Corneal Neuropathic Pain: A Patient and Physician Perspective.

Corneal neuropathic pain (CNP) is a debilitating condition characterized by pain in the absence of a noxious stimulus. Symptoms such as ocular stinging, burning, photophobia, irritation, and a deep aching pain can be severe despite a seemingly normal ocular surface on examination. CNP may develop due to either peripheral or central sensitization. Peripheral sensitization develops due to aberrant regeneration of corneal nociceptors and nerve fibers as a result of corneal injury or disease of peripheral corneal nerves. Whereas, central sensitization develops due to upregulation of excitatory neurotransmitters as a result of chronic inflammation, which leads to amplification of neuronal response to stimuli. Unfortunately, due to the disparity in severity of symptomology and the observable signs on examination, patients' symptoms are commonly thought to be "psychological" or "functional", and patients report feeling ignored and neglected. Additionally, diagnosis is often delayed which adversely affects patient outcomes. Research to date has focused on the scientific aspects of corneal neuropathic pain: its pathophysiology, epidemiology, investigations, and management. Research into the patient personal experience and the challenges faced by individual patients and their clinicians is lacking. We present the patient and physician perspective on the journey of both patients in order to provide insights into the challenges faced by patients and physicians in the diagnosis, assessment, and management of corneal neuropathic pain.

Corneal neuropathic pain is a rare disease that causes patients to experience severe eye pain without a painful stimulus. Patients often experience one or more symptoms, such as ocular stinging, burning, irritation, photophobia, and deep aching pains even though ophthalmologists cannot see any abnormality when they examine the eyes. Due to a significant lack of awareness and understanding of the condition, diagnosis is often delayed and patients are left feeling ignored or neglected. Research to date has focused on the scientific aspects of corneal neuropathic pain such as what causes it, the tests doctors use to diagnose it, and the best treatments to manage it. In this article, we present the patient and physician perspective, an understanding of which we believe can improve the care that patients receive and can promote awareness and understanding of the condition, facilitating early diagnosis and treatment.

Minimally invasive corneal neurotization for neurotrophic keratopathy: The potential effect of age, denervation chronicity and lesion location.

Purpose: Neurotrophic keratopathy (NK) is an uncommon but challenging clinical condition characterized by altered corneal nerves and sensation leading to corneal damage. Corneal neurotization, a surgical technique that aims to "re-innervate" the cornea, has gained increasing popularity in view of the potential to permanently improve or even restore the normal corneal sensation. In this study, we aimed to report the outcomes of two cases of NK that underwent indirect minimally invasive corneal neurotization (MICN) with a sural nerve autograft, and to provide plausible explanations for the observed clinical outcomes. Observations: This was an interventional case series of two patients who underwent MICN for severe unilateral NK. The MICN technique was adapted from the technique originally described by Elbaz et al., in 2014. Clinical severity of NK was graded according to Mackie's grading system. Corneal sensation was measured using the Cochet-Bonnet esthesiometer (0-60mm) and corneal nerves were examined using in vivo confocal microscopy (IVCM) with Heidelberg HRT3 Rostock Corneal Module. Patient 1 was a 70-year-old man with a right grade III NK following trigeminal nerve decompression for trigeminal neuralgia. Patient 2 was a 62-year-old man with a left grade II NK following a left-sided acoustic neuroma resection. The denervation time was 23 years for both patients. Following the MICN surgery, none of the patients achieved sustained improvement in the corneal sensation (though patient 1 achieved a transient improvement in central corneal sensation to 20mm at 4 months' postoperative before returning to 0mm at 6 months' postoperative). IVCM did not reveal any changes in the corneal nerve density and morphology post-MICN. Conclusions and Importance: Based on our observations and the literature, we postulate that long denervation time, proximal injury to the trigeminal nerve and older patient age may serve as poor prognostic factors for MICN. As CN is being increasingly adopted in clinical practice for treating NK, understanding of these potential factors will facilitate better risk-benefit stratification and patient counselling. Future larger studies are required to elucidate these findings.

Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

INTRODUCTION: We aimed to determine the expression of inflammatory cytokines in the tears of patients with unilateral total limbal stem cell deficiency (TLSCD) caused by chemical burns before and after autologous cultivated limbal epithelial stem cell transplantation (CLET). METHODS: Tear samples were collected from both eyes of 23 patients with unilateral TLSCD and 11 healthy controls, at fixed timepoints before and after CLET. Dissolved molecules were extracted from Schirmer's strips using a standardised method and analysed on an array plate of ten inflammatory cytokines (V-Plex Proinflammatory Panel 1 Human Kit, MSD). RESULTS: IL1ß expression was significantly elevated in the TLSCD eye compared with the unaffected eye at baseline (p < 0.0001) but decreased to normal 3 months post-CLET (p = 0.22). IL6 and IL8 were unaffected at baseline but significantly elevated in the TLSCD eyes at 1 month post-CLET (p = 0.001 and p < 0.0001, respectively). IL6 returned to normal at 3 months and IL8 at 6 months post-CLET. There was a significant renewed increase in IL1ß, IL6 and IL8 expression at 12 months post-CLET (p < 0.0001, p = 0.0001 and p = 0.0003, respectively). IFNγ, IL10 and IL12p70 expression were significantly reduced in both eyes of patients with unilateral TLSCD at all timepoints. CONCLUSION: IL1ß is a specific marker of inflammation in TLSCD eyes that could be therapeutically targeted pre-CLET to improve stem cell engraftment. At 12 months post-CLET the spike in levels of IL1ß, IL6 and IL8 coincides with cessation of topical steroids, suggesting ongoing subclinical inflammation. We therefore recommend not discontinuing topical steroid treatment in cases where penetrating keratoplasty is indicated; however, further investigation is needed to ascertain this. TRIAL REGISTRATION: European Union Drug Regulating Authorities Clinical Trials Database (EuDRACT 2011-000608-16); ISRCTN (International Standard Randomised Controlled Trial Number (isrctn51772481).

National survey of corneal cross-linking (CXL) practice patterns in the United Kingdom during 2019.

OBJECTIVE: To provide an insight into trends in corneal cross-linking (CXL) practice in the UK, including criteria for progression of corneal ectasia, identification of patients for CXL, the CXL procedure itself and post-operative management. METHODS: All ophthalmologist members of the UK Cross-linking (UK-CXL) Consortium were invited to complete an online survey about CXL practice for the year 2019. The data collected was anonymised by site and analysed with descriptive statistics. RESULTS: Responses were received from 16 individual CXL centres (16/38; 42% response rate) and the data represented ~2,000 CXL procedures performed in the UK in 2019. The commonest indication for CXL was progressive keratoconus. Between centres, there were variations in diagnostic evaluation, patient selection for CXL, the CXL procedure and the pre- and post-operative monitoring of patients. CONCLUSION: Consistent with the wide number of CXL treatment techniques described in the published literature world-wide, variations in the monitoring of corneal ectasia, indications for CXL, CXL practice and post-CXL follow-up were found to exist between UK-based CXL centres.

Long-Term Graft Survival and Decline in Endothelial Cell Density Following Penetrating Keratoplasty with Organ-Cultured Corneas.

INTRODUCTION: Endothelial cell density (ECD) changes long after penetrating keratoplasty (PKP) of organ-cultured corneas have been little studied. We aim to calculate the point when ECD decline stabilises following PKP with organ culture stored corneas. METHODS: This is an observational study of first-ever PKPs and first-ever re-grafts, performed over 17 years under a single surgeon. ECDs were acquired at 3 and 6 months, 1 year post-graft and annually thereafter by specular microscopy. Time-dependent ECD data was fitted to a log-biexponential model. RESULTS: We studied 465 first-ever grafts and 128 re-grafts. Mean recipient age was 59 years (range 0-96 years; SD 22). Median follow-up was 5.7 (range 0.2-17.1) years. Probability of ED at 5 years in first grafts and re-grafts was 4.4% (2.6-7.1%) and 14.8% (8.3-23.2%). In first grafts, ECD loss reached 0.6% per annum at 7.9 (6.2-9.6) years post-operatively. The half-lives of ECD loss during the immediate post-operative period for first grafts, re-grafts, dystrophies, ectasias, and previous ocular surgery are 20.1 (14.9-30.9), 12.8 (6.9-79.4), 19.5 (13.1-37.7), 26.2 (16.2-68), and 11.6 (6.7-41.3) months, respectively. The half-life during this rapid phase of ECD loss has an inverse correlation with graft survival at 10 years (r = - 0.89, p = 0.02). CONCLUSIONS: Rate of endothelial decompensation is higher in first grafts than re-grafts. ECD decline stabilises 7.9 years post-operatively in first grafts but then becomes lower than the physiological loss expected. Further work is needed to verify whether organ-cultured grafts reach physiological levels of ECD loss faster than hypothermically stored grafts.

Practice patterns of corneal transplantation in Europe: first report by the European Cornea and Cell Transplantation Registry.

PURPOSE: To report practice patterns of corneal transplantation in Europe. SETTING: Corneal clinics in 10 European member states (MS), the United Kingdom, and Switzerland. DESIGN: Multinational registry study. METHODS: Corneal transplant procedures registered in the European Cornea and Cell Transplantation Registry were identified. Preoperative donor and recipient characteristics, indication and reason for transplantation, and surgical techniques were analyzed. RESULTS: A total of 12 913 corneal transplants were identified from 10 European Union MS, the United Kingdom, and Switzerland. Most countries were self-sufficient with regard to donor tissue. Fuchs endothelial corneal dystrophy was the most common indication (41%, n = 5325), followed by regraft (16%, n = 2108), pseudophakic bullous keratopathy (12%, n = 1594), and keratoconus (12%, n = 1506). Descemet stripping automated endothelial keratoplasty (DSAEK, 46%, n = 5918) was the most commonly performed technique, followed by penetrating keratoplasty (30%, n = 3886) and Descemet membrane endothelial keratoplasty (9%, n = 1838). Vision improvement was the main reason for corneal transplantation (90%, n = 11 591). Surgical technique and reason for transplantation differed between indications. CONCLUSIONS: This report provides the most comprehensive overview of corneal transplantation practice patterns in Europe to date. Fuchs endothelial dystrophy is the most common indication, vision improvement the leading reason, and DSAEK the predominant technique for corneal transplantation.

Effects of whole globe enucleation versus in situ corneoscleral excision on donor cornea tissue quality: a systematic review protocol.

OBJECTIVE: The objective of this review is to systemically compare the effects of whole globe enucleation versus in situ corneoscleral excision on donor cornea tissue quality. INTRODUCTION: Corneal transplantation serves as a sight-restoring surgery for corneal diseases, but the treatment is limited by the persistent shortage of donor corneas globally. Whole globe enucleation and in situ corneoscleral excision are the two methods for eye retrieval. Although studies have reported a higher acceptance rate for corneal donation among donors' relatives with in situ corneoscleral excision than whole globe enucleation, there are concerns regarding the impact on donor cornea tissue quality with in situ corneoscleral excision. Currently, there is limited high-quality evidence comparing the two methods. INCLUSION CRITERIA: We will consider prospective and retrospective comparative studies that examine the effects of whole globe enucleation and in situ corneoscleral excision on donor cornea tissue quality. There will be no restrictions on the recipients' characteristics, including age, sex, ocular comorbidities, or potential visual acuity after corneal transplantation. METHODS: Electronic databases, including (but not limited to) MEDLINE, Embase, Cochrane CENTRAL, ClinicalTrials,gov, and ISRCTN registry will be searched, with no restriction to the language used or date of publication. Retrieval of full-text studies, assessment of methodological quality, and data extraction will be performed independently by two reviewers. A meta-analysis, using fixed or random effects, will be performed for the included randomized controlled trials when there are sufficient similarities in the reporting of outcome measures. If meta-analysis is not possible, the pre-specified outcomes will be narratively synthesized. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO (CRD42020210575).

Outcomes of corneal transplantation in Europe: report by the European Cornea and Cell Transplantation Registry.

PURPOSE: To analyze real-world graft survival and visual acuity outcomes of corneal transplantation in Europe. SETTING: Corneal clinics in 10 European Union member states, the United Kingdom, and Switzerland. DESIGN: Multinational registry study. METHODS: All corneal transplant procedures registered in the European Cornea and Cell Transplantation Registry (ECCTR) were identified. Graft survival of primary corneal transplants were analyzed using Kaplan-Meier survival curves with log-rank test and Cox regression. Corrected distance visual acuities (CDVAs) are reported at baseline and 2 years postoperatively using the Lundström distribution matrix. RESULTS: A total of 12 913 corneal transplants were identified. Overall, 32-year graft survival of corneal transplants was high (89%) but differed between indications, ranging from 98% in keratoconus and 80% for trauma. Overall, CDVA improved postoperatively, but the risk for losing vision ranged from 7% (baseline vision ≤0.1 Snellen) to 58% (baseline vision ≥1.0 Snellen). CONCLUSIONS: This report provides a comprehensive overview of graft survival and visual outcomes of corneal transplantation in Europe. In addition, it provides real-world estimates of outcomes for a variety of indications and surgical techniques to support benchmarking and demonstrates the relationship between baseline and postoperative vision.

Response of human oral mucosal epithelial cells to different storage temperatures: A structural and transcriptional study.

PURPOSE: Seeking to improve the access to regenerative medicine, this study investigated the structural and transcriptional effects of storage temperature on human oral mucosal epithelial cells (OMECs). METHODS: Cells were stored at four different temperatures (4°C, 12°C, 24°C and 37°C) for two weeks. Then, the morphology, cell viability and differential gene expression were examined using light and scanning electron microscopy, trypan blue exclusion test and TaqMan gene expression array cards, respectively. RESULTS: Cells stored at 4°C had the most similar morphology to non-stored controls with the highest viability rate (58%), whereas the 37°C group was most dissimilar with no living cells. The genes involved in stress-induced growth arrest (GADD45B) and cell proliferation inhibition (TGFB2) were upregulated at 12°C and 24°C. Upregulation was also observed in multifunctional genes responsible for morphology, growth, adhesion and motility such as EFEMP1 (12°C) and EPHA4 (4°C-24°C). Among genes used as differentiation markers, PPARA and TP53 (along with its associated gene CDKN1A) were downregulated in all temperature conditions, whereas KRT1 and KRT10 were either unchanged (4°C) or downregulated (24°C and 12°C; and 24°C, respectively), except for upregulation at 12°C for KRT1. CONCLUSIONS: Cells stored at 12°C and 24°C were stressed, although the expression levels of some adhesion-, growth- and apoptosis-related genes were favourable. Collectively, this study suggests that 4°C is the optimal storage temperature for maintenance of structure, viability and function of OMECs after two weeks.

Recent Advances in Stem Cell Therapy for Limbal Stem Cell Deficiency: A Narrative Review.

Destruction of the limbus and depletion of limbal stem cells (LSCs), the adult progenitors of the corneal epithelium, leads to limbal stem cell deficiency (LSCD). LSCD is a rare, progressive ocular surface disorder which results in conjunctivalisation and neovascularisation of the corneal surface. Many strategies have been used in the treatment of LSCD, the common goal of which is to regenerate a self-renewing, transparent, and uniform epithelium on the corneal surface. The development of these techniques has frequently resulted from collaboration between stem cell translational scientists and ophthalmologists. Direct transplantation of autologous or allogeneic limbal tissue from a healthy donor eye is regarded by many as the technique of choice. Expansion of harvested LSCs in vitro allows smaller biopsies to be taken from the donor eye and is considered safer and more acceptable to patients. This technique may be utilised in unilateral cases (autologous) or bilateral cases (living related donor). Recently developed, simple limbal epithelial transplant (SLET) can be performed with equally small biopsies but does not require in vitro cell culture facilities. In the case of bilateral LSCD, where autologous limbal tissue is not available, autologous oral mucosa epithelium can be expanded in vitro and transplanted to the diseased eye. Data on long-term outcomes (over 5 years of follow-up) for many of these procedures is needed, and it remains unclear how they produce a self-renewing epithelium without recreating the vital stem cell niche. Bioengineering techniques offer the ability to re-create the physical characteristics of the stem cell niche, while induced pluripotent stem cells offer an unlimited supply of autologous LSCs. In vivo confocal microscopy and anterior segment OCT will complement impression cytology in the diagnosis, staging, and follow-up of LSCD. In this review we analyse recent advances in the pathology, diagnosis, and treatment of LSCD.

Hypothermically Stored Adipose-Derived Mesenchymal Stromal Cell Alginate Bandages Facilitate Use of Paracrine Molecules for Corneal Wound Healing.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) may alleviate corneal injury through the secretion of therapeutic factors delivered at the injury site. We aimed to investigate the therapeutic factors secreted from hypothermically stored, alginate-encapsulated Ad-MSCs' bandages in in vitro and in vivo corneal wounds. Ad-MSCs were encapsulated in 1.2% w/v alginate gels to form bandages and stored at 15 °C for 72 h before assessing cell viability and co-culture with corneal scratch wounds. Genes of interest, including HGF, TSG-6, and IGF were identified by qPCR and a human cytokine array kit used to profile the therapeutic factors secreted. In vivo, bandages were applied to adult male mice corneas following epithelial debridement. Bandages were shown to maintain Ad-MSCs viability during storage and able to indirectly improve corneal wound healing in vivo. Soluble protein concentration and paracrine factors such as TSG-6, HGF, IL-8, and MCP-1 release were greatest following hypothermic storage. In vivo, Ad-MSCs bandages-treated groups reduced immune cell infiltration when compared to untreated groups. In conclusion, bandages were shown to maintain Ad-MSCs ability to produce a cocktail of key therapeutic factors following storage and that these soluble factors can improve in vitro and in vivo corneal wound healing.

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.

A case of ocular cystinosis associated with two potentially severe CTNS mutations.

BACKGROUND: Ocular cystinosis is a rare autosomal recessive disorder caused by one severe and one mild mutation in the CTNS gene. It is characterised by cystine deposition within the cornea and conjunctiva however, the kidneys are not affected. We report a case of ocular cystinosis caused by two potentially severe CTNS mutations and discuss the possible mechanism of renal sparing. METHODS: This is an observational case report of the proband and her unaffected relatives. All subjects underwent ophthalmic examination, whilst in the proband, In vivo laser scanning confocal microscopy was used to demonstrate cystine crystals within her corneas and conjunctiva. Genetic diagnosis was confirmed by DNA sequencing of the proband and the segregation of the mutations was established in her relatives. RT-PCR of leukocyte RNA was undertaken to determine if aberrant splicing of the CTNS gene was taking place Results: The proband was found to have cystine crystals limited to the anterior corneal stroma and the conjunctiva. Sequencing of the proband's CTNS gene found her to be a compound heterozygote for a 27bp deletion in exon8/intron 8 (c.559_561 + 24del) and a novel c.635C>T variant in exon 9 that is predicted be pathogenic and to result in the substitution of alanine with valine at amino acid position 212 (p.Ala212Val), which is within the 3rd transmembrane spanning domain of the CTNS protein. Examination of the proband's leukocyte RNA failed to demonstrate any aberrant CTNS gene splicing. CONCLUSION: We present a case of ocular cystinosis caused by two potentially severe CTNS gene mutations. The lack of renal involvement may be due to localised (ocular) aberrant CTNS RNA splicing.

Contact Transscleral Cyclodiode Laser Treatment for Refractory Glaucoma After Penetrating Keratoplasty: Retrospective Long-term Outcomes.

PRéCIS:: This is a retrospective study with long-term follow-up using transscleral cyclodiode laser photocoagulation (TCP) with low complication rate and good graft survival and intraocular pressure (IOP) control. Selective 180-degree TCP may offer a good IOP control with reduced complication rates. PURPOSE: To study the long-term safety and efficacy of contact TCP in eyes with refractory glaucoma after penetrating keratoplasty (PKP). PATIENTS AND METHODS: All consecutive patients who were treated with TCP for refractory glaucoma following PKP between March 1996 and February 2017 in a tertiary corneal transplantation service in the United Kingdom. Only patients with a follow-up of 5 years were included. Eligible patients were identified through the corneal transplantation service database. Medical records and database data were retrospectively analyzed and compared at 5 years from baseline. RESULTS: In total, 28 eyes of 28 patients presented with a mean IOP of 30.4 mm Hg (SD, 7.5) at baseline despite maximally tolerated topical and systemic antiglaucoma medications. IOP was reduced significantly to 12.8 mm Hg (SD, 3.6) (P<0.0001) at 5 years with 100% of patients with a successfully controlled IOP (defined as ≤21 mm Hg). All patients had a clear graft at the beginning of the study period and at 5 years 60.7% (n=17) still presented clear grafts. The average number of topical glaucoma medication was reduced from 2.8 (SD, 0.8) to 1.7 (SD, 1.2) (P=0.019) at 5 years. Visual acuity remained stable in 67.9% of patients at 5-year endpoint. No complications (ie, hypotony or phthisis bulbi) were reported during the study period and the corneal graft failure rate remained low at 5 years (39.3%). CONCLUSIONS: Cyclodiode laser treatment with initial selective 180-degree protocol seems to be an efficient therapeutic option in the management of patients with refractory glaucoma post-PKP when compared with other surgical alternatives. A selective 180-degree protocol seems to potentially reduce the rate of complications. Further randomized controlled studies are needed to compare outcomes with modified treatment's protocols and glaucoma drainage device.

Acute Chemical Eye Injury and Limbal Stem Cell Deficiency-A Prospective Study in the United Kingdom.

PURPOSE: To analyze the incidence, nature, outcomes, and complications of acute chemical eye injuries, including the incidence of limbal stem cell deficiency (LSCD) and to compare the 2 main classifications for ocular chemical injuries: Roper-Hall (RH) and Dua. METHODS: This is a prospective, consecutive, interventional single-center study between April and October 2009 of all new patients with acute chemical eye injury presenting to the Royal Victoria Infirmary eye emergency department (EED). RESULTS: Of 11,683 patients who attended the EED, 98 patients (110 eyes) presented with acute chemical eye injury (60% male). This represents an estimated annual incidence of 5.6 new cases per 100,000 population. Mean age was 36.5 years (1-78; SD 17.1 years), including 7 children (age <10 years). Fifty-one patients (52%) had work-related injuries. The most common chemical agent was alkali (78%). All 4 RH grade IV cases were unilateral, assault with ammonia, and required early amniotic membrane transplantation as per the protocol, but despite full treatment, they developed total LSCD in the affected eye. CONCLUSIONS: Acute chemical eye injuries are rare. Male patients in the working age group are more prone to work-related chemical injuries, whereas young children tend to have domestic injuries. Grade I, II, and III RH and Dua chemical injuries had a very good prognosis with topical treatment only, whereas RH grade IV (Dua grade IV-VI), mainly assaults with ammonia, progressed to total/severe LSCD despite appropriate management including early amniotic membrane transplantation. The Dua classification includes conjunctival involvement, having a greater value in predicting the final clinical outcome when grading chemical eye injuries.

Alginate encapsulated multipotent adult progenitor cells promote corneal stromal cell activation via release of soluble factors.

To reduce the increasing need for corneal transplantation, attempts are currently aiming to restore corneal clarity, one potent source of cells are multipotent adult progenitor cells (MAPC®). These cells release a powerful cocktail of paracrine factors that can guide wound healing and tissue regeneration. However, their role in corneal regeneration has been overlooked. Thus, we sought to explore the potential of combining the cytoprotective storage feature of alginate, with MAPC to generate a storable cell-laden gel for corneal wound healing. 72 hours following hypothermic storage, alginate encapsulation was shown to maintain MAPC viability at either 4 or 15°C. Encapsulated MAPC (2 x106 cells/mL) stored at 15°C presented the optimum temperature that allowed for cell recovery. These cells had the ability to reattach to tissue culture plastic whilst exhibiting normal phenotype and this was maintained in serum-free and xenobiotic-free medium. Furthermore, corneal stromal cells presented a significant decrease in scratch-wounds in the presence of alginate encapsulated MAPC compared to a no-cell control (p = 0.018). This study shows that immobilization of MAPC within an alginate hydrogel does not hinder their ability to affect a secondary cell population via soluble factors and that these effects are successfully retained following hypothermic storage.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

Neurotrophic keratopathy.

Neurotrophic Keratopathy (NK) refers to a condition where corneal epitheliopathy leading to frank epithelial defect with or without stromal ulceration (melting) is associated with reduced or absent corneal sensations. Sensory nerves serve nociceptor and trophic functions, which can be affected independently or simultaneously. Loss of trophic function and consequent epithelial breakdown exposes the stroma making it susceptible to enzymatic degradation. Nerve pathology can range from attrition to aberrant re-generation with corresponding symptoms from anaesthesia to hyperaesthesia/allodynia. Many systemic and ocular conditions, including surgery and preserved medications can lead to NK. NK can be mild (epithelium and tear film changes), moderate (non-healing epithelial defect) or severe (stromal melting and perforation). Moderate and severe NK can profoundly affect vision and adversely impact on the quality of life. Medical management with lubricating agents from artificial tears to serum/plasma drops, anti-inflammatory agents, antibiotics and anti-proteases all provide non-specific relief, which may be temporary. Contact lenses, punctal plugs, lid closure with botulinum toxin and surgical interventions like tarsorrhaphy, conjunctival flaps and amniotic membrane provide greater success but often at the cost of obscuring sight. Corneal surgery in a dry ocular surface with reduced sensation is at high risk of failure. The recent advent of biologicals such as biopolymers mimicking heparan sulfate; coenzyme Q10 and antisense oligonucleotide that suppress connexin 43 expression, all offer promise. Recombinant nerve growth factor (cenegermin), recently approved for human use targets the nerve pathology and has the potential of addressing the underlying deficit and becoming a specific therapy for NK.

Outcomes of Penetrating Keratoplasty Following Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

The purpose of this study is to investigate the outcomes of penetrating keratoplasty (PKP) following autologous cultivated limbal epithelial stem cell transplantation (CLET). A prospective, single center, interventional cohort study investigating patients with unilateral total limbal stem cell deficiency (LSCD) treated with CLET who underwent PKP. Patients with confirmed corneal re-epithelialization > 6 months post-CLET, and with best-corrected visual acuity (BCVA) <0.3 logMAR were offered PKP. CLET survival assessed by slit lamp, corneal impression cytology (CIC), and in vivo confocal microscopy. Confirmation of corneal re-epithelialization by histological and immunocytochemical (ICC) examination of trephined corneal buttons. Mean change in best-corrected visual acuity (logMAR) following PKP and PKP survival at 12 months were calculated. Twenty patients underwent PKP. Mean time of PKP was 19 months (range 11-41 months, SD 7.26) post-CLET. Median follow-up time post-PKP was 15 months (range 1-32, SD 10.2). CIC and ICC of all corneas confirmed corneal re-epithelialization before PKP. Mean pre-PKP BCVA was 1.46 (range 0.3-2.7, SD 0.94) improving to a mean post-PKP BCVA of 0.74 (range 0-2.7, SD 0.87); mean improvement in BCVA post-PKP of 36 letters (95% CI 15.0-57.1, p = .002). Kaplan-Meier mean graft survival was 90.9% (95% CI 50.8-98.7) at 12 months. We recommend a two-stage approach with CLET followed by PKP >12 months later. Patients experienced a significant improvement in BCVA following PKP. PKP did not have a detrimental effect on CLET survival. PKP survival post-CLET is better than that reported for high risk PKP. Stem Cells 2018;36:925-931.

Progressive idiopathic unilateral corneal endothelial failure of unknown aetiology in phakic eyes.

AIM: To describe a cohort of patients with irreversible unilateral bullous keratopathy (BK) of undetermined aetiology. METHOD: Retrospective, single-centre case series in a tertiary corneal referral centre. RESULTS: Eleven consecutive patients (nine females; mean age 71.7 years) presented from 1999 to 2009 with acute onset unilateral visual loss. At presentation, the best-corrected visual acuity of the affected eyes was 6/9 or worse with mean central corneal thickness (CCT) of 684 (SD 66) µm. Specular microscopy was not possible in the affected eyes. There was no other ocular pathology in the affected eye. The fellow eye remained normal throughout the study (mean endothelial cell density (ECD) of 1980 (SD 736) cells/mm2 and CCT of 536 (SD 34) µm). Topical steroid, antiviral treatments (both topical and systemic) or a combination of both did not yield any improvement. After a mean follow-up of 82.2 months, eight eyes had penetrating keratoplasty (PK). One required two regrafts. Histology showed typical BK features, with endothelial cell (EC) loss and thickened Descemet's membrane (DM). Transmission electron microscopy revealed DM abnormalities in a non-consistent pattern, featuring variable collagen deposition posterior to the non-banded zone. The ECs were degenerated, reduced or absent. Neither viruses nor pseudoexfoliation material was identified. CONCLUSION: While medical treatment is not beneficial, PK appears to offer good results. Non-guttate Fuchs' corneal endothelial dystrophy merits consideration but it would be unusual to see an exclusively unilateral presentation. DM thickening is reflective of a chronic EC loss but the cause of this loss remains elusive.