISA; endothelial cell morphology and area density were assessed on days 0, 10, and 20. RESULTS Spiking of organ culture media with endotoxin led to a substantial increase in the level of IL-8, and a smaller one in that of IL-6, but none of the other cytokines were detected. In five of the six stimulated corneas, these changes coincided with an increased incidence of endothelial cell loss, compared with that incurred by the fellow control, and the surviving population also evinced signs of degeneration not seen in the latter. CONCLUSION Endotoxin induced increases in the levels of IL-6 and IL-8 appear to be correlated with endothelial cell loss. Since no adverse effects of this toxin on long term cultured monolayers of human corneal endothelial cells have been previously observed, the damage incurred in corneal organ culture may well be attributable to the influence of cytokines produced by other corneal cells or a non-intrinsic (passenger) cell population, such as macrophages, Langerhans cells, or lymphocytes present under these latter conditions.

Detection of neurone-specific enolase in long-term cultures of human corneal endothelium.

Abstractu2002· Background: Human corneal endothelial cells cultivated in monolayer culture for protracted periods undergo morphological dedifferentiation, whereby they assume a more fibroblast-like appearance. These cultures may also become overgrown with contaminating stromal fibroblasts and/or with keratocytes, when non-selective media are employed, thus rendering identification of actual endothelial cells difficult on a strictly morphological basis. · Methods: The endothelium of the human cornea stains for neurone-specific enolase (NSE) in situ, and we therefore wished to study the expression of this marker in primary and long-term monolayer cultures of these cells. Ten such cultures were established, six being stained for NSE at the primary and first-passage stage, the other four for 6, 8, 10 and 12 months. The NSE-staining pattern manifested in co-cultures of corneal endothelium and fibroblasts or keratocytes (first to fifth passage cultures) was also investigated, and co-cultures established from each of the latter two cell types served as controls. · Results: In monolayers of corneal endothelium which had retained their cobblestone-like morphology, NSE could be demonstrated even after more than 20 passages, which amounted to 1 year in culture. Dedifferentiated or degenerating endothelial cells stained poorly and inhomogeneously. Control cultures of fibroblasts or keratocytes were consistently NSE-negative, and when each of these cell types was co-cultured separately with corneal endothelium, only the latter expressed the marker protein.u2002· Conclusion: Since antibodies against NSE are commercially available, practical use may be made of this marker protein for confirming corneal endothelial status in long-term cultures.

Endophthalmitis nach Kataraktoperation Disponierende Faktoren, Erreger und Therapie

ZusammenfassungHintergrund: Die infektiös bedingte exogene Endophthalmitis stellt eine ernste Komplikation nach Kataraktoperation dar. Sie ist trotz moderner medikamentöser und chirurgischer Behandlungsmethoden oftmals schwer therapierbar.nPatienten und Methoden: In einer retrospektiven Studie wurden alle Patienten (n=36) untersucht, die zwischen Januar 1989 und Dezember 1997 an der Universitäts-Augenklinik Hamburg-Eppendorf (UKE) aufgrund einer Endophthalmitis nach Kataraktextraktion behandelt worden waren.nErgebnisse: Von 36 Patienten wurden der Klinik 29 (80,6%) von außerhalb zugewiesen. 14 Patienten (38,9%) hatten eine Endophthalmitis nach ambulanter Kataraktextraktion entwickelt. 7 Patienten (19,4%) waren stationär in der Augenklinik UKE operiert worden. Bei 29 der Patienten (80,6%) wurde eine Vitrektomie vorgenommen. Ein Erreger konnte aus 50% der Vorderkammer- bzw. Glaskörperproben isoliert werden, am häufigsten Koagulase-negative Staphylokokken (n=4). Zu den prädisponierenden Faktoren der Endophthalmitis zählten Diabetes mellitus (27,8%), perioperativer Glaskörperverlust (19,4%), systemische Kortikosteroidtherapie (13,9%) und Wunddehiszenz (11,1%). Von 27 nachuntersuchten Patienten (75%) erreichten 16 (59,3%) einen Visus von 0,05 oder besser (durchschnittlicher Visus 0,5). Bei 4 Patienten (13,8%) mußte eine Enukleation vorgenommen werden.nSchlussfolgerung: In der vorliegenden Studie konnte bei etwa 60% der Patienten mit einer postoperativen Endophthalmitis das Auge erhalten und eine optische Rehabilitation durch chirurgische Intervention erzielt werden.SummaryBackground: Infectious exogenous endophthalmitis is a serious complication after cataract surgery. Despite modern pharmacological and surgical methods, its treatment is still difficult.nPatients and methods: In a retrospective study the records of all patients treated for endophthalmitis following cataract extraction at the Department of Ophthalmology of the University Hospital in Hamburg between January 1989 and December 1997 were assessed.nResults: Of 36 patients treated for endophthalmitis, 29 (80.6%) had been referred. In 14 (38.9%) of these 29 patients endophthalmitis had occurred after outpatient cataract surgery. Seven patients (19.4%) had been treated as inpatients at the University Eye Hospital Eppendorf. Vitrectomy was performed in 80.6% of the cases. An infectious agent was isolated from 50% of diagnostic probes. The most common organism isolated were coagulase-negative staphylococci (4 cases). Predisposing factors for the development of endophthalmitis were diabetes (27.8%), intraoperative loss of vitreous (19.4%), application of systemic steroids (13.9%) and wound dehiscence (11.1%). Of 27 patients (75%) followed up, 16 (59.3%) had a final visual acuity of 20/400 or better (mean 20/40). An enucleation had to be performed in 4 patients (13.8%).nConclusion: In this study, approximately 60% of patients with endophthalmitis following cataract extraction had their globes preserved and a good visual outcome after appropriate surgical interventions.

Effect of perfluorodecalin on human retinal pigment epithelium and human corneal endothelium in vitro.

Abstractu2002· Background:Perfluorocarbon liquids are useful intraoperative tools in complicated vitreoretinal surgery. They are usually removed at the end of the procedure, but small amounts may remain in the eye. Recently, contradictory results have been reported on the damage in association with residual perfluorocarbon liquids in the eye. This study examined the effects of perfluorodecalin on human retinal pigment epithelium and corneal endothelium in vitro.u2002· Methods:Vitality and proliferative capacity of cell cultures were measured after incubation with perfluorodecalin. Vitality of cell cultures were measured using the Life-Dead assay. Cell proliferation was determined by measuring incorporation of 5-bromo-2’-deoxyuridine into cellular DNA. Furthermore, endothelium of organ-cultured human corneas was examined after incubation with perfluorodecalin by photodocumentation.u2002· Results:Both cell types showed less extinctions in the Life-Dead assay after incubation with perfluorodecalin. After removing perfluorodecalin from the cultures, cells showed the same capacity of proliferation as the control cells. Compared to control corneas, perfluorodecalin induced a decrease in endothelial cell density. In four corneas, endothelial cell necrosis was observed.u2002· Conclusion:Decreasing extinctions in the Life-Dead assay after incubation with perfluorodecalin can be interpreted as showing a decreasing amount of vital cells. Because cell proliferation showed no significant changes the results suggest that perfluorodecalin may not be directly toxic to cells in vitro. It may exert an indirect or mechanical effect on cell function by impeding the normal metabolic exchange between endothelium and medium. Based on these results perfluorodecalin should be completely removed after operation.

A sensitive method for testing the quality of organ culture media and of individual medium components in a cornea bank

Abstractu2002· Background: It has been suggested that variations in the quality of organ culture preservation media are responsible for variations in early postoperative graft morphology. Spates of such variations have been observed repeatedly for short periods. This paper reports the results of a series of grafts with low postoperative clearing observed during a period of 6 weeks. Simultaneously, preoperative phase-contrast microscopy evaluation of the corneal endothelium revealed that an unusually large proportion of donor corneae were unsuitable for transplantation. · Methods: The corneal storage media were therefore rigorously screened, paying particular attention to specific components and properties of the medium, including L-glutamine, amphotericin B, water quality, pH, and the glassware used. Possible toxic effects were identified by means of a sensitive growth assay performed using isolated human corneal endothelial cells.u2002· Results: The evaluation demonstrated that both the water quality and the L-glutamine which had been used for preparation of the medium were substandard during the period in which poor clinical results were obtained.u2002· Conclusion: It is recommended that cornea banks undertaking long-term organ culture use standardized protocols and carefully monitored equipment. The quality of the basal media and supplements should be routinely checked.

Endothelial cell death in organ-cultured donor corneae : the influence of traumatic versus nontraumatic cause of death

Abstract• Background: Donors who have suffered a traumatic death are, on average, younger than those who have died from other causes, and the time from death to enucleation (DET) also tends to be shorter. Corneae obtained from such donors are therefore considered particularly suitable for grafting. One of the reasons for excluding a donor cornea from transplantation is the occurrence of endothelial cell necrosis during organ culture, and we investigated whether the incidence of this phenomenon bears a relationship to death by traumatic or nontraumatic means. • Methods: Data from 2125 donor corneae were collected using standardized evaluation protocols between January 1991 and December 1995 and included information on cause of death, age and DET, as well as endothelial cell loss and necrosis. Traumatic deaths were recorded in 346 cases, nontraumatic deaths in the other 1779 cases. Since differences in age (P = 0.006) but not in DET occurred within each of these groups, a more refined comparison, with matched data (< 35 years), was also undertaken. • Results: Forty (11.6%) of the 346 corneae derived from traumatic death donors manifested total or partial endothelial cell death in organ culture. The corresponding figure in the nontraumatic death group was only 105/1779 (5.8%;P = 0.0002). After matching for age, endothelial cell death during culturing was revealed in 18 (13.5%) of the 133 of the traumatic death corneae and in 3 (2.6%) of the 115 nontraumatic death ones (P = 0.004); the overall incidence of endothelial cell death (total or partial) during organ culture was 6.8% (145/2125). Endothelial cell loss during culturing of the 227 age-matched donor corneae which still had an intact endothelial monolayer at the end of the incubation period was 340 ± 388 cells/mm2 in traumatic death corneae (n = 115) and 255 ± 318 cells/mm2 in nontraumatic death ones (n = 112;P = 0.051). • Conclusion: Corneae obtained from traumatic death donors were more liable to undergo total or partial endothelial cell death during organ culture than were those procured from nontraumatic death ones. However, in corneae which survived the period of culture, there was no significant difference in endothelial cell loss between the two groups. Whilst the mechanism underlying this increased susceptibility of traumatic death corneae to cell death remains elusive, the data gleaned from this investigation nonetheless emphasize the potential importance of being able to perform meaningful in vitro viability tests on donor corneae; this is possible only under organ culture conditions.

Combined immunosuppressive and surgical therapy of necrotic keratitis in rheumatoid arthritis

SummaryIntroduction: Areactive forms of keratitis in patients with seropositive rheumatoid arthritis are inflammations threatening the visual acuity and integrity of the eye. They commonly occur in a rheumatologically inactive interval and have a poor prognosis. A retrospective evaluation of medicamentous and surgical strategies for a curative therapy with optical rehabilitation is necessary to optimise the treatment of patients with necrotic sclerokeratitis.nPatients and methods: A total of 27 eyes of 22 patients (14 women and 8 men, ranging in age at the time of operation from 40 to 88 years; mean 68.7 years) with seropositive rheumatoid arthritis and secondary Sjögrens syndrome were reviewed retrospectively. There were 17 eyes with necrotic keratitis and 9 eyes with necrotic sclerokeratitis. In one eye, necrotic sclerokeratitis with bacterial transmigrating keratitis and hypopyon occurred. Operations: In 8 cases we performed a perforating mini-keratoplasty, in 16 cases a tectonic and optical perforating keratoplasty, in 3 cases a tectonic sclerokeratoplasty, in 9 patients a combined keratoplasty and cataract extraction with posterior chamber lens implantation and in 1 case a partial conjunctival plasty. Follow-up ranged from 7 months to 4 years (average 2.8 years).nResults: In all eyes, a sufficient tectonic and primary curative effect was achieved only under cyclophosphamide immunosuppression. In 3 cases, a rekeratoplasty had to be performed because of recurrent keratitis after changing the systemic cyclophosphamide therapy to methotrexate, glucocorticosteroids or non-steroid antiphlogistic agents. Visual acuity outcome was depending on the eccentricity of the keratoplasty and earlier affections of the eye. Postoperatively, the visual acuity improved in 23 eyes. In 3 cases, no change of visual acuity was achieved. Visual acuity deteriorated in one case from counting fingers to hand motions. Peri- and postoperative complications during the follow-up period were corneal infiltration around sutures in 4 eyes, graft rejecting reactions in 3 cases, and sicca syndrome in 6 cases.nConclusions: The intensive cooperation of ophthalmologists and rheumatologists enables the successful treatment of apparently hopeless situations in necrotic sclerokeratitis in patients with seropositive rheumatoid arthritis. The rate of complications under an immunosuppressive therapy with cyclophosphamide was found at average 2.8 years follow-up to be low. The indication for the combined therapy depends on the ophthalmological findings; rheumatologists and ophthalmologists should decide on the appropriate dosage for the systemic cyclophosphamide therapy. Topical glucocorticosteroid therapy alone is contra-indicated.ZusammenfassungHintergrund: Areaktive Keratitiden bei chronischer Polyarthritis (cP) sind den Visus und die Integrität des Auges bedrohende Entzündungen, die meist in einem rheumatologisch inaktiven Intervall auftreten und eine schlechte Prognose haben. Die retrospektive Auswertung medikamentöser und chirurgischer Strategien zur kurativen Therapie mit optischer Rehabilitation ist für die Optimierung der Behandlung dieser Patienten wichtig.nPatienten und Methoden: Untersucht wurden 22 Patienten mit 27 erkrankten Augen bei bekannter seropositiver chronischer Polyarthritis und sekundärem Sjögren-Syndrom. 14 Patienten waren weiblich, 8 männlich. Das Patientenalter betrug zum Zeitpunkt der Operation 40–88 Jahre (Mittelwert: 68,7 Jahre). 17 Augen hatten eine nekrotisierende Keratitis, 9 Augen eine nekrotisierende Sklerokeratitis und ein Auge eine Sklerokeratitis mit bakterieller Durchwanderungskeratitis und Hypopyon. Es wurden eine tektonische und optische perforierende Keratoplastik in 16 Fällen, eine tektonische Mini-Keratoplastik in 8 Fällen und eine tektonische Sklerokeratoplastik in 3 Fällen durchgeführt. Kombinierte Eingriffe mit simultaner Hinterkammerlinsenimplantation erfolgten davon 9 mal, mit partieller Bindehautdeckung einmal. Der Nachbeobachtungszeitraum lag zwischen 7 Monaten und 4 Jahren, im Durchschnitt bei 2,8 Jahren.nErgebnisse: Ein tektonischer und primär kurativer Effekt wurde bei allen Augen nur unter Immunsuppression mit Cyclophosphamid erzielt, in 3 Fällen mußte nach Umstellung der systemischen Therapie von Cyclophosphamid auf Methotrexat, Glukokortikoide oder nichtsteroidale Antiphlogistika bei rezidivierender Keratitis eine Re-Keratoplastik durchgeführt werden. Der Visusverlauf war abhängig von der Exzentrizität der Keratoplastik und von Vorerkrankungen. In 23 Fällen stieg der Visus postoperativ an. In 3 Fällen blieb der Visus unverändert, in einem Fall fiel er postoperativ von Fingerzählen auf Handbewegungen ab. An peri- und postoperativen Komplikationen traten im Nachbeobachtungszeitraum 4× Fadeninfiltrate, 3× Transplantatreaktionen und 6× eine Sicca-Problematik auf.nSchlußfolgerungen: Durch eine intensive interdisziplinäre Kooperation von Ophthalmologen und Rheumatologen können aussichtslos erscheinende Situationen am Auge beherrschbar werden. Die Häufigkeit von Komplikationen unter Immunsuppression mit Cyclophosphamid ist zumindest mittelfristig im Nachbeobachtungszeitraum von durchschnittlich 2,8 Jahren als gering einzuschätzen. Die Indikation zur systemischen Therapie hängt vom augenärztlichen Befund ab, die Dosierung der systemischen Immunsuppression erfolgt durch Rheumatologen und Ophthalmologen. Eine alleinige topische Glukokortikoidtherapie ist kontraindiziert.

Pathogenese der choroidalen Neovaskularisation

ZusammenfassungDie altersbedingte Makuladegeneration (AMD) ist im Alter die häufigste Erblindungsursache in den westlichen Industrieländern. Die Pathogenese ist bislang nicht hinreichend geklärt, und zufrieden stellende Therapien sind nicht vorhanden.Die Dysfunktion des retinalen Pigmentepithels (RPE) scheint eine zentrale Rolle zu spielen und die Entwicklung in Richtung einer choroidalen Atrophie oder einer Proliferation im Sinne einer choroidalen Neovaskularisationsmembran (CNV) zu determinieren. Die vorliegende Übersichtsarbeit behandelt die derzeit diskutierten Konzepte zur Entstehung choroidaler Atrophie und Neovaskularisationen. Darüber hinaus werden die gängigen Tiermodelle sowie mögliche therapeutische Ansatzpunkte erläutert.AbstractAge-related macular degeneration (AMD) is the leading cause of blindness in the developed world but the pathogenesis remains poorly understood.Malfunction of the retinal pigment epithelium (RPE) plays a central role in the disease and leads to either choriodal arophy or proliferation.This article reviews the current concepts of the development of choriodal atrophy and neovascularisation. Furthermore, available animal models and potential therapeutical targets are discussed.