Corneoscleral Rims Research Articles

The value of corneoscleral rim cultures in keratoplasty: a systematic review and cost-effectiveness analysis.

PurposeThis study evaluated the performance of donor corneoscleral rim cultures for predicting infection after corneal transplantation, and determines if there is a correlation between positive corneoscleral rim cultures and postkeratoplasty infection.Design and data sourcesThis was a systematic review, prognostic accuracy analysis, and cost-effectiveness analysis. Databases searched were: Medline (Ovid), Embase (Ovid), CINAHL, Cochrane Library, Web of Science, and BioSis Previews. Grey literature was also explored.Materials and methodsA systematic review was conducted to locate published and unpublished studies. All studies examining corneal button contamination and its association with endophthalmitis and keratitis posttransplantation were included. Extracted data were used to calculate sensitivity, specificity, positive predictive value, and negative predictive value. Cost data from the London Laboratory Services Group in London, ON were used to calculate the cost-effectiveness of culturing donor rim cultures.ResultsOf 7,870 grafts, 954 had a positive rim culture (12.1%), with 12 patients going on to develop keratitis or endophthalmitis (1.3%). The prevalence of keratitis and endophthalmitis in this study was 0.15%, and the positive predictive value 1.5%. Of the 12 infections, nine were fungal and three bacterial. The estimated cost of a positive and negative test result was CAD$45.99 and $14.15, respectively. The cost to run all 7,870 tests was estimated to be $141,735.86, with an incremental cost-effectiveness ratio of $40,215.70.ConclusionThere was a significant divergence between bacterial and fungal rim-culture results. Bacterial cultures predicted clinical infection poorly, did not change management, and were expensive. Fungal cultures predicted clinical infection in over 10% of patients, had the potential to change management, and were 40% less expensive than full rim culturing (bacterial and fungal tests). Fungal rim cultures may be considered in areas where fungal infection rates are high.

Development and Assessment of a Novel Canine Ex Vivo Corneal Model

ABSTRACTPurpose: To develop a novel ex vivo extended culture model of canine corneal epithelial cell wound healing.Materials and Methods: Canine corneoscleral rims (CSR) were obtained and, after preparation for culture, were placed on a nutating scaffold and incubated in physiological conditions. In experiment 1, eight CSR in a serum-containing antimicrobial-fortified medium were monitored for epithelial integrity and bacterial infection up to 28 days in culture. CSR were assessed histologically at the end of the culture period end points 0, 7, 14, and 28 days with accompanying scanning electron microscopic (SEM) and transmission electron microscopic (TEM) evaluation. Samples for microbial culture were obtained at days 0, 3, 7, 14, and 28. In experiment 2, uniform 8-mm-diameter superficial corneal epithelial wounds were created and monitored for re-epithelialization in the same culture conditions or in a serum-free protein equivalent medium, with four CSR per group. Standardized digital images were obtained with cobalt filter at the time of fluorescein staining and media change every six hours. Image J imaging software was used to measure the area of fluorescein retention. Re-epithelialization rates were calculated and CSR then fixed for immunohistochemistry (IHC).Results: All corneas survived to end points as described in experiment 1 with no evidence of contamination or compromised epithelial integrity. Histologically, a multilayered epithelium was maintained and corneal edema was not appreciated until day 14. SEM examination revealed epithelial cell layer confluence and migrating epithelial cells of normal cellular morphology with normal cell–cell interactions on TEM. In experiment 2, all eight corneas healed with a healing rate of 0.702 ± 0.130 mm2/h (1.25 mm/day epithelial cell migration rate) and were positive in IHC evaluation for markers of corneal fibrosis.Conclusion: This ex vivo canine corneal wound healing model is an appropriate and clinically relevant tool for assessment and modulation of epithelial wound healing.

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

PurposeIn the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models.MethodsExcised rabbit and human corneoscleral rims maintained in organ culture were infected using 108 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared.ResultsWe observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p < 0.05) in the majority of organisms from infected corneas. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P. aeruginosa throughout the entire cornea, with less infiltration observed for S. aureus, C. albicans and F. solani. The models also supported dual infections.ConclusionsBoth scalpel wounding and injection methods are suitable for inducing infection of ex vivo rabbit and human cornea models. These simple and reproducible models will be useful as an alternative to in vitro and in vivo models for investigating the detection and treatment of microbial keratitis, particularly when this might be due to two infective organisms.

Incidence and Outcomes of Positive Donor Corneoscleral Rim Fungal Cultures after Keratoplasty

To determine the incidence of positive corneoscleral donor rim fungal cultures after keratoplasty and to report clinical outcomes of grafts with culture-positive donor rims. Retrospective cohort study. Consecutive donor corneas and keratoplasty recipients at a single tertiary referral center over 20 years. Patient charts were reviewed to determine the incidence of positive donor rim fungal cultures and clinical outcomes of all grafts using contaminated tissue. The primary outcome measures were positive donor rim fungal culture results and the development of postkeratoplasty fungal infection using corresponding corneal tissue. The secondary outcome measure was the impact of postoperative prophylaxis on donor tissue-associated infections. A total of 3414 keratoplasty cases were included in the statistical analysis. Seventy-one cases (2.1%) were associated with a fungal culture-positive donor rim. Candida species were cultured in 40 cases (56.3%). There was a higher incidence of positive rim cultures over the last 5 years of the analytic period compared with the first 15 years (P= 0.018). Fungal keratitis developed in 4 cases (5.6%), and all patients required further surgical intervention to achieve cure. There were no cases of fungal endophthalmitis. Empiric antimycotic prophylaxis initiated at the time of positive culture result reduced the incidence of keratitis from 15.8% in untreated cases to 1.9% in treated cases (P= 0.056). Positive donor rim fungal cultures are uncommon, but carry an unacceptably high risk of postoperative fungal infection. This risk may be reduced with prophylactic antimycotic therapy when culture-positive donor rims are identified.

Dual-purpose corneal tissue for anterior lamellar keratoplasty and Descemet’s membrane endothelial keratoplasty

ObjectiveTo assess the intraoperative issues and surgical outcomes of preparing a single-donor corneal tissue for same-day use in both deep anterior lamellar keratoplasty (DALK) and Descemet’s membrane endothelial keratoplasty (DMEK). DesignConsecutive retrospective case series. ParticipantsTen eyes of 10 patients who underwent DALK (5 patients) or DMEK (5 patients) surgery using dual-purpose corneal tissue. MethodsFive dual-purpose corneoscleral rims were used to prepare tissue for 5 DMEK and 5 DALK procedures. The submerged cornea using backgrounds away technique was first used to harvest the 5 DMEK grafts, and the remaining tissue was used for the 5 DALK grafts. Tissue preparation and operative use occurred on the same day. Tissue preparation challenges, intraoperative complications, and visual recovery were assessed. ResultsThere were no difficulties in preparing the 5 dual-purpose tissues, and all 10 lamellar transplants were completed successfully. At the 6-month follow-up, the mean best-corrected distance visual acuity improved from 20/250 to 20/80 in the DALK patients, and from 20/300 to 20/25 in the DMEK patients. Postoperative complications after DALK included retained viscoelastic agent at the interface in 1 patient and a double anterior chamber managed with rebubbling in another. After DMEK, a peripheral partial graft detachment occurred in 1 patient and was managed successfully with rebubbling. All corneas demonstrated clarity on slit-lamp examination. ConclusionsSingle-donor corneal tissue can be effectively used for both DALK and DMEK, and may represent a more efficient use of corneal tissue. Complications with the preparation of dual-purpose tissue were not encountered.

Heterologous, Fresh, Human Donor Sclera as Patch Graft Material in Glaucoma Drainage Device Surgery.

To determine the safety and efficacy of fresh, human sclera allografts as a patch graft material in glaucoma drainage device (GDD) surgery. Retrospective, noncomparative, interventional, consecutive case series. All GDD cases operated between 2008 and 2013 in which fresh human corneoscleral rims were used immediately after the central corneal button was used for penetrating or endothelial keratoplasty. Surgery was performed by 2 surgeons at 2 facilities. The Ahmed Glaucoma Valve (FP-7) was used exclusively in this cohort. Sixty-four eyes of 60 patients were identified; demographic data were recorded along with intraocular pressure (IOP), medication requirements, visual acuity, complications, and subsequent interventions. Incidence of complications. IOP and medication requirements at the last follow-up. Quilified success utilizing Tube Versus Trabeculectomy study criteria. The mean age of the cohort was 66.2±19.1 years; the average preoperative IOP was 33.2±11.1 mm Hg on 4.2±1.3 IOP-lowering agents before GDD surgery. IOP decreased significantly to 14.1±4.7 mm Hg (P<0.001) on 1.6±1.2 IOP-lowering agents (P<0.001) after an average follow-up of 18.2±15.4 months. There were no cases of early or late blebitis or endophthalmitis, and there was 1 case of conjunctival erosion and tube/plate exposure (1.6%) occurring 30 days after surgery. Qualified success was estimated as 90.5% and 81% at 1 and 2 years, respectively, using Tube Versus Trabeculectomy study criteria. Heterologous, fresh, human donor sclera appears to be a safe material for GDD tube coverage. It provides a cost-efficient alternative compared with traditional patch graft materials associated with a low risk of pathogen transmission.

Biophysico-functional compatibility of Seoul National University (SNU) miniature pig cornea as xenocorneal graft for the use of human clinical trial.

Xenocorneal transplantation is one of the solutions for shortage of donor cornea, and remarkable advances have been made in pig-to-rhesus studies from the immunological perspective. Most successful preclinical trials have been carried out with corneas of the Seoul National University (SNU) miniature pig (SNU pig, genetically unmodified) as donor tissues; however, there has been no biophysico-functional evaluation of the SNU pig cornea as a substitute for human cornea. The purpose of this study was to investigate the biophysical and functional compatibility of SNU pig cornea for use in human clinical trials. Ninety-three eyeballs obtained from 51 SNU pigs were used to evaluate the physical properties and changes in porcine corneal endothelial cells (PCECs) depending on preservation time and storage condition before surgery, proliferative and functional characteristics of PCECs, and the microbiologic safety of porcine cornea. Corneal diameters and curvatures, axial length, anterior chamber depth, and central corneal thickness were measured and compared with previously reported human data. Corneal endothelial cell density (ECD) was serially measured with a confocal microscope during 7 days of preservation in the same storage solution used for human corneas. Corneal endothelial cell proliferation and immunofluorescence staining of Na- and K-dependent ATPase in PCECs were evaluated after 7 days of preservation. The corneoscleral rims of SNU pigs were cultured for gram-positive bacteria, gram-negative bacteria, and fungi to evaluate their microbiological safety. Corneal diameter and thickness in SNU pigs was larger than human and corneal curvature was flatter; however, they were within surgically operable ranges. Mean ECD (day 0) and ECD loss after 7 days of preservation were 2625 ± 81 cells/mm(2) and 7.60 ± 1.53%, respectively, which is comparable to human ECD and ECD loss in the same conditions. The ECD of SNU pigs was inversely decreased with aging (R(2) = 0.4034, P = 0.001), and the estimated ages of pigs whose mean ECD would be more than 2500 and 2200 cells/mm(2) or more were 48 and 72 months or less, respectively. Mean doubling time of the endothelial cells was 52 to 96 h depending on the method used. The Na- and K-dependent ATPase pump in SNU pig cornea was well maintained for 7 days. No cultured microorganisms were found upon using the modified European Eye Bank Association protocol, which included additional antiseptic management during the enucleation procedure. In conclusion, SNU pig cornea is feasible for xenocorneal transplantation using the same preservation protocol as human with respect to biophysical and functional properties and can be stored for up to 7 days for transplantation in human clinical trials. An age limitation of donor pigs may be required for qualified corneal products to be used in human trials.

Correlation Between Corneal Button Size and Intraocular Pressure During Femtosecond Laser–Assisted Keratoplasty

To evaluate changes in intraocular pressure (IOP) in recipient and donor eyes during femtosecond laser-assisted keratoplasty (FLAK) and to assess for differences in the diameter of trephinated corneal buttons according to changes in pressure. Twenty porcine whole eyes (recipient model) and 20 porcine-corneoscleral rims (donor model) were prepared, and anterior chamber pressures were measured using a fiberoptic sensing device (Opsens, Quebec, Canada) during the femtosecond laser corneal cutting process. To determine the diameter of corneal buttons, 10 porcine whole eyes (recipient model) and 12 corneoscleral rims (donor model) of each baseline IOP were cut with the femtosecond laser programmed to the following pattern: "vertical side cut"; 1200 μm (depth), 8 mm (diameter). Digital photographs were obtained using microscopy and subsequently analyzed. The IOP (mean ± SD) for the recipient model was 10.2 (±0.9) mm Hg at baseline and ranged from 96.6 (±4.5) to ∼138.4 (±3.8) mm Hg during the corneal cutting process. This shows that the maximum IOP during FLAK increased 13.5 times compared with baseline. In the donor model, the mean pressure elevation from baseline artificial anterior chamber (AAC) pressure to corneal cutting was 15.8 (±5.4) mm Hg. This showed a positive correlation with baseline IOP [correlation coefficient (CC) = 0.827, P = 0.006]. As the baseline IOP in the recipient eye increased, trephinated corneal button size was reduced by up to 3.9% in diameter (CC = -0.945, P = 0.015). In addition, in donor eyes, the diameter was decreased by up to 11.7% as the baseline AAC pressure increased (CC = -0.934, P = 0.006). During the FLAK procedure, the IOP increases in both recipient and donor eyes. The diameter of the trephinated donor and recipient corneal buttons was decreased as the initial baseline IOP increased. Ophthalmic surgeons can determine the AAC pressure based on the baseline IOP in the recipient patient.

Effect of culture medium on propagation and phenotype of corneal stroma–derived stem cells

Background aimsThe limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. MethodsPrimary human corneal stroma–derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. ResultsAll media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. ConclusionsCulture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.

Three‐dimensional structure of the mammalian limbal stem cell niche

PurposeAlthough the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its 3D architecture remains incomplete.MethodsThe LSC niche was explored on freshly excised corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full‐field optical coherence microscopy (FFOCM).ResultsLimbal crypt features were detected in 90% of human corneoscleral rims, extending between the palisades of Vogt as radial (74%) and/or rounded (23%) forms, often branching off to or becoming interconnected by sub‐scleral radially or circumferentially oriented crypts (56%). [kg1] Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss‐crossing invaginations beneath the sclera. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of limbal volume in humans, 9% in pigs and 7% in mice. FFOCM combined with fluorescence showed presence of p63‐α+ cells and cytokeratin‐3 + cells in the limbal crypts. LSC density increased with percentage limbal volume occupied by crypts. Colony Forming Efficiency increased with limbal crypt volume. [kg1] See how to rephrase all this.ConclusionsCrypt architecture in the three species appears associated with eye exposure to light. Clone production correlated strongly with the limbal crypt volume in humans indicating that limbal crypts constitute a niche for adult limbal stem cells. FFOCM imaging could assist in assessment of the richness of the limbal crypts and targeted biopsy for cell culture.

An Immunohistochemical Study of Inflammatory Cell Changes and Matrix Remodeling With and Without Acute Hydrops in Keratoconus.

To determine the inflammatory cell and matrix changes in advanced keratoconus, including acute hydrops, using immunohistochemical analysis. The corneal tissue from eight subjects with keratoconus undergoing corneal transplantation (three keratoconic buttons, five buttons post acute hydrops—one of them with extensive neovascularization following hydrops) was compared with tissue from two normal corneoscleral rims (n = 10). The corneas were sectioned and analyzed with specific markers for macrophages, lymphocytes, dendritic cells, and scar associated matrix molecules laminin, fibronectin, tenascin-C, and type III collagen. Populations of cells using markers for macrophages, leucocytes and antigen presenting cells were found to be associated with the epithelium and stroma of keratoconic tissue. Populations of these cells appeared decreased in hydrops-associated keratoconus except for a large increase in leucocytes in the stroma and endothelium associated with neovascularization. Extracellular matrix deposition was found to be uniquely demonstrated in localized areas of the stroma, corresponding to the site of hydrops involvement. Immunohistochemical analysis revealed a chronic, inflammatory process with recruitment of immunoinflammatory cells and deposition of scar tissue in keratoconus. The inflammatory markers were somewhat attenuated in hydrops-associated keratoconus corneas and thus inflammation was not considered to be a major factor in the development of acute corneal hydrops.

Three-dimensional structure of the mammalian limbal stem cell niche

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

Impact of the radius of the injector system on the cell viability in descemet membrane endothelial keratoplasty: an ex vivo experimental study.

To evaluate the impact of the radius of the injector system on the viability of the endothelial cells in Descemet membrane endothelial keratoplasty (DMEK). Descemet membranes (DMs) of 30 corneoscleral rims were used in a paired design and divided into three groups (A, B, C). The right corneas were used as control (without shooting through the injector after DM preparation) and the left cornea for simulation of the implantation step (shooting through a glass injector of different radii). The injector in Group A had a diameter of 0.5 mm, group B 0.9 mm and group C 1.4 mm. Prior to preparation, endothelial cell count was measured and endothelial cell quality assessed. Quantification of potential damage was achieved by staining with a viability bioassay (calcein acetoxymethyl and ethidium homodimer-1) and DAPI. Apoptosis was evaluated by TUNEL (terminal dUTP nick-end labelling) staining. The average percentage of the endothelial cell death ratios was 4.9 ± 3.3% in group A (n = 5), 3.7 ± 3.7% in group B (n = 5) and 3.9 ± 3.6% in group C (n = 5) with no significant difference compared to the control. The average percentage of apoptotic endothelial cells was 0.05 ± 0.05%, 0.60 ± 0.53% and 0.15 ± 0.26% in groups A, B and C, respectively, with no significant difference compared to the control. Prepared grafts for transplantation in DMEK procedures may be injected through a smaller sized injector system of up to 0.5 mm with no significant increase in cell loss. Clinical studies are necessary to draw a final conclusion.

Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in vitro.

Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behavior, and long-term fate after transplantation. The aim of this research was to evaluate, in vitro, quantum dot (Qdot) technology as a tool for tracking transplanted HLECs. In vitro cultured HLECs were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony-forming efficiency assays and expression of CK3, P63alpha, and ABCG2. Sheets of cultured HLECs labeled with Qdot nanocrystals were transplanted onto decellularized human corneoscleral rims in an organ culture model and observed to investigate the behavior of transplanted cells. Quantum dot labeling had no detrimental effect on HLEC viability or function in vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks after transplantation in our organ culture model. In addition, it was possible to label and observe epithelial cells in intact human corneas by using the Rostock corneal module adapted for use with the Heidelberg HRA. This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks after transplantation; however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterization in in vivo models are required.

Ultrastructural Changes in Human Trabecular Meshwork Tissue after Laser Trabeculoplasty.

Purpose. To compare morphologic changes in human trabecular meshwork (TM) after selective laser trabeculoplasty (SLT) and argon laser trabeculoplasty (ALT). Design. Laboratory evaluation of ex vivo human eye TM after laser trabeculoplasty. Methods. Corneoscleral rims from human cadaver eyes were sectioned and treated with varying powers of either SLT or ALT. Specimens were examined using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Results. TEM of SLT at all powers resulted in disrupted TM cells with cracked and extracellular pigment granules. SEM of SLT samples treated at high power revealed tissue destruction with scrolling of trabecular beams. SEM of ALT-treated tissue showed increasing destruction with exposure to higher power. The presence or absence of “champagne” bubbles during SLT did not alter the histologic findings. Conclusions. SLT-treated human TM revealed disruption of TM cells with cracked, extracellular pigment granules, particularly at higher treatment powers. Tissue scrolling was noted at very high SLT energy levels. ALT-treated tissue showed significant damage to both the superficial and deeper TM tissues in a dose-dependent fashion. Further studies are needed to guide titration of treatment power to maximize the IOP-lowering effect while minimizing both energy delivered and damage to target tissues.

Human limbal biopsy-derived stromal stem cells prevent corneal scarring.

Conventional allograft therapy for corneal scarring is widespread and successful, but donor tissue is not universally available, and some grafts fail owing to rejection and complications such as endothelial failure. We investigated direct treatment of corneal scarring using autologous stem cells, a therapy that, if successful, could reduce the need for corneal grafts. Mesenchymal cells were expanded from small superficial, clinically replicable limbal biopsies of human cadaveric corneo-scleral rims. Limbal biopsy-derived stromal cells (LBSCs) expanded rapidly in media containing human serum, were highly clonogenic, and could generate spheres expressing stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). Human LBSCs differentiated into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS) and organized a thick lamellar stroma-like tissue containing aligned collagen and keratan sulfate proteoglycans when cultured on aligned nanofiber substrata. When engrafted into mouse corneal wounds, LBSCs prevented formation of light-scattering scar tissue containing fibrotic matrix components. The presence of LBSCs induced regeneration of ablated stroma with tissue exhibiting lamellar structure and collagen organization indistinguishable from that of native tissue. Because the limbus can be easily biopsied from either eye of an affected individual and LBSCs capable of corneal stromal remodeling can be expanded under xeno-free autologous conditions, these cells present a potential for autologous stem cell-based treatment of corneal stromal blindness.

Peripheral deep anterior lamellar keratoplasty using a cryopreserved donor cornea for Terrien’s marginal degeneration

To evaluate the clinical efficacy of peripheral deep anterior lamellar keratoplasty (DALK) using a cryopreserved donor cornea for Terrien's marginal degeneration (TMD). Thirty-one eyes of 27 patients with TMD underwent peripheral DALK using cryopreserved donor corneas. According to the distance between the inner edge of the lesion and the limbus, a ring-shaped or D-shaped DALK was performed. All grafts were stored at -20 °C. Cryopreserved corneoscleral rims were prepared for ring-shaped grafts and cryopreserved whole eyeballs were prepared for D-shaped grafts. The general conditions, intraoperative performance, postoperative corneal reconstruction, astigmatism, best corrected visual acuity (BCVA), and various complications were analyzed. Ring-shaped DALK was performed in 28 eyes and D-shaped DALK was performed in 3 eyes. Postoperative follow-up time was (28.4±24.8) months. There was evidence of inflammation before surgery in 12 eyes (38.7%) and intraoperative perforation occurred in 13 eyes (41.9%). The corneal structures of all eyes were reconstructed. Postoperative astigmatism and BCVA showed improvement (both P=0.00) except for cases that underwent D-shaped DALK. Ten eyes (32.2%) developed transient ocular hypertension and one eye (3.2%) developed secondary glaucoma. No primary disease recurrence or corneal allograft rejection was observed. Peripheral DALK for TMD using cryopreserved donor tissue is an effective technique that eliminates rejection and extends the use of donor eyes. Inflammatory history or intraoperative perforation has no adverse effect on graft recovery. However, D-shaped DALK did not achieve good visual outcomes.

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells.

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-β signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell-cell junctions and instead activates Ras homologue gene family A (RhoA)-Rho-associated protein kinase (ROCK)-canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest-like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.

Risk factors for donor endothelial loss in eye bank-prepared posterior lamellar corneal tissue for descemet stripping automated endothelial keratoplasty.

The aim of this study was to investigate donor, tissue, and precut procedure risk factors for endothelial cell density (ECD) loss in posterior lamellar corneal tissue preparation by an eye bank for Descemet stripping automated endothelial keratoplasty. A total of 259 corneoscleral rims precut by the Singapore Eye Bank from October 2011 to August 2013 were evaluated. Donor characteristics, tissue characteristics, and precut procedure parameters were analyzed. The mean donor age was 57.18 ± 11.35 years, and the mean cutting transition time was 4.16 ± 0.75 seconds. The mean ECD was 2826 ± 225 and 2787 ± 224 cells per square millimeter before and after precutting, respectively, with an average ECD change of -1.38% ± 3.28%. The precutting procedure failure rate was 1.2%. Mutivariate regression analysis showed that an older donor age, a higher ECD before cutting, and a slower cutting transition speed were significant factors. Corneas with an ECD >2800 cells per square millimeter before precutting, cutting transition time >5.5 seconds, and corneas with donor age >65 years were significantly more likely to have greater than 5% ECD loss after precutting (odds ratio, 6.42, 1.66, and 1.62; 95% confidence interval, 1.44-29.43, 1.45-2.72, and 1.66-5.82, respectively). Donor source, death-to-preservation time (range, 0.67-10.88 hours), death-to-precutting time (range, 0-7 days), and graft thickness (range, 43-232 μm) were not statistically significant factors. The ECD loss in the precut tissue prepared by the eye bank was very low. The risk factors identified provide better understanding of how to improve the quality and safety profiles when preparing graft tissue for Descemet stripping automated endothelial keratoplasty.

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.