Long-Term Outcomes of Keratoprosthesis With Biological Haptic: A Review of 25 Years of Single-Center Surgeries.

PURPOSE: To study the outcome of eyes that underwent surgery for keratoprosthesis with a biological haptic, osteo-odonto-keratoprosthesis (OOKP) or tibia keratoprosthesis, by a single surgeon over a time span of more than 25 years. METHODS: One hundred thirty eyes that had received a keratoprosthesis with a biological haptic between 1994 and 2022 by a single surgeon were included in this retrospective analysis. Main outcome parameters were postoperative best corrected visual acuity, postoperative refractive error, postoperative complications, anatomical and functional survival of the prosthesis as well as comparison of subgroups of the 2 different types (OOKP n = 78; tibia keratoprosthesis n = 52) of keratoprostheses, and subgroup analysis of different indications for surgery. Patients were examined every 6 months. RESULTS: The longest follow-up was 25.8 years. Reasons for implantation were graft-vs-host disease (6.9%), vascularized corneas and dry eye (22.9%), physical or chemical burns (29.8%), Stevens-Johnson syndrome (9.9%), and ocular cicatricial pemphigoid (30.5%). The functional success rate with postoperative visual acuity of better than 0.7 log MAR was achieved by 56.9%. The OOKP subgroup showed a better mean visual outcome. 14 keratoprostheses (10.7%) had to be explanted over the whole time span. In the time leading to explantation, refraction showed a statistically significant myopic shift when compared with the non-explanted prosthesis. Anatomical survival rates were better for the OOKP in the first 12 years after implantation. CONCLUSIONS: The study shows that keratoprosthesis with a biological haptic has favorable long-term outcomes. The retention rate stayed very high with excellent functional outcomes.

Characterisation of ectasia after penetrating keratoplasty in keratoconus eyes using anterior segment optical coherence tomography.

BACKGROUND/AIMS: Ectasia of the cornea can occur decades after penetrating keratoplasty (PK), especially in keratoconus eyes. The purpose of this study was to characterise ectasia after PK by morphological findings in anterior segment optical coherence tomography (AS-OCT). METHODS: In this retrospective, single-centre case series, 50 eyes of 32 patients with a history of PK at an average of 25±10 years earlier were included. The eyes were classified either as ectatic (n=35) or as non-ectatic (n=15). The main parameters included central corneal thickness (CCT), lowest corneal thickness at the interface (LCTI), anterior chamber depth, graft-host interface angle at the thinnest point and host cornea-iris angle. Furthermore, steep and flat keratometry readings obtained by AS-OCT (CASIA-2, Tomey) and Scheimpflug tomography (Pentacam, Oculus) were assessed. OCT findings were correlated with clinical grading of ectasia. RESULTS: There was a highly significant difference in LCTI, graft-host interface angle and anterior chamber depth (in pseudophakic eyes) between the groups. The ratio calculated by the quotient of LCTI divided by CCT was significantly lower in ectatic than non-ectatic eyes (p<0.001). In eyes with an LCTI/CCT ratio of ≤0.7, the OR for the occurrence of a clinical detectable ectasia was 2.4 (CI 1.5 to 3.7). Steep keratometry values were significantly higher in ectatic eyes. CONCLUSION: AS-OCT is a helpful tool to recognise and quantify ectasia in post-PK eyes objectively.

Risk factors for ocular hypotony after XEN Gel Stent implantation.

PURPOSE: To investigate the incidence of postoperative hypotony, and risk factors for the development of hypotony in eyes who had undergone XEN Gel Stent implantation. METHODS: In this retrospective, single-centre case series, medical records of 170 consecutive eyes who had undergone XEN Gel Stent implantation with or without simultaneous phacoemulsification for primary or secondary open angle glaucoma were analysed. Primary outcome parameters were the incidence of postoperative hypotony and potential risk factors for its development, and secondary parameters were pre- and postoperative visual acuity, intraocular pressure (IOP), and number of IOP-lowering eye drops. RESULTS: Postoperative hypotony ≤ 6 mmHg occurred in 57% of eyes. Hypotony was without complications in 70.1%, 13.4% had transient complications with spontaneous resolution, and 16.5% had complications requiring treatment. Mean visual acuity logMAR before surgery accounted for 0.47 ± 0.46 in all eyes and 0.47 ± 0.48 at the 4-week visit. There was no significant difference of BCVA in the group of eyes with and without postoperative hypotony before and after surgery. The mean IOP before surgery was 24.6 ± 8.4 mmHg and decreased significantly to 18.4 ± 10.2 after 4 weeks. Eyes with an axial length over 24.3 mm had a threefold increased risk for postoperative hypotony (OR 3.226, 95% confidence interval 1.121-9.279). This risk was decreased in eyes with simultaneous cataract surgery (OR 0.483, 95% confidence interval 0.258-0.903). CONCLUSION: In our sample, postoperative hypotony was a common complication after XEN Gel Stent implantation, but serious, persistent complications were rare. A longer axial length predisposes the eye for the development of hypotony.

Post-COVID-19 Syndrome: Retinal Microcirculation as a Potential Marker for Chronic Fatigue.

Post-COVID-19 syndrome (PCS) is characterized by persisting sequelae after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). PCS can affect patients with all COVID-19 disease severities. As previous studies have revealed impaired blood flow as a provoking factor triggering PCS, it was the aim of the present study to investigate the potential association between self-reported chronic fatigue and retinal microcirculation in patients with PCS, potentially indicating an objective biomarker. A prospective study was performed, including 201 subjects: 173 patients with PCS and 28 controls. Retinal microcirculation was visualized by OCT angiography (OCT-A) and quantified using the Erlangen-Angio-Tool as macula and peripapillary vessel density (VD). Chronic fatigue (CF) was assessed according to the variables of Bell's score, age and gender. VDs in the superficial vascular plexus (SVP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP) were analyzed, considering the repetitions (12 times). Seropositivity for autoantibodies targeting G protein-coupled receptors (GPCR-AAbs) was determined by an established cardiomyocyte bioassay. Taking account of the repetitions, a mixed model was performed to detect possible differences in the least square means between the different groups included in the analysis. An age effect in relation to VD was observed between patients and controls (p < 0.0001). Gender analysis showed that women with PCS showed lower VD levels in the SVP compared to male patients (p = 0.0015). The PCS patients showed significantly lower VDs in the ICP as compared to the controls (p = 0.0001 (CI: 0.32; 1)). Moreover, considering PCS patients, the mixed model revealed a significant difference between those with chronic fatigue (CF) and those without CF with respect to VDs in the SVP (p = 0.0033 (CI: −4.5; −0.92)). The model included variables of age, gender and Bell's score, representing a subjective marker for CF. Consequently, retinal microcirculation might serve as an objective biomarker in subjectively reported chronic fatigue in patients with PCS.

Identification, Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma.

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells.

PURPOSE: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS: Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS: LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION: These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

Low- vs. high-dose radiotherapy in Graves' ophthalmopathy: a retrospective comparison of long-term results.

PURPOSE: Radiotherapy represents an effective treatment option in Graves' ophthalmopathy (GO), leading to palliation of clinical symptoms. However, there are only a limited number of trials comparing the effectiveness of low- vs. high-dose radiotherapy. METHODS: We analyzed 127 patients treated with radiotherapy for stage 3/4 GO (NOSPECS classification). Patients were treated with single doses of 2.0 Gy (cumulative dose 20 Gy) until 2007, afterwards a single dose of 0.8 Gy (cumulative dose 4.8 Gy) was applied. With a median follow-up-time of 9.0 years, the treatment efficacy (overall improvement, sense of eye pressure, lid edema, ocular motility, exophthalmos, subjective vision, and diplopia) and adverse effects were analyzed by a standardized survey. RESULTS: Overall, 63.8% described improvement of symptoms after radiotherapy. No significant differences in overall treatment response and improvement of main outcome measures between low- or high-dose radiotherapy treatments are detectable, while low-dose radiotherapy leads significantly more often to retreatment (13.1% vs. 1.7%, p = 0.016). The main independent predictor of treatment response is the presence of lid edema (odds ratio, OR, 3.53; p = 0.006). CONCLUSION: At long-term follow-up, the majority of patients reported palliation of symptoms with limited adverse effects, suggesting clinical effectiveness of radiotherapy for amelioration of GO symptoms independent of low- or high-dose radiotherapy.

Influence of corneal guttae and nuclear cataract on contrast sensitivity.

AIMS: To evaluate the contrast sensitivity in patients with nuclear cataract and corneal guttae compared to patients with nuclear cataract without guttae. METHODS: In this retrospective, single-centre case series, 50 eyes of 50 patients fulfilling the inclusion criteria were enrolled. Patients with corneal guttae and nuclear cataract (n=25, study group) underwent triple Descemet membrane endothelial keratoplasty (DMEK). Patients with nuclear cataract and healthy corneas underwent cataract surgery (n=25, control group). Inclusion criteria were preoperative best-corrected visual acuity ≥20/40, no corneal oedema and similar lens opacity (nuclear opalescence 2.0-2.9). Outcome measures included MARS letter and OPTEC 6500P contrast sensitivity test, corneal volume, central corneal thickness and anterior and posterior corneal densitometry. RESULTS: Preoperative MARS letter and OPTEC 6500P contrast sensitivity was significantly worse in the study group (MARS: p<0.001; OPTEC 6500P: p<0.007 at low spatial frequencies in daylight with and without glare and nightlight without glare). After surgery, there was no significant difference in MARS letter contrast sensitivity between groups (p=0.225). OPTEC 6500P contrast sensitivity remained significantly lower in the study group in daylight and nightlight with and without glare at most spatial frequencies (p<0.01) postoperatively. Preoperative and postoperative corneal volume, central corneal thickness and anterior corneal densitometry were equal in both groups (p>0.05). Posterior densitometry was significantly higher in the study group than in the control group preoperatively (p<0.001) but turned into equal values postoperatively (p=0.07). CONCLUSIONS: Corneal guttae cause an additional significant decrease in contrast sensitivity in eyes with nuclear cataract. This is in favour of performing a triple DMEK even in eyes with a visual acuity of ≥20/40.

Refractive Outcomes After Descemet Membrane Endothelial Keratoplasty + Cataract/Intraocular Lens Triple Procedure: A Fellow Eye Comparison.

PURPOSE: Descemet membrane endothelial keratoplasty is often combined with phacoemulsification and intraocular lens implantation (DMEK + cataract/IOL triple procedure) in phakic patients. This procedure results in a refractive shift that is difficult to predict. The aim of this study was to evaluate the hypothesis that the refractive shift in the second eye follows the shift in the first eye. METHODS: In this retrospective, single-center, consecutive case series, the refractive outcomes of 254 eyes of 127 patients who underwent DMEK + cataract/IOL triple procedure in both eyes for Fuchs endothelial corneal dystrophy have been analyzed. Main outcome measures were spherical equivalent outcome (shift calculations), best spectacle-corrected visual acuity, central corneal thickness, and posterior simulated keratometry. RESULTS: The mean best spectacle-corrected visual acuity before surgery was 0.51 ± 0.24 and increased to 0.19 ± 0.15 (logMAR) after surgery (P < 0.001). After surgery, a mean hyperopic shift of 0.98 ± 0.89 D was observed. The refractive shift was 1.03 ± 0.93 D and 0.92 ± 1.02 D, in the first and second eyes, respectively (P = 0.435). In a paired analysis, the mean difference of the refractive shift between the first and second eyes was 0.49 ± 0.43 D. CONCLUSIONS: In our fellow eye comparison, the refractive shift after DMEK + cataract/IOL triple procedure in the second eye was comparable with the shift in the first eye. As a consequence, the refractive outcome of the first eye might serve as a reference for optimizing the refractive target in the second eye. Further studies investigating the influence of corneal parameters on refractive shift are needed for a more predictable lens power selection.

Global Consensus on the Management of Limbal Stem Cell Deficiency.

PURPOSE: In recent decades, the medical and surgical treatment of limbal stem cell deficiency (LSCD) has evolved significantly through the incorporation of innovative pharmacological strategies, surgical techniques, bioengineering, and cell therapy. With such a wide variety of options, there is a need to establish a global consensus on the preferred approaches for the medical and surgical treatment of LSCD. METHODS: An international LSCD Working Group was established by the Cornea Society in 2012 and divided into subcommittees. Four face-to-face meetings, frequent email discussions, and teleconferences were conducted since then to reach agreement on a strategic plan and methods after a comprehensive literature search. A writing group drafted the current study. RESULTS: A consensus in the medical and surgical management of LSCD was reached by the Working Group. Optimization of the ocular surface by eyelid and conjunctival reconstruction, antiinflammatory therapy, dry eye and meibomian gland dysfunction treatment, minimization of ocular surface toxicity from medications, topical medications that promote epithelialization, and use of a scleral lens is considered essential before surgical treatment of LSCD. Depending on the laterality, cause, and stage of LSCD, surgical strategies including conjunctival epitheliectomy, amniotic membrane transplantation, transplantation of limbal stem cells using different techniques and sources (allogeneic vs. autologous vs. ex vivo-cultivated), transplantation of oral mucosal epithelium, and keratoprosthesis can be performed as treatment. A stepwise flowchart for use in treatment decision-making was established. CONCLUSIONS: This global consensus provides an up-to-date and comprehensive framework for the management of LSCD.

Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes.

Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell-cell and cell-matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.

Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells.

Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.

Supplementation of organ culture medium with dextran is not required in pre-stripped human donor tissue for DMEK surgery.

To assess corneal endothelial cell (CEC) quantity and quality in eye-bank prepared lamellar grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) from organ cultured donor corneas that have not undergone de-swelling by media supplementation with dextran. Prior to graft preparation, corneas that had not undergone de-swelling (n = 30) were placed into fresh storage medium without dextran (KMI). Corneas in the control group (n = 30) were placed in dehydration medium containing 5% dextran (KMII). Subtotal stripping of Descemet's membrane (DM) was performed manually. Following graft preparation, 10 corneas of each group were cultured further in their respective medium for 24 h, 72 h, or 120 h, respectively. Before and after DM stripping, as well as at the end of culture, CEC numbers were obtained. At the end of culture, CEC morphology was graded using a scoring system and CEC metabolism was assessed by detection of adenosine triphosphate. At 24 h after DM stripping, mean CEC counts (in cells/mm2) were 2204 in corneas stored in KMII, and 2391 in corneas stored in KMI (p = 0.003). This corresponds to a mean relative CEC loss of 12.4% with dextran versus 9.7% without dextran (p = 0.04). At 72 h, CEC counts were 1946 in KMII, and 2289 in KMI (p = 0.004). This corresponds to CEC loss of 23% with dextran versus 14% without dextran (p = 0.009). At 120 h, CEC counts were 2047 in KMII, and 2230 in KMI (p = 0.14). This corresponds to CEC loss of 22.7% with dextran versus 17.2% without dextran (p = 0.14). Also, at 120 h after DM stripping, 6/10 corneas fell below a threshold of 2000 cells/mm2 if stored in medium containing dextran, versus 1/9 corneas if stored without dextran (p = 0.003). Morphological assessment of CEC quality revealed equal scores for cell polymorphism (median = 1), granulation (median = 0) and segmentation (median = 1) in all groups. Lower ATP/protein ratios were observed in corneas stored in medium without dextran at 24 h (p < 0.001), 72 h (p < 0.001), and 120 h (p = 0.02). Abandoning the use of dextran in corneas destined for DMEK surgery leads to increased CEC counts and thereby serves to reduce tissue loss.

Can we predict the refractive outcome after triple Descemet membrane endothelial keratoplasty?

PURPOSE:: To analyze and correlate corneal parameters with refractive shift after Descemet membrane endothelial keratoplasty combined with cataract surgery (triple Descemet membrane endothelial keratoplasty). METHODS:: This single-center retrospective observational case series included 152 eyes of 152 consecutive patients undergoing triple Descemet membrane endothelial keratoplasty in the first eye for Fuchs endothelial corneal dystrophy. Patients were examined preoperatively, as well as at 3, 6, and 12 months after surgery. The main outcome measures were: refractive shift (predicted refractive outcome based on intraocular lens calculation compared to actual postoperative refractive outcome), central corneal thickness, corneal volume, anterior and posterior corneal curvature, and corneal densitometry. These parameters were analyzed and correlated with the refractive shift after surgery. RESULTS:: After 3 months from surgery, a mean refractive shift of +1.12 ± 1.10 D was observed and remained stable until the last follow-up at 12 months (+1.24 ± 1.07 D). Correlation analysis showed a weak but significant positive correlation between refractive shift and preoperative posterior curvature (rho = 0.314; p = 0.002) or preoperative posterior densitometry (rho = 0.227; p = 0.008). No correlation was found between refractive shift and preoperative central corneal thickness, corneal volume, anterior curvature, or anterior/mid-cornea densitometry. CONCLUSION:: Changes of the posterior cornea may have an influence on the refractive shift. Patients with flatter posterior corneal curvature or higher posterior corneal density seem to exhibit a higher hyperopic shift. The weak correlations indicate a poor predictive value of any preoperative parameter used in our study.

Fungal Interface Keratitis After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To evaluate the incidence, clinical course, and management of fungal interface keratitis (IK) after Descemet membrane endothelial keratoplasty (DMEK). METHODS: This is a single-center retrospective observational case series of 3950 eyes undergoing DMEK. Six eyes with fungal IK were detected and analyzed. Analysis included graft storage condition, incidence of fungal IK, identification of the pathogenic agent, topical/systemic and surgical treatment regimen, and best-corrected visual acuity. RESULTS: Fungal IK after DMEK occurred in 6 of 3950 cases (0.15%). Corneal grafts were either stored in Optisol-GS (n = 4) or in organ culture (n = 2). In all cases, Candida species were isolated (Candida tropicalis, Candida albicans, Candida orthopsilosis, and Candida guilliermondii). Four eyes developed fungal IK during the early postoperative period (3-5 d) and 2 eyes later at 16 to 42 days after surgery. All patients received topical and systemic antifungal treatment and intracameral application of antifungal agents. In the case of an early infection, graft removal was performed in 3 of 4 patients. Late infections were eradicated without graft exchange. Recurrence of fungal infection was observed in 1 case after early IK and in both cases after late IK. Final visual acuity ranged from 20/200 to 20/20. CONCLUSIONS: Fungal IK is a rare complication after DMEK. Based on our experience, we believe that treatment of early fungal IK with aggressive presentation should include both immediate graft exchange and intracameral application of voriconazole and amphotericin, in addition to topical and systemic antifungal treatment. Graft exchange seems not to be mandatory in late infections.

Assessment of Corneal Endothelium during Continued Organ Culture of Pre-Stripped Human Donor Tissue for DMEK Surgery.

PURPOSE: To measure corneal endothelial cell (EC) quality and quantity following Descemet membrane (DM) stripping of human donor corneas and continued storage in organ culture medium containing dextran. METHODS: DM stripping was performed in 30 organ cultured, corneoscleral discs. Corneas were divided into 3 groups of 10 corneas each. Baseline mean EC density (cells/mm2) was 2,372 (SD ± 259) in group 1, 2,540 (SD ± 266) in group 2, and 2,665 (SD ± 263) in group 3. Following subtotal DM stripping, culture was continued at 31°C for 24 hours (group 1), 72 hours (group 2), and 120 hours (group 3), respectively. EC density was measured before stripping and at the end of culture. At the end of culture, corneal EC morphology was graded using a scoring system and EC viability was measured by detection of adenosine triphosphate. RESULTS: At the end of culture, mean EC density was 2,159 (SD ± 293) in group 1, 1,946 (SD ± 182) in group 2, and 2,047 (SD ± 225) in group 3. This constitutes an EC loss of 9,1% (SD ± 5,3%) in group 1, 23,0 % (SD ± 6,5%) in group 2, and 22,7% (SD ± 9,1%) in group 3 (p < 0.001). After completion of follow-up, all groups contained corneas with EC counts < 2,000 cells/mm2. Cell morphology scores did not differ between the three experimental groups. EC viability measurements showed a tendency toward lower readings with extended length of culture. CONCLUSIONS: Corneal EC loss does occur following DM stripping and continued organ culture. EC loss increases with storage past 24 hours, but donor corneas may fall below 2,000 cells/mm2 independently of storage duration. The use of eye bank prepared donor lamellae for Descemet Membrane Endothelial Keratoplasty (DMEK) may increase patient safety by offering standardized quality control before tissue release.

Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells.

Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.

Trends in Corneal Transplantation from 2001 to 2016 in Germany: A Report of the DOG-Section Cornea and its Keratoplasty Registry.

PURPOSE: The purpose of this retrospective panel study was to provide an overview of absolute numbers and of trends in the types of and indications for corneal transplantation in Germany from 2001 to 2016. METHODS: A questionnaire about absolute numbers, types of transplantation, and indications was sent to 111 ophthalmologic departments in Germany, out of which 94 (85%) provided their data. RESULTS: Since the year 2001, the number of corneal transplantations has increased by 1.5-fold, from 4730 penetrating keratoplasties (PKPs) in 2001 to 7325 penetrating and lamellar keratoplasties in 2016. The shift from penetrating to lamellar procedures began in 2006. In 2014, lamellar procedures (231 [4%] anterior and 2883 [49%] posterior lamellar keratoplasties) surpassed PKPs (2721, 47%) for the first time. Main indications for keratoplasty in Germany (2016) are Fuchs endothelial corneal dystrophy (46%), pseudophakic corneal decompensation (bullous keratopathy, 13%), repeated keratoplasty after graft failure (11%), keratoconus (8%), and corneal scarring (6%; others: 16%). The number of Descemet membrane endothelial keratoplasties (DMEKs) was 12 times higher (3850, 53%) than Descemet stripping automated endothelial keratoplasties (DSAEKs, 319, 4.4%) in 2016. The proportion of deep anterior lamellar keratoplasties (DALKs) never exceeded 6% (269 in 2011). CONCLUSIONS: The number of keratoplasties in Germany has increased from 2001 to 2016. Since 2014, posterior lamellar keratoplasties have surpassed PKPs. There was a constant increase of DMEKs, with a 12-fold higher number compared to DSAEKs in 2016. The shorter recovery time after DMEK seems to contribute to the trend toward earlier operative intervention in corneal endothelial diseases.

Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier.

The goal of this protocol is to establish a procedure for cultivating stem cells on a fibrin carrier to allow for eventual transplantation to the eye. The ability to transfer stem cells to a patient is critical for treatment for a variety of disorders and wound repair. We took hair follicle stem cells from the vibrissae of transgenic mice expressing a dual reporter gene under the control of the Tet-on system and the keratin 12 promoter (Meyer- Blazejewska et al., 2011 ). A clonal growth assay was performed to enrich for stem cells. Once holoclones formed they were transferred onto a fibrin carrier and cultivated to obtain a confluent epithelial cell layer. Limbal stem cell deficient (LSCD) mice were used as the transplant recipient in order to test for successful grafting and eventual differentiation into a corneal epithelial phenotype.