Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.

P188, P237, P338, and P407 performed similarly in preserving ECs, improving EC visualization, deswelling corneal stroma and inducing moderate injuries to corneal ultrastructure. They appear superior to dextran for corneal deswelling in OC.

Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (β-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas β-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions’ integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with β-gal ranged from 0.1 to 54% of the transfected ECs (mean ± SD: 7 ± 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.

Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

Purpose: A reliable viability test for corneal endothelium is necessary for the assessment of new processes likely to affect it (storage media, lamellar cutting methods...). Aim: to improve the classical calcein‐AM (C)/Ethidium homodimer (E) double staining (live/dead assay) by adding Hoechst and by using a 3D microscope and image analysis that allow a quantitative assessment on the whole endothelium. As an example of application, the assessment of the actual endothelial viability immediately before graft is given. Methods: The endothelial side was incubated 45 min at 31°C with 200 μL of C (2μM), E (5μM), H (10μM). After flat mount, images of the whole endothelial area (81mm²) were taken using a microscope equipped with a motorized stage (IX81, Olympus) and a x4 objective. Z stacks were taken and an extended focal imaging algorithm allowed taking account of corneal folds. Endothelial cell density (ECD) and mortality were determined by automatic counting of H+ and E+ nuclei. Area of C+ cells was measured. Results: This upgraded staining on the whole endothelium allowed the exhaustive assessment of ECD, cell mortality and metabolic activity. On corneas immediately before graft, it highlighted a dramatic lower number of viable cells than expected with the routine assessment of ECD only, done 48H before graft in the eye bank. Conclusions: The triple HEC staining takes account of the heterogeneous pattern of lesions within the whole endothelial layer and is therefore far less biased than assessment done on small cell samples. Its use improves the assessment of all new processes likely to affect the endothelium ex vivo.

Purpose In eye banks, corneal quality control requires an accurate endothelial cell density (ECD). ECs are counted after osmotic preparation (NaCl/sucrose) that makes cells visible. Nevertheless, endothelial images remain often poorly contrasted, non uniform, with a high background noise. Aim: to replace this technique by a non toxic ‘endothelial dye’ Methods 30 dyes were pre‐selected. Ability to stain EC was assessed on porcine, bovine and rabbit corneas. They were exposed (1 min) to pure or diluted dyes and observed under a light microscope using bright field and 4 standard fluorescent filters. Toxicity was assessed in vitro on EC cultures (live/dead assay, Hoechst Ethidium Calcein). Using the same assay, the less toxic dyes were further assessed on keratocytes cultures and on animal corneas organ cultured for 24H after exposure to dyes. Controls: exposure to BSS only or 3% H2O2 Results Only Ledermycin, Hemalun, Rose Bengal, Nuclear Red, Congo red, Evans Blue, and Chicago Sky Blue (CSB) stained ECs, and only the 3 diazoïc dyes (Congo red, Evans Blue, and CSB) stained EC borders using fluorescence (Em554/Ex568nm). Both blue dyes also stained the nuclei of dead cells. Hemalun and Rose Bengal were highly toxic. In vitro and ex vivo toxicity of CSB was negligible. The other dyes had an intermediate toxicity Conclusion Diazoïc dyes and especially CSB could be non toxic dyes for ECs borders. They could improve the accuracy of ECD determination. Their ability to stain human endothelium and their safety (using an animal graft model) have to be assessed as well as their contribution in ECD determination versus the classical osmotic endothelial preparation

Purpose: To develop an image analysis device devoted to the measurement of donor corneal transparency (T), folding (F) and clear corneal diameter excluding gerontoxon (G) during organ culture Methods: High res digital images of a retroilluminated test chart comprising parallel lines viewed through the cornea were analysed using a dedicated software. T (%) was a ratio of local contrast of the test chart and F (%) was a ratio of mean of line profiles, each measured with and without the cornea. Area of clear cornea outside gerontoxon was calculated from a circle delineated by the observer. On each image, 3 independent experts classified T, F and G according to 3‐level score (++/+/0) which served as reference. 179 human corneas (fresh, stored and deswelled) were consecutively analysed to include a broad spectrum of T and F Results: The device was able to discriminate between the 3 classes of T, F and G established by the experts. T was 20±4, 36±8 and 51±10% respectively for transparency deemed poor, average and excellent (P<0.01). F was 29±10, 22±6, and 17±6% respectively for folding deemed high, average and minimal (P<0.01). Diameter of clear central cornea was 7.7±1.1, 8.9±0.8, 10.4±0.6 mm respectively for gerontoxon deemed severe, moderate and absent (P<0.01) Conclusions: This simple and original device provides reliable quantitative objective measures of T, F and G. It will help standardizing quality assessment of corneas among eye banks. A trial designed to assess the ability of the device to select corneas before delivery in the setting of the routine practice of our cornea bank is ongoing. Grants :Etab Français des Greffes 2004 and PHRC/AOL 2007