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1.  New Instruments for Lenticule Extraction in Small Incision Lenticule Extraction (SMILE) 
PLoS ONE  2014;9(12):e113774.
Small incision lenticule extraction (SMILE) is an alternative to Laser-Assisted in situ Keratomileusis (LASIK) for correction of myopia. In cases where surgeons inadvertently dissect the posterior surface first, identification of the anterior surface and subsequent removal become difficult since the anterior surface of the lenticule is compacted against the anterior stromal surface. This may result in incomplete lenticule removal, and a remnant of intrastromal lenticule in SMILE may lead to visual sequelae. In order to aid surgeons in lenticule removal, we have designed and developed 5 novel SMILE lenticule strippers to locate and extract the lenticules more easily. The aim of this study was to investigate and compare the efficacy and quality of these lenticule strippers in assisting SMILE. Thirty porcine eyes were used. The ease of extraction and removal of the lenticule with different strippers was graded by an experienced SMILE surgeon, the extracted lenticule circularity was evaluated by calculating the lenticule circularity, and the intactness of the extracted lenticule edge was assessed using scanning electron microscopy. We found these novel strippers can be of great help to improve the safety and quality of SMILE surgery, particularly in those cases of difficult lenticule extraction.
doi:10.1371/journal.pone.0113774
PMCID: PMC4249972  PMID: 25436451
2.  Effectiveness of Antimicrobial Peptide Immobilization for Preventing Perioperative Cornea Implant-Associated Bacterial Infection 
Titanium (Ti) is a promising candidate biomaterial for an artificial corneal skirt. Antimicrobial peptide (AMP) immobilization may improve the bactericidal effect of the Ti substrate. In this study, we tested the bactericidal efficacy of a functionalized Ti surface in a rabbit keratitis model. A corneal stromal pocket was created by a femtosecond laser. The Ti films were then inserted into the pocket, and Staphylococcus aureus or Pseudomonas aeruginosa was inoculated into the pocket above the implant films. The corneas with Ti-AMP implants were compared with the corneas implanted with unprotected Ti by slit lamp observation and anterior segment optical coherence tomography (AS-OCT). Inflammatory responses were evaluated by bacterium counting, hematoxylin-eosin staining, and immunostaining. There was a lower incidence and a lesser extent of infection on rabbit corneas with Ti-AMP implants than on those with unprotected Ti implants. The bactericidal effect of AMP against S. aureus was comparable to that of postoperative prophylactic antibiotic treatment; hence, SESB2V AMP bound to the Ti implant provided functional activity in vivo, but its efficacy was greater against S. aureus than against P. aeruginosa. This work suggests that SESB2V AMP can be successfully functionalized in a rabbit keratitis model to prevent perioperative corneal infection.
doi:10.1128/AAC.02859-14
PMCID: PMC4135834  PMID: 24957820
3.  Determinants of Posterior Corneal Biometric Measurements in a Multi-Ethnic Asian Population 
PLoS ONE  2014;9(7):e101483.
Purpose
To describe the corneal and anterior segment determinants of posterior corneal arc length (PCAL) and posterior corneal curvature (PCC).
Methods
Cross-sectional, population-based study of 1069 subjects (1069 eyes) aged 40–80 years, from three major Asian ethnic groups. All underwent anterior segment optical coherence tomography imaging and analysis with Zhongshan Angle Assessment Program. Our main outcome measures were determinants of PCAL and PCC using adjusted, multivariate linear regression analysis, adjusted for confounders to obtain the estimated marginal means (EMM) with standard error (SE).
Results
The overall mean (± SD) of PCC was: 6.51±0.39 mm; and PCAL was: 12.52±0.59 mm. Malays had a relatively longer PCAL (EMM = 12.74 mm, SE = 0.04 mm) than Chinese (EMM = 12.48 mm, SE = 0.03 mm, P<0.001), and Indians (EMM = 12.42 mm, SE = 0.03 mm, P<0.001). Anterior segment parameters had weak-moderate correlations with PCAL, which included: anterior chamber depth (ACD) (r = 0.55, P<0.001), PCC (r = 0.27, P<0.001), anterior corneal curvature (ACC) (r = 0.14, P<0.001) and central corneal thickness (CCT) (r = −0.07, P = 0.023). In multivariate analysis, anterior segment parameters explained only 37.6% of the variance of PCAL, with ACD being the most important determinant (partial R2  = 0.300; P<0.001). The determinants of PCC included ACC, PCAL and CCT (explaining 72.1% variation of PCC), with ACC being the most important determinant (partial R2  = 0.683; P<0.001).
Conclusion
There was moderate correlation of PCAL with ACD, but anterior segment parameters accounted for only a small proportion of the variation in PCAL. The significant differences in PCAL and PCC amongst different Asian ethnic groups suggests that there is a need to consider this factor when planning for anterior segment surgeries such as endothelial keratoplasty.
doi:10.1371/journal.pone.0101483
PMCID: PMC4090002  PMID: 25006679
4.  A Cost-Minimization Analysis of Tissue-Engineered Constructs for Corneal Endothelial Transplantation 
PLoS ONE  2014;9(6):e100563.
Corneal endothelial transplantation or endothelial keratoplasty has become the preferred choice of transplantation for patients with corneal blindness due to endothelial dysfunction. Currently, there is a worldwide shortage of transplantable tissue, and demand is expected to increase further with aging populations. Tissue-engineered alternatives are being developed, and are likely to be available soon. However, the cost of these constructs may impair their widespread use. A cost-minimization analysis comparing tissue-engineered constructs to donor tissue procured from eye banks for endothelial keratoplasty was performed. Both initial investment costs and recurring costs were considered in the analysis to arrive at a final tissue cost per transplant. The clinical outcomes of endothelial keratoplasty with tissue-engineered constructs and with donor tissue procured from eye banks were assumed to be equivalent. One-way and probabilistic sensitivity analyses were performed to simulate various possible scenarios, and to determine the robustness of the results. A tissue engineering strategy was cheaper in both investment cost and recurring cost. Tissue-engineered constructs for endothelial keratoplasty could be produced at a cost of US$880 per transplant. In contrast, utilizing donor tissue procured from eye banks for endothelial keratoplasty required US$3,710 per transplant. Sensitivity analyses performed further support the results of this cost-minimization analysis across a wide range of possible scenarios. The use of tissue-engineered constructs for endothelial keratoplasty could potentially increase the supply of transplantable tissue and bring the costs of corneal endothelial transplantation down, making this intervention accessible to a larger group of patients. Tissue-engineering strategies for corneal epithelial constructs or other tissue types, such as pancreatic islet cells, should also be subject to similar pharmacoeconomic analyses.
doi:10.1371/journal.pone.0100563
PMCID: PMC4065108  PMID: 24949869
5.  Correction: Hevin Plays a Pivotal Role in Corneal Wound Healing 
PLoS ONE  2014;9(1):10.1371/annotation/03bcd6d6-0b9c-4591-a3f3-165201253234.
doi:10.1371/annotation/03bcd6d6-0b9c-4591-a3f3-165201253234
PMCID: PMC3879390
6.  LASIK following Small Incision Lenticule Extraction (SMILE) Lenticule Re-Implantation: A Feasibility Study of a Novel Method for Treatment of Presbyopia 
PLoS ONE  2013;8(12):e83046.
Presbyopia remains a major visual impairment for patients, who have previously undergone laser refractive correction and enjoyed unaided distance vision prior to the onset of presbyopia. Corneal stromal volume restoration through small incision lenticule extraction (SMILE) lenticule re-implantation presents an opportunity for restoring the patients’ non-dominant eye to previous low myopia to achieve a monovision. In this study, we investigated the feasibility of performing LASIK after lenticule re-implantation as a method to create presbyopic monovision. A -6.00D SMILE correction was performed in 9 rabbit eyes. The lenticules were cryopreserved for 14 days and re-implanted. Five weeks later, 3 of these eyes underwent LASIK for -5.00D correction (RL group); 3 underwent LASIK flap creation, which was not lifted (RN); and no further procedures were performed on the remaining 3 eyes. These groups were compared with 3 eyes that underwent standard LASIK for a -5.00D correction (LO); 3 that underwent creation of non-lifted flap (LN); and 3 non-operated eyes. Rabbits were euthanized 1 day post-surgery. Tissue responses were analyzed by immunohistochemistry, slit lamp and in vivo confocal microscopy (IVCM). Intrastromal irregularities and elevated reflectivity levels of the excimer-ablated plane were observed on slit lamp and IVCM, respectively in the RL group. The results were comparable (P = 0.310) to IVCM findings in the LO group. RL and LO groups showed similar fibronectin expression levels, number of CD11b-positive cells (P = 0.304) and apoptotic cells (P = 0.198). There was no difference between the RN and LN groups in reflectivity levels (P = 0.627), fibronectin expression levels, CD11b-positive cells (P = 0.135) and apoptotic cells (P = 0.128). LASIK can be performed following lenticule re-implantation to create presbyopic monovision. The tissue responses elicited after performing LASIK on corneas that have undergone SMILE and subsequent lenticule re-implantation are similar to primary procedure.
doi:10.1371/journal.pone.0083046
PMCID: PMC3859649  PMID: 24349429
7.  Hevin Plays a Pivotal Role in Corneal Wound Healing 
PLoS ONE  2013;8(11):e81544.
Background
Hevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin-/-) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser.
Methodology/Principal Findings
Wild type (WT) and hevin-/- mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin-/- mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice.
Conclusions/Significance
Hevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin-/- mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.
doi:10.1371/journal.pone.0081544
PMCID: PMC3841198  PMID: 24303054
8.  Cataract surgery following KAMRA presbyopic implant 
Intrastromal corneal inlays are an emerging treatment for presbyopic patients. The KAMRA™ small aperture inlay was the first such inlay to receive Conformité Européenne (CE) marking in 2005. It has been shown to improve uncorrected near and intermediate visual acuity without adversely affecting uncorrected distance visual acuity. Due to the age of presbyopic patients, they may eventually develop cataracts. In two such cases, we found that cataract surgery with the KAMRA implant left in place was not technically more difficult, and that the surgical procedure could be improved by additional ocular rotations to improve visualization. Biometry readings were reliable, and it appeared that the SRK/T formula was accurate for calculation of intraocular lens power. Cataract surgery with the KAMRA implant left in situ is a viable option for patients.
doi:10.2147/OPTH.S52182
PMCID: PMC3788815  PMID: 24092969
cataract surgery; KAMRA; corneal inlay; AcuTarget; presbyopia
9.  De novo ocular hypertension after Descemet stripping endothelial keratoplasty: comparative 3-year incidence, risk factors, and outcomes 
Objective
To compare the 3-year incidence of de novo ocular hypertension (OHT) after Descemet stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty (PK). For DSAEK, to evaluate predictors for OHT and 2-year outcomes after OHT development.
Methods
This was a review of the prospective Singapore Corneal Transplant Study at a single tertiary referral center. Consecutive DSAEKs and PKs for Fuchs’ endothelial dystrophy (FED) and pseudophakic bullous keratopathy (PBK) in eyes without pre-existing glaucoma were analyzed. OHT incidence after DSAEK and PK were compared using Kaplan–Meier survival analysis, and OHT risk factors identified using Cox proportional regression. OHT was defined: intraocular pressure (IOP) ≥ 24 mmHg or ≥ 10 mmHg from baseline. Secondary outcomes 2 years after OHT development in DSAEK were rates of glaucoma medical therapy failure, IOP success, graft failure and rejection, and best-spectacle corrected visual acuity (BSCVA).
Results
There were 108 (96.4%) DSAEKs and 216 (96%) PKs. The 1-, 2- and 3-year de novo OHT incidence was not significantly different between DSAEK (36.1%, 47.2%, 47.2%, respectively) and PK (35.7%, 44.9%, 45.8%, respectively; P = 0.914). OHT incidence did not differ in subgroup analyses of multiple clinical variables (P > 0.1). OHT predictors after DSAEK were: fellow eye glaucoma (hazard ratio [HR] 3.20, P = 0.004), age <60 years (HR 2.41, P = 0.016), concurrent goniosynechiolysis (HR 3.29, P = 0.021), post-graft complications or procedures (HR 2.85, P = 0.006). Two years after OHT onset, 29.7% of DSAEKs failed glaucoma medical therapy requiring trabeculectomy. Complete and qualified IOP success was achieved in 23.5% and 76.5%, respectively. Graft failure developed in 9.8% and graft rejection in 5.9%. At 6 months, 1, and 2 years from OHT onset, 86.3%, 88.3%, and 92.1% achieved BSCVA 20/40, respectively.
Conclusion
DSAEK and PK have comparable OHT risks. A significant 30% of DSAEK eyes with OHT require filtration surgery. Effective IOP control and good graft and visual outcomes are achieved with treatment.
doi:10.2147/OPTH.S50584
PMCID: PMC3788681  PMID: 24092962
DSAEK; glaucoma; ocular hypertension; risk factors
10.  A Biodegradable, Sustained-Released, Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model 
PLoS ONE  2013;8(8):e70419.
Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.
doi:10.1371/journal.pone.0070419
PMCID: PMC3734265  PMID: 23940573
11.  High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells 
PLoS ONE  2013;8(7):e67546.
Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.
doi:10.1371/journal.pone.0067546
PMCID: PMC3699644  PMID: 23844023
12.  Transmembrane water-flux through SLC4A11: a route defective in genetic corneal diseases 
Human Molecular Genetics  2013;22(22):4579-4590.
Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11−/− mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.
doi:10.1093/hmg/ddt307
PMCID: PMC3889808  PMID: 23813972
13.  Reversible Femtosecond Laser-Assisted Myopia Correction: A Non-Human Primate Study of Lenticule Re-Implantation after Refractive Lenticule Extraction 
PLoS ONE  2013;8(6):e67058.
LASIK (laser-assisted in situ keratomileusis) is a common laser refractive procedure for myopia and astigmatism, involving permanent removal of anterior corneal stromal tissue by excimer ablation beneath a hinged flap. Correction of refractive error is achieved by the resulting change in the curvature of the cornea and is limited by central corneal thickness, as a thin residual stromal bed may result in biomechanical instability of the cornea. A recently developed alternative to LASIK called Refractive Lenticule Extraction (ReLEx) utilizes solely a femtosecond laser (FSL) to incise an intrastromal refractive lenticule (RL), which results in reshaping the corneal curvature and correcting the myopia and/or astigmatism. As the RL is extracted intact in the ReLEx, we hypothesized that it could be cryopreserved and re-implanted at a later date to restore corneal stromal volume, in the event of keratectasia, making ReLEx a potentially reversible procedure, unlike LASIK. In this study, we re-implanted cryopreserved RLs in a non-human primate model of ReLEx. Mild intrastromal haze, noted during the first 2 weeks after re-implantation, subsided after 8 weeks. Refractive parameters including corneal thickness, anterior curvature and refractive error indices were restored to near pre-operative values after the re-implantation. Immunohistochemistry revealed no myofibroblast formation or abnormal collagen type I expression after 8 weeks, and a significant attenuation of fibronectin and tenascin expression from week 8 to 16 after re-implantation. In addition, keratocyte re-population could be found along the implanted RL interfaces. Our findings suggest that RL cryopreservation and re-implantation after ReLEx appears feasible, suggesting the possibility of potential reversibility of the procedure, and possible future uses of RLs in treating other corneal disorders and refractive errors.
doi:10.1371/journal.pone.0067058
PMCID: PMC3691223  PMID: 23826194
14.  Comparison of Anterior Segment Optical Tomography Parameters Measured Using a Semi-Automatic Software to Standard Clinical Instruments 
PLoS ONE  2013;8(6):e65559.
Objective
To compare anterior segment parameters measured using a semi-automatic software (Zhongshan Angle Assessment Program, ZAP) applied to anterior segment optical coherence tomography (AS-OCT) images, with commonly used instruments.
Methods
Cross-sectional study of a total of 1069 subjects (1069 eyes) from three population-based studies of adults aged 40–80 years. All subjects underwent AS-OCT imaging and ZAP software was applied to determine anterior chamber depth (ACD), central corneal thickness (CCT), anterior and keratometry (K) – readings. These were compared to auto-refraction, keratometry and ocular biometry measured using an IOLMaster, ultrasound pachymeter and auto-refractor respectively. Agreements between AS-OCT (ZAP) and clinical instrument modalities were described using Bland-Altman, 95% limits of agreement (LOA).
Results
The mean age of our subjects was 56.9±9.5 years and 50.9% were male. The mean AS-OCT (ZAP) parameters of our study cohort were: ACD 3.29±0.35 mm, CCT 560.75±35.07 µm; K-reading 46.79±2.72 D. There was good agreement between the measurements from ZAP analysis and each instrument and no violations in the assumptions of the LOA; albeit with a systematic bias for each comparison: AS-OCT consistently measured a deeper ACD compared to IOLMaster (95% LOA −0.24, 0.55); and a thicker CCT for the AS-OCT compared to ultrasound pachymetry (16.8±0.53 µm 95% LOA −17.3, 50.8). AS-OCT had good agreement with auto-refractor with at least 95% of the measurements within the prediction interval (P value <0.001).
Conclusion
This study demonstrates that there is good agreement between the measurements from the AS-OCT (ZAP) and conventional tools. However, small systematic biases remain that suggest that these measurement tools may not be interchanged.
doi:10.1371/journal.pone.0065559
PMCID: PMC3672133  PMID: 23750265
15.  Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro 
BMC Research Notes  2013;6:176.
Background
Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.
Results
Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.
Conclusions
Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.
doi:10.1186/1756-0500-6-176
PMCID: PMC3659058  PMID: 23641909
Human corneal endothelium; Human corneal endothelial cells; Primary cell culture; Cell density
16.  Comparison of corneal sensitivity, tear function and corneal staining following laser in situ keratomileusis with two femtosecond laser platforms 
Purpose
To evaluate longitudinal changes in corneal sensitivity, tear function, and corneal staining in patients who underwent laser in situ keratomileusis (LASIK) using two different femtosecond lasers.
Methods
In a prospective, randomized clinical trial, contralateral eyes of 45 patients underwent flap creation by either VisuMax or IntraLase™ femtosecond laser. Corneal sensitivity, tear break up time (TBUT), Schirmer’s test, and corneal fluorescein staining were assessed preoperatively and at 1 week, 1 month, and 3 months postoperatively.
Results
There were no statistical differences in any clinical outcome measure between the two femtosecond lasers (P > 0.05), although there was a trend towards slightly lower reductions for corneal sensitivity and TBUT in VisuMax-operated eyes. Overall, corneal sensitivity was significantly reduced at 1 week (P < 0.05), 1 month (P < 0 .001), and 3 months (P < 0.001) postoperatively. A significantly greater reduction of corneal sensitivity was noted in eyes with a myopic spherical equivalent of −6.00 diopters (D) to −11.25 D as compared with eyes that had a relatively lower level of myopia of less than −6.00 D (P < 0.001). TBUT and Schirmer’s test values were significantly diminished at 1 week postoperatively (P < 0.04). Overall, corneal staining was significantly increased at 1 week postoperatively (P < 0.001). The level of myopia did not significantly affect postoperative changes in TBUT, Schirmer’s test values, or corneal staining (P > 0.05).
Conclusion
This study showed that changes in corneal sensitivity, tear function, and corneal staining were statistically similar in LASIK using VisuMax and IntraLase femtosecond lasers for flap creation. However, the trend towards faster recovery of corneal sensitivity and TBUT observed in VisuMax-operated eyes may be attributable to improved technical specifications.
doi:10.2147/OPTH.S42266
PMCID: PMC3616690  PMID: 23576858
femtosecond laser; corneal sensitivity; tear film break-up time; Schirmer’s; corneal staining
17.  Fibrin glue for Gundersen flap surgery 
Purpose
To evaluate the feasibility of fibrin glue in Gundersen flap surgery.
Design
Prospective case series.
Study subjects
Seven eyes of seven subjects who had undergone Gundersen flap surgery from 2009 to 2011 at the Singapore National Eye Centre, Singapore.
Methods
Review of case records for outcomes after Gundersen flap surgery.
Main outcome measures
Surgical success was defined as achieving a stable ocular surface. Complications to be noted included flap retraction or exposure of underlying corneal surface.
Results
Surgical success was achieved in all eyes with significant reduction in ocular surface inflammation. No retractions were noted and recovery was uncomplicated.
Conclusion
Fibrin glue application is a viable alternative to sutures in Gundersen flap surgery. It reduces surgical downtime, gives faster ocular surface rehabilitation, and offers similar outcomes to conventional conjunctival flap surgery.
doi:10.2147/OPTH.S42105
PMCID: PMC3593825  PMID: 23493670
Gundersen; conjunctival flap; fibrin glue
18.  In vivo biocompatibility of two PEG/PAA interpenetrating polymer networks as corneal inlays following deep stromal pocket implantation 
This study compared the effects of implanting two interpenetrating polymer networks (IPNs) into rabbit corneas. The first (Implant 1) was based on PEG-diacrylate, the second (Implant 2) was based on PEG-diacrylamide. There were inserted into deep stromal pockets created using a manual surgical technique for either 3 or 6 months. The implanted corneas were compared with normal and sham-operated corneas through slit lamp observation, anterior segment optical coherence tomography, in vivo confocal scanning and histological examination. Corneas with Implant 1 (based on PEG-diacrylate) developed diffuse haze, ulcers and opacities within 3 months, while corneas with Implant 2 (based on PEG-diacrylamide) remained clear at 6 months. They also exhibited normal numbers of epithelial cell layers, without any immune cell infiltration, inflammation, oedema or neovascularisation at post-operative 6 month. Morphological studies showed transient epithelial layer thinning over the hydrogel inserted area and elevated keratocyte activity at 3 months; however, the epithelium thickness and keratocyte morphology were improved at 6 months. Implant 2 exhibited superior in vivo biocompatibility and higher optical clarity than Implant 1. PEG-diacrylamide-based IPN hydrogel is therefore a potential candidate for corneal inlays to correct refractive error.
doi:10.1007/s10856-012-4848-3
PMCID: PMC3620449  PMID: 23354737
19.  A mouse model of corneal endothelial decompensation using cryoinjury 
Molecular Vision  2013;19:1222-1230.
Purpose
To develop a mouse model of bullous keratoplasty and evaluate the safety and efficacy of cryoinjury-induced corneal endothelial decompensation.
Methods
Transcorneal freezing was performed on the right eye of each mouse. One cycle of cryoinjury was performed in 18 eyes (group A), and three cycles were performed in 17 eyes (group B). Pachymetry and intraocular pressure (IOP) measurements were done preoperatively, as well as at 1, 3, 7, 14, and 21 days after cryoinjury. At each post-cryoinjury time point, three mice from each group were euthanized, and the corneas underwent histology and electron microscopy.
Results
In both groups, significant corneal edema was noted at post-cryoinjury day 1, which was maintained throughout the study period. IOP remained within normal range in group A, but increased significantly with time in group B (p=0.011 at day 1, 0.038 at day 3, 0.026 at day 14, and 0.008 at day 21). In group B, serious complications including hyphema (one case), severe iridocorneal adhesion (15 cases), and total cataract (three cases) were detected, while only mild iridocorneal adhesion (four cases) and cataract (three cases) were noted in group A. Live/dead cell assay, hematoxylin and eosin staining, and scanning electron microscopy revealed successful ablation of corneal endothelial cells and absence of regeneration in both groups. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assay showed that apoptosis was mainly confined to the posterior stroma and endothelium in group A, while severe apoptosis was observed throughout all layers of the cornea in group B.
Conclusions
One cycle of cryoinjury was safer than three, while both were equally effective in inducing bullous keratopathy. This cryoinjury mouse model of bullous keratopathy was a consistently reproducible model that can be used for further studies on endothelial cell damage and rescue therapy.
PMCID: PMC3675054  PMID: 23761724
20.  Plastic Compressed Collagen as a Novel Carrier for Expanded Human Corneal Endothelial Cells for Transplantation 
PLoS ONE  2012;7(11):e50993.
Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.
doi:10.1371/journal.pone.0050993
PMCID: PMC3511456  PMID: 23226443
21.  Sweating of Descemet’s membrane during deep anterior lamellar keratoplasty in absence of perforation 
We report a case of spontaneous Descemet’s membrane sweating of aqueous humor during a manual deep anterior lamellar keratoplasty (DALK) without perforation of Descemet’s membrane. An 81-year-old female developed a neurotrophic central ulcer with descemetocele in the right eye, and her visual acuity was count fingers at 30 cm. She was unresponsive to medical treatment, and an uneventful manual DALK was performed. Six months after surgery, unaided visual acuity improved to 6/30. Seven months after surgery, the patient had a decrease in visual acuity to count fingers in the same eye. She was diagnosed as having corneal melting with a central descemetocele in the previous lamellar graft. A repeat manual DALK graft was performed. Lamellar dissection was performed starting from the edge of descemetocele, proceeding to the corneal periphery and maintaining the surgical plane of the previous DALK. During the surgical procedure, continuous and localized sweating of aqueous through Descemet’s membrane was observed in the area of the descemetocele. After drying of the recipient bed, no visible perforation of Descemet’s membrane was found. After removal of the previous DALK graft, a new stromal lamellar graft was sutured. The surgery was concluded without complications. One day after surgery, the graft was clear, with no detachment of Descemet’s membrane. If Descemet’s membrane sweating is observed during DALK and there is no visible perforation, the reason may be a hidden micron perforation in an intact Descemet’s membrane. It is recommended to continue with surgery maintaining maximum diligence and low intraocular pressure to prevent extension of micron perforation.
doi:10.2147/OPTH.S36268
PMCID: PMC3460700  PMID: 23055660
deep anterior lamellar keratoplasty; perforation; Descemet’s membrane; sweating
22.  Reproducibility and age-related changes of ocular parametric measurements in rabbits 
Background
The rabbit is a common animal model for ophthalmic research, especially corneal research. Ocular structures grow rapidly during the early stages of life. It is unclear when the rabbit cornea becomes mature and stabilized. We investigated the changes of keratometry, refractive state and central corneal thickness (CCT) with age. In addition, we studied the intra- and inter-observer reproducibility of anterior chamber depth (ACD) and anterior chamber width (ACW) measurements in rabbits using anterior segment-optical coherence tomography (AS-OCT).
Results
The growth of New Zealand White rabbits (n = 16) were monitored from age 1 to 12 months old. Corneal keratometric and refractive values were obtained using an autorefractor/keratometer, and CCT was measured using an AS-OCT. Keratometry and CCT changed rapidly from 1 to 7 months and appeared to be stabilizing after 8 months. The reduction of corneal curvature was approximately 1.36 diopter (D)/month from age 1 to 7 months, but the change decelerated to 0.30 D/month from age 8 to 12 months. An increase of 10 μm/month in CCT was observed from age 1 to 7 months, but the gain was reduced to less than 1 μm/month from age 8 to 12 months. There was a hyperopic shift over the span of 12 months, albeit the increase in spherical equivalent was slow and gradual. Rabbits of random age were then selected for 2 repeated ACD and ACW measurements by 2 independent and masked observers. Bland-Altman plots revealed a good agreement of ACD and ACW measurements inter- and intra-observer and the ranges of 95% limit of agreement were acceptable from a clinical perspective.
Conclusions
Corneal keratometry, spherical equivalent refraction and CCT changed significantly during the first few months of life of rabbits. Young rabbits have been used in a large number of eye research studies. In certain settings, the ocular parametric changes are an important aspect to note as they may alter the findings made in a rabbit experimental model. In this study, we have also demonstrated for the first time a good between observer reproducibility of measurements of ocular parameters in an animal model by using an AS-OCT.
doi:10.1186/1746-6148-8-138
PMCID: PMC3514359  PMID: 22901963
Cornea; Rabbit; Refractive; Keratometry; Anterior chamber; Reproducibility
23.  Reproducibility of Corneal Graft Thickness measurements with COLGATE in patients who have undergone DSAEK (Descemet Stripping Automated Endothelial Keratoplasty) 
BMC Medical Imaging  2012;12:25.
Background
The CorneaL GrAft Thickness Evaluation (COLGATE) system was recently developed to facilitate the evaluation of corneal graft thickness from OCT images. Graft thickness measurement can be a surrogate indicator for detecting graft failure or success. The purpose of this study was to determine the reproducibility of the COLGATE system in measuring DSAEK graft area between two observers.
Methods
This was a prospective case series in which 50 anterior segment OCT images of patients who had undergone DSAEK in either eye were analysed. Two observers (MW, AC) independently obtained the image analysis for the graft area using both semi automated and automated method. One week later, each observer repeated the analysis for the same set of images. Bland-Altman analysis was performed to analyze inter and intra observer agreement.
Results
There was strong intraobserver correlation between the 2 semi automated readings obtained by both observers. (r = 0.936 and r = 0.962). Intraobserver ICC for observer 1 was 0.936 (95% CI 0.890 to 0.963) and 0.967 (95% CI 0.942 to 0.981) for observer 2. Likewise, there was also strong interobserver correlation (r = 0.913 and r = 0.969). The interobserver ICC for the first measurements was 0.911 (95% CI 0.849 to 0.949) and 0.968 (95% CI 0.945 to 0.982) for the second. There was statistical difference between the automatic and the semi automated readings for both observers (p = 0.006, p = 0.003). The automatic readings gave consistently higher values than the semi automated readings especially in thin grafts.
Conclusion
The analysis from the COLGATE programme can be reproducible between different observers. Care must be taken when interpreting the automated analysis as they tend to over estimate measurements.
doi:10.1186/1471-2342-12-25
PMCID: PMC3443444  PMID: 22853647
Anterior segment optical coherence tomography; Descemet Stripping Automated Endothelial Keratoplasty; Graft thickness
24.  Comparison of outcomes of conventional WaveLight® Allegretto Wave® and Technolas® excimer lasers in myopic laser in situ keratomileusis 
Objective
To compare the results of laser in situ keratomileusis for myopia using WaveLight® Allegretto Wave® Eye-Q® and Technolas® 217z excimer lasers.
Method
A retrospective, comparative case series of 442 eyes matched for age and myopia: half each were treated with Allegretto’s wavefront-optimized algorithm and Technolas PlanoScan. Outcome measures were postoperative mean logarithm of the minimum angle of resolution (logMAR) uncorrected visual acuity (UCVA), manifest refraction spherical equivalent (MRSE), cylinder, safety and efficacy indices, refractive predictability, and optical zone size selection. Refractive predictability of a subgroup treated for −2.50 to −4.0 diopter (D) was analyzed separately.
Results
At mean follow-up of 80.5 days, mean logMAR UCVA, mean MRSE and mean postoperative cylinder were 0.02 ± 0.07 (range −0.12 to 0.30), 0.27 ± 0.36 D (range −1.25 to 1.50 D) and −0.33 ± 0.30 D (range 0.00 to −1.50 D) for Allegretto versus 0.02 ± 0.08 (range −0.12 to 0.40), 0.095 ± 0.47 D (range −1.25 to 1.13 D) and −0.44 ± 0.5 2 D (range 0.00 to −2.25 D) for Technolas (P = 0.98, 0.80 and 0.006). Mean safety and efficacy indices were 1.05 ± 0.13 (0.75–1.33) and 0.97 ± 0.13 (0.50–1.33) for Allegretto and 1.07 ± 0.14 (0.75–1.49) and 0.97 ± 0.17 (0.40–1.49) for Technolas (P = 0.23 and 0.69). Proportions of eyes achieving postoperative MRSE within ±1.0 D, ±0.5 D, and ±0.25 D were 98.2%, 91.9% and 75.6% for Allegretto and 99.1%, 97.8% and 72.4% for Technolas (P = 0.68, 0.20 and 0.51). Mean optical zone size selected was 6.48 ± 0.10 mm (range 6.0–6.5 mm) for Allegretto and 6.38 ± 0.19 mm (range 5.6–6.6 mm) for Technolas (P < 0.001). Of the subgroup with treatment between −2.5 and −4.0 D, 86.8% and 58.5% of eyes treated with Allegretto achieved postoperative MRSE within ±0.50 D and ±0.25 D versus 70.4% and 44.4% for Technolas (P = 0.006 and 0.057).
Conclusion
No differences were seen in postoperative mean logMAR UCVA, MRSE, safety and efficacy indices between the two lasers. Allegretto produced less residual astigmatism, possibly improved refractive predictability, and required smaller optical zone selection.
doi:10.2147/OPTH.S29660
PMCID: PMC3413344  PMID: 22888213
LASIK; myopia; laser vision correction; conventional laser algorithm
25.  Correlation of anterior segment optical coherence tomography measurements with graft trephine diameter following descemet stripping automated endothelial keratoplasty 
BMC Medical Imaging  2012;12:19.
Background
To assess repeatability of the Zhongshan Assessment Program (ZAP) software measurement of Anterior Segment Optical Coherence Tomography (ASOCT) images and correlate with graft trephine diameter following Descemet Stripping Automated Endothelial Keratoplasty (DSAEK)
Methods
Retrospectively evaluated interventional case series. 121 consecutive eyes undergoing DSAEK over a 26 month period underwent ASOCT imaging 1month after their surgery. ASOCT images were processed using ZAP software which measured the graft and cornea parameters including anterior and posterior graft arc length and cord length, posterior cornea arc length (PCAL) and anterior chamber width.
Results
The graft measurements showed good repeatability on ASOCT using ZAP with high intra class coefficient and small variation in the coefficient of variation. On ASOCT, the mean recipient PCAL was 12.99+/−0.69mm and the anterior chamber width was 11.16+/−0.57mm. The mean Graft anterior arc length was 9.69+/−0.66mm and the mean Graft anterior cord length was 8.92+/−2.94mm. The mean graft posterior arc length was 9.24+/−0.75mm and the mean graft posterior cord length was 8.15+/−0.57mm. Graft posterior arc length (rho=0.788, p< 0.001) correlated best with intra-operative graft trephine diameter. The mean ratio of posterior graft arc length to PCAL was 0.712 +/− 0.056.
Conclusions
We have validated the repeatability of the ZAP software for DSAEK graft measurements from ASOCT images and shown that the graft arc length parameters calculated from the ASOCT images correlate well with the intra-operative graft trephine diameter. This software may help surgeons determine the optimal DSAEK graft size based on pre-operative ASOCT measurements of the recipient eye.
doi:10.1186/1471-2342-12-19
PMCID: PMC3431259  PMID: 22824516

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