Is Keratoconus an Inflammatory Disease? The Implication of Inflammatory Pathways.

PURPOSE: Keratoconus is considered a non-inflammatory condition. Recently however, increased proinflammatory cytokines have been detected in the tears of keratoconic patients and clinical and immunohistochemical observations reported infiltration of matured dendritic cells and leukocytes. Our laboratory utilized cytokine antibody arrays to elucidate the inflammatory aspects of keratoconus. METHODS: Protein was extracted from 42 corneal buttons (14 keratoconic and 28 non-keratoconic) and incubated with cytokine antibody arrays scanning 120 cytokines. Mann Whitney U test with a p-value of <0.05 was considered significant. RESULTS: Pathways for wound healing, neuroprotection, angiogenesis, and inflammation were activated in keratoconic samples with 23 cytokines showing significant elevation. Fifteen were expressed only in keratoconus with 8 cytokines elevated 1.7-42-fold. CONCLUSION: This study identified elevated inflammatory pathways covering immune responses in keratoconus. Our results support the evidence for inflammatory pathway activation in keratoconus and a possible redefinition of keratoconus as a chronic inflammatory corneal disease.

Differences in sphere-forming cells from keratoconic and normal corneal tissue: Implications for keratoconus pathogenesis.

Keratoconus is primarily an anterior corneal disorder of unclear aetiology. Stem cells may play a role in the perpetuation of keratoconus, although this has yet to be definitively established. Sphere-forming cells from normal human donor corneas have previously been shown to be a heterogenous mix of epithelial, stromal, stem and progenitor cell components which have potential for treatment of corneal dystrophies. Our work set out to isolate and characterise sphere-forming cells from human keratoconic tissue. Keratoconic donor corneas were successfully used to culture sphere-forming cells in vitro. Time lapse imaging of these spheres on a collagen surface over 8 days revealed keratoconic spheres lack the ability to maintain a central core and have diminished ability to repopulate the surface. Immunocytochemistry showed positive labelling for the stem cell marker 'Adenosine triphosphate-binding cassette sub-family B member 5 (ABCB5)' indicating stem cell retention and the myofibroblast marker alpha smooth muscle actin indicating wound repair while droplet digital Polymerase Chain Reaction confirmed an increase in expression of stem and stromal cell markers in keratoconic spheres compared to spheres cultured from normal donors at day 7 post-placement. Keratoconic sphere-forming cells showed a diminished repopulation ability, a faster wound healing response and lack of central core retention. These results suggest stem cells in keratoconus may be in an elevated state of wound repair and unable to respond appropriately to further injury in corneal maintenance. Sphere forming cell populations in keratoconus appear to be different to those isolated from normal corneas and this may be an important consideration in unearthing keratoconus aetiology.

Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy.

BACKGROUND: Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue. AIM: To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue. METHODS: Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells. These spheres were implanted into incisions created in full thickness and onto the surface of 10 µm thin sections of keratoconic and normal stromal tissues in vitro. Tissue sections were used to maximise use of limited keratoconic tissue available for research. Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d (D14) from implantation. Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment. RESULTS: Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10 µm thin en face tissue sections. No observable differences were seen in sphere migration, proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14. Spheres stained positively with Calcein-AM up to D14. Cell migration increased from day 0 to D14, occurring radially in three dimensions from the sphere and in alignment with tissue edges. Cell proliferation marker, EdU, was detected at day 10. Implanted spheres stained positively for putative stem cell markers ∆Np63α and ABCB5, while ABCG2, ABCB5, ∆Np63 and p63α were detectable by droplet digital PCR up to D14. Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin (myofibroblast marker) in some migrated cells. Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers, indicating an appropriate response to repopulating diseased tissue. CONCLUSION: Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.

Auckland Cataract Study IV: Practical application of NZCRS cataract risk stratification to reduce phacoemulsification complications.

IMPORTANCE: Reduction of intraoperative complications in phacoemulsification cataract surgery. BACKGROUND: To assess practicability of a risk stratification system, the New Zealand Cataract Risk Stratification (NZCRS) system, in a major teaching hospital service, without investigator oversight, to ascertain whether benefits identified in research studies are maintained in busy clinical practice. DESIGN: Prospective cohort study in a major public teaching hospital. PARTICIPANTS: Five hundred cases of phacoemulsification cataract surgery. METHODS: NZCRS system inserted into 621 consecutive preoperative cataract patient files. Recommendation to allocate higher-risk cases to experienced surgeons. MAIN OUTCOME MEASURES: NZCRS system uptake and adherence, appropriate identification of high risk cases and intraoperative complication rates. RESULTS: NZCRS scores calculated in 500 of 621 (80.5%) cases and 98 (19.6%) scored as "high risk." Cataract surgery (N = 500) performed by: 12 Registrars (20%), 4 Fellows (7.2%), 26 Consultants (72.8%). Risk scores adhered to in 99%. Overall intraoperative complications (3.0%) included iris prolapse 1.6% and posterior capsule tear 0.8%. No statistical difference in complication rates identified between surgeon grades. Mean best-corrected visual acuity was 6/10 (20/32). Postoperatively, cystoid macular oedema occurred in 3.2%. Rescoring by an experienced investigator noted a greater number of "high risk scores" (31.6% vs 19.6%) related to differences in subjective scoring of anterior chamber depth and cataract density. CONCLUSIONS AND RELEVANCE: Practical uptake of cataract risk stratification was promising in this study with NZCRS calculated in 80.5% with 99% adherence to scoring recommendations. Compared to baseline studies, in the day-to-day clinical setting, a continued, decreasing trend in frequency and severity of intraoperative complications was noted. Subjective variability of risk scoring may be further improved by better, objective, standardization.

Molecular evidence for the role of inflammation in dry eye disease.

Ocular surface inflammation is propagated by a complex series of molecular processes and has been implicated in the pathogenesis of dry eye disease (DED), either as a causal or a downstream effect of ocular surface disease. A state of hyperosmolarity elicits an acute immune response in DED, leading to subsequent activation of the adaptive immune response. This cascade incites dysregulation of the immune system, triggering a vicious cycle of events that causes damage to the ocular surface. Symptoms associated with these events include burning, irritation, redness, photophobia and blurred vision. The chronic nature of the disease process can cause permanent alterations to the ocular surface and adnexa. An increasing investment in treatment options, and positive outcomes with novel therapies that have received subsequent regulatory approval, lends further support to the role of inflammation in DED. This review highlights the nature and function of a range of fundamental inflammatory molecules in DED to provide the clinician with an appreciation for the ways in which these factors might be manipulated in DED management.

Auckland Cataract Study III: Refining Preoperative Assessment With Cataract Risk Stratification to Reduce Intraoperative Complications.

PURPOSE: To assess intraoperative complications of phacoemulsification surgery in public teaching hospital settings using modified preoperative risk stratification systems. DESIGN: Prospective cohort study. METHODS: Preoperative risk stratification of 500 consecutive cataract cases using the New Zealand Cataract Risk Stratification (NZCRS) scoring system. Recommended allocation of higher-risk phacoemulsification procedures to experienced surgeons in public teaching hospital setting. MAIN OUTCOME MEASURE: Intraoperative complications relative to adherence to stratification recommendations. RESULTS: NZCRS classified 192 cases (38%) as high-risk, recommended for fellows or consultants (attendings). Primary surgeons were residents (n = 142, 28%), fellows (n = 88, 18%), and consultants (n = 270, 54%). Overall rate (N = 500) of any intraoperative complication was 5.0%. Where NZCRS scoring recommendations were observed (n = 448) the intraoperative complication rate was 4.5% but in "nonadherence" cases (n = 52 residents operating on higher-risk cases) this nearly doubled (9.6%). Postoperative complications occurred in 5.2%, primarily cystoid macular edema (3.7%). Postoperatively, mean unaided visual acuity was 6/12 (20/40) and best-corrected visual acuity improved from 6/20 (20/63) preoperatively to 6/10 (20/32) postoperatively (P < .05). CONCLUSIONS: The NZCRS system aids identification of higher-risk cataract cases and appropriate case-to-surgeon allocation and may increase surgeon awareness of risk factors. Compared to 2 previous studies under similar conditions in the same institution, the NZCRS system was associated with a 40% reduction in intraoperative complications (8.4% to 5%). The rate of posterior capsular tear was 0.6% (P = .035) compared to 2.6% in baseline phase and 1.4% in a prior risk stratification phase. Risk stratification seems to reduce intraoperative phacoemulsification complications in public teaching hospital settings.

Use of a Purpose-Built Impression Cytology Device for Gene Expression Quantification at the Ocular Surface Using Quantitative PCR and Droplet Digital PCR.

PURPOSE: To describe an impression cytology (IC) technique using a purpose-built, sterile, EYEPRIM IC device that can be coupled with a TRIzol reagent-based RNA extraction protocol to yield sufficient RNA for gene expression analysis. METHODS: IC samples using the EYEPRIM device were collected from the bulbar conjunctiva, with and without topical anesthesia, and evaluated for RNA yield, the absence of polymerase chain reaction (PCR) inhibitors, and the ability to detect biomarkers by quantitative real-time PCR and droplet digital PCR. A technique for collecting IC samples in the clinic, while preserving RNA, and a protocol for subsequent laboratory analysis of RNA were developed. RESULTS: The extracted RNA was free of PCR inhibitors and could be synthesized into complementary DNA and used for successful relative quantification of ocular surface biomarkers by quantitative real-time PCR. For gene targets present in low abundance, complementary DNA could also be used for quantification by the relatively new and emerging method of droplet digital PCR. The described method was successfully used to evaluate 3 biomarkers in a clinical trial assessing the tolerability of a proprietary eyelid therapy in 92 IC samples from a study population of 46 participants. CONCLUSIONS: IC is a recognized technique for ocular surface cell evaluation and protein biomarker quantification but is infrequently used for quantifying gene expression. The EYEPRIM device allows ease of use and impression-to-impression consistency while accurate gene expression data offers a highly specific and sensitive method of disease characterization for clinician scientists to use in diagnosis.

The Sheep Cornea: Structural and Clinical Characteristics.

PURPOSE: The aim of this study was to perform qualitative and quantitative analyses to characterize the corneas of young, healthy sheep. MATERIALS AND METHODS: Eight healthy male sheep, 10 months to 1 year of age, were included as experimental subjects. Central corneal thickness was measured using a handheld pachymeter, and an Easygraph corneal topographer provided topographic maps. Microstructural imaging of corneal layers was achieved by using the Heidelberg Retina Tomograph III Rostock Corneal Module in vivo corneal microscope (IVCM). An Ocular Response Analyzer (ORA) provided quantitative measurements of intraocular pressure (IOP), corneal hysteresis (CH), and corneal resistance factor. Tissue histology and immunohistochemistry were carried out to obtain detail on the corneal layers. RESULTS: Light microscopy and immunohistochemical labeling revealed a stratified epithelium, a limbus with numerous limbal crypts, a thick basement membrane, a thin Bowman's layer, a thick corneal stroma with a dense population of keratocytes, and a thick, hyper-reflective Descemet's membrane. Using IVCM, the cell density of the basal layer was noted to be significantly higher than that of other epithelial cell types. The density of keratocytes was significantly higher (P value = 0.0223) in the anterior compared to the posterior stroma. The endothelial cells were organized in a characteristic honeycomb pattern. The mean and standard deviation values for central corneal pachymetry were 623.14 ± 19.5 µm and 616.37 ± 34.87 µm for the left and right eyes, respectively. ORA-derived mean values for IOPcc and CH for the left and right eyes were 14.93 ± 1.73 mm Hg and 15.16 ± 2.02 mm Hg and 3.56 ± 0.72 mm Hg and 3.73 ± 0.49 mm Hg, respectively. CONCLUSIONS: The anatomical and clinical characteristics of the sheep cornea, as outlined in this study, make the sheep a suitable and relevant model for corneal research. This study provides researchers with important data on the suitability of sheep as a model for ophthalmic experiments.

Extreme Descemet's membrane rupture with hydrops in keratoconus: Clinical and histological manifestations.

PURPOSE: To study the clinical and histological manifestations of an extreme Descemet's membrane rupture as a result of keratoconus. OBSERVATIONS: Using Periodic acid-Schiff assay to study a keratoconic cornea with an extreme rupture showed that the ruptured Descemet's membrane had retracted and folded into scrolls and ridges. The dimensions of the rupture were estimated to be 3.7mm2, and the central cornea was extremely thinned with a thickness of only 260µm. Stromal scarring and loosely packed lamellae were present anterior to the scrolls and ridges. Antibodies targetting the major components of Descemet's membrane, Laminin and type IV collagen, displayed intense labelling adjacent to the scrolls where the stroma was denuded and differential expression patterns lined the ridges. Environmental scanning electron microscopy showed possible collagen deposition at the site of rupture. CONCLUSIONS AND IMPORTANCE: The specific staining patterns of laminin and type IV collagen suggest these components have an important role in re-endothelisation of the cornea. This is the first known report of spatial resolution of the topography of the Descemet's membrane rupture established by environmental scanning electron microscopic image montage.

Randomized double-masked trial of eyelid cleansing treatments for blepharitis.

PURPOSE: To compare the efficacy of a dedicated eyelid cleanser and diluted baby shampoo in the management of blepharitis. METHODS: Forty-three participants with clinical blepharitis signs were enrolled in a prospective, randomized, double-masked, paired-eye trial. A dedicated eyelid cleanser (TheraTears® SteriLid®) was applied to the eyelids of one eye (randomized) and diluted baby shampoo (Johnson's® No More Tears®) to the fellow eye, twice daily for 4 weeks. Tear film parameters, ocular surface characteristics, symptomology and cytology markers were assessed at baseline and day 28. RESULTS: Baseline measurements did not differ between treatments (all p > 0.05). The eyelid cleanser was preferred over baby shampoo by the majority of participants (p < 0.001). Improvements in the tear lipid layer, inferior lid wiper epitheliopathy (LWE), cylindrical collarettes, and MMP-9 expression were limited to the dedicated eyelid cleanser (all p < 0.05), and a greater decrease in SANDE symptoms score was also observed (p = 0.04). Meibomian gland capping and MUC5AC expression worsened with baby shampoo treatment (both p < 0.05). SPEED symptoms score, superior LWE, seborrhoeic lash crusting, and trichiasis decreased significantly following application of both treatments (all p < 0.05), but did not differ between treatments (all p > 0.05). CONCLUSION: Clinical improvements in blepharitis occurred with both treatments. However, only the dedicated eyelid cleanser proved effective in reducing ocular surface inflammation, and was the preferred therapy. Long term impact of decreased goblet cell function secondary to baby shampoo treatment requires further exploration.

Aberrant Patterns of Key Epithelial Basement Membrane Components in Keratoconus.

PURPOSE: In the cornea, the epithelial basement membrane (BM) plays an important role in maintaining corneal integrity and homeostasis. Aberrations in this vital structure are associated with several corneal pathologies including keratoconus. The aim of this study was to investigate the expression of key structural components of the epithelial BM in keratoconic corneas and to identify and describe any aberrant patterns. METHODS: Immunohistochemical labeling of key BM components including fibronectin, laminin, and type IV and VII collagen was performed in healthy and keratoconic corneas. RESULTS: Clear changes in the BM components in the keratoconic corneas were seen with the key structural components either being absent or forming a discontinuous pattern. Another aberrant pattern, the expression of BM proteins, particularly fibronectin, laminin, and type IV collagen, in the anterior stroma of keratoconic corneas was also observed. CONCLUSIONS: These results indicate the activation of keratocytes into the fibroblast and myofibroblast wound phenotypes and the potential source of corneal scarring commonly observed in keratoconic corneas. Our data also support the hypothesis of dysregulated collagen synthesis and breakdown in the keratoconic cornea, in particular, the BM, and suggest a role for the BM in initiation and progression of keratoconus.

The Auckland Cataract Study II: Reducing Complications by Preoperative Risk Stratification and Case Allocation in a Teaching Hospital.

PURPOSE: To assess the effect of preoperative risk stratification for phacoemulsification surgery on intraoperative complications in a teaching hospital. DESIGN: Prospective cohort study. METHODS: Prospective assessment of consecutive phacoemulsification cases (N = 500) enabled calculation of a risk score (M-score of 0-8) using a risk stratification system. M-scores of >3 were allocated to senior surgeons. All surgeries were performed in a public teaching hospital setting, Auckland, New Zealand, in early 2016. Postoperatively, data were reviewed for complications and corrected distance visual acuity (CDVA). Results were compared to a prospective study (N = 500, phase 1) performed prior to formal introduction of risk stratification. RESULTS: Intraoperative complications increased with increasing M-scores (P = .044). Median M-score for complicated cases was higher (P = .022). Odds ratio (OR) for a complication increased 1.269 per unit increase in M-score (95% confidence interval [CI] 1.007-1.599, P = .043). Overall rate of any intraoperative complication was 5.0%. Intraoperative complication rates decreased from 8.4% to 5.0% (OR = 0.576, P = .043) comparing phase 1 and phase 2 (formal introduction of risk stratification). The severity of complications also reduced. A significant decrease in complications for M = 0 (ie, minimal risk cases) was also identified comparing the current study (3.1%) to phase 1 (7.2%), P = .034. There was no change in postoperative complication risks (OR 0.812, P = .434) or in mean postoperative CDVA (20/30, P = .484) comparing current with phase 1 outcomes. CONCLUSION: A simple preoperative risk stratification system, based on standard patient information gathered at preoperative consultation, appears to reduce intraoperative complications and support safer surgical training by appropriate allocation of higher-risk cases.

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue.

Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue ( Mathan et al., 2016 ). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.

New Zealand trends in corneal transplantation over the 25†years 1991-2015.

AIMS: To report the 25-year longitudinal trends in indications and corneal transplantation techniques in New Zealand. METHODS: Statistical analysis of prospectively acquired New Zealand National Eye Bank (NZNEB) electronic database from 1991 to 2015 inclusive. Subjects were recipients of corneal transplants in 62 centres supplied by the NZNEB. Main outcome measures were indications, recipient age and transplantation techniques. RESULTS: From January 1991 to December 2015, NZNEB supplied tissue for 5574 corneal transplants, increasing annually from 89 (1991) to 290 (2015). Penetrating keratoplasty remained the most commonly performed technique throughout the 25-year period, although it decreased from 98.9% of all transplants in 1991 to 60.3% in 2015. There was a corresponding increase in deep anterior lamellar and endothelial keratoplasty over the most recent decade from 2.5% to 7.2% and 4.9% to 31.4%, respectively. Keratoconus remained the leading indication for keratoplasty through to 2015 (34.5%). Regrafts (23.1%) and Fuchs endothelial corneal dystrophy (17.0%) have become more common indications, while bullous keratopathy has become less common (10.8%). There was a bimodal distribution in age with peaks at 20-29 and 60-79 years. There was a reduction in recipients under age 40 and corresponding increase in the percentage of recipients aged 40-69. CONCLUSION: Changing indications and increasing uptake of lamellar keratoplasty have been significant international trends over the last 25 years. However, New Zealand's corneal disease and population characteristics create unique longitudinal trends, with keratoconus remaining the leading indication and penetrating keratoplasty the leading technique from 1991 to 2015.

Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells.

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies.

The Auckland Cataract Study: Assessing Preoperative Risk Stratification Systems for Phacoemulsification Surgery in a Teaching Hospital.

PURPOSE: To evaluate 2 preoperative risk stratification systems for assessing the risk of complications in phacoemulsification cataract surgery, performed by residents, fellows, and attending physicians in a public teaching hospital. DESIGN: Cohort study. METHODS: One observer assessed the clinical data of 500 consecutive cases, prior to phacoemulsification cataract surgery performed between April and June 2015 at Greenlane Clinical Centre, Auckland, New Zealand. Preoperatively 2 risk scores were calculated for each case using the Muhtaseb and Buckinghamshire risk stratification systems. Complications, intraoperative and postoperative, and visual outcomes were analyzed in relation to these risk scores. RESULTS: Intraoperative complication rates increased with higher risk scores using the Muhtaseb or Buckinghamshire stratification system (P = .001 and P = .003, respectively, n = 500). The odds ratios for residents and fellows were not significantly different from attending physicians after case-mix adjustment according to risk scores (P > .05). Postoperative complication rates increased with higher Buckinghamshire risk scores but not with Muhtaseb scores (P = .014 and P = .094, respectively, n = 476). Postoperative corrected-distance visual acuity was poorer with higher risk scores (P < .001 for both, n = 476). CONCLUSION: This study confirms that the risk of intraoperative complications increases with higher preoperative risk scores. Furthermore, higher risk scores correlate with poorer postoperative visual acuity and the Buckinghamshire risk score also correlates with postoperative complications. Therefore, preoperative assessment using such risk stratification systems could assist individual informed consent, preoperative surgical planning, safe allocation of cases to trainees, and more meaningful analyses of outcomes for individual surgeons and institutions.

Keratocytes are induced to produce collagen type II: A new strategy for in vivo corneal matrix regeneration.

The stroma, the middle layer of the cornea, is a connective tissue making up most of the corneal thickness. The stromal extracellular matrix (ECM) consists of highly organised lamellae which are made up of tightly packed fibrils primarily composed of collagens type I and V. This layer is interspersed with keratocytes, mesenchymal cells of neural crest origin. We have previously shown that adult corneal keratocytes exhibit phenotypic plasticity and can be induced into a neuronal phenotype. In the current study we evaluated the potential of keratocytes to produce collagen type II via phenotypic reprogramming with exogenous chondrogenic factors. The cornea presents a challenge to tissue engineers owing to its high level of organisation and the phenotypic instability of keratocytes. Traditional approaches based on a scar model do not support the engineering of functional stromal tissue. Type II collagen is not found in the adult cornea but is reported to be expressed during corneal development, raising the possibility of using such an approach to regenerate the corneal ECM. Keratocytes in culture and within intact normal and diseased tissue were induced to produce collagen type II upon treatment with transforming growth factor Beta3 (TGFß3) and dexamethasone. In vivo treatment of rat corneas also resulted in collagen type II deposition and a threefold increase in corneal hardness and elasticity. Furthermore, the treatment of corneas and subsequent deposition of collagen type II did not cause opacity, fibrosis or scarring. The induction of keratocytes with specific exogenous factors and resulting deposition of type II collagen in the stroma can potentially be controlled by withdrawal of the factors. This might be a promising new approach for in vivo corneal regeneration strategies aimed at increasing corneal integrity in diseases associated with weakened ectatic corneal tissue such as keratoconus.

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.

BACKGROUND: The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue. METHODS: Sphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue. RESULTS: Spheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation. CONCLUSION: These observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

A COL17A1 Splice-Altering Mutation Is Prevalent in Inherited Recurrent Corneal Erosions.

PURPOSE: Corneal dystrophies are a genetically heterogeneous group of disorders. We previously described a family with an autosomal dominant epithelial recurrent erosion dystrophy (ERED). We aimed to identify the underlying genetic cause of ERED in this family and 3 additional ERED families. We sought to characterize the potential function of the candidate genes using the human and zebrafish cornea. DESIGN: Case series study of 4 white families with a similar ERED. An experimental study was performed on human and zebrafish tissue to examine the putative biological function of candidate genes. PARTICIPANTS: Four ERED families, including 28 affected and 17 unaffected individuals. METHODS: HumanLinkage-12 arrays (Illumina, San Diego, CA) were used to genotype 17 family members. Next-generation exome sequencing was performed on an uncle-niece pair. Segregation of potential causative mutations was confirmed using Sanger sequencing. Protein expression was determined using immunohistochemistry in human and zebrafish cornea. Gene expression in zebrafish was assessed using whole-mount in situ hybridization. Morpholino-induced transient gene knockdown was performed in zebrafish embryos. MAIN OUTCOME MEASURES: Linkage microarray, exome analysis, DNA sequence analysis, immunohistochemistry, in situ hybridization, and morpholino-induced genetic knockdown results. RESULTS: Linkage microarray analysis identified a candidate region on chromosome chr10:12,576,562-112,763,135, and exploration of exome sequencing data identified 8 putative pathogenic variants in this linkage region. Two variants segregated in 06NZ-TRB1 with ERED: COL17A1 c.3156C→T and DNAJC9 c.334G→A. The COL17A1 c.3156C→T variant segregated in all 4 ERED families. We showed biologically relevant expression of these proteins in human cornea. Both proteins are expressed in the cornea of zebrafish embryos and adults. Zebrafish lacking Col17a1a and Dnajc9 during development show no gross corneal phenotype. CONCLUSIONS: The COL17A1 c.3156C→T variant is the likely causative mutation in our recurrent corneal erosion families, and its presence in 4 independent families suggests that it is prevalent in ERED. This same COL17A1 c.3156C→T variant recently was identified in a separate pedigree with ERED. Our study expands the phenotypic spectrum of COL17A1 disease from autosomal recessive epidermolysis bullosa to autosomal dominant ERED and identifies COL17A1 as a key protein in maintaining integrity of the corneal epithelium.