Alteration of Gene Expression in Pathological Keratinization of the Ocular Surface.

Purpose: To investigate the molecular mechanism of pathological keratinization in the chronic phase of ocular surface (OS) diseases. Methods: In this study, a comprehensive gene expression analysis was performed using oligonucleotide microarrays on OS epithelial cells obtained from three patients with pathological keratinization (Stevens-Johnson syndrome [n = 1 patient], ocular cicatricial pemphigoid [n = 1 patient], and anterior staphyloma [n = 1 patient]). The controls were three patients with conjunctivochalasis. The expression in some transcripts was confirmed using quantitative real-time PCR. Results: Compared to the controls, 3118 genes were significantly upregulated by a factor of 2 or more than one-half in the pathological keratinized epithelial cells (analysis of variance P < 0.05). Genes involved in keratinization, lipid metabolism, and oxidoreductase were upregulated, while genes involved in cellular response, as well as known transcription factors (TFs), were downregulated. Those genes were further analyzed with respect to TFs and retinoic acid (RA) through gene ontology analysis and known reports. The expression of TFs MYBL2, FOXM1, and SREBF2, was upregulated, and the TF ELF3 was significantly downregulated. The expression of AKR1B15, RDH12, and CRABP2 (i.e., genes related to RA, which is known to suppress keratinization) was increased more than twentyfold, whereas the expression of genes RARB and RARRES3 was decreased by 1/50. CRABP2, RARB, and RARRES3 expression changes were also confirmed by qRT-PCR. Conclusions: In pathological keratinized ocular surfaces, common transcript changes, including abnormalities in vitamin A metabolism, are involved in the mechanism of pathological keratinization.

Topical and oral peroxisome proliferator-activated receptor-α agonist ameliorates diabetic corneal neuropathy.

Diabetic corneal neuropathy (DCN) is a common diabetic ocular complication with limited treatment options. In this study, we investigated the effects of topical and oral fenofibrate, a peroxisome proliferator-activated receptor-α agonist, on the amelioration of DCN using diabetic mice (n = 120). Ocular surface assessments, corneal nerve and cell imaging analysis, tear proteomics and its associated biological pathways, immuno-histochemistry and western blot on PPARα expression, were studied before and 12 weeks after treatment. At 12 weeks, PPARα expression markedly restored after topical and oral fenofibrate. Topical fenofibrate significantly improved corneal nerve fibre density (CNFD) and tortuosity coefficient. Likewise, oral fenofibrate significantly improved CNFD. Both topical and oral forms significantly improved corneal sensitivity. Additionally, topical and oral fenofibrate significantly alleviated diabetic keratopathy, with fenofibrate eye drops demonstrating earlier therapeutic effects. Both topical and oral fenofibrate significantly increased corneal ß-III tubulin expression. Topical fenofibrate reduced neuroinflammation by significantly increasing the levels of nerve growth factor and substance P. It also significantly increased ß-III-tubulin and reduced CDC42 mRNA expression in trigeminal ganglions. Proteomic analysis showed that neurotrophin signalling and anti-inflammation reactions were significantly up-regulated after fenofibrate treatment, whether applied topically or orally. This study concluded that both topical and oral fenofibrate ameliorate DCN, while topical fenofibrate significantly reduces neuroinflammation.

Amelioration of Fibrosis via S1P Inhibition Is Regulated by Inactivation of TGF-ß and SPL Pathways in the Human Cornea.

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.

Objective Analysis of Corneal Nerves and Dendritic Cells. / Objektive Analyse von Hornhautnerven und dendritischen Zellen.

Corneal nerves and dendritic cells are increasingly being visualised to serve as clinical parameters in the diagnosis of ocular surface diseases using intravital confocal microscopy. In this review, different methods of image analysis are presented. The use of deep learning algorithms, which enable automated pattern recognition, is explained in detail using our own developments and compared with other established methods.

The inferocentral whorl region and its directional patterns in the corneal sub-basal nerve plexus: A review.

There has been a growing application of in vivo confocal microscopy (IVCM) in the examination of corneal microstructure, including different corneal layers and corneal nerve fibers in health and in pathological conditions. Corneal nerves forming the sub-basal nerve plexus (SBNP) beneath the corneal basal epithelial cell layer in particular have been intensively researched in health and disease as a marker for corneal neurophysioanatomical and degenerative changes. One intriguing feature in the SBNP that is found inferior to the corneal apex, is a whorl-like pattern (or vortex) of nerves, which represents an anatomical landmark. Evidence has indicated that the architecture of this 'whorl region' is dynamic, changing with time in healthy individuals but also in disease conditions such as in diabetic neuropathy and keratoconus. This review summarizes the known information regarding the characteristics and significance of the whorl region of nerves in the corneal SBNP, as a potential area of high relevance for future disease monitoring and diagnostics.

Clinical and pathologic characterization of a mouse model of graded limbal stem cell deficiency.

Limbal stem cell deficiency (LSCD) is a clinically challenging eye disease caused by damage to limbal stem cells (LSCs). Currently, the international consensus classifies LSCD into three clinical stages based on the disease severity. However, no existing animal models attempt to replicate the varying degrees of LSCD observed in clinical cases. The present study demonstrates an easy-to-create, reproducible, and reliable mouse model of graded LSCD. To achieve mild, moderate, or severe LSCD, filter paper rings with a variety of central angles (90°, 180°, or 270°) are utilized to deliver alkali burns to different sizes of the limbal area (1, 2, or 3 quarters). The animal model has successfully resulted in the development of clinical signs and pathological manifestations in escalating severity that are similarly observed in the three clinical stages of LSCD. Our study thus provides new insights into distinct pathological features underlying different grades of LSCD and serves as a new tool for further exploring the disease mechanisms and developing new effective therapeutics for repairing damaged LSCs.

Nanohydroxyapatite Coating Attenuates Fibrotic and Immune Responses to Promote Keratoprosthesis Biointegration in Advanced Ocular Surface Disorders.

Keratoprosthesis (KPro) implantation is frequently the only recourse for patients with severe corneal disease. However, problems arise due to inadequate biointegration of the KPro, particularly the PMMA optical cylinder, such as tissue detachment, tissue melting, or eye-threatening infection in the interface. Here, using the AuroKPro as a model prosthesis, a surface functionalization approach─coating the optical cylinder with nanohydroxyapatite (nHAp)─was trialed in rabbit eyes with and without a proceeding chemical injury. In chemically injured eyes, which simulated total limbal epithelial stem cell deficiency, clear benefits were conferred by the coating. The total modified Hackett-McDonald score and area of tissue apposition differences 12 weeks after implantation were 5.0 and 22.5%, respectively. Mechanical push-in tests revealed that 31.8% greater work was required to detach the tissues. These differences were less marked in uninjured eyes, which showed total score and tissue apposition differences of 2.5 and 11.5%, respectively, and a work difference of 23.5%. The improved biointegration could be contributed by the attenuated expression of fibronectin (p = 0.036), collagen 3A1 (p = 0.033), and α-smooth muscle actin (p = 0.045)─proteins typically upregulated during nonadherent fibrous capsule envelopment of bioinert material─adjacent to the optical cylinders. The coating also appeared to induce a less immunogenic milieu in the ocular surface tissue, evidenced by the markedly lower expression of tear proteins associated with immune and stimulus responses. Collectively, the level of these tear proteins in eyes with coated prostheses was 1.1 ± 13.0% of naïve eyes: substantially lower than with noncoated KPros (246.5 ± 79.3% of naïve, p = 0.038). Together, our results indicated that nHAp coating may reduce the risk of prosthesis failure in severely injured eyes, which are representative of the cohort of KPro patients.

Topical insulin for refractory persistent corneal epithelial defects.

The aim was clinical evaluation of the efficacy of topical insulin eye drops in patients with refractory persistent epithelial defects (PEDs). This prospective non-randomized investigation was conducted to examine the efficacy of insulin eye drops in treating patients with PEDs that did not respond to conventional therapy. A total of twenty-three patients were included in the study, and they were administered insulin eye drops formulated as 1 U/mL, four times a day. The rate of epithelial defect resolution and time to complete corneal re-epithelialization were considered primary outcome measures. The relative prognostic impact of initial wound size and other parameters, including age, sex, smoking, diabetes, and hypertension were also analyzed. The results showed that during follow-up (maximum 50 days), a total of 16 patients (69.6%) achieved improvement. Insulin eye drops significantly reduced the corneal wounding area in 75% of patients with small epithelial defects (5.5 mm2 or less) during 20 days. Only 61% of patients with moderate epithelial defects (5.51-16 mm2) showed a significant recovery in 20-30 days. Also, 71% of patients with a defect size greater than 16 mm2, demonstrated a significant improvement in the rate of corneal epithelial wound healing in about 50 days. In conclusion topical insulin reduces the PED area and accelerates the ocular surface epithelium wound healing.

Corneal fibrosis: From in vitro models to current and upcoming drug and gene medicines.

Fibrotic diseases are characterised by myofibroblast differentiation, uncontrolled pathological extracellular matrix accumulation, tissue contraction, scar formation and, ultimately tissue / organ dysfunction. The cornea, the transparent tissue located on the anterior chamber of the eye, is extremely susceptible to fibrotic diseases, which cause loss of corneal transparency and are often associated with blindness. Although topical corticosteroids and antimetabolites are extensively used in the management of corneal fibrosis, they are associated with glaucoma, cataract formation, corneoscleral melting and infection, imposing the need of far more effective therapies. Herein, we summarise and discuss shortfalls and recent advances in in vitro models (e.g. transforming growth factor-ß (TGF-ß) / ascorbic acid / interleukin (IL) induced) and drug (e.g. TGF-ß inhibitors, epigenetic modulators) and gene (e.g. gene editing, gene silencing) therapeutic strategies in the corneal fibrosis context. Emerging therapeutical agents (e.g. neutralising antibodies, ligand traps, receptor kinase inhibitors, antisense oligonucleotides) that have shown promise in clinical setting but have not yet assessed in corneal fibrosis context are also discussed.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Associations between corneal curvature and other anterior segment biometrics in young myopic adults.

To investigate the associations between corneal curvature (CC) and other anterior segment biometrics in young myopic adults. In this retrospective multi-center study, 7893 young myopic adults were included. CC and other anterior segment biometrics were measured by Scheimpflug imaging (Pentacam). CC was defined as SimK at central 3 mm area, and other anterior segment biometrics included white-to-white corneal diameter (WTW), central corneal thickness (CCT), corneal volume (CV) at 3 mm, 5 mm, and 7 mm area, anterior corneal astigmatism (ACA), posterior corneal astigmatism (PCA), anterior corneal eccentricity (ACE) and asphericity (ACAP), posterior corneal eccentricity (PCE) and asphericity (PCAP), anterior chamber depth (ACD), and anterior chamber volume (ACV). Univariate regression analyses were used to assess the associations between CC and other anterior segment biometrics, and multivariate regression analyses were further performed to adjusted for age, gender and spherical equivalent. CC was higher in patients of female gender and higher myopia (all P < 0.05). Eyes in higher CC quartiles had lower WTW, thinner CCT, lower CV at 3 mm and 5 mm, lower ACD, and lower ACV (all P < 0.001), but had larger ACA, larger PCA, less PCE and less PCAP (all P < 0.001), compared to eyes in lower CC quartiles. The trends of CV at 7 mm, ACE and ACAP were inconsistent in different CC quartiles. After adjusting for age, gender and spherical equivalent with multivariate linear regression, CC was positively correlated to CV at 7 mm (ßs = 0.069), ACA (ßs = 0.194), PCA (ßs = 0.187), ACE (ßs = 0.072), PCAP (ßs = 0.087), and ACD (ßs = 0.027) (all P < 0.05), but was negatively correlated to WTW (ßs = - 0.432), CCT (ßs = - 0.087), CV-3 mm (ßs = - 0.066), ACAP (ßs = - 0.043), PCE (ßs = - 0.062), and ACV (ßs = - 0.188) (all P < 0.05). CC was associated with most of the other anterior segment biometrics in young myopic adults. These associations are important for better understanding of the interactions between different anterior segment structures in young myopic patients, and are also useful for the exploration of the pathogenesis of myopia.

Advances in Organoid Technology: A Focus on Corneal Limbal Organoids.

Organoid technology provides a versatile platform for simulating organogenesis, investigating disease pathogenesis, and exploring therapeutic interventions. Among various types of organoids that have been developed, corneal limbal organoids, the three-dimensional miniaturized corneas which are derived from either pluripotent stem cells or limbal epithelial stem cells, are particularly promising for clinical translation. This narrative review summarized the state-of-the-art in corneal limbal organoids research including the cultivation methods, clinical relevance and its limitations and challenges. The potential of corneal limbal organoids in mimicking corneal development, disease modelling, drug screening, and regenerative medicine was discussed. Technical improvements in cultivation techniques, imaging modalities, and gene editing tools are anticipated to overcome current limitations and further promote its clinical potential. Despite challenges and difficulties, the development of corneal limbal organoids opens a new era of regenerative medicine and provides a potential source of stem cell replacement therapies for challenging corneal diseases with the establishment of an in vitro corneal limbal organoid bank.

SMAD4-Dependent Signaling Pathway Involves in the Pathogenesis of TGFBR2-Related CE-like Phenotype.

(1) Background: Our previous data indicated that disturbance of the Transforming Growth Factor beta (TGFB) signaling pathway via its Type-2 Receptor (TGFBR2) can cause a Corneal Ectasia (CE)-like phenotype. The purpose of this study is to elucidate whether the SMAD4-dependent signaling pathway is involved in the TGFBR2-related CE-like pathogenesis. (2) Methods: Smad4 was designed to be conditionally knocked out from keratocytes. Novel triple transgenic mice, KerartTA; Tet-O-Cre; Smad4flox/flox (Smad4kera-cko), were administered with doxycycline (Dox). Optical Coherence Tomography (OCT) was performed to examine Central Corneal Thickness (CCT), Corneal Radius, Anterior Chamber and CE-like phenotype and compared to the littermate Control group (Smad4Ctrl). (3) Results: The OCT revealed normal cornea in the Smad4Ctrl and a CE-like phenotype in the Smad4kera-cko cornea, in which the overall CCT in Smad4kera-cko was thinner than that of Smad4Ctrl at P42 (n = 6, p < 0.0001) and showed no significant difference when compared to that in Tgfbr2kera-cko. Furthermore, the measurements of the Anterior Chamber and Corneal Radius indicated a substantial ectatic cornea in the Smad4kera-cko compared to Smad4Ctrl. The H&E staining of Smad4kera-cko mimics the finding in the Tgfbr2kera-cko. The positive immunostaining of cornea-specific marker K12 indicating the cell fate of cornea epithelium remained unchanged in Smad4kera-cko and the Proliferating Cell Nuclear Antigen (PCNA) immunostaining further indicated an enhanced proliferation in the Smad4kera-cko. Both immunostainings recapitulated the finding in Tgfbr2kera-cko. The Masson's Trichrome staining revealed decreased collagen formation in the corneal stroma from both Smad4kera-cko and Tgfbr2kera-cko. The collagen type 1 (Col1a1) immunostaining further confirmed the reduction in collagen type 1 formation in Smad4kera-cko. (4) Conclusions: The aforementioned phenotypes in the Smad4kera-cko strain indicated that the SMAD4-dependent signaling pathway is involved in the pathogenesis of the CE-like phenotype observed in Tgfbr2kera-cko.

[Severe Conjunctival and Corneal Epithelial Dysplasia: A Case Series]. / Schwere konjunktivale und korneale epitheliale Dysplasie: eine Fallserie.

BACKGROUND/OBJECTIVES: Ocular surface squamous neoplasia (OSSN) are among the most frequent non-pigmented malignancies of the ocular surface. They have a wide range of histological characteristics - ranging from mild epithelial dysplasia to invasive carcinoma of the squamous cells of the cornea. They may be restricted to the conjunctiva or also involve the cornea. As there are no leading symptoms in the early stages, diagnosis may be very delayed in patients who do not receive regular ophthalmological treatment. The present case series describes clinical and histological data on OSSN and includes clinical and histological data on OSSN, including possible clinical presentations, important risk factors, special histological and cytological features and therapeutic options. METHODS: Retrospective case series of patients with histologically confirmed severe epithelial dysplasia of the conjunctiva and cornea consistent with OSSN who presented to the Department of Ophthalmology in Basel University Hospital. The analysis covered demographic data, symptoms, diagnostic testing (photo documentation, brush biopsy), treatment and cytological and/or histological material and findings. RESULTS: We report on five patients aged between 41 and 92 years at the time of diagnosis. The histological findings in all patients included severe epithelial dysplasia, but with a heterogenous clinical presentation. In all cases, the lesion started in the conjunctiva, but traversed the limbus and extended to the cornea. The primary treatment was always surgical removal. In one patient, this had to be repeated several times due to recurrent metaplasia and was complemented by subsequent mitomycin C therapy. The clinical outcome ranged between total restitution of the original state to inevitable enucleation. CONCLUSION: The clinical presentation of OSSN is highly heterogenous, so that the initial diagnosis is difficult. There are no official guidelines for treatment, so that the treatment of choice varied between clinics. Regular ophthalmological follow-ups are recommended, even after complete surgical excision. Possible relevant concomitant diseases and risk factors must be identified before therapy.

Chronic inflammation as a proposed risk factor for ocular surface squamous neoplasia.

PURPOSE: Chronic inflammation is a predisposing factor for metaplastic changes and ultimately dysplasia. We describe cases of OSSN occurring in the setting of chronic ocular surface inflammation. METHODS: Sixteen eyes from 14 individuals were included from one ocular oncology clinic between 2010 and 2023. Patients presented with ocular surface squamous neoplasia (OSSN) in the setting of chronic inflammation. The diagnosis of OSSN was made using anterior segment high-resolution optical coherence tomography (HR-OCT) and confirmed by histopathological analysis in all cases. RESULTS: Median age on presentation was 61 [IQR 47.5-69.2] years. Eleven (86%) individuals were male and five (36%) identified as White Hispanic. Ten eyes were referred with ocular surface diagnoses including pannus (n = 4), scarring (n = 3), pterygium (n = 2), and herpetic keratitis (n = 1). Only six eyes were referred as possible neoplasia. All individuals had a history of ocular surface inflammation. The most common inflammatory conditions were ocular rosacea (seven individuals) and atopic keratoconjunctivitis (AKC) (five individuals). Two individuals were found to have bilateral OSSN, one in the setting of ocular rosacea and the other in the setting of AKC. All 16 eyes from 14 individuals were suspected to have OSSN based on HR-OCT findings which guided the location of the incisional biopsies that subsequently confirmed histopathological diagnosis in all cases. CONCLUSION: OSSN may arise in the setting of chronic inflammation on the ocular surface. Identification of the tumor can be challenging in these cases, and HR-OCT can be a key diagnostic tool in detecting OSSN.

Effects of Diabetes Mellitus on Corneal Immune Cell Activation and the Development of Keratopathy.

Diabetes mellitus (DM) is one of the most prevalent diseases globally, and its prevalence is rapidly increasing. Most patients with a long-term history of DM present with some degree of keratopathy (DK). Despite its high incidence, the underlying inflammatory mechanism of DK has not been elucidated yet. For further insights into the underlying immunopathologic processes, we utilized streptozotocin-induced mice to model type 1 DM (T1D) and B6.Cg-Lepob/J mice to model type 2 DM (T2D). We evaluated the animals for the development of clinical manifestations of DK. Four weeks post-induction, the total frequencies of corneal CD45+CD11b+Ly-6G- myeloid cells, with enhanced gene and protein expression levels for the proinflammatory cytokines TNF-α and IL-1ß, were higher in both T1D and T2D animals. Additionally, the frequencies of myeloid cells/mm2 in the sub-basal neural plexus (SBNP) were significantly higher in T1D and T2D compared to non-diabetic mice. DK clinical manifestations were observed four weeks post-induction, including significantly lower tear production, corneal sensitivity, and epitheliopathy. Nerve density in the SBNP and intraepithelial terminal endings per 40x field were lower in both models compared to the normal controls. The findings of this study indicate that DM alters the immune quiescent state of the cornea during disease onset, which may be associated with the progressive development of the clinical manifestations of DK.

Allogeneic limbo-deep anterior lamellar keratoplasty (Limbo-DALK)-A novel surgical technique in corneal stromal disease and limbal stem cell deficiency.

PURPOSE: To describe a novel corneal surgical technique combining Deep Anterior Lamellar Keratoplasty (DALK) with grafting of allogeneic limbus (Limbo-DALK) for the treatment of eyes with corneal stromal pathology and limbal stem cell deficiency (LSCD). METHODS: Clinical records of six Limbo-DALKs performed in five patients diagnosed with LSCD and corneal stromal pathology requiring keratoplasty were retrospectively reviewed. All patients were diagnosed with LSCD due to various pathologies including thermal and chemical burns, congenital aniridia or chronic inflammatory ocular surface disease. Parameters analysed included demographics, diagnoses, clinical history, thickness measurements using anterior segment OCT, visual acuity, and epithelial status. Regular follow-up visits were scheduled at 6 weeks as well as 3, 6, 9, and 12 and 18 months postoperatively. Main outcome measures were time to graft epithelialisation and the occurrence of corneal endothelial decompensation. RESULTS: Two grafts showed complete epithelial closure at 2 days, two at 14 days. In one eye, complete epithelial closure was not achieved after the first Limbo-DALK, but was achieved one month after the second Limbo-DALK. No endothelial decompensation occurred except in one patient with silicone oil associated keratopathy. Endothelial graft rejection was not observed in any of the grafts. CONCLUSION: Based on the data from this pilot series, limbo-DALK appears to be a viable surgical approach for eyes with severe LSCD and corneal stromal pathology, suitable for emergency situations (e.g. corneal ulceration with impending corneal perforation), while minimising the risk of corneal endothelial decompensation.