Optimizing the deswelling process of organ-cultured corneas.

PURPOSE: During deswelling of organ-cultured human corneas, endothelial cell loss occurs. Therefore, it is necessary to minimize the deswelling time and achieving an optimal central corneal thickness (CCT) of approximately 550 microm at the same time. We investigated the minimal deswelling time necessary and analyzed endothelial cell loss. METHODS: Fifty-eight human corneas were stored between 13 and 81 days in organ culture. CCT was measured by optical coherence tomography. Measurements were performed before preparation, during culturing, before deswelling, and after varying deswelling periods (1-72 hours) using 5% dextran. Additionally, vital staining was performed in 6 human corneas to assess endothelial cell loss between 24 and 30 hours of deswelling. To evaluate absolute cell loss, endothelial cells were counted on human corneal pairs after 24 and 30 hours of deswelling. RESULTS: After organ culture, mean CCT was 1194 microm. After 24 hours of deswelling in dextran-containing medium, mean CCT was 600 microm, whereas after 30 hours, mean CCT was 510 microm and hardly any corneas showed a CCT of more than 550 microm. Almost no further decrease in CCT was observed thereafter. No factors could be identified predicting the necessary deswelling time; however, paired corneas showed significant correlation of deswelling characteristics. We did not see any differences in endothelial cell loss 24 and 30 hours of deswelling or the ratio of living to dead endothelial cell counts. CONCLUSIONS: Deswelling for 24 hours does not provide an optimal corneal thickness. Because endothelial cell loss does not increase between 24 and 30 hours of deswelling, a period of 30 hours is more suitable for obtaining sufficient corneal thickness.

The protective effect of quercetin against oxidative stress in the human RPE in vitro.

PURPOSE: To investigate the possible protective effect of the dietary antioxidant quercetin on retinal pigment epithelial (RPE) cell dysfunction and cellular senescence occurring in age-related macular degeneration (AMD). The major flavonoid quercetin was studied on RPE cells in vitro. METHODS: Cultured human RPE cells were incubated with different concentrations of quercetin for 24 hours. Cells were then treated with 150 to 300 microM hydrogen peroxide for 2 hours. Mitochondrial function was measured by using MTT assay and cell vitality by live-dead staining assay. Intracellular levels of glutathione were determined by using a glutathione assay kit. Apoptosis was quantified by a caspase-3 assay, and cellular senescence was quantified by beta-galactosidase staining. Expression of the senescence-associated transmembrane protein caveolin-1 was investigated by Northern and Western blot analyses. RESULTS: Hydrogen peroxide treatment caused significant decreases in mitochondrial function (52%) and in cell vitality (71%), whereas preincubation with 50 microM quercetin diminished this decrease in a dose-dependent manner. Quercetin treatment did not show any notable effect on intracellular levels of glutathione in either used concentration of quercetin. Hydrogen peroxide-induced activation of caspase-3 was reduced by 50 microM quercetin, from 1.9- to 1.4-fold, compared with untreated control (P < 0.001). Hydrogen peroxide caused a large (>90%) dose-dependent increase in beta-galactosidase-positive cells, whereas in the untreated control only single cells expressed this enzyme (<5%). This increase in cellular senescence was significantly attenuated by quercetin in a dose-dependent manner. The highest attenuation was reached at 50 microM quercetin. Quercetin caused a significant dose-dependent reduction of caveolin-1 mRNA 48 hours after treatment with hydrogen peroxide. After 96 hours of incubation, caveolin-1 protein levels were also reduced. CONCLUSIONS: The data demonstrate that quercetin is able to protect RPE cells from oxidative damage and cellular senescence in vitro in a dose-dependent manner. The authors suggest that this increase in antioxidative capacity is--among other mechanisms, such as the intracellular redox state--also mediated by inhibiting the upregulation of caveolin-1. Downregulation of caveolin-1 may be important for the retinal pigment epithelium to prevent apoptotic cell death in response to cellular stress, a condition implicated in the early pathogenesis of AMD. Therefore, the authors believe that the use of antioxidative dietary flavonoids such as quercetin is a promising approach in the prevention of early AMD.

Tele-screening for diabetic retinopathy with the retinal thickness analyzer.

OBJECTIVE: To compare the effectiveness of tele-screening using a novel enhanced retinal thickness analyzer (RTA) with onsite routine ophthalmologic examination for diabetic retinopathy. RESEARCH DESIGN AND METHODS: A consecutive series of 31 eyes from diabetic patients were included. All underwent ophthalmologic examination, including stereoscopic dilated funduscopy and scanning with the RTA. The RTA reports consisted of a wide-angle, red-free fundus photograph and a macular-region retinal thickness map. Reports were graded by three independent graders in a masked manner. The diagnoses of proliferative retinopathy, macular edema, and treatment decisions made by the RTA graders and the clinical examiner were compared. RESULTS: On clinical examination 5 of 31 eyes were diagnosed with proliferative diabetic retinopathy (PDR). All five were referred for treatment by two graders and four eyes by one grader. All eyes with PDR and 12 of the 26 eyes with nonproliferative diabetic retinopathy showed severe macular edema. Seven of the 12 eyes with macular edema were clinically eligible for focal laser treatment, and all of them were detected by all RTA graders. Macular thickening was detected in eight eyes by RTA where no treatment was necessary, as judged by clinical examination. Thus, sensitivity was 93% (mean) for detecting PDR and 100% for detecting macular edema, with a specificity of 58-96% depending on the grader. The RTA did not allow valid assessment due to poor image quality in only one case. CONCLUSIONS: Screening for diabetic retinopathy with a combination of wide-angle fundus photography and macular thickness mapping by an objective method, such as optical coherence tomography or the RTA, offers the prerequisites for establishing a successful tele-screening program.

Consenting to cornea donation: influencing factors.

BACKGROUND: The purpose of the study was to determine factors influencing the decision of the next relative being asked for consent to cornea donation in a prospective, non-comparative case series. METHODS: Interviews with the relatives of 264 potential cornea donors to the eye bank of the Ludwig-Maximilians-Universität, Munich, were documented following a standardized protocol. The influence of sociological factors on the frequency of obtained consents was investigated by uni- and multivariate analysis. RESULTS: During a 9-month period, 214 relatives of 264 possible donors were interviewed for consent to cornea donation. In total, 144 consents (67%) and 70 refusals (33%) were obtained; 50 relatives could not be reached. Analysis showed a higher consent rate for donors with a university degree (72%) versus those without (68%). Consent was more often obtained from relatives of donors that were divorced (71%) than from unmarried ones (63%). Husband and wife (71%) consented more frequently to cornea donation than children of age (62%) or parents (58%). Examination of residential area postal codes indicated more frequent refusals from donors of rural (56% consent rate) than urban Munich populations (67% consent rate). The situation was the opposite concerning the residential area of the consenting relative. Postal codes of both the donor and the relative were statistically significant factors in a multiple logistic regression model. There was relatively high willingness to donate in suicides (70%), but not significantly different from other causes like natural deaths (70%) and traffic accidents (66%). CONCLUSIONS: Some socioeconomic factors such as - in particular - the permanent address of the potential donors and their relatives reflecting a different background and status play an important role in the willingness to donate. A better understanding of this decision making can help the physician to ask more empathically for consent and inform the public more specifically about the donation process.

Sterile structural imaging of donor cornea by optical coherence tomography.

PURPOSE: The purpose of the study was to demonstrate a new noncontact method for sterile measurement of structure and thickness of donor cornea with use of optical coherence tomography (OCT). METHODS: A commercially available OCT instrument designed for retinal measurements was used for noncontact assessment of human corneas. Structural changes occurring during organ culture were evaluated in 29 corneas. Comparison with histology was performed, and the ability of OCT to detect corneal scars and corneal thickness was investigated. RESULTS: Corneal epithelium, stroma, and posterior curvature, as well as thickness, can be measured by standard OCT while the cornea remains in its storage bottle. Epithelial changes leading to a reduction of epithelial thickness, stromal structural changes, and hydration folds can be visualized. OCT scans correlate well with histology. Preexisting and developing corneal scars can be detected by OCT. CONCLUSIONS: Corneal structural imaging can be performed under sterile conditions by OCT. This provides a method for improvement of corneal storage and a screening method for signs of photorefractive surgery and scarring in donor cornea.