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PURPOSE The posterior cornea is rotationally asymmetric, and Descemet membrane endothelial keratoplasty (DMEK) grafts preferentially scroll vertically. This prospective study assessed whether graft attachment after DMEK differed depending on the rotational alignment of the donor graft in the recipient eye. METHODS Pseudo-randomization and blinding of the graft orientation in the recipient's eye were possible by procedural separation: (1) The eye bank recorded the position of an orientation marker in the donor cornea; (2) the surgeon preparing the DMEK graft recorded an upside-down marker relative to the eye bank marker; and (3) the surgeon assessed the position of the upside-down marker in the recipient after DMEK. Surgeons were masked towards the eye bank marker. Using mixed-effects models, we assessed graft attachment relative to the rotational alignment of the donor graft. RESULTS Postoperatively, the graft was not fully attached in 59 of 179 eyes (33%). A second air fill (rebubbling) was performed in 11%. The graft axis was in line with the recipient cornea axis in 40%, oblique in 28% and orthogonal in 32%. We did not detect an elevated risk of incomplete attachment (odds ratio [OR], 1.16; 95% CI, 0.61-2.20), risk of rebubbling (OR, 1.25; 95% CI, 0.47-3.31) or larger areas of graft detachment in non-aligned grafts compared to aligned grafts. CONCLUSION Rotational alignment was not strongly associated with the risk of incomplete graft attachment, although modestly elevated risks cannot be ruled out. Efforts are needed to reduce the need for rebubbling after DMEK and to identify modifiable risk factors for graft detachment.

Purpose To study the morphology of human corneal nerves in eye bank organ-cultured corneas and in corneal grafts post-transplantation. Methods Thirty-seven organ-cultured corneas were divided into: Group-A, anterior 300-400 μm of 20 corneas used for Descemets stripping endothelial keratoplasty, and Group-B, 17 full-thickness corneas unsuitable for transplantation. Corneas whole mounts were stained for nerves using acetylcholinesterase technique and examined by NanoZoomer digital pathology microscope. Central and sub-Bowman's stromal nerves and the sub-basal nerve plexus including perforation sites and terminal bulbs were studied. Ten eyes were imaged following penetrating keratoplasty using in-vivo confocal microscopy (IVCM) for the presence of sub-basal and stromal nerves at 1, 4-5 and 7-8 weeks postoperatively (five eyes) and in all the other five eyes, the final follow-up was at 12 weeks. Results Fifteen of twenty (75%) corneas had stromal nerves in Group-A and 15 of 17 (88.2%) in Group-B. Average number of stromal nerves entering peripherally were 9.1 (range: 1-36). 7.5 in Group-A and 10.8 in Group-B. Central stromal nerves were seen in eight samples in Group-A and nine in Group-B. Many stromal nerves terminated abruptly without demonstrable continuity through Bowman's membrane. No terminal bulbs or sub-basal nerves were detected. In-vivo confocal microscopy (IVCM) showed 4 of 5 in 9 of 10 (90%) donor corneas had stromal nerves 1 week postoperatively, which remained present in 8 of 10 (80%) corneas at 4-5 weeks and in 9 of 10 (90%) at 7-8 weeks postoperatively. All 5 corneas analysed at 12 weeks showed the same stromal nerves from 1 to 12 weeks postoperatively. Sub-basal nerves were absent in all corneas over the 12-week study period. Conclusion This study provides further insight into the behaviour of corneal nerves in transplanted corneas. Corneal stromal nerves/nerve-sheaths are preserved in organ-cultured eye bank eyes and persist post-transplantation up to 3 months. These could provide directional guidance to regenerating nerves from host stroma.

Demographische und historische Analyse der Hamburger Hornhautbank uber 30 Jahre Purpose: To evaluate donor demographics, trends in donor tissue procurement and tissue storage over a long period. Methods: A retrospective, longitudinal, descriptive analysis was undertaken of data from the Hamburg Eye Bank Data Base (HEB-DB) that had been collected between 1981 and 2010. Data on 54 parameters of cornea donors [including clinical history, age, death cause, gender and death-to-explantation interval (DEI)]and of cultivated corneas (endothelial quality and development in culture, cultivation period, microbiological contamination) were retrieved. These data were analysed statistically, focusing on the historical development of the eye bank. Results: At the time of retrieval (June 2010), the HEB-DB contained data on 10 943 corneas (5503 donors). Most donors were men (65%) and had died from cardiopulmonary (n = 801) ⁄ cerebral (n = 261) failure or as the result of a polytraumatic accident ⁄ suicide (n = 602). Within these years, donor age,DEI and storage time increased. The percentage of stored corneas suitable forntransplantation displayed a variable but increasing trend; in 2007, almost 75% of the stored corneas were transplanted. Between 1995 and June 2010, the median microbiological contamination rate was 5.3%. A change in the procurement procedure from enucleation to corneoscleral explantation in 2008 led to a briefly increased contamination rate. Conclusion: Donor demographic data run parallel to the general demographic development. Our analysis indicates a dynamic development of the eye bank over the last 30 years and emphasizes the need for an active quality management in coping with the challenges of modern eye banking.

Purpose: To describe a standardized ‘no-touch’ harvesting technique of anterior and Descemet membrane (DM) grafts for use in deep anterior lamellar keratoplasty (DALK) and Descemet membrane endothelial keratoplasty (DMEK), which provides undamaged anterior and posterior corneal grafts. Methods: A retrospective evaluation was performed of our standard method for harvesting DM grafts and DALK grafts (Technique I; n = 31) versus a newly designed ‘no-touch’ technique (Technique II; n = 31), in which a peripheral ring of trabecular meshwork tissue is left in-situ, and the DM graft is trephined on an underlying soft contact lens. Endothelial cell density (ECD) before and immediately after DM stripping was used as the main outcome parameter. Results: Endothelial cell density did not differ within Techniques I and II (before versus after DM stripping) (p = 0.75 and p = 0.54, respectively) or among Techniques I and II (p = 0.61). With the latter technique, anterior corneal grafts and posterior DM grafts could be harvested with negligible damage to the endothelial cell layer or the posterior stromal bed. All 93 grafts (62 DM grafts) were eligible for transplantation, and six months post-operatively all transplants used were functional. Conclusion: The new technique offers the following advantages: (i) production of ‘undamaged’ grafts for DALK and DMEK, (ii) better controlled tissue handling of the thin DM graft during DM stripping and (iii) an increase in availability of corneal grafts obtained from the same donor tissue pool.

Central corneal cell density was evaluated from photographs obtained by a clinical non-contact specular microscope (Zeiss) in 48 patients at a mean post-operative time of 7 1/2 months after penetrating keratoplasty for a number of different causes, keratoconus being the most frequent indication. Cell density decrease on this occasion was compared to pre-operative data from the Eye Bank in Arhus, Denmark, which supplied corneo-scleral preparations transported by ferry and train to Linköping, Sweden. A mean decrease in cell density of 4.4% was recorded during bank time (11-27 days). A further mean decrease from final preparation to post-operative photography of 36.5% was found. Mean total cell density decrease amounted to 39.4%, which compares well with results found in other studies. Among several factors studied, the only one associated with significantly (P less than 0.02) greater cell density decrease than other factors was long post-mortal time periods (death to bank preparation). Mean final endothelial cell density was 1899 cells/mm2, which should correspond to a good functional reserve of donor endothelium. Neither the long bank time, nor the transportation seemed to be of major importance with respect to donor material quality. Foreign material supply can be a good solution to local corneal shortage.

Endothelial pump activity of rabbit corneas kept under eye bank conditions was examined. Different swelling rates for medium with and without ouabain were used for the calculation of endothelial pump rate. After an initial weight increase during 24 h, the control corneas kept approximately constant weight for 1 week. The ouabain-treated corneas increased continually, and during the time interval 24-48 h the increase was 41% of start weight. This corresponds to a pump activity of 2.3% of the weight of the normal corneal stroma per h, or to 9 microns of the corneal thickness per h. Each endothelial cell pumped fluid corresponding to 200-300% of the cell volume per h between day 1 (24 h) and day 8. The pump activity was gradually reduced after 8 days. The average weight was the same in both groups after 7 weeks.

The influence of endothelial cell pumping and of cell barrier functions on cultivated corneas has been studied by comparing the rate of weight increase in freeze-treated, ouabain-treated and fresh corneas. Inhibition of the endothelial cell pump activity increased the swelling rate from 0.5% of start weight per h (fresh corneas) to about 2% per h (ouabain-treated corneas), as measured after 24 h in culture medium. The cell barrier effect was estimated by comparing the swelling rate of ouabain-treated corneas after 24 h, and the swelling rate of freeze-treated corneas with the same swelling pressure. The values are 2 and 9.5% per h, respectively. The swelling rate of freeze-treated corneas with different button diameters was found to increase with decreasing corneal button diameter.

Purpose The purpose of this study was to identify ultrastructural changes associated with ectasia and to determine the association between lamellar count and corneal thinning. Methods Five surgically removed keratoconic corneal buttons and four, non-keratoconic, normal eye bank control corneas were processed for transmission electron microscopy using an established protocol, ensuring minimal tissue distortion. A sequence of overlapping digital images, spanning the full apical cone corneal thickness, was assembled. A seamless digital montage was printed at 5000× magnification. Lamellae were counted in the anterior–posterior orientation, along a linear line, using established criteria for identification of individual lamellae. Results The stromal thickness estimated as a 95% confidence interval for the mean, CI (0.95), in the keratoconic corneas was 372 ± 62 μm, while in the normal cornea, it was 446 ± 89 μm. All keratoconic corneas showed ultrastructural evidence of lamellar splitting and a loss of interweaving anterior lamellae. In the keratoconic corneas, the median total linear stromal lamellar absolute count tangential to the corneal surface was 362, (25th percentile; 75th percentile) = (355; 365) lamellae and in the normal cornea, 246, (25th percentile; 75th percentile) = (239; 251). The linear lamellar density in the keratoconic corneas was estimated as CI (0.95) 117 ± 22 and 86 ±19 lamellae per 100 μm in the anterior and posterior portion of the stroma, respectively. In normal cornea, the linear lamellar density was estimated as CI (0.95) 51 ± 8 and 80 ± 20 lamellae per 100 μm. The mean difference of linear lamellar count between the anterior and the posterior portion of the cornea was estimated as CI (0.95) 31 ± 23 for keratoconic corneas and −29 ± 28 for the normal corneas. Conclusions The current morphometric analysis of ultrastructural changes suggests that ectasia and thinning in keratoconus is associated with lamellar splitting into multiple bundles of collagen fibrils and loss of anterior lamellae. These structural changes, possibly in addition to lateral shifting of lamellae due to the pressure gradient over the cornea, are a potential explanation to the central loss of mass.

Purpose it is universally admitted that a very early important postoperative decrease in ECD occurs after all types of corneal graft, generally attributed to surgical traumatism. This presumed cell loss is calculated between eye bank ECD (ebECD) and post op cell count done in recipients by specular microscopy (SM). Aim: to redefine the very early postop cell loss. Methods 1/Prospective series of consecutive penetrating keratoplasty for all-comer indications with post-op follow-up by SM (SP3000-P) performed as soon as possible and at least at D15, M1 and M3. This series provided reasonable quantification of the gap between ebECD and very early postop ECD in patients. 2/Ten pairs of corneas with comparable left/right ebECD were randomized: one cornea was grafted, the other was stored similarly and its viable ECD was measured after triple labelling with Hoechst/Ethidium/calcein-AM (IOVS2011 Pipparelli) and quantification with CorneaJ (Cornea2014 Bernard). Graft and vECD measurement were done simultaneously. Relationship between vECD and early postop ECD were studied. Results For series 1, a gap of 623 (98-1479) cells/mm2 (median (range)) between ebECD and early postop was measured. For series 2, vECD and early postop ECD of paired corneas were well correlated (r=0.7, P

Corneal tissue shortage has become a major concern worldwide, which has motivated the search for alternative solutions to eye bank human eyes for corneal transplantation. Biocompatibility and functionality of stromal lamellar grafts tissue-engineered in vitro from corneal stromal cells using self-assembly approach, without the addition of exogenous material or scaffold, and transplanted in vivo in the cornea of an experimental model open the way to a new therapeutic modality for the rehabilitation of diseased corneas.

Purpose In France, the cutting of endothelial graft for DSAEK is almost exclusively performed by surgeons directly in the operating room whereas in the US more than 2/3 are now precut by eye banks (EB) technicians using 4°C storage. Among the French Blood Center EB, the EB of St-Etienne received the first agreement from the French National Agency for Medicines and Health products for precutting and distribution of corneas. Aim: to report our first experience with DSAEK precut by technicians using organ-cultured (OC) corneas Methods OC corneas with an initial endothelial cell density (ECD) >2200 cells/mm2 (by image analysis) were precut by two trained technicians using a Moria microkeratome. Corneas were stored in CMax (Eurobio) and deswelled in CJet (5% Dextran T500) 20h prior to pre-cutting. Central corneal thickness (CCT) was measure by ultrasound before and after cut with a single pass of the 350μm head. Microbiological testing was done with blood culture bottles 24h after precutting Results Twelve corneas with initial ECD of 2724±269 cells/mm2 (mean±SD), 19±4 days of OC and CCT of 548±50 μm were cut and sent to 4 centers (Feb to May 2013). Final CCT was 194±33μm (166 to 277). All surgeries were uneventful but rebubbling was necessary in 6 cases and 1 graft with eccentric trephination by the surgeon did never adhere Conclusion Precutting of OC corneas by EB allows quality and microbiology controls. Three improvements are ongoing: OCT thickness map before, during and after cut, use of the microkeratome double-pass technique, and ink marking of the centre to guide final trephination by surgeons. Grants: ONDAINE, AOL CHU 2012

Development of new surgical techniques for corneal transplantation induces major modifications in the graft selection process in eye banks. Quality of the donor corneal tissue must currently be assessed according to the scheduled surgical procedure. Not only the endothelial layer should be assessed by conventional techniques (trypan blue staining, light microscopy, specular microscopy) but also the corneal stroma and epithelium. This latter task requires development of new techniques such as high definition optical coherence tomography. Full field-OCT, in providing a detailed study of corneal structures with ultrahigh resolution (1 µm in all directions), permits detecting corneal conditions that are currently difficult to identify in eye banks, such as keratoconus, absence of Bowman’s layer, or corneal scars. It permits evaluating precisely Descemet’s membrane condition. It seems to be a promising technique in complement to current assessment methods of human donor corneas. Proper evaluation of the donor tissue by the new technologies implies formation of the eye bank staff. The staff has to get close relationships with the surgeons and to be attentive to the special requirements related to the scheduled surgical technique.

Purpose Quantification of dioptric power (DP) and transparency (T) of stored corneas remain almost unmet in daily eye bank practice. Complementary to T, DP could help screening donors having had refractive surgery and selecting the best donor/recipient refractive matching. During the development of an innovative corneal bioreactor (BR) (patented) for preclinical research and long-term storage, we took into account the possibility to integrate devices to perform these new quality controls. Aim: to present a device, named “frontofoco-graft-meter” (FGM) able to quantify DP and T of corneas in BR Methods measure of DP was based on the principle of magnification measurement and for T on the transmitted light over incoming light ratio. The FGM comprised a light source + specific chart made of parallel black lines + digital camera. Human corneas in the BR were pressurised at 15mmHg, with medium circulation in endothelial chamber, whereas epithelial chamber was filled with air during measurements. DP was compared to the power map provided by OCT (Tomey, Casia), and T measures to results provided by our previous device called ‘transparometer’ (based on the analysis of modulation transfer function). Measurements were done by 3 operators in a masked fashion Results For DP, agreement with OCT was good (within ±1 Dioptry). For T, measures were comparable with both devices. Inter-rater agreement was good for DP and T. FGM took a limited place and could easily be integrate in eye banks Conclusion the new FGM provides objective and reproducible QC of DP and T of corneas placed in the BR. Grants: ABM 2012, ANSM 2012, EFS 2012

Purpose The rate of DSAEK failure decreases during learning curve. It has been previously reported that one of the main cause of rapid DSAEK failure was endothelial cell (EC) loss (Zhang, Arch Ophthalmol 2010). Aim: to report a DSAEK immediate failure and its innovative assessment with determination of the viability of residual EC Methods In the eye bank, a cornea (donor: 44 years old, organ-culture: 23 days, ECD the day before cut: 2963 cells/mm2, deswelling 21h) was precut with a single pass of a 350μm head (Moria microkeratome). Lamella thickness was 199μm (ultrasound). The recipient had a Fuchs dystrophy. In the operating room, trephination with a Baron punch was eccentric but insertion was uneventful. The graft failed despite 2 rebubbling. A penetrating keratoplasty was performed 2 months later. During surgery the explanted lamella was immediately incubated with Hoechst 33342-Ethidium-Calcein-AM (HEC triple staining, Pipparelli, IOVS 2011) to determine the viability of residual EC. The thickness map of the lamella was then performed with an OCT (Casia, Tomey). The posterior surface of the recipient corneal button was observed with a macroscope Results The eccentric trephination caused a important step on 90° of circumference. Unplanned retention of Descemet membrane with guttae was found on the posterior face of the recipient cornea. The viable ECD of the explanted lamella was 585 cells/mm2. Keratocytes of both the lamella and the recipient cornea were viable Conclusion Allogenic EC and keratocytes survive on a detached lamella at least during 2 months in the anterior chamber of the recipient. Grant: AOL 2012

Purpose Spatial structure of collagen lamellae of the human cornea is thought to be an important determinant of corneal rigidity. Analysis of the low-frequency (1–107 Hz) dielectric spectra of the normal and keratoconus human cornea in the temperature range of 140-300 K and for different hydration states were examined. Methods For the measurements, the 100 – 200 μm specimens containing the middle layers of corneal stroma were used. The 9 specimens of healthy, control corneas from Eye Bank were obtained with second microkeratom section during Ultra Thin-DSAEK. The 12 specimens of keratoconus corneas were manually prepared from corneas removed during penetrating keratoplasty. Results Experimental results were interpreted in terms of ionic diffusion and space charge polarization according to the Sawada’s theory. The new presentation of the dielectric spectra, i.e.: (delta eps’/delta ln f)*f, was used. The presented method and the Sawada’s expression were applied to the analysis of the changes in spatial molecular structure of collagen fibril network in human cornea. Conclusion Fitting procedure of the theoretical function to the experimental data allowed us to determine two diffusive relaxation regions with two structural distance parameters ds, describing spatial arrangement of collagen fibrils in cornea.

Purpose To assess the viable endothelial cell density (vECD) of posterior lamellar grafts, stored in organ culture, precut by eye bank technicians, and sent to a distant center. Methods Paired corneas with an ECD >2200 cells/mm2 at the standard assessment with the SambaCornea analyser (TribVn, France), were stored in CorneaMax (Eurobio, France) and deswelled in CorneaJet (5% Dextran T500) for 6 to 24 hours prior to pre-cutting. They were randomly assigned as full-thickness grafts or as precut posterior transplants for DSAEK using a microkeratome with a 350μm head (Moria, France). Corneas were sent to a distant center where the vECD was assessed by using the triple Hoechst/Ethidium/Calcein (HEC) staining and fluorescent image analysis of the entire posterior surface: calculation of the area covered by living cells (H+/C+/E-) coupled with counting of nuclei in large areas allowed to determine the vECD, i.e. the useful cell pool (Pipparelli. IOVS 2011.52:6018). DSAEK and controls data were compared with non-parametric tests. Results In both groups, the vECD was lower than the standard ECD determined by the eye bank (for 20±7% for full thickness grafts, 34±16% for DSAEK), because living cells never completely covered the entire posterior surface. vECD were respectively of 2006+/-349 vs 1661+/-464 (P=0.075). Conclusion The HEC triple staining combined with image analysis provides a unique accurate assessment of the endothelial quality by giving the vECD. This laboratory technique allows a reliable assessment of the endothelial quality of tissues supplied to surgeons, precut or not. It can also be used to assess any methods liable to modify EC.

Purpose Pneumodissection or 'big-bubble' technique (BBT) is used to separate Descemet’s membrane (DM) and stroma in both deep anterior lamellar keratoplasty and Descemet’s membrane endothelial keratoplasty. It has been reported that BBT may produce two typer of bubble outlines: clear or white margin. It seems that the components of the posterior lamella obtained with each bubble rims also differ. Our aim is to compare BBT dissection plane using histological and electron microscopy (EM) analysis on eye-bank corneas. Methods BBT was carried out on donor corneas unsuitable for transplantation for reasons other than corneal disease.Corneoscleral discs were mounted on an artificial anterior chamber and BBT was carried out using a 27G needle. 5 samples which had a bubble with white margin and 5 with clear margin were fixed in formalin and sent for histology followed by EM. Results Samples in which the edge of the bubble had a white margin showed variable residual stroma adherent to DM. When the edge of the bubble was clear no residual stroma was seen attached to a bare, smooth DM. The anterior lamellae of the cases without residual stroma were also analysed. These showed no evidence of DM elements excluding the unlikely possibility of intra-Descemet’s dissection plane. The findings were confirmed by EM in all cases. These showed intact banded DM in all posterior lamellae but only those with a white bubble outline had attached residual stromal collagen. Conclusion In eye bank corneas the histological elements of the posterior lamella dissected using BBT can be predicted depending on the margin of the bubble obtained. Assumptions that pneumodissection in BBT routinely bares DM need to be revisited.

Purpose Reasons for endothelial cell density (ECD) decrease after corneal grafts remain unclear. We recently demonstrated that eye bank ECD (ebECD) overestimates the pool of viable EC really grafted to patients (Pipparelli.IOVS2011;52:6018) because standard ebECD cannot take account of acellular areas or area covered by dying EC. Autograft is a very rare situation where ECD can be easily determined by specular microscopy just before graft and almost all grafted EC are living. Aim: to compare EC decrease after simultaneous autograft and organ cultured (OC) allograft in the same patient Methods A 71 year-old woman presented with pseudophakic corneal edema in left eye and amblyopic right eye with a normal cornea. She underwent two penetrating keratoplasties (PK): autograft of the left eye and allograft of the right eye with an OC cornea. Follow-up at day (D)1, 5, 15, 20 and 30 comprised slit lamp examination and non-contact central specular microscopy (Topcon SP2000P) Results Both grafts were uneventful. For the allograft, ebECD 48h before graft was 2787 cells/mm² and postop ECD decreased of 39%, 40%, 40% and 43% at D5, 15, 20 and 30. For the autograft, pregraft specular ECD was 2017 cells/mm² and postop ECD were 1941, 1934, 1924, 1909 and 1900 cells/mm² respectively at D1, 5, 15, 20 and 30 Conclusion This exceptional case confirms a dramatic discrepancy between ebECD and very early postop ECD, likely to be explained mainly by an overestimation of the true EC pool of viable cells by standard eye banks count, rather than by overmortality due to peri-operative events, since almost no early cell loss is observed during autograft

Purpose To evaluate utilization of corneal tissue procured by the eye bank of a tertiary care centre in north India, to determine factors that were responsible for labeling corneas unsuitable for transplantation, and to identify methods to improve donor tissue utilization. Methods We retrospectively reviewed records from our eye bank from May 2005 to April 2010. We compared donor demographics, death- enucleation interval, and biologic contamination of utilized and non- utilized corneal tissues. Results 3951 corneas were received during the study period. Of these, 2561 donor corneas (65%) were used for surgical purposes. Of the 1390 corneas not utilized for transplantation, 703 corneas (50.6%) were excluded due to poor tissue quality. Other causes were logistical reasons, such as no suitable recipient, in 267 (19.2%), bacterial growth on culture in128 (9.2%), contraindication in the donor’s medical history in 102(7.3%), and serological results in 83(6%). Mean age of donors in the utilized cornea group was 58.9 years (± 21.57 years) as compared to 67.52 years (± 19.82 years) in the non-utilized cornea group. The difference was statistically significant (P=0.000). Mean death-enucleation interval was comparable in the 2 groups (5.38 hours versus 4.62 hours; P=0.54). Conclusion Increase in age of donors results in poorer tissue quality and emerges as a significant factor in non–utilization of donor corneas for transplantation.

Purpose Corneal endothelium of organ cultured cornea forms a 3D surface because of corneal curvature and of the Descemetic folds (of hundreds of μm depth) that appear during storage. Eye bank technicians have therefore a risk of endothelial cell density (ECD) overestimation when counting on an image projection. Our aim is to develop a 3D reconstruction from a Z stack of images in order to take account of the local slope and increase ECD reliability Methods Z stacks of 35 images were acquired with a motorized light microscope (BX41, Olympus) through a x10 objective. After routine eye bank preparation with saline, EC appeared with a clear cytoplasm and dark borders but could locally show a contrast reversal. In each section, the best-focused pixels were determined using focus measurement algorithms. Depth map and texture image were then separately generated and the 3D reconstruction was obtained by projecting the texture image onto the depth map. A new algorithm, based on projections onto eigenbases, was developed and compared to 5 existing algorithms. Comparison was done using 1/ simulated images with additive noises to assess the reliability and noise robustness 2/ true endothelial image Results The new algorithm exhibited a more robust behaviour in presence of noise than the other algorithms. 3D endothelial reconstructions were also visually better, especially in case of local contrast reversal Conclusion Using a technology easily transposable into an eye bank, a reliable 3D reconstruction is possible from routine images that are often of average quality with poor contrast and high background noise. Our next step is to integrate the local slope information to correct local ECD

Purpose We developed and evaluated a new hybrid tissue-polymer drug delivery system using lyophilized cornea and synthetic hydrogel. The feasibility of this system as a carrier and a drug delivery system for an artificial cornea (Boston Keratoprosthesis) was evaluated in vitro while non-modified cornea tissue was used as a control. Methods Corneal tissue from the eye bank was first lyophilized. The tissue-polymer hybrid was synthesized by reconstituting the lyophilized cornea in a norfloxacin-loaded hydrogel solution followed by polymerization. Four different tissue-hydrogel compositions of varying hydrophobicity were synthesized and characterized over one month for the swelling and the drug release profile. Unmodified cornea tissue was used as a control. The mechanical strength and suture characteristics of the hybrid system and unmodified cornea were evaluated as a carrier for Boston K-pro using an artificial anterior chamber. Results Both the hybrid-system and the control show excellent mechanical properties as carriers for the Boston K-pro. They withstood similar challenges of intrachamber pressures (50-70 mmHg) for wound stability. In vitro drug release analysis demonstrates a longer and more controlled drug release profile for the hybrid system as compared to the control. The most hydrophobic hybrid construct shows a release that is above the Minimum Inhibitory Concentration 90 of Staphylococcus epidermidis for the first two days. Conclusion The new hybrid tissue-polymer system shows sufficient mechanical stability to serve as a carrier for the Boston K-pro. This system has the potential to simplify storage and distribution of donor tissue as a carrier for artificial cornea in developing countries where donor tissue is otherwise not readily available.

Purpose To evaluate the efficacy of a new storage medium able to avoid swelling the donor cornea before the microkeratome cut in eye bank routine setting Methods The Eusol-C (Alchimia s.r.l.,Italy) storage medium and a modified storage medium (only for experimental use,Alchimia) were used to store two different sets of donor corneas (n=12).Before the microkeratome cut,corneas were stored in Eusol-C (n=6) while the controlateral donor corneas were stored in the modified medium (M.M.) (n=6).Donor lenticules were created with a full pass of microkeratome blade which resulted in a posterior lamella and a free cap.Endothelial cell density,cellular viability and corneal thickness by AS-OCT (VisanteTM,Carl Zeiss) were evaluated before dissection,4,24 and 48 hours after dissection. Results Endothelial cell density and cellular viability were well preserved during the time course in both storage medium.Pachymetry values, after the microkeratome cut did not show significant variations during the time course.Conversely significant differences were found between the two groups (p

Purpose to describe a manual technique for the preparation of thin endothelial grafts or DMEK and to report clinical results in patients with Fuchs dystrophy. Methods We report here a series of 92 patients with Fuchs dystrophy undergoing endothelial keratoplasty. A manual dissection is performed on an artificial chamber to obtain posterior graft. After completing the learning curve, it is possible to select the depth of dissection: from pure descemet membrane isolation, to very thin posterior graft. Results Visual acuity is significantly higher after DMEK (0.7) than after thin posterior grafts (0.58), 6 months after surgery. However, endothelial density is much lower with 1190 cel/mm² after DMEK compared to thin posterior grafts which have 1870 cell/mm² 6 months after grafting. Conclusion Manual dissection is more difficult to perform than automated techniques but allow preparation of much thinner grafts. Among these, pure descemet membrane grafts have the best visual rehabilitation but less endothelial cells due to surgical manipulations.

Purpose Discussion of the evolution of a technique suggested by Jose I. Barraquer in 1947, later completed by Strampelli and adopted and modified by other corneal surgeons. The original procedure was based on the clinical observation that regeneration of the corneal epithelial cells is dependant on the state of the limbus. This explained the bad prognosis of chemical burns and severe limbus trauma. At present it is well known that the stem cells located in the corneal limbus are responsible for epithelial regeneration. Penetrating keratoplasty limited to the cornea does not supply stem cells. Methods The technique consists in obtaining and grafting limbus tissue, in form of an annular or partial conjunctivosclerocorneal limbal graft preferably from the fellow eye or a living donor, or an eye bank eye. A case of chemical burn operated in 1981 is presented to illustrate the original technique of Strampelli. To illustrate the modern approach a case of chemical trauma with superior and inferior symblepharon and total vascularized leucoma operated in 1999 (limbal donor graft)taking advantage of the modern advances available at present (improved instrumentation, pharmacological inhibition of homograft reaction)is shown. Results The results were encouraging.The patient operated in 1981 died in 1988 with good vision (0.6).In the case operated in 1999, three months after the limbal transplant, re-epithelization of the cornea was correct and an 8 mm penetrating keratoplasty combined with cataract extraction and IOL implantation was performed. Corrected vision 5 years postoperatively was 0.35. Conclusion Modern investigation and technology have converted a procedure based on surgical inspiration more than half a century ago into a valuable option in cases of irreversible damage of the corneal limbus.

magnitude and direction of vector forces during eye rotations. The usual chronic course of AMD with exacerbations and remissions and often progressive, bilateral appearance are suggestive of intermittent traumatic effects. Physiological vitreous changes and eye rotations occur in everyone, but vitreous structures will only touch the retina in the presence of elongated cisterns and ⁄or significant vitreous movements, which process might be facilitated by vitreomacular adhesions. Our hypothesis that mechanical stress might contribute to the pathogenesis of AMD is supported by the clinical findings of others, who used spectral domain optical coherence tomography to evaluate the vitreoretinal interface: a positive relationship between a persistent attachment of the posterior vitreous and early signs of AMD was found (Schulze et al. 2012) as well as a complete correspondence between the adhesion and subretinal neovascularization (Krebs et al. 2011). We realize that observational and anatomical, experimental studies have their limitations, and further histopathological as well as case–control studies are required to confirm our hypothesis. We thank Peter J. de Lint, vitreoretinal surgeon in the Medical Center Alkmaar; the Corneal Eye Bank, Amsterdam; the Jan Worst Research Group, Haren; the ophthalmic photography units of the VU University Medical Center, Amsterdam; and theMedical Center Alkmaar, the Netherlands.