Paired Corneas Research Articles

Preclinical validation of an innovative corneal bioreactor versus organ culture for long term storage: a randomized controled study

PurposeThe organ culture (OC) is a passive corneal storage, gold standard in Europe for decades. The bioreactor (BR) is a sterile transparent device that allows active storage: transcorneal pressure gradient and fluid renewal. Aim: To compare quality of human corneas stored for 1 month in our BR versus (vs) OC.MethodsRandomized controled preclinical study, authorized by the French Biomedicine Agency and an IRB, including only fresh scientific paired human corneas >2000cells/mm2 and <10% difference between both corneas at day (D)2. Immediately at retrieval, one cornea was randomized and stored in OC and the other in BR, both with the same commercial medium (CorneaMax). At D26 only OC corneas were transferred to a deswelling medium (CorneaJet). We calculated that 49 pairs were needed to show a difference of 500 viable cells/mm2 between groups. Assessments were performed at D2, 26, 28. Main criterion: viable ECD (vECD) determined by triple staining Hoechst/Ethidium/Calcein at D28, blind of storage method. Secondary criteria: average transparency (T), central corneal thickness (CCT) and microbiology.ResultsWe retrieved 60 pairs, included 52 pairs at D2, analyzed 50 pairs (1 contamination per group). At D2 ECD in BR was 2607±340 vs 2576±357cells/mm2 in OC (p=0.243). At D26 ECD was >2000cells/mm2 for 92% of corneas in BR vs 58% in OC (p<0.001). At D28 vECD was higher in BR 2188±379 vs 1687±389cells/mm2 in OC, with 76% of corneas >2000 viable cells/mm2 in BR vs 22% in OC (p<0.001); T didn't differ and CCT was higher in BR with 684±52 vs 608±53μm in OC (p<0.001).ConclusionsRestoring IOP and renewing medium significantly improves endothelial cell survival while avoiding deleterious deswelling necessary in OC: after 1 month‐storage, the BR provides much more corneas suitable for transplantation and higher quality grafts that could survive longer in recipients.

Recombinant human serum albumin for corneal preservation.

To study the performance of a completely synthetic organ culture (OC) preservation system containing recombinant human serum albumin (rHSA) for preservation of human donor corneas. Twenty-four paired donor corneas were randomly collected, and one cornea from each donor was preserved in synthetic (experimental) and serum-based media (control). The tissues were assessed at day 0; after 6days of preservation at room temperature (RT) in Cornea Trans® and Cornea Prep II® ; after 28days at 31°C in Cornea Syn® [with rHSA] and Cornea Max® [with foetal calf serum (FCS)] and; 4-day post deswelling in Cornea Trans® and Cornea Jet® . Thickness was determined with optical coherence tomography (OCT) and transparency with a validated, custom device. Morphology, endothelial cell density (ECD) and mortality were observed after treating the tissues with Trypan blue and sucrose. Glucose uptake by the cells was analysed. Data were compared using non-parametric paired Wilcoxon tests with p<0.05 deemed significant. Histology using periodic acid-schiff (PAS), expressions of p63, CK12, αSMA and ZO-1 were analysed, and cell apoptosis postpreservation was studied. Corneas stored in synthetic media showed a higher and statistically significant value as compared to serum-based media in terms of viable endothelial cell density (VECD), mortality, morphology and glucose uptake postpreservation. Histology showed presence of all the layers, all the markers were expressed, and no apoptosis was observed in either media. The new synthetic preservation system containing rHSA (and other confidential constituents) showed better preservation effects than traditional media containing serum.

Single-Pass Dissection of Ultrathin Organ-Cultured Endothelial Lamellae Using an Innovative Microkeratome System.

To determine the feasibility, dissection accuracy, and endothelial viability of ultrathin endothelial lamellae harvested from organ-cultured corneas using a single-pass with an innovative motor-driven linear microkeratome system. Forty-eight (n = 48) paired organ-cultured human corneas were randomly assigned to dissection (study eyes, n = 24) with fellow eyes serving as the control (fellow eyes, n = 24). After organ culture and deswelling in a medium containing 6% dextran, endothelial lamellae with a target thickness ≤100 μm were dissected using a motor-driven linear microkeratome system (SLc, Gebauer, Neuhausen, Germany) equipped with 400-μm (n = 4), 450-μm (n = 10), 500-μm (n = 5), or 550-μm (n = 5) heads. Central corneal thickness (CCT) and posterior and anterior lamellar thicknesses were measured using ultrasound pachymetry (Pachette 3; DGH Technology Inc, PA) and anterior segment optical coherence tomography (Casia SS-1000; Tomey, Nagoya, Japan). Endothelial viability [endothelial cell density (ECD)] was measured using trypan vital staining. CCT measured 595 ± 66 μm (n = 48) on arrival, 846 ± 131 μm (n = 48) after organ culture, and 565 ± 58 μm (n = 48) after deturgescence. CCT did not differ between study and control eyes. Posterior lamellar thickness measured 88 ± 18 μm (n = 24) immediately after dissection, 126 ± 30 μm (n = 24) 1 hour after dissection, and 131 ± 41 μm (n = 24) 2.3 ± 0.6 days after dissection. ECD measured 2637 ± 264 cells per square millimeter (n = 48) on arrival, 2524 ± 232 cells per square millimeter (n = 48) after organ culture, 2493 ± 253 cells per square millimeter (n = 48) after dissection, and 2311 ± 218 cells per square millimeter (n = 48) 2.3 ± 0.6 days after dissection. ECD did not differ between study and control eyes at all time points. Single-pass motor-driven linear microkeratome dissection provides an accurate and safe alternative for harvesting ultrathin endothelial lamellae from organ-cultured donor corneas.

Designing an innovative bioreactor destined to improve the endothelial viability of stored corneas

PurposeLoss of intraocular pressure (IOP) after death and after corneo‐scleral procurement triggers a stromal swelling causing posterior folds in which endothelial mortality is increased. We hypothesized that restoration of IOP after procurement will be key for long‐term corneal storage with preserved endothelial functions.MethodsWe designed two bioreactors (BR) (for human and porcine corneas) and their control units. The BR presented as a sterile disposable cassette comprising a corneal chamber with transparent windows surrounded by the fluidic system. Sterile sampling sites were added in the circuit for monitoring physiochemical parameters as well as sterility without risk of contamination.ResultsAfter insertion of the corneo‐scleral rim, the system was closed and the cassette connected to its control unit, comprising a peristaltic pump driven by a microcontroller. In a comparative study (ongoing) on paired corneas, the BR maintained a normal corneal thickness and transparency during the storage, while reducing the number of endothelial folds and increasing the endothelial viability.ConclusionsThe innovative BR restores IOP while ensuring a continuous medium renewal in a fully transparent chamber. The BR will be available for eye banks, research laboratories, contract research organizations, and cosmetic industry. GRANTS: AP looks for from ANSM, EFS, UJM.

Randomized, prospective, single-masked clinical trial of endothelial keratoplasty performance with 2 donor cornea 4°C storage solutions and associated chambers.

The aim of this study was to compare endothelial cell loss and graft success 6 months after endothelial keratoplasty (EK) with paired donor corneas stored in Optisol GS and Life4°C solutions and their associated storage chambers. Donor pairs were stored, one in Optisol GS and the other in Life4°C, and prepared for Descemet stripping automated EK or Descemet membrane EK. Matched pairs of recipients with Fuchs dystrophy were randomized to 1 member of each donor pair. Clarity of recipient stroma, intraocular pressure, and complications were followed for 6 months. Central endothelial images of the donor cornea at screening and 3 and 6 months after EK were analyzed by a masked central reading center. Dual grading of endothelial cell density was performed using the center method. Differences in endothelial cell density and cell loss were examined by paired analysis. Thirty-two pairs were enrolled, and 27 were analyzed (5 had improper matching or loss to follow-up). Donor age was 59 ± 14 years, median death-to-surgery time was 4 days (range, 2-9 days), 6 recipient pairs had Descemet membrane EK, and 21 had Descemet stripping automated EK. Recipient age was comparable in the Optisol GS and Life4°C groups (70 vs. 68 years, respectively, P = 0.46). Six-month central endothelial cell loss did not differ significantly between the Life4°C and Optisol GS groups (18 ± 18% vs. 20 ± 20%, respectively, P = 0.55). All recipient corneas were clear at 6 months in both groups. Endothelial cell loss and graft success were comparable at 6 months for paired donor corneas stored in Optisol GS and Life4°C.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01657500.

Surgeon graft less viable endothelial cells than the eye bank cell count suggests

AbstractPurpose 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&lt;0.05), with a difference of only 143 (5‐359) cells between them.Conclusion These 2 complementary series demonstrated that ebECD provided to surgeons overestimate the number of viable CEC grafted to patients. Taking account only the vECD at the end of storage (just before graft) and the postop ECD measured as early as possible, we show that the presumed initial very important cell loss is fictive and that the model of postop ECD decrease should be revisited.

Deformation response of paired donor corneas to an air puff: Intact whole globe versus mounted corneoscleral rim

To evaluate the influence of ocular shell biomechanical characteristics on corneal deformation response to an air puff. The Ohio State University, Columbus, Ohio, USA. Experimental study. Twenty-four eyes of 12 human donors were obtained in matched pairs. One eye was secured in a purpose-designed whole globe mount (whole-globe group). The cornea from the fellow eye was placed in a Barron artificial anterior chamber (artificial-chamber group). The corneas were mounted sequentially and connected to a pressure-control system. Deformation data were acquired using the Corvis ST, a dynamic Scheimpflug analyzer. Internal pressure was set to 10, 20, 30, 40, and 50 mm Hg; at least 4 examinations were performed at each pressure. Statistically significantly higher maximum deformation amplitude was observed in the whole-globe group than in the artificial-chamber group at all pressures. The mean amplitude differences were 1.006 mm ± 0.238 [SD], 0.614 ± 0.137 mm, 0.384 ± 0.099 mm, 0.229 ± 0.087 mm, and 0.133 ± 0.068 mm at 10, 20, 30, 40, and 50 mm Hg, respectively (P<.0001, P<.0001, P<.0001, P<.0001, and P<.0002, respectively). Nonlinear regression of the deformation amplitude differences between pairs showed a significant decrease with increasing pressure (P<.0001, R(2) = 0.8385). The deformation response to an air puff was affected by the type of mount used, with a stiffer shell producing a stiffer corneal response and decreasing differences at higher internal pressures. In vivo air-puff examinations may be affected by scleral stiffness in addition to the cornea. Dr. Roberts is a consultant to Oculus Optikgeräte GmbH and Ziemer Ophthalmic Systems AG and has received research funding from Carl Zeiss Meditec AG and travel funds from Sooft Italia. No other author has a financial or proprietary interest in any material or method mentioned.

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro

BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Evaluation of interface quality in organ-cultured lamellar corneal transplants.

BackgroundWith increasing numbers of lamellar keratoplasties, eye banks are challenged to deliver precut lamellar donor tissue. In Europe, the most common technique of corneal storage is organ culture which requires a deswelling process before surgical processing. The aim of this study was to investigate the influence of different deswelling times on the cutting plane quality after microkeratome-assisted lamellar dissection.MethodsEight paired donor corneas (16 specimens) not suitable for transplantation were organ cultured under standard conditions at the Eye Bank of the Ludwig-Maximilians Universität, Munich, Germany. Pairs of corneal buttons were analyzed during the deswelling process in dextrane-containing medium. While one cornea was cut at an early time point during the deswelling process and put back into deswelling medium thereafter, the partner cornea was completely deswollen and dissected after 72 hours. Specimens were then further processed for scanning electron microscopy. Surface quality was assessed both digitally using Scanning Probe Imaging Processing software, and manually by three blinded graders.ResultsThe corneal buttons processed at the beginning of the deswelling process had a smoother surface when compared to the partner cornea that was cut at the end of the deswelling process. In our setting, no relevant difference was detectable between manual and automated microkeratome dissection.ConclusionFor lamellar keratoplasty, organ-cultured corneas should be processed at an early stage during the deswelling process. We interpret the smoother dissection plane during early deswelling as a result of mechanical properties in a highly hydrated cornea.

Poloxamines for Deswelling of Organ-Cultured Corneas

Background: Poloxamines are amphiphilic tetrofunctional block copolymers composed of four polyoxyethylene-polyoxypropylene arms joined to a central ethylene diamine bridge. Their safe profile allows diverse pharmaceutical and biomedical applications. Aim: To assess their use for corneal deswelling using a porcine model of organ culture (OC). Methods: Five poloxamines (T90R4, T904, T908, T1107 and T1307) were dissolved in a standard commercial OC medium (control) to reach 350 mosm kg^–1. In vitro cytotoxicity was tested using MTT assay on human corneal epithelial and endothelial cell (EC) lines and on primary human corneal fibroblasts. Paired porcine corneas stored in OC for 3 days were assigned for 48 h to a poloxamine medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, mortality, stromal thickness and transparency were evaluated before and after deswelling. Post-deswelling, EC viability/mortality was determined using a fluorescent live/dead assay. Results: Besides similar corneal thickness reduction and transparency improvement, T908, T1107 and T1307 decreased EC loss (5.4 ± 1.7% vs. 9.9 ± 2.6% in controls (p < 0.001)) and mortality, improved EC morphometry and reduced endothelial lesions compared to dextran. Conclusion: On this porcine model, poloxamines T908, T1107 and T1307 appear as good candidates to replace dextran for the deswelling. Experiments on human corneas are now necessary to confirm their efficiency and safety profile in OC.

Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas

Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability.Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage.The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.

Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

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.

Evaluation of Megacell™ MEM as a Storage Medium for Corneas Destined for Transplantation

Purpose: Gibco’s Minimum Essential Medium with Earle’s salts and HEPES supplemented with glutamine, antibiotics (EB MEM) and 2% foetal calf serum (FCS) is used in European eye banks to store corneas. Although FCS is important to endothelial cell survival in this medium, it is a potential biohazard. Megacell MEM, formulated to reduce the FCS requirement of cells by a factor of 5, has therefore been evaluated as a corneal storage medium. Methods: Corneal stromal and epithelial cells were incubated in Megacell MEM (serum-free or 2% FCS) to assess their viability in these media. Endothelial cell densities of paired corneas held in either EB MEM 2% FCS or Megacell MEM (serum-free or 2% FCS) were measured over 5 weeks. Discs subsequently punched from the centre of these corneas were weighed, dried and reweighed to determine hydration levels. Results: Both corneal stromal and epithelial cells proliferated in Megacell MEM 2% FCS. Relative to EB MEM, 2% FCS Megacell MEM prolonged the viability of corneal endothelial cells and improved their morphological appearance, irrespective of whether it contained FCS or not. This was independent of corneal swelling. Conclusion: Serum-free Megacell MEM is a better storage medium than EB MEM 2% FCS for corneas destined for transplantation

Experimental assessment of endothelial viability of grafts

Abstract Purpose Endothelial quality of penetrating keratoplasty or posterior lamellar grafts (be they manually, mechanically or femto laser dissected) remains of paramount importance for the graft survival in recipients, especially when the recipient endothelial cell peripheral reserve is weak or absent (bullous keratopathy) Methods 1/Review of the literature on the available methods to measure the endothelial quality. 2/presentation of a new concept: experimental quantification of the actual endothelial cell density of cell deemed viable, thus representing the actual number of cells capable of surviving in the recipient eye. This method uses a live/dead assay coupled with the microscopy analysis of the whole endothelium. A comparative series of graft pre‐cut for DMEK versus intact organ cultured paired corneas will illustrate our method Results For the first time the exact number of viable endothelial cells can be directly measured. The notion of viable endothelial cell density is introduced. Though it cannot be a routine technique but only a laboratory one, the method can be ‐and perhaps should be‐ applied for all kind of graft preparation during its experimental development. Lamellar posterior grafts are good candidates. As expected, the viable ECD is lower than the ECD determined with the routine method used in eye banks, even the most precise ones Conclusion The new notion of viable ECD may participate in our understanding of the dramatic endothelial cell loss ineluctably observed shortly after all types of grafts

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

Optimizing the Deswelling Process of Organ-Cultured Corneas

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. 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. 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. 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.

Functional CNTF Receptor α Subunit Restored by Its Recombinant in Corneal Endothelial Cells in Stored Human Donor Corneas: Connexin-43 Upregulation

Ciliary neurotrophic factor (CNTF) is undergoing testing in human clinical trials to rescue degenerating retina, whereas studies show that the CNTF-binding alpha-subunit of the CNTF receptor (CNTFRalpha) is released from injured tissues. Here, the recombinant human (rh) CNTFRalpha was shown to restore functional CNTFRalpha in human corneal endothelial (CE) cells that lost endogenous CNTFRalpha during corneal storage In CE cells of stored human donor corneas, endogenous CNTFRalpha levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFRalpha (8.3 nM) in augmenting the CNTF (0.83 nM) effect was tested. Paired human donor corneas were used as vehicle versus CNTF-treated or CNTF- versus (rhCNTFRalpha+CNTF)-treated (24 hours, 37 degrees C), followed by analysis of CE cell connexin-43 mRNA and protein by semiquantitative RT-PCR and Western blot analysis, respectively. After 90-minute incubation with stored human corneas, rhCNTFRalpha incorporation into the CE membrane fraction was demonstrated by Western blot analysis CE cell CNTFRalpha levels decreased as corneal storage time increased. CE cell connexin-43 mRNA levels in CNTF-treated and (rhCNTFRalpha+CNTF)-treated paired corneas averaged (mean+/-SEM) 0.26+/-0.08 and 0.58+/-0.21, respectively (P=0.029; eight pairs; storage time >or= 25 days). rhCNTFRalpha augmentation was confirmed at the protein level. In corneas with short storage times (<or= 9 days) that retained abundant endogenous CNTFRalpha, rhCNTFRalpha decreased the effectiveness of CNTF. rhCNTFRalpha was incorporated into CE membranes rhCNTFRalpha acted as a surrogate to the lost endogenous membrane-bound CNTFRalpha in CNTF signaling, suggesting the potential of an adjuvant rhCNTFRalpha therapy in CNTF-therapy.

Evaluation of Endothelial Mucin Layer Thickness After Phacoemulsification With Next Generation Ophthalmic Irrigating Solution

To evaluate human corneal endothelial mucin layer thickness and ultrastructure after phacoemulsification and irrigation-aspiration with either next generation ophthalmic irrigating solution (NGOIS) or BSS PLUS. Paired human corneas were mounted in an artificial anterior chamber, exposed to 3 minutes of continuous ultrasound (US) at 80% power using the Alcon SERIES 20000 LEGACY surgical system (n = 9) or to 2 minutes of pulsed US at 50% power, 50% of the time at 20 pps using the Alcon INFINITI Vision System (n = 5), and irrigated with 250 mL of either NGOIS or BSS PLUS. A control group of paired corneas did not undergo phacoemulsification or irrigation-aspiration (n = 5). Corneas were divided and fixed for mucin staining or transmission electron microscopy. Mucin layer thickness was measured on the transmission electron microscopy prints. The mucin layer thickness in the continuous phaco group was 0.77 +/- 0.02 microm (mean +/- SE) with NGOIS and 0.51 +/- 0.01 microm with BSS PLUS (t test, P < 0.001). The mucin layer thickness in the pulsed phaco group was 0.79 +/- 0.02 microm with NGOIS and 0.54 +/- 0.01 microm with BSS PLUS (P < 0.001). The mucin layer thickness in the untreated control group was 0.72 +/- 0.02 microm. The endothelial ultrastructure was normal in all corneas. In this in vitro corneal model, NGOIS, due to its lower surface tension and higher viscosity, preserved endothelial mucin layer thickness better than BSS PLUS with both the INFINITI Vision System (pulsed US) and the LEGACY surgical system (continuous US).

Descemet-stripping Automated Endothelial Keratoplasty

To compare the effect of storing Descemet-stripping automated endothelial keratoplasty (DSAEK) tissue with anterior lamellar corneal tissue (ALCT)-on versus -off. An in vitro model was used with corneoscleral rims and DSAEK quality paired corneas. After microkeratome-assisted excision of ALCT, 4 pairs of corneas (8 eyes) were stored with the ALCT left on the stroma (on) and the others with ALCT off the stroma (off) for 24 hours in Optisol GS solution. A vital dye assay was used to identify devitalized and necrotic endothelial cells with alizarin red S and trypan blue. Corneal endothelial cell damage was observed in the ALCT-off specimens, whereas almost no staining was observed in the ALCT-on samples. In addition, the ALCT-off donor corneas were clinically edematous and opaque, whereas the ALCT-on corneas were clear. Moreover, Descemet membranes of ALCT-off samples were found to be loose and easily detached from the stroma, with many Descemet striae observed in the specimens. This study shows that endothelial damage occurs in ALCT-off corneas. We hypothesize that the absence of the Bowman layer may contribute to the damage, because it has been shown that the Bowman layer provides a barrier function. These data suggest that it is important to keep the ALCT/Bowman layer on the stromal side of the DSAEK graft as long as possible to avoid stromal swelling and endothelial damage.

VIP and VIP Gene Silencing Modulation of Differentiation Marker N-Cadherin and Cell Shape of Corneal Endothelium in Human Corneas Ex Vivo

Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and is present in the aqueous humor, which bathes CE cells in vivo. This study demonstrated the role of CE cell VIP in maintaining the expression level of a CE differentiation marker, N-cadherin, and the hexagonal cell shape. To determine the most effective VIP concentration, bovine corneoscleral explants were treated with 0 (control) and 10(-12) to 10(-6) M VIP. Paired human corneas (nine donors) from an eye bank were used as control; the other corneas were treated with VIP. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semiquantitative RT-PCR (for mRNA) and Western blot analysis/immunocytochemistry (for protein), whereas alizarin red S staining revealed CE cell shape. VIP concentration dependently increased bovine CE cell N-cadherin mRNA levels, with the maximal effect observed between 10(-10) (1.47 +/- 0.06-fold; P = 0.002) and 10(-8) M VIP (1.48 +/- 0.18-fold; P = 0.012). VIP (10(-8) M) treatment increased N-cadherin protein levels in bovine and human CE cells to 1.98 +/- 0.28-fold (P = 0.005) and 1.17 +/- 0.10 (range, 0.91-1.87)-fold (P = 0.050) of their respective controls. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape and decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the antiapoptotic Bcl-2 protein. Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium in situ.