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.

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.

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.

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.

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.

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.

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.

Background

Small incision lenticule extraction or SMILE is a novel form of ‘flapless’ corneal refractive surgery that was adapted from refractive lenticule extraction (ReLEx). SMILE uses only one femtosecond laser to complete the refractive surgery, potentially reducing surgical time, side effects, and cost. If successful, SMILE could potentially replace the current, widely practiced laser in-situ keratomileusis or LASIK. The aim of this study is to evaluate whether SMILE is non-inferior to LASIK in terms of refractive outcomes at 3 months post-operatively.

Methods/Design

Single tertiary center, parallel group, single-masked, paired-eye design, non-inferiority, randomized controlled trial. Participants who are eligible for LASIK will be enrolled for study after informed consent. Each participant will be randomized to receive SMILE and LASIK in each eye. Our primary hypothesis (stated as null) in this non-inferiority trial would be that SMILE differs from LASIK in adults (>21 years old) with myopia (> −3.00 diopter (D)) at a tertiary eye center in terms of refractive predictability at 3 months post-operatively. Our secondary hypothesis (stated as null) in this non-inferiority trial would be that SMILE differs from LASIK in adults (>21 years old) with myopia (> −3.00 D) at a tertiary eye center in terms of other refractive outcomes (efficacy, safety, higher-order aberrations) at 3 months post-operatively. Our primary outcome is refractive predictability, which is one of several standard refractive outcomes, defined as the proportion of eyes achieving a postoperative spherical equivalent (SE) within ±0.50 D of the intended target. Randomization will be performed using random allocation sequence generated by a computer with no blocks or restrictions, and implemented by concealing the number-coded surgery within sealed envelopes until just before the procedure. In this single-masked trial, subjects and their caregivers will be masked to the assigned treatment in each eye.

Discussion

This novel trial will provide information on whether SMILE has comparable, if not superior, refractive outcomes compared to the established LASIK for myopia, thus providing evidence for translation into clinical practice.

Trial registration

Clinicaltrials.gov NCT01216475.

Background

Corneal transplantation has rapidly evolved from full-thickness penetrating keratoplasty (PK) to selective tissue corneal transplantation, where only the diseased portions of the patient's corneal tissue are replaced with healthy donor tissue. Descemet's membrane endothelial keratoplasty (DMEK) performed in patients with corneal endothelial dysfunction is one such example where only a single layer of endothelial cells with its basement membrane (10–15 µm in thickness), Descemet's membrane (DM) is replaced. It is challenging to replace this membrane due to its intrinsic property to roll in an aqueous environment. The main objective of this study was to determine the effects of fibrin glue (FG) on the biomechanical properties of DM using atomic force microscopy (AFM) and relates these properties to membrane folding propensity.

Methodology/Principal Findings

Fibrin glue was sprayed using the EasySpray applicator system, and the biomechanical properties of human DM were determined by AFM. We studied the changes in the “rolling up” tendency of DM by examining the changes in the elasticity and flexural rigidity after the application of FG. Surface topography was assessed using scanning electron microscopy (SEM) and AFM imaging. Treatment with FG not only stabilized and stiffened DM but also led to a significant increase in hysteresis of the glue-treated membrane. In addition, flexural or bending rigidity values also increased in FG-treated membranes.

Conclusions/Significance

Our results suggest that fibrin glue provides rigidity to the DM/endothelial cell complex that may aid in subsequent manipulation by maintaining tissue integrity.

Background

The purpose of this study was to compare endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty in Asian eyes.

Methods

This was a retrospective study of patients from our prospective Singapore Corneal Transplant Study cohort who received corneal transplantation in 2006–2008. We compared eyes that underwent DSAEK or penetrating keratoplasty for Fuchs’ endothelial dystrophy or pseudophakic and aphakic bullous keratopathy. Clinical data, and donor and recipient characteristics were recorded. Of 241 patients who met our inclusion criteria, 68 underwent DSAEK and 173 underwent penetrating keratoplasty. The main outcome measure was endothelial cell loss at 1 year. Secondary outcome measures were graft survival and visual outcomes at 1-year follow-up.

Results

There were no significant differences in baseline characteristics of patients between the treatment groups. Percent endothelial cell loss at 1-year follow-up was greater in penetrating keratoplasty eyes (40.9% ± 2.9%) compared with DSAEK eyes (22.4% ± 2.3%; P < 0.001). DSAEK-treated eyes had significantly superior uncorrected visual acuity (mean difference = 0.42 ± 0.0059; P < 0.001) and best spectacle-corrected visual acuity (mean difference = 0.14 ± 0.032; P < 0.001) as compared with penetrating keratoplasty-treated eyes. Penetrating keratoplasty-treated eyes had worse astigmatism as compared with DSAEK-treated eyes (−3.0 ± 2.1 versus −1.7 ± 0.8; P < 0.001). Graft survival at 1 year was comparable in both groups, ie, 66/68 (97.0%) DSAEK-treated eyes versus 158/173 (92.0%) of penetrating keratoplasty-treated eyes had clear grafts (P = 0.479).

Conclusion

We report lower percent endothelial cell loss comparing DSAEK and penetrating keratoplasty at 1-year follow-up in Asian eyes, with comparable graft survival rates in both groups.

Purpose

To evaluate the intraoperative changes in the donor lenticule, recipient cornea, and the reduction of interface fluid thickness during Descemet’s stripping and automated endothelial keratoplasty with EndoGlide™ (Angiotech Pharmaceuticals Inc, Vancouver, Canada) donor insertion, using intraoperative spectral-domain optical coherence tomography.

Methods

Prospective observational case series of patients underwent Descemet’s stripping and automated endothelial keratoplasty using the EndoGlide inserter. Spectral-domain optical coherence tomography (iVue; Optovue Inc, Fremont, CA) with a handheld probe was used to image the cornea and anterior chamber. Standardized software was used to measure interface fluid gap, host cornea, and donor lenticule thicknesses during the following surgical stages of Descemet’s stripping and automated endothelial keratoplasty: (1) after donor insertion and immediately before full air tamponade; (2) after air tamponade and expression of fluid from venting incisions; (3) at 6 minutes of air tamponade; and (4) at 10 minutes of air tamponade.

Results

Ten patients with a mean age of 74.9 ± 11.8 years were recruited. Spectral-domain optical coherence tomography measurements of the interface fluid gap after fluid was expressed through the venting incisions (P < 0.001), at 6 minutes of air tamponade (P < 0.001) and at 10 minutes of air tamponade (P < 0.001 and P = 0.001, respectively), were significantly decreased compared to the measurements immediately before air tamponade. Donor thickness increased significantly at 6 minutes of air tamponade (P = 0.004) but reduced by 10 minutes compared to immediately before air tamponade.

Conclusion

Significant intraoperative changes in the donor, recipient cornea, and interface fluid thickness occurred following endothelial keratoplasty donor insertion.

The culture of human corneal endothelial cells (CECs) is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs) that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS) technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

Purpose

To investigate the role of Tafazzin (TAZ) protein in regulating the proliferation of normal human conjunctiva epithelial cells and epithelial cells from pterygium tissue.

Methods

Conjunctiva epithelial cells were cultured in keratinocytes growth medium and treated with transformation growth factor β (TGFβ) to analyze the expression and translocation of TAZ protein by immunostaining and BrdU analysis. Immortalized conjunctiva epithelial cells (NHC) were treated with TGFβ, targeting siRNA, TGFβ receptor antibody or TGFβ receptor inhibitor, to study the involvement of TAZ and TGFβ signaling pathway in conjunctiva cell proliferation by cell adhesion assay. Conjunctiva tissues from a normal human eye and an eye with pterygium disease were collected for histological analyses and western blot to evaluate the TAZ protein expression in vivo.

Results

TAZ expression was upregulated in mitotic conjunctiva epithelial cells, proliferating conjunctiva epithelial cells, TGFβ treated conjunctiva epithelial cells and human pterygium epithelium. TAZ siRNA induced less conjunctiva epithelial cell growth. Moreover, TGFβ receptor antibody and TGFβ receptor inhibitor rescued this anti-proliferative effect of TAZ siRNA.

Conclusions

TAZ is involved in human conjunctiva epithelial cells proliferation via regulating TGFβ signaling pathway.

Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability.

Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage.

The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.

Background

Descemet’s stripping automated endothelial keratoplasty (DSAEK) has been shown to have superior refractive and visual results compared with penetrating keratoplasty, but higher rates of primary graft failure (PGF). This paper presents donor and surgical risk factors for PGF in DSAEK cases in Asian eyes.

Design

Retrospective case-control study.

Participants

All consecutive patients who underwent DSAEK at a tertiary referral teaching hospital from March 2006–December 2008.

Methods

Donor details analyzed were: age of donor, cause of donor death, death to harvesting time, donor storage time, distribution distance of tissue, preoperative endothelial cell count. Surgical factors analyzed were: donor diameter, donor thickness, and method of donor insertion. These risk factors in cases of PGF were compared with patients with successful DSAEK as the control group.

Main outcome measure

PGF.

Results

A total of 124 DSAEK procedures were performed. Six DSAEK procedures (five eyes of five patients; one eye with two failures) resulted in PGF (4.8%). Significant risk factors were found for PGF to include graft insertion using a folding technique (odds ratio [OR], 34.03; 95% confidence interval [CI], 3.75–314.32; P = 0.0017) and a small donor diameter (OR, 39.94; 95% CI, 2.18–732.17; P = 0.013).

Conclusion

The results of this study suggest that in Asian eyes with shallow anterior chambers, surgical trauma relating to the technique of donor insertion, and the use of a small donor are major risk factors for PGF following DSAEK.

Purpose:

To describe a novel technique of using Spectral-domain (SD) anterior segment optical coherence tomography (SD-OCT) in the evaluation of corneal epithelial healing under a therapeutic contact lens (TCL) after lamellar keratoplasty and Epi-LASIK procedures.

Design:

Prospective, non-comparative, observational case series.

Methods:

Ten eyes of eight patients undergoing lamellar corneal transplantation and Epi-LASIK procedures at the Singapore National Eye Centre were included in the study. Ultra-high resolution SD-OCT scans of the cornea with a TCL in-situ were performed sequentially on the first, third and fifth day after procedure, with the RTVue (Optovue, Inc, Fremont, CA, USA), and the image findings were correlated with the clinical picture. Complete epithelial healing was verified with removal of TCL and fluorescein staining.

Results:

5 eyes underwent Descemet’s stripping automated endothelial keratoplasty (DSAEK), 1 eye underwent deep anterior lamellar keratoplasty (DALK) and 4 eyes underwent Epi-LASIK. All eyes had complete epithelial healing with TCL in-situ by the third post-operative day. SD-OCT images were able to demonstrate the epithelial layer distinctly under the TCL in all cases.

Conclusions:

SD-OCT is a valuable imaging tool for monitoring the progression of epithelial healing with TCL in situ in patients following corneal surgical procedures.

Purpose

To study the expression and cellular distribution of multiple S100A genes and proteins in normal corneal-limbal epithelium and ocular surface squamous cell carcinoma (SCC) tissue.

Methods

Normal corneal-limbal tissue was obtained from the Lions Eye Bank, Tampa, FL. Ocular surface SCC tissues were excised from patients undergoing surgery at Singapore National Eye Centre. S100A mRNA expression was measured by quantitative PCR. S100 protein distribution was determined by immunofluorescent staining analysis.

Results

Twelve S100 mRNAs were identified in human corneal and limbal epithelial cells. S100A2, A6, A8, A9, A10, and A11 mRNA was expressed at high level, while S100A1, A3, A4, A5, A6, A7, and A12 mRNA expression was low. The intracellular localization of S100A2, A6, A8, A9, A10 and A11 protein was determined in normal corneal-limbal and SCC tissues. S100A2 and S100A10 proteins were enriched in basal limbal epithelial cells of the normal tissue. S100A8 and S100A9 were found only at the surface of peripheral corneal and limbal epithelium. S100A6 was uniformly found at the plasma membrane of corneal and limbal epithelial cells. S100A11 was found at the supralayer limbal epithelial cells adjacent to the conjunctiva. SCC tissue showed typical pathological changes with expression of cytokeartin (CK) 14 and CK4 in the epithelial cells. All SCC epithelial cells were positive of S100A2, S100A10, S100A6 and S100A11 staining. Intracellular staining of S100A8 and S100A9 was found in several layers of SCC epithelium. Expression of S100A2 and S100A10 decreased dramatically in cultured limbal epithelial cells with increased passaging, which was accompanied by a small increase of S100A9 mRNA, with no changes of S100A8 gene expression. Serum and growth hormone depletion of the culture serum caused a small reduction of S100A2 and S100A10 gene expression, which was accompanied by a small increase of S100A9 mRNA while no changes of S100A8 expression was measured.

Conclusions

Normal corneal and limbal epithelial cells express a broad spectrum of S100 genes and proteins. Ocular surface SCC express high levels of S100A2, S100A10, S100A8 and S100A9 proteins. The expression of S100A2 and S100A10 is associated with limbal epithelial cell proliferation and differentiation.

Purpose

To assess and compare keratocyte viability and collagen structure in cornea stroma lenticules collected immediately after refractive lenticule extraction (ReLEx) and one month after cryopreservation.

Methods

The fresh and cryopreserved human stroma lenticules procured after ReLEx were processed for ultrastructural analysis of keratocytes and collagen fibrils with transmission electron microscopy (TEM), apoptotic cell detection with deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL) assay, and cultured for keratocyte-specific gene expression analysis using reverse transcriptase polymerase chain reaction (RT–PCR).

Results

The periphery of the lenticule had greater TUNEL-positive cells compared to the center of the lenticule in both fresh and cryopreserved groups. There was an increase in TUNEL-positive cells after cryopreservation, which was significantly higher in the center of the lenticule, but not in the periphery. TEM showed apoptotic, necrotic and viable quiescent keratocytes in fresh and cryopreserved lenticules. Collagen analysis with TEM showed a well preserved and well aligned structure in fresh and cryopreserved lenticules; without significant change in the total number of collagen fibrils but with an increased collagen fibril density (CFD) after cryopreservation. In vitro, isolated keratocytes derived from fresh and cryopreserved lenticules exhibited a typical fibroblastic phenotype. RT–PCR showed a positive gene expression for keratocan (KERA) and aldehyde dehydrogenase 3A1 (ALDH3A1) in cells isolated from fresh and cryopreserved lenticules.

Conclusions

The stromal lenticules extracted from ReLEx surgery remain viable after cryopreservation. Although they showed a decrease in CFD, the collagen architecture was preserved and there was good cellular viability.

Dacryocystorhinostomy (DCR) remains the surgery of choice for the treatment of epiphora secondary to nasolacrimal duct (NLD) obstruction. It involves creating a direct soft‐tissue anastomosis between the lacrimal sac and the ipsilateral nasal cavity, via an osteotomy created by removal of the floor of the lacrimal fossa and surrounding bone. Successful surgery clearly requires the presence of a nasal space and absence of this poses a surgical challenge.

We describe three patients with absent nasal cavity on the side of lacrimal obstruction, where DCR was performed by the creation of an anastomosis between the lacrimal sac and the contralateral nasal space.

Purpose

To investigate the effect of intracameral injection of fibrin tissue sealant on the anterior segment structures in a rabbit model.

Methods

One eye of 10 rabbits received an intracameral injection of fibrin tissue sealant with a thrombin concentration of 500 IU (TISSEEL), and the fellow eye received an intracameral injection of balanced salt solution as a control. The rabbits were followed up with serial slit-lamp examinations, photography, high resolution anterior segment optical coherence tomography scans with pachymetry measurement, and intraocular pressure (IOP) monitoring until complete dissolution of the fibrin sealant. Corneal endothelial cell viability was evaluated using live/dead cell assays. Apoptosis of the cornea and trabecular meshwork were evaluated using TUNEL assays. Ultra-structural examinations of the cornea and trabecular meshwork were performed using electron microscopy. Histology of the trabecular meshwork and iris were analyzed using light microscopy.

Results

The quantity of the intracameral fibrin sealant was shown to be significantly correlated with increased IOP and pachymetry post-operatively. Complete dissolution of the fibrin sealant occurred between 15 and 30 days. Live/dead cell assays showed no decrease in viability of the corneal endothelium, and TUNEL assays showed no increase in apoptosis of the corneal epithelium, stroma, endothelium, or trabecular meshwork in the eyes with the fibrin sealant. Light and electron microscopy of the anterior segment structures were unremarkable.

Conclusion

The intracameral use of fibrin glue was associated with a transient increase in IOP and pachymetry. However, there was no evidence of toxicity or structural damage to the corneal endothelium, trabecular meshwork, or iris.

Purpose

To analyze for the presence of lipids in conjunctival fibroblasts of a patient with Schnyder corneal dystrophy (SCD).

Methods

A proband with SCD was identified, and the pedigree was examined. The proband underwent an automated lamellar therapeutic keratoplasty (ALTK). At the same time, the proband underwent a skin and conjunctival biopsy. Specimens were sent for histological and ultrastructural examination. Conjunctival fibroblasts were cultured from the biopsy specimen and stained with filipin.

Results

The proband showed no evidence of recurrence following the ALTK procedure. Histopathological examination showed no evidence of hydrophobic lipids in the conjunctival or dermal fibroblasts. Lipid particles were detected in the cornea. Ultrastructural examination showed no lipid particles in the conjunctival fibroblasts. Cultured fibroblasts showed no evidence of intracellular unesterified cholesterol unless low density lipoprotein (LDL) was added to the culture medium.

Conclusions

There was no evidence of lipid deposition in conjunctival or skin fibroblasts in our patient with SCD. The evidence suggests local factors are responsible for the lipid deposition in the cornea.

Aim

Keratoprosthesis (KPro) devices are prone to long-term corrosion and microbiological assault. The authors aimed to compare the inflammatory response and material dissolution properties of two candidate KPro skirt materials, hydroxyapatite (HA) and titania (TiO2) in a simulated in vitro cornea inflammation environment.

Methods

Lipopolysaccharide-stimulated cytokine secretions were evaluated with human corneal fibroblasts on both HA and TiO2. Material specimens were subjected to electrochemical and long-term incubation test with artificial tear fluid (ATF) of various acidities. Topography and surface roughness of material discs were analysed by scanning electron microscopy and atomic force microscopy.

Results

There were less cytokines secreted from human corneal fibroblasts seeded on TiO2 substrates as compared with HA. TiO2 was more resistant to the corrosion effect caused by acidic ATF in contrast to HA. Moreover, the elemental composition of TiO2 was more stable than HA after long-term incubation with ATF.

Conclusions

TiO2 is more resistant to inflammatory degradation and has a higher corrosion resistance as compared with HA, and in this regard may be a suitable material to replace HA as an osteo-odonto-keratoprosthesis skirt. This would reduce resorption rates for KPro surgery.