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Corneal crosslinking is a U.S. Food and Drug Administration--approved therapy to stiffen the cornea and prevent progression of corneal ectasia in patients with keratoconus. The standard procedure involves removal of the corneal epithelium (epithelial-off) prior to treatment. Variations to the standard procedure include accelerated crosslinking and transepithelial procedures. This study reviewed what is known regarding the risk for infection after epithelial-off crosslinking, the spectrum of pathogens, and clinical outcomes. 26 publications were identified. All eyes were fit with a bandage contact lens postoperatively. Available data indicate that the overall frequency of infectious keratitis after epithelium-off crosslinking is low. Bacterial infections are the most common, with a mean time of presentation of 4.8 days postoperatively. The use of steroids and bandage contact lenses in the immediate postoperative period and/or a history of atopic or herpetic disease were associated with infection. These patients require intense postoperative care with prophylactic antiviral therapy when appropriate.

Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3−/−) knockout mice. The corneal stroma in F3−/− mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3−/− mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3−/− mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease. • Fibulins are expressed throughout the body at varying levels. • Fibulin-3 has a tissue-specific pattern of expression within the eye. • Lack of fibulin-3 leads to structural deformities in the cornea. • The retina and RPE remain structurally and functionally healthy in the absence of fibulin-3 in both mice and humans.

Assessment of donor suitability and criteria development for tissue donation evaluation which appropriately addresses the risk factors for disease transmission, especially high risk for Hepatitis B or C, HIV or other transmissible diseases as defined by the Food and Drug Administration, FDA, is a continuing concern for tissue banks. The relationship of drug use, especially IV drugs, has been determined to be associated with an increased possibility of reactive serology (Centers for Disease Control and Prevention (USCDC) in Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention. Hepatitis C questions and answers for health professionals. https://www.cdc.gov/hepatitis/hcv/hcvfaq.htm; Centers for Disease Control and Prevention (USCDC) in infectious diseases, opioids and injection drug use, 2018. https://www.cdc.gov/pwid/opioid-use.html; HIH National Institute on Drug Abuse in Health Consequences of Drug Misuse, 2017. https://www.drugabuse.gov/related-topics/health-consequences-drug-misuse). Therefore, prior drug use determined by medical social history screening frequently results in deferral of a potential donor even when the route of drug administration has not been determined to be intravenous. Because of the association of drug use in numerous cases, which come under Medical Examiner jurisdiction, a possible rule out of a number of otherwise suitable medical examiner cases could occur. This retrospective review of medical examiner cases, tissue bank referrals and tissue donors in a 3-year period examines the relationship, if any, between reactive serology and positive toxicology results. These results would appear to indicate assessment of donor medical social history screening is effective in reducing recovery of high-risk donors.

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

Purpose To compare the effectiveness of femtosecond laser-assisted cataract surgery (FLACS) and conventional phacoemulsification cataract surgery (CPS) by resident surgeons. Setting Parkland Memorial Health and Hospital System, Dallas, Texas, USA. Design Prospective randomized study. Methods All surgeries to be performed by postgraduate year 3 and year 4 residents from October 2015 through June 2017 were eligible for inclusion. Patients were required to complete postoperative day 1, week 1, month 1, and month 3 visits. Specular microscopy was performed preoperatively and postoperatively. Surgeries were filmed, and each step was timed and compared. Surgeon and patient surveys were filled out postoperatively. Results Of the 135 eyes of 96 subjects enrolled in the study, 64 eyes received FLACS and 71 eyes received CPS. There was no significant difference in corrected distance visual acuity (CDVA), either preoperatively or at the postoperative day 1, week 1, month 1, or month 3 visits (P = .469, .539, .701, .777, and .777, respectively). Cumulated dissipated energy and irrigation fluid usage were not different between FLACS and CPS (P = .521 and .368, respectively), nor was there a difference in the reduction of endothelial cell counts postoperatively (P = .881). Wound creation (P = .014), cortical cleanup (P = .009), and IOL implantation (P = .031) were faster in the CPS group. Survey results indicated that the overall patient experience was similar for FLACS and CPS. Conclusions This first prospective randomized trial evaluating resident-performed FLACS shows that, in resident hands, FLACS provides similar results to CPS regarding visual acuity, endothelial cell loss, operative time, patient satisfaction, and surgical complication rate.

Purpose To investigate the long-term corneal changes in patients with Fuchs endothelial corneal dystrophy contributing to superior postoperative visual outcomes after Descemet membrane endothelial keratoplasty (DMEK) compared with Descemet stripping automated endothelial keratoplasty (DSAEK). Methods Using retrospective analysis, we evaluated 9 patients with Fuchs endothelial corneal dystrophy who underwent DSAEK in 1 eye and DMEK in the fellow eye. Patients were genotyped for the triplet repeat expansion in the TCF4 gene and imaged using optical coherence tomography, Scheimpflug imaging, and in vivo confocal microscopy through focusing. Results Eight of 9 subjects were genotyped, and all were found to harbor the triplet repeat expansion. The average time between endothelial keratoplasty and imaging was 76 ± 22 and 37 ± 9 months after DSAEK and DMEK, respectively. The mean best spectacle-corrected visual acuity (logMAR) was 0.04 ± 0.05 and 0.11 ± 0.03 in the DMEK eyes versus DSAEK eyes (P = 0.02), respectively. Posterior corneal higher order aberrations were less in the DMEK eyes compared with fellow DSAEK eyes (0.25 ± 0.06 and 0.66 ± 0.25, respectively, P ≤ 0.01). Using confocal microscopy through focusing, we found that the persistent anterior stromal haze was correlated between the right and left eyes (R = 0.73, P ≤ 0.05), but total stromal backscattering was higher for the DSAEK eyes (P ≤ 0.05). Conclusions DSAEK inherently results in higher total stromal backscattering (haze) compared with DMEK because of the addition of stromal tissue. Lower higher order aberrations of the posterior cornea and lower total stromal backscattering (haze) may both contribute to superior visual outcomes after DMEK compared with DSAEK.

We previously reported that corneal fibroblasts within 3D fibrin matrices secrete, bind, and organize fibronectin into tracks that facilitate cell spreading and migration. Other cells use these fibronectin tracks as conduits, which leads to the development of an interconnected cell/fibronectin network. In this study, we investigate how cell-induced reorganization of fibrin correlates with fibronectin track formation in response to two growth factors present during wound healing: PDGF BB, which stimulates cell spreading and migration, and TGF&beta, 1, which stimulates cellular contraction and myofibroblast transformation. Both PDGF BB and TGF&beta, 1 stimulated global fibrin matrix contraction (p &lt, 0.005), however, the cell and matrix patterning were different. We found that, during PDGF BB-induced cell spreading, fibronectin was organized simultaneously with the generation of tractional forces at the leading edge of pseudopodia. Over time this led to the formation of an interconnected network consisting of cells, fibronectin and compacted fibrin tracks. Following culture in TGF&beta, 1, cells were less motile, produced significant local fibrin reorganization, and formed fewer cellular connections as compared to PDGF BB (p &lt, 0.005). Although bands of compacted fibrin tracks developed in between neighboring cells, fibronectin labeling was not generally present along these tracks, and the correlation between fibrin and fibronectin labeling was significantly less than that observed in PDGF BB (p &lt, 0.001). Taken together, our results show that cell-induced extracellular matrix (ECM) reorganization can occur independently from fibronectin patterning. Nonetheless, both events seem to be coordinated, as corneal fibroblasts in PDGF BB secrete and organize fibronectin as they preferentially spread along compacted fibrin tracks between cells, producing an interconnected network in which cells, fibronectin and compacted fibrin tracks are highly correlated. This mechanism of patterning could contribute to the formation of organized cellular networks that have been observed following corneal injury and refractive surgery.

We previously reported that fibroblasts migrating within 3-D collagen matrices move independently, whereas fibroblasts within 3-D fibrin matrices form an interconnected network. Similar networks have been identified previously during in vivo corneal wound healing. In this study, we investigate the role of fibronectin in mediating this mechanism of collective cell spreading, migration and patterning.To assess cell spreading, corneal fibroblasts were plated within fibrillar collagen or fibrin matrices. To assess migration, compacted cell-populated collagen matrices were nested inside cell-free fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without RGD peptide, anti-α5 or anti-fibronectin blocking antibodies. In some experiments, LifeAct and fluorescent fibronectin were used to allow dynamic assessment of cell-induced fibronectin reorganization. 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, F-actin, α5 integrin and fibronectin organization.Corneal fibroblasts within 3-D fibrin matrices formed an interconnected network that was lined with cell-secreted fibronectin. Live cell imaging demonstrated that fibronectin tracks were formed at the leading edge of spreading and migrating cells. Furthermore, fibroblasts preferentially migrated through fibronectin tracks laid down by other cells. Interfering with cell-fibronectin binding with RGD, anti α5 integrin or anti fibronectin antibodies inhibited cell spreading and migration through fibrin, but did not affect cell behavior in collagen.In this study, a novel mode of cell patterning was identified in which corneal fibroblasts secrete and attach to fibronectin via α5β1 integrin to facilitate spreading and migration within 3-D fibrin matrices, resulting in the formation of localized fibronectin tracks. Other cells use these fibronectin tracks as conduits, resulting in an interconnected cell-fibronectin network.

This study utilizes high resolution multi-dimensional imaging to identify temporal and spatial changes in cell/extracellular matrix (ECM) patterning mediating cell migration, fibrosis, remodeling and regeneration during wound healing. Photorefractive keratectomy (PRK) was performed on rabbits. In some cases, 5([4,6-dichlorotriazin-2yl]-amino)fluorescein (DTAF) was applied immediately after surgery to differentiate native vs. cell-secreted collagen. Corneas were assessed 3–180 days postoperatively using in vivo confocal microscopy, and cell/ECM patterning was evaluated in situ using multiphoton and second harmonic generation (SHG) imaging. 7 days post-PRK, migrating fibroblasts below the ablation site were co-aligned with the stromal lamellae. At day 21, randomly patterned myofibroblasts developed on top of the ablation site; whereas cells underneath were elongated, co-aligned with collagen, and lacked stress fibers. Over time, fibrotic tissue was remodeled into more transparent stromal lamellae. By day 180, stromal thickness was almost completely restored. Stromal regrowth occurred primarily below the ablation interface, and was characterized by co-localization of gaps in DTAF labeling with elongated cells and SHG collagen signaling. Punctate F-actin labeling was detected along cells co-aligned with DTAF and non-DTAF labeled collagen, suggesting cell-ECM interactions. Overall, collagen lamellae appear to provide a template for fibroblast patterning during wound healing that mediates stromal repopulation, regeneration and remodeling.

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike.

Pseudomonas aeruginosa is a pathogenic gram-negative organism that has the ability to cause blinding corneal infections following trauma and during contact lens wear. In this study, we investigated the directional movement and orientation of an invasive corneal isolate of P. aeruginosa in the corneal stroma during infection of ex vivo and in vivo rabbit corneas using multiphoton fluorescence and second harmonic generation (SHG) imaging. Ex vivo, rabbit corneas were subject to three partial thickness wounds prior to inoculation. In vivo, New Zealand white rabbits were fit with P. aeruginosa laden contact lenses in the absence of a penetrating wound. At all time points tested, infiltration of the corneal stroma by P. aeruginosa revealed a high degree of alignment between the bacteria and collagen lamellae ex vivo (p In vivo, P. aeruginosa traveled throughout the stroma in discrete regions or bands. Within each region, the bacteria showed good alignment with collagen lamellae (P = 0.002). Interestingly, in both the in vitro and in vivo models, P. aeruginosa did not appear to cross the corneal limbus. Taken together, our findings suggest that P. aeruginosa exploits the precise spacing of collagen lamellae in the central cornea to facilitate spread throughout the stroma.

Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation by were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.

Purpose—The purpose of this study was to develop and test hardware and software modifications to allow quantitative full-thickness corneal imaging using the HRT Rostock Corneal Module. Methods—A PC-controlled motor drive with positional feedback was integrated into the system to allow automated focusing through the entire cornea. The left eyes of ten New Zealand White rabbits were scanned from endothelium to epithelium. Image sequences were read into a customdeveloped program for depth calculation and measurement of sub-layer thicknesses. 3-D visualizations were also generated using Imaris. In six rabbits, stack images were registered, and depth-dependent counts of keratocyte nuclei were made using Metamorph. Results—The mean epithelial and corneal thicknesses measured in the rabbit were 47 ± 5 μm and 373 ± 25 μm, respectively (N = 10 corneas); coefficients of variation for repeated scans were 8.2% and 2.1%. Corneal thickness measured using ultrasonic pachymetry was 374 ± 17 μm. The mean overall keratocyte density measured in the rabbit was 43,246 ± 5,603 cells/mm 3 in vivo (N = 6 corneas). There was a gradual decrease in keratocyte density from the anterior to posterior cornea (R = 0.99), consistent with previous data generated in vitro. Conclusions—This modified system allows high resolution 3-D image stacks to be collected from the full thickness rabbit cornea in vivo. These datasets can be used for interactive visualization of corneal cell layers, measurement of sub-layer thickness, and depth-dependent keratocyte density measurements. Overall, the modifications significantly expand the potential quantitative research applications of the HRT-RCM microscope.

Extracellular matrix (ECM) supplies both physical and chemical signals to keratocytes which can impact their differentiation to fibroblasts and/or myofibroblasts. It also provides a substrate through which they migrate during wound repair. We have previously shown that following transcorneal freeze injury (FI), migrating corneal fibroblasts align parallel to the stromal lamellae during wound repopulation. In this study, we compare cell and ECM patterning both within and on top of the stroma at different time points following lamellar keratectomy (LK) in the rabbit. Twelve rabbits received LK in one eye. Rabbits were monitored using in vivo confocal microscopy at 3, 7, 21 and 60 days after injury. A subset of animals was sacrificed at each time point to further investigate cell and matrix patterning. Tissue was fixed and labeled in situ with Alexa Fluor 488 phalloidin (for F-actin), and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging (for collagen). Immediately following LK, cell death occurred in the corneal stroma directly beneath the injury. At 7 and 21 days after the procedures, analysis of fluorescence (F-actin) and SHG results (collagen) indicated that fibroblasts were co-aligned with the collagen lamellae within this region. In contrast, stromal cells accumulating on top of the stromal wound bed were randomly arranged, contained more prominent stress fibers, and expressed alpha smooth muscle actin (α-SMA) and fibronectin. At 60 days, cells and matrix in this region had become co-aligned into lamellar-like structures; cells were elongated but did not express stress fibers. Corneal haze measured using in vivo confocal microscopy peaked at 21 days after LK, and was significantly reduced by 60 days. Cell morphology and patterning observed in vivo was similar to that observed in situ. Our results suggest that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after LK injury in the rabbit. In contrast, stromal cells accumulating on top of the stromal wound bed initially align randomly and produce a fibrotic ECM. Remarkably, over time, these cells appear to remodel the ECM to produce a lamellar structure that is similar to the native corneal stroma.

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, alpha-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P

Hyaluronan (HA) is a glycosaminoglycan. Despite its high molecular weight, Brown et al. has shown that topically applied HA rapidly disseminated throughout the epidermis and reached the dermis in both mouse and human skin [1]. Importantly, the majority of cutaneous malignant melanomas are positive for the HA receptor, CD44 [2]. We envisioned that HA conjugated to anti-cancer drugs would be useful for: 1) Transport of the drug into the skin after topical application, and 2) Targeted delivery of the drug to CD44 positive malignant melanoma cells. To test this concept, we conjugated doxorubicin (DOX) to HA and tested its impact on B16-F10 melanoma cells in vitro and in vivo. We used DOX because of its intrinsic fluorescence and the availability of anti-DOX antibodies. Thus, DOX-HA could be evaluated in vitro and in vivo. B16-F10 melanoma cells expressed CD44 as assessed by FACS (Fig. 1A). The binding of CD44 to HA and DOX-HA was evaluated using cell adhesion assays. As shown in Fig. 1B, panel 1, B16-F10 melanoma cells showed significantly better binding to wells coated with HA as compared to the wells lacking the HA substrate. An anti-CD44 monoclonal antibody significantly (P

Purpose To report the clinical and visual outcomes of a series of patients implanted with the PermaVision intracorneal lens (ReVision Optics Inc, Lake Forest, California, USA) for correction of hyperopia. Design Prospective, nonrandomized, clinical trial study. Methods Fifteen patients were implanted with the intracorneal lens for correction of spherical hyperopic refractive errors ranging from 1.00 to 5.00 diopters (D) (mean, 2.45 D). All eyes were correctable to 20/20 before surgery. Patients were followed up to 3 years with measurements of residual refractive error, uncorrected visual acuity (UCVA), and best spectacle-corrected visual acuity (BSCVA). Results For patients with follow-up of 1 year or more, the mean logarithm of the minimum angle of resolution BSCVA was 0.15 (20/28 Snellen equivalent; range, 0.1 to 0.6) with an average change of −2.5 lines of BSCVA (range, 1 to −6 lines). Only 3 eyes (25%) achieved 20/20 or better UCVA. Two patients experienced early adverse events and 1 patient experienced a late-onset adverse event requiring eventual removal of the implant. Nine patients elected to have the implant removed, with dissatisfaction with the quality of vision as the most frequent reason for removal. Conclusions The quality of visual and refractive outcomes with this device was less than satisfactory for most of the patients in our series. Although the problems of early onset migration or extrusion of the device are possible, late-onset ulceration noted in one of our patients most likely represents a neurotropic melt. Further research to improve the safety and quality of refractive result with intracorneal implants is needed.

The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 microm thick fibrillar collagen matrices and cultured for 1-2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent.

PURPOSE: To report effects of the PermaVision intracorneal lens at the cellular level using in vivo confocal microscopy. METHODS: Four eyes implanted with intracorneal lenses beneath an IntraLase flap for correction of hyperopia were evaluated preoperatively and 1 to 6 months postoperatively. RESULTS: Intracorneal lenses were tolerated in three eyes with little or no haze observed clinically and good visual results. Minimal keratocyte activation was detected by confocal microscopy, and cell density was decreased posterior to the implants. Epithelial thinning was observed 1 month after implantation. Thickness stabilized by 6 months but remained thinner than baseline (33±2 µm vs 48±8 µm, P CONCLUSIONS: Implantation of intracorneal lenses can induce side effects of epithelial thinning, keratocyte loss, and keratocyte activation. [J Refract Surg. 2007;23:410-413.]

Purpose To compare the response of the cornea to laser in situ keratomileusis (LASIK) with flap creation using the IntraLase FS15 or FS30 femtosecond laser (IntraLase Corp.). Setting Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. Methods Twenty-three patients (31 eyes) who had LASIK with flap creation using the FS15 or FS30 laser were assessed by clinical examination and confocal microscopy in a nonrandomized parallel treatment group comparative trial. Eight FS15 patients (15 eyes) were examined preoperatively and 3 months postoperatively, and 14 FS30 patients (15 eyes) were examined 3 months postoperatively. Results No patient in either group had clinically significant flap interface haze. One FS15 eye and 1 FS30 eye had significant keratocyte activation at the flap interface. The mean difference between the actual flap thickness and intended flap thickness was 16.8 μm ± 11.1 (SD) and 13.9 ± 7.1 μm in the FS15 group and FS30 group, respectively ( P = .49). The mean measured interface reflectivity was 156.4 ± 88.6 confocal backscatter units (CBU) and 104.8 ± 91.2 CBU, respectively ( P = .15). The mean density of interface particles was 21.4 ± 14.8 particles/mm 2 in the FS15 group and 11.0 ± 7.1 particles/mm 2 in the FS30 group ( P Conclusions Both the FS15 and FS30 lasers provided more reproducible flap thickness and fewer interface particles than previously observed using microkeratomes. The response of corneal keratocytes to intra-LASIK was reduced compared with previous results in which higher raster energies were used. Compared with the FS15, there was an apparent reduction in overall interface reflectivity and fewer interface particles with the FS30 laser.

Objective To describe a newly recognized clinical syndrome in Acanthamoeba keratitis consisting of severe reactive ischemic posterior segment vascular inflammation. Design Noncomparative, retrospective, single-institution observational case series. Participants Five eyes of 5 patients with Acanthamoeba keratitis. Methods A retrospective review of the records of patients diagnosed with Acanthamoeba keratitis between January 1, 1995, and December 1, 2005, was conducted to identify those who underwent eventual enucleation. Five enucleated eyes of 118 eyes with Acanthamoeba keratitis were identified. Main Outcome Measures History, clinical examination results, available laboratory study results, and histopathologic examination results. Results Histopathologic examination showed Acanthamoeba cysts in the cornea in 4 eyes, whereas it failed to demonstrate amebic cysts or trophozoites in the posterior segment of all eyes studied and unexpectedly revealed chronic chorioretinal inflammation with perivascular lymphocytic infiltration and diffuse neuroretinal ischemia in 4 of 5 eyes. Retinal artery thrombosis was present in 3 of the 4 involved eyes, and central retinal artery and vein thrombosis was found in 1 eye. Hematologic studies in 3 patients showed abnormal anticardiolipin antibody levels in 1 patient and factor V Leiden deficiency in another. Conclusions Prolonged Acanthamoeba keratitis can result in a severe sterile ischemic posterior segment inflammation that is potentially blinding, especially in patients with underlying hypercoagulation disorders.

Purpose To examine the safety and efficacy of topical anti-interleukin-1 (anti-IL-1) following photorefractive keratectomy (PRK) in rabbit eyes. Setting Joint Warfighter Refractive Surgery Center, Lackland Air Force Base, Texas, USA. Design Experimental study. Methods After standard PRK, 48 eyes of 24 New Zealand white rabbits were divided into 4 treatment arms and 1 control arm. Eyes in the treatment arms were randomized to receive fluorometholone 0.1% or an anti-IL-1 suspension (2.50 mg, 1.25 mg, or 0.25 mg doses) plus standard moxifloxacin, balanced salt solution (BSS), and an ocular lubricant (Systane) 4 times a day. Control eyes received only moxifloxacin, balanced salt solution, and ocular lubricant. Results No adverse events were observed with anti-IL-1. The safety of anti-IL-1 was affirmed because there was no statistically significant difference in time to epithelial closure, foam-layer histology and thickness, or final stromal thickness measurements between the anti-IL-1 and the steroid or control groups. No increase in haze was observed with anti-IL-1. There was a trend toward decreased haze with anti-IL-1 at several data points compared with the control and steroid groups. Finally, there was a trend toward less haze in all metrics at almost every timepoint for the 2.50 mg anti-IL-1 group compared with lesser concentrations. Conclusions Anti-IL-1 therapy might be a safe, effective alternative to steroids for haze prevention after PRK. Of the doses studied, 2.50 mg of anti-IL-1 4 times a day appeared to be most effective. Further studies in human eyes are needed. Financial Disclosure Dr. Reilly has been a consultant to Alcon Laboratories, Inc., and Abbott Medical Optics, Inc. None of the authors has a financial or proprietary interest in any material or method mentioned.

The goal of this study was to investigate how alterations in extracellular matrix (ECM) biophysical properties modulate corneal keratocyte phenotypes in response to specific wound healing cytokines and Rho GTPases. Rabbit corneal keratocytes were plated within standard collagen matrices (2.5 mg/mL) or compressed collagen matrices (~100 mg/mL) and cultured in serum-free media, PDGF BB, IGF, FGF2 or TGFβ1, with or without the Rac1 inhibitor NSC23766 and/or the Rho kinase inhibitor Y-27632. After 1 to 4 days, cells were labeled for F-actin and imaged using confocal microscopy. Keratocytes within standard collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in serum-free media, PDGF, IGF and FGF, but developed stress fibers in TGFβ1. Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in serum-free media, PDGF and IGF, but developed stress fibers in both FGF and TGFβ1. The Rac inhibitor had no significant impact on growth factor responses in compliant matrices. Within compressed collagen matrices however, the Rac inhibitor induced fibroblastic transformation in serum-free media, PDGF and IGF. Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition. Overall, keratocyte growth factor responses appear to be regulated by both the interplay between Rho and Rac signaling, and the structural and mechanical properties of the ECM.

A new depth encoding system (DES) is presented, which makes it possible to calculate, display, and record the z-axis position continuously during in vivo imaging using tandem scanning confocal microscopy (TSCM). In order to verify the accuracy of the DES for calculating the position of the focal plane in the cornea both in vitro and in vivo, we compared TSCM measurements of corneal thickness to measurements made using an ultrasonic pachymeter (UP, a standard clinical instrument) in both enucleated rabbit, cat, and human eyes (n = 15), and in both human patients (n = 7). Very close agreement was found between the UP and TSCM measurements in enucleated eyes; the mean percent difference was 0.50 +/- 2.58% (mean +/- SD, not significant). A significant correlation (R = 0.995, n = 15, p < 0.01) was found between UP and TSCM measurements. These results verify that the theoretical equation for calculating focal depth provided by the TSCM manufacturer is accurate for corneal imaging. Similarly, close agreement was found between the in vivo UP and TSCM measurements; the mean percent differences was 1.67 +/- 1.38% (not significant), confirming that z-axis drift can be minimized with proper applanation of the objective. These results confirm the accuracy of the DES for imaging of the cornea both ex vivo and in vivo. This system should be of great utility for applications where quantitation of the three-dimensional location of cellular structures is needed.

Purpose The antiapoptotic protein Bcl-2 and its proapoptotic counterpart Bax may play an important role in the regulation of corneal epithelial renewal. To understand the physiologic importance of Bcl-2 and Bax in the healthy cornea, epithelial and stromal thickness, proliferation, and surface cell exfoliation rates were examined in the central cornea of genetically altered mice overexpressing Bcl-2 and lacking Bax. Methods Adult Bcl-2 transgenic (n = 55) and Bax knockout (n = 82) mice and wild-type controls were clinically prescreened at no less than 2 months of age for any ocular developmental abnormalities. Polymerase chain reaction was used to confirm genotype. Corneal epithelial renewal was examined by in vivo tandem scanning confocal microscopy to measure total corneal and sublayer thickness and by immunohistochemistry on whole mount corneal tissues to determine basal epithelial cell proliferation using 5-bromo-2-deoxyuridine (BrdU) and surface cell exfoliation with a Calcein AM-Ethidium homodimer assay (live/dead). Stained corneas were scanned with a laser scanning confocal microscope, and images were digitized, and cell counts were obtained. Levels of Bcl-2 protein were assessed by Western blots. Results No significant changes in proliferation or cell death were found in either group compared to wild-type littermate controls; however, epithelial and stromal thicknesses were greater in the Bcl-2 transgenic group compared to wild-type and decreased in the Bax knockout. Western blotting confirmed that there was no change in the level of Bcl-2 expression in the corneal epithelium in the Bcl-2 transgenic or Bax knockout strains. Conclusions The significant difference in epithelial and stromal thickness suggests a functional role for Bcl-2 and Bax in the maintenance of corneal homeostasis; however, the lack of significant alterations in proliferation, cell exfoliation, and levels of Bcl-2 protein in the adult corneal epithelium suggest that these phenotypic changes are a result of a new stable homeostatic equilibrium (proliferation + migration = shedding). Additional experiments to delineate the role of Bcl-2 and Bax during development and in the adult cornea are necessary.

To investigate the morphological effects of Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%) on lens epithelial cells (LECs).Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA, and the Laboratory of Ultrastructural Morphology, Zoological Institute, University of Liège, Liège, Belgium.Human LECs collected via capsulorhexis were examined by light microscopy (LM) and transmission electron microscopy (TEM). Lens epithelial cells from rabbit capsulorhexis samples were studied by LM and TEM following exposure to Provisc (sodium hyaluronate 1.0%) or Viscoat ophthalmic viscoelastic device (OVD). Since Viscoat is hypertonic (340 mOsm), hypertonic, isotonic, and hypotonic solutions were compared to investigate a possible mechanism for the observed effects. The effects of Provisc and Viscoat on rabbit LECs in the intact lens were also compared.Human LECs gathered via capsulorhexis following exposure to Viscoat were generally thinner than control samples and often had condensed nuclei and increased intracellular vacuolization. Rabbit capsular tissue exposed in situ to Viscoat demonstrated changes similar to those seen in humans. Cells exposed to Provisc were similar to cells in untreated controls in humans and rabbits. Corneal endothelial cells exposed to either agent were unaffected. Experiments with hypertonic and hypotonic buffers induced some of the changes noted with Viscoat, but the effects were less severe. Lens epithelial cells in intact rabbit lenses exposed to Viscoat appeared similar to LECs in the control samples.Light microscopy and TEM of human lens capsule tissue suggest that Viscoat induces significant morphological changes in LECs during cataract surgery. The changes may underlie the improved visualization of these cells that has been reported during cataract surgery. Corneal endothelial cells were unaffected by exposure to Viscoat. Studies in a rabbit model suggest that the hyperosmolarity of Viscoat may play a partial role in the LEC changes.

Purpose. To determine recovery time needed for the corneal epithelium to return to a normal proliferation rate following 24-hour rigid gas-permeable (RGP) lens wear. Methods. An RGP lens (Dk/t = 10) was fitted at 9:00 AM on one randomly chosen eye of each rabbit (N = 16) and removed 24 hours later while the other eye served as a control. Following contact lens removal, the rabbits were injected at four different time intervals (1.2, 4, and 7 days) with 5-bromo-deoxyuridine (BrdU) at 9:00 AM to label dividing corneal epithelial cells. All animals were sacrificed 24 hours after BrdU injection, and corneas were stained for BrdU. A series of continuously adjacent digital images of the whole-mount epithelium was collected from superior limbus to central cornea, and all BrdU-labeled epithelial cell pairs on each digitized image were counted. Results. On day I following lens removal, the number of BrdU-labeled corneal epithelium cells was reduced by 50% centrally (C) and 36% peripherally (P) (P 0.001) compared with control eyes. At day 2, there was no statistically significant difference between control and experimental corneas (P = 0.1175). At day 4, a marked significant increase in BrdU-labeled cells was noted, averaging 83% (C) and 96% (P) (P < (1.001). At day 7. BrdU-labeling returned to control values, although it was still slightly elevated (14% Cl and 5% [P], P = 0. 045). Conclusions. The corneal epithelium in the rabbit required at least 1 full week to recover to its stable baseline proliferation rate following only 24 hours of RGP lens wear. Interestingly, hyperproliferation was noted within the limbal, peripherals and mid-peripheral corneal epithelium 4 days after lens removal.

Numerous studies have shown that fibroblasts play an important role in corneal wound healing, however, the dynamic cellular events underlying wound tissue organization and contraction remain unclear. The purpose of this study was to develop a system to enable live cell imaging of corneal wound healing fibroblasts in situ. To this end, concentrated preparations of an RD114 pseudotyped MLV-based vector expressing the enhanced green fluorescent protein (EGFP) were evaluated in vitro for gene transfer efficiency using cultured rabbit corneal keratocytes. Primary rabbit keratocytes were efficiently labeled in vitro (up to 50% EGFP + ) at a low multiplicity of infection (MOI=10). To evaluate this gene transfer vector in vivo, rabbit corneal fibroblasts were transduced by direct application of vector supernatant to injured corneas following lamellar keratectomy. Fluorescent fibroblasts were then visualized in situ using epifluorescence microscopy and multiphoton confocal microscopy of excised fresh tissue at multiple time points from 14 days to four months following gene transfer. Fourteen days post-transduction, labeled fibroblasts expressing EGFP were readily detectable by fluorescence microscopy. Detectable fluorescence was noted up to eight weeks post-transduction. Labeled fibroblasts were detected in clusters located predominantly along the margin circumscribing the wound and to a lesser extent within the wound area. Cell growth in clusters was suggestive of the expansion of individual transduced clones. High-resolution imaging showed fluorescent fibroblasts to have a broad, flattened, dendritic morphology, distinct from the spindle shape of cultured fibroblasts. Utilizing multiphoton confocal microscopy, three-dimensional imaging of viable, labeled cells showed wound healing fibroblasts to be extensively interconnected and multi-layered within the corneal wound. These results demonstrate that rabbit corneal fibroblasts can be efficiently transduced in vitro and in vivo using RD114 pseudotyped MLV-based vectors and that these vectors direct long-term transgene expression without apparent toxicity, pathogenesis or perturbation of native fibroblast morphology. Our data further suggest that, in vivo, wound-healing fibroblasts have a defined life span within the wound.

Purpose The purpose of this study is to assess the effects of commercially available contact lens wetting solutions on bacterial binding and cell exfoliation rates in human corneal epithelium. Methods The effects of four contact lens care solutions were tested: ReNu Multi Plus (Bausch & Lomb, Rochester, NY) multipurpose solution; OPTI-FREE Express (Alcon, Ft. Worth, TX) multipurpose solution; Complete Blink-N-Clean (Allergan, Irvine, CA) lens drops; and Lens Plus (Allergan) rewetting drops. Study design Prospective, double-masked, randomized crossover clinical trial (N = 20 subjects). Outcome measures Measures of outcome included binding of Pseudomonas aeruginosa (PA) to exfoliated corneal epithelial cells, and the rate of surface cell exfoliation. Cells were collected at the baseline (pretreatment) examination and 4 days later, after subjects used the assigned solution six times daily and once again immediately before cell collection (posttreatment). Following cell collection, patients underwent 1 week of recovery, during which no drops were used, and random cross-over assignment to the next test solution. Results Use of test solutions increased PA binding, with a range of + 11.9% to + 58.2%. Analyzed together, PA binding increased significantly (+ 29%; P = 0.02, paired t-test); Lens Plus solution alone raised PA binding levels significantly (P = 0.022, 2-way ANOVA, Student-Newman-Keuls [SNK] test). Exfoliation rates were decreased from -7% to -52.7%. Analyzed together, cell exfoliation decreased significantly (P = 0.004; Wilcoxon signed rank test). Individual use of OPTI-FREE decreased exfoliation significantly (P = 0.019: 2-way ANOVA, SNK test). Conclusions Topical application of common commercial contact lens care solutions increases PA binding and reduces corneal surface cell exfoliation. Similar effects have also been reported with contact lens wear. Taken together, the data suggest that the use of lens solution itself may play a role in increasing PA binding to corneal epithelial cells and, hence, might potentially contribute inadvertently to increased risk for lens-related microbial keratitis.

Purpose. Confocal microscopy through focusing (CMTF) of the cornea produces a three-dimensional display of corneal structure and intensity profiles that allow objective measurements of corneal sublayer thickness. This study investigated the feasibility of using in vivo CMTF to measure postlens tear thickness (PLTT). Methods. Two rabbits and one human were evaluated in this study. Both rigid gas-permeable (RGP) and silicone hydrogel contact lenses were used. After contact lens insertion, CMTF scans were performed by rapidly focusing from the tip of the objective, through the contact lens, PLTT, and full thickness of the cornea, and into the anterior chamber of the eye. PLTT was calculated by measuring the distance between the posterior surface of the lens and the anterior surface of the epithelium. Results. With a steep RGP lenses, the position of the posterior lens surface and the superficial epithelial surface was easily visualized using three-dimensional CMTF, and the PLTT could be measured. However, when a properly fit RGP or silicone hydrogel contact lens was used, the strong signal from the posterior surface of the lens washed out the signal from the front surface of the cornea in most cases, and the PLTT could not be directly assessed. We also found that the PLTT was sensitive to applanation of the confocal objective lens. Conclusion. The data suggest that the tear film under a properly fit contact lens is less than the 9-μm axial resolution of our confocal microscope. Furthermore, the potential for undetected eye movement and accidental touching of the contact lens raises questions regarding the overall reliability of the measurements.

Objective To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear. Design Prospective, randomized, double-masked, single-center, 13-month clinical trial. Participants One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98). One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n=55, 30 nights). Testing Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW. Main outcome measures (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH). Results Daily wear with control lens increased PA binding from 5.90 ± 2.60 to 7.81 ± 3.04 bacteria per cell ( P P P = 0.0017), but not after 1 month of subsequent EW ( P = 0.533). One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear ( P P Conclusions Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs. 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year. These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.

To measure flap thickness in laser in situ keratomileusis (LASIK) patients using in vivo confocal microscopy through-focusing (CMTF) and compare measured versus intended flap thickness achieved by 2 microkeratomes, the Automated Corneal Shaper(R) (ACS) (Chiron BauschLomb) and the Hansatome (BauschLomb).Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.Twenty-seven eyes of 27 patients were examined by in vivo CMTF 3 to 12 months after LASIK was performed with the ACS (12 patients) or Hansatome (15 patients) microkeratome. The central cornea was scanned, and the epithelium, flap, stroma, and total corneal thickness were measured. Normalized flap thickness (NFT) was also calculated to account for changes in epithelial thickness that may have occurred postoperatively.The mean posterior stromal thickness was 341.1 microm +/- 53.9 (SD) (range 233 to 431 microm) in the ACS group and 320.3 +/- 42.3 microm (range 258 to 382 microm) in the Hansatome group. The mean nonnormalized flap thickness was 132.7 +/- 12.5 microm (range 11 to 151 microm) in the ACS group and 167.4 +/- 21.4 microm (range 141 to 209 microm) in the Hansatome group. The NFT was 129.6 +/- 9.5 microm and 158.4 +/- 22.1 microm, respectively. Both microkeratomes cut significantly less than intended (P.05); however, the ACS cut a thinner-than-intended thickness in all cases, and the Hansatome cut thicker than intended in 13% of cases. The Hansatome also showed significantly greater variability in flap thickness than the ACS (P.05).A significant difference in precision was noted between the 2 microkeratomes. The findings emphasize the importance of performing thickness measurements and the usefulness of in vivo CMTF in making these determinations to ensure the safety and effectiveness of LASIK.

This report reviews and integrates our recent clinical and laboratory findings of the effects of daily (DW)/overnight (EW) contact lens wear on corneal epithelial homeostasis. In a prospective, double-masked human clinical study, three lens types (high and hyper Dk/t soft; hyper Dk/t RGP) were used to assess the effects of daily and overnight lens wear on corneal epithelial thickness, cell surface size, Pseudomonas aeruginosa (PA) binding to exfoliated epithelial cells and exfoliation rates. Concomitantly, we studied in a rabbit model, the effects of overnight lens wear on corneal epithelial surface cell viability (live/dead assay, TUNEL-labeling, Annexin-V staining, Bcl-2 expression), basal cell proliferation and migration patterns. The comprehensive results obtained yield important new insights on both the normal homeostasis of the corneal epithelium and the biological changes induced by contact lens wear.

Objective To determine effects of lens type and oxygen transmissibility on human corneal epithelium during extended wear (EW). Design Prospective, randomized, double-masked, single-center, parallel treatment groups, 1-year clinical trial. Participants One hundred seventy-eight patients completed the study: (1) high-O 2 soft lens (6-night [N] EW) (n = 27); (2) hyper-O 2 soft lens (6N-EW, n=33) or (30N-EW, n=66); and (3) hyper-O 2 rigid gas-permeable lens (RGP) (30N-EW, n=52). Intervention Irrigation chamber to collect exfoliated corneal surface cells, confocal microscopy, and tear collection at baseline, 1, 3, 6, 9, 12 months of EW. Main outcome measures (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness; (3) superficial epithelial cell area; (4) epithelial surface cell exfoliation; and (5) tear lactate dehydrogenase. Results Quantitative evidence demonstrated increased binding of PA to human exfoliated corneal epithelial cells during the first 3 months of soft lens EW; the control high-O 2 test lens showed significantly higher bacterial binding ( P P 2 soft lens for all outcome measures. Importantly, hyper-O 2 RGP lens wear did not show significantly increased PA binding during 1 year. Conclusions This study establishes three important new findings: (1) hyper-O 2 soft lens EW produces significantly less PA binding than the lower O 2 soft lens with no significant difference in PA binding with 6N versus 30N EW of the hyper-O 2 soft lens; (2) there is a remarkable adaptive recovery after 6 months with all soft lens wear with gradual return to prelens PA binding levels and partial recovery of other outcome measures for all test lenses EW except surface cell size; (3) 30N EW of the hyper-O 2 RGP lens produced no significant increases in PA binding over 1 year. Taken together, these results suggest that introduction of new hyper-O 2 transmissible lens materials into clinical use may offer safer EW, and future epidemiologic studies of ulcerative infectious keratitis should consider both lens type and time in lens EW in any incidence/risk analysis.

Purpose To establish a rabbit model of infectious Pseudomonas aeruginosa keratitis using ultrahigh oxygen transmissible rigid lenses and characterize the frequency and severity of infection when compared to a non-oxygen transmissible lens material. Methods Rabbits were fit with rigid lenses composed of ultrahigh and non-oxygen transmissible materials. Prior to wear, lenses were inoculated with an invasive corneal isolate of P. aeruginosa stably conjugated to green fluorescent protein (GFP). Corneas were examined before and after lens wear using a modified Heidelberg Rostock Tomograph in vivo confocal microscope. Viable bacteria adherent to unworn and worn lenses were assessed by standard plate counts. The presence of P. aeruginosa-GFP and myeloperoxidase-labeled neutrophils in infected corneal tissue was evaluated using laser scanning confocal microscopy. Results The frequency and severity of infectious keratitis was significantly greater with inoculated ultrahigh oxygen transmissible lenses. Infection severity was associated with increasing neutrophil infiltration and in severe cases, corneal melting. In vivo confocal microscopic analysis of control corneas following lens wear confirmed that hypoxic lens wear was associated with mechanical surface damage, whereas no ocular surface damage was evident in the high-oxygen lens group. Conclusions These data indicate that in the absence of adequate tear clearance, the presence of P. aeruginosa trapped under the lens overrides the protective effects of oxygen on surface epithelial cells. These findings also suggest that alternative pathophysiological mechanisms exist whereby changes under the lens in the absence of frank hypoxic damage result in P. aeruginosa infection in the otherwise healthy corneal epithelium.

To determine the effects of lens type and oxygen transmissibility on human corneal epithelium during daily lens wear (DW).Prospective, randomized, double-masked, single-center, parallel treatment groups clinical trial.Two hundred forty-six patients fitted with: (1) high oxygen-transmissible soft lenses (n = 36), (2) hyper oxygen-transmissible soft lenses (n = 135), and (3) hyper oxygen-transmissible rigid gas-permeable (RGP) lenses (n = 75).Irrigation chamber to collect exfoliated epithelial surface cells, confocal microscopy, and tear collection at baseline, 2 weeks, and 4 weeks of DW.(1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal epithelial surface cells, (2) central epithelial thickness, (3) superficial epithelial cell area, (4) epithelial surface cell exfoliation, and (5) tear lactate dehydrogenase (LDH).Four weeks of DW with the high oxygen-transmissible soft lens significantly increased PA binding from baseline 6.55 +/- 3.01 to 8.75 +/- 3.05 bacteria per epithelial cell (P0.01). By contrast, hyper oxygen-transmissible soft lens wear increased binding significantly less (6.13 +/- 2.45 to 7.62 +/- 3.06; P0.01), whereas hyper oxygen-transmissible RGP lens wear demonstrated no significant changes (5.91 +/- 2.40 to 6.13 +/- 2.17; P = 0.533). No significant change in central epithelial thickness was found after 4 weeks of DW in either soft lens; however, the epithelial thickness decreased by 9.8% (P0.001) with RGP lens wear. Epithelial cell surface area increased 3.3% and 4.1% with the high and hyper oxygen-transmissible soft lenses, respectively, and 10.5% with the hyper oxygen-transmissible RGP lens (P0.001). Epithelial desquamation significantly decreased in all groups (P0.001). Tear LDH levels increased for all test lenses (P0.001).Increased PA binding induced by wear of a conventional soft lens material is significantly greater than that induced by the new hyper oxygen-transmissible soft silicone hydrogel lens during DW. However, both soft materials showed significant increases in PA binding as compared with baseline controls. By contrast, hyper oxygen-transmissible RGP lens DW did not increase PA binding significantly. Taken together, these findings suggest for the first time both an oxygen effect as well as a difference between soft and rigid lens types on PA binding in DW.

Measurement of sublayer thickness and transparency at cellular level in the living animal are critical to understanding the role of specific transgenes and transgene products in controlling corneal development and maintenance of transparency. Using two different transgenic mouse strains having altered corneal clarity, we have evaluated the ability of in vivo confocal microscopy to measure corneal haze and localize light scattering structures. Projection of 2-D and 3-D image information identified the nature and location of light scattering within the cornea and allowed correlation of unique structural differences to transgene expression. Our findings suggest that in vivo confocal microscopy can be used to identify the effects of transgene expression on mouse corneal transparency.

Purpose To determine differences of corneal wound healing and haze after photorefractive keratectomy (PRK) using either mechanical epithelial debridement or laser-scrape epithelial removal in human subjects. Design A 6-month randomized, masked, prospective, paired-eye clinical study. Participants Twenty eyes in 10 myopic patients treated between March 1999 and May 1999. Intervention Photorefractive keratectomy treatments with two different epithelial removal techniques. Continuous z-scan of confocal image, termed confocal microscopy through focusing (CMTF), was performed before surgery and at 3 weeks, 6 weeks, 3 months, and 6 months after surgery. Main outcomes measures Epithelial and stromal thickness measurement, achieved stromal ablation depth, and objective assessment of corneal light-backscattering (corneal haze) were obtained from digital image analysis of the CMTF scans. Manifest refraction was also measured. Student's paired t test or two-way repeated-measures analysis of variance after rank transformation were performed to evaluate statistical differences between groups. Results Comparison of the mean posttreatment spherical equivalent between the two techniques showed no statistically significant difference. In preoperative corneas, mean epithelial thickness was 50.08 ± 3.70 μm in the mechanical debridement group and 50.49 ± 4.01 μm in laser-scrape group (not significant). For both groups, the epithelium was significantly thinner at 3 weeks, but returned to preoperative values by 6 months, with no difference between groups. Planned stromal ablation depth by PRK was 59.38 ± 11.48 μm (39–73 μm; n=8) in the mechanical group and 57.75 ± 7.21 μm (48–70 μm; n=8) in the laser-scrape group. Achieved stromal ablation depth was not significantly different between the two groups. Most importantly, in both groups CMTF-measured corneal haze increased significantly after surgery, peaked at 3 months, and then decreased at 6 months, with no significant difference between groups. (Power = 0.96). Conclusions There is no significant difference in the corneal wound healing response between mechanical epithelial debridement versus laser-scrape technique in human myopic eyes undergoing PRK.

PURPOSE: To examine the healing response of laser in situ keratomileusis flap margin in vivo. METHODS: Forty-three eyes of 43 patients who had undergone myopic (n = 39) or hyperopic (n = 4) laser in situ keratomileusis were examined once after surgery. The flap margin was imaged by in vivo confocal microscopy at various depths, and the wound healing response, flap alignment, and complications were evaluated. Ten eyes were examined on day 3 postoperatively, 13 eyes at 1 to 2 weeks, 10 eyes at 1 to 2 months, five eyes at 3 months, and five eyes at 6 months or later. RESULTS: At 3 days after laser in situ keratomileusis, the surface epithelium and basal epithelium appeared normal. Keratocyte activation was strongest at 1 to 2 weeks and 1 to 2 months, and an increased amount of haze was observed correspondingly. Intrastromal epithelial cells forming a plug could occasionally be perceived in the wound gape. Wound constriction was completed in most cases by 3 to 6 months or later. Good alignment was observed in 12 of 43 flaps (27.9%) and moderate and poor alignment in 17 of 43 flaps (39.5%) and 13 of 43 flaps (30.2%), respectively. Poor alignment was not associated with lamellar epithelial ingrowth. Epithelial ingrowth was associated with dense haze at the interface. Diffuse lamellar keratitis was imaged in two corneas after hyperopic laser in situ keratomileusis. CONCLUSIONS: The laser in situ keratomileusis incision wound at the flap margin appears to heal after the sequence observed in incisional wounds in nonhuman primates. Complications, such as lamellar epithelial in growth and diffuse lamellar keratitis, were often observed, particularly after hyperopic laser in situ keratomileusis.

Purpose To evaluate the mechanism(s) producing refractive instability and corneal haze development after photorefractive keratectomy (PRK). Design Prospective, nonrandomized, comparative case series, self-controlled. Participants Seventeen eyes of 17 patients with low- to moderate-grade myopia (−2.88 to −9.13 diopters [D]) were included. Methods Surgical intervention was a standardized, 6-mm diameter PRK procedure using the Meditec MEL 60 excimer laser (Aesculap-Meditec, Heroldsberg, Germany). The photoablation center was evaluated before surgery and at 1, 3, 6, 9, and 12 months after PRK using rapid, continuous z-scans of confocal images, termed confocal microscopy through focusing (CMTF). Main outcome measures Simultaneous epithelial and stromal thickness analysis and objective assessment of corneal light backscattering were obtained from digital image analysis of the CMTF scans. Corneal reinnervation and anterior stromal keratocyte density and wound healing morphologic features were evaluated on high resolution, in vivo confocal images. Manifest refraction was measured and corneal clarity was graded by slit-lamp biomicroscopy. Results Epithelial thickness averaged 45 ± 10 μm at 1 month, 50 ± 8 μm at 3 months, and 52 ± 6 μm at 12 months after PRK, as compared with 51 ± 4 μm before surgery, demonstrating complete restoration of the preoperative thickness without compensatory hyperplasia. Interestingly, epithelial rethickening had no significant correlation with refractive regression. By contrast, stromal regrowth (from 1–12 months) averaged 6 ± 12 μm (range, 27 μm thinning–22 μm rethickening) and correlated closely ( r = 0.84, P r = 0.63, P cellular-based reflections, as opposed to extracellular matrix deposition, are the major origin of increased corneal light scattering after PRK. Conclusions Taken together, these data indicate that keratocyte-mediated regrowth of the photoablated stroma appears to be the main cause of myopic regression in humans treated with a 6-mm diameter PRK, whereas hyperopic shifts appear to be a direct consequence of stromal thinning. By contrast, the corneal epithelium appeared to restore its preoperative thickness without contributing significantly to the refractive changes after PRK. Finally, this study also provides strong evidence that the development of haze after PRK is directly associated with increased cellular reflectivity from high numbers of wound healing keratocytes.

Objective To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. Design Observational case series Participants Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. Methods Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. Main outcome measures The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowman's layer, stromal keratocytes, and endothelium was analyzed. Results The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowman's layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. Conclusions In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.

This special issue of Exp Cell Research brings together a series of review articles providing fresh perspectives on cell–matrix interactions and descriptions of new tools being developed to study cell mechanics within native ECM and fibrillar protein gels. Mechanical interactions between cells and extracellular matrices (ECM) play a defining role in a range of fundamental biological processes such as developmental morphogenesis, wound healing and cancer metastasis. During embryonic development, physical forces exerted by cells organize ECM into specific spatial patterns that provide structural support, produce mechanical guidance and give form and organization to vertebrate tissue. Following injury or surgery, cell migration, wound contraction and remodeling of connective tissue matrix are also dependent on mechanical interactions between fibroblasts and ECM fibrils. Similarly, migration and invasion of tumor cells is regulated by the complex interplay between ECM stiffness and porosity, cell–cell adhesion, protease activity and the pattern and amount of sub-cellular force generation. An understanding of cell–matrix mechanical interactions is also critical in the field of tissue engineering, where it is necessary to modulate ECM remodeling to produce a matrix with desired architecture and mechanical properties while limiting excessive contraction. While 2-D studies have provided important insights into many aspects of cell mechanical behavior, cells reside within 3-D extracellular matrices in vivo, and ECM dimensionality has been shown to effect cell morphology, mechanical activity, and adhesion organization and composition. This issue includes overviews of specific model systems, imaging techniques, and computational analyses that can be used to quantitatively study 3-D cell motility and mechanical behavior. Also included are reviews on how the complex structural and mechanical properties of 3-D matrices and their boundary conditions can impact individual and collective cell behavior. The manuscripts underscore the need for understanding the non-linear, fibrillar nature of the ECM in modulating the fine balance between cell motility and contraction in these matrices. Throughout the issue, examples of how recent findings in 3-D mechanobiology relate to tissue function in both health and in disease are provided. Protein matrices have been used to study the forces exerted by cells on the ECM and each other for over 50 years, and, as demonstrated in this special issue, advances in imaging technologies, live cell labeling techniques, matrix mechanical characterization and computational models continue to expand our ability to study mechanobiology at the cellular level in a biofidelic environment.

The purpose of this study was to assess the ability of in vivo confocal microscopy (CM) to provide noninvasively derived histopathologic correlates of surfactant-induced eye irritation from which specific pathologic mechanisms can be identified and later evaluated in alternative in vitro models. Rats and rabbits, divided into groups of 5, received 10 μl of an anionic or cationic surfactant in one eye with the other eye used as a control. At specified times, eyes were examined and scored for ocular irritancy using a penlight and slit-lamp. Subsequently, corneas were evaluated by in vivo CM to evaluate epithelial layer thickness and surface epithelial cell area, corneal thickness, depth of necrosis, inflammation, fibrosis, and endothelial injury. At 3 hr, the anionic surfactant produced slight irritation with peak scores of 12.4 and 8.0 out of a possible 110 in the rats and rabbits, respectively. In vivo CM revealed changes limited to the corneal epithelium that decreased in thickness to 78% in rats and 81% in rabbits at 3 hr. This decrease in the thickness correlated with a significant decrease in surface epithelial cell area from 2,061 ± 395 μm2 to 567 ± 330 μm2 in the rats and 1,523 ± 185 μm2 to 934 ± 71 μm2 in the rabbits (p < 0.005 and 0.005, respectively). The cationic surfactant produced severe irritation in both the rats and rabbits with peak scores of 85.4 and 80.2 occurring at day 2, respectively. In vivo CM in the rats showed complete loss of corneal epithelium, lysis of keratocytes, and loss of corneal endothelium. In the rabbits, injury appeared limited to the anterior cornea with complete loss of epithelium and loss of keratocytes extending to 52% of the corneal thickness. These findings establish the application of noninvasive, in vivo CM to qualitatively and quantitatively characterize the pathobiology of ocular irritation in situ. This information will be important in the development and evaluation of mechanistically based in vitro alternatives for ocular irritancy testing.

Purpose. For 14 years, the U. S. Food and Drug Administration (FDA) and eye care practitioners have strongly discouraged patients from sleeping in contact lenses. In the past 9 months however, the FDA has approved three new hyper-oxygen transmissible lenses for up to 30-night extended wear. Is this a great advance or another clinical triumph of hope over experience? What should the public know? What should patients do? Methods. Our research group has studied all three new lenses in prospective, randomized, masked, parallel clinical trials in a single center. As an outcome measure, we looked at whether lens wear caused more bacterial binding to surface corneal cells. Results. Compared with conventional lens use, the new lenses caused no or only small increases in bacterial binding in either daily or extended wear. Furthermore, the increases seen stratified with known infection risks by both lens type (hard or soft) and wearing schedule. Indeed, early epidemiologic reports indicate that this new generation of lenses may reduce lens-related microbial infection risks by 10- to 40-fold. Conclusion. This represents a true clinical paradigm shift of the first magnitude in safety of contact lens wear. Taken together, this data suggest that patients will soon replace their current, conventional lenses with this new generation of materials for any schedule of wear.

This in-vitro study compared the toxicity of bimatoprost 0.01% containing benzalkonium chloride (BAK) 0.02% with other commercial BAK-free or BAK-containing prostaglandin analogs.Six test solutions were evaluated: travoprost 0.004% with polyquaternium-1 0.001% (PQ), PQ, bimatoprost 0.01% with BAK 0.02%, latanoprost 0.005% with BAK 0.02%, tafluprost 0.0015% preservative free (PF), and BAK 0.02%. Phosphate-buffered saline (PBS) was the live control and 70% methanol was the dead control. Confluent human corneal epithelial cells were incubated with test solutions (diluted 1:5 or 1:10 with PBS) or control solutions for 10 or 25 min, after which cells were fluorescently labeled to distinguish live and dead cells. Data were expressed as a percentage of PBS live-cell fluorescence for automated readouts. Live and dead cells were manually counted for numeric analyses.For 1:5 and 1:10 dilutions using automated readout, cells exposed to bimatoprost with BAK, latanoprost with BAK, and BAK alone demonstrated significant reductions in the live cell signal compared with PBS, travoprost with PQ, and PQ alone (all P0.001). They also demonstrated significantly greater toxicity than tafluprost PF for 1:5 dilutions (all P0.001) and 1:10 dilutions (P ≤ 0.02), except for 1:10-diluted bimatoprost with BAK (P = 0.41). For 1:5 dilutions using manual cell count, cells exposed to bimatoprost with BAK demonstrated significant reductions in the percentage of live cells compared with PBS (P = 0.02). For 1:10 dilutions using manual cell count, cells exposed to bimatoprost with BAK, latanoprost with BAK, and BAK alone demonstrated significantly greater toxicity than PBS, travoprost with PQ, PQ alone, and tafluprost PF (all P ≤ 0.03). No significant differences were observed among PBS, travoprost with PQ, and PQ alone under any test conditions (P ≤ 0.63).This study demonstrated that BAKcontaining solutions, including bimatoprost 0.01% with BAK, were toxic to human corneal epithelial cells, whereas BAK-free solutions showed little to no evidence of toxicity.

Extracellular matrix (ECM) supplies both physical and chemical signals to cells and provides a substrate through which fibroblasts migrate during wound repair. To directly assess how ECM composition regulates this process, we used a nested 3D matrix model in which cell-populated collagen buttons were embedded in cell-free collagen or fibrin matrices. Time-lapse microscopy was used to record the dynamic pattern of cell migration into the outer matrices, and 3-D confocal imaging was used to assess cell connectivity and cytoskeletal organization. Corneal fibroblasts stimulated with PDGF migrated more rapidly into collagen as compared to fibrin. In addition, the pattern of fibroblast migration into fibrin and collagen ECMs was strikingly different. Corneal fibroblasts migrating into collagen matrices developed dendritic processes and moved independently, whereas cells migrating into fibrin matrices had a more fusiform morphology and formed an interconnected meshwork. A similar pattern was observed when using dermal fibroblasts, suggesting that this response in not unique to corneal cells. We next cultured corneal fibroblasts within and on top of standard collagen and fibrin matrices to assess the impact of ECM composition on the cell spreading response. Similar differences in cell morphology and connectivity were observed – cells remained separated on collagen but coalesced into clusters on fibrin. Cadherin was localized to junctions between interconnected cells, whereas fibronectin was present both between cells and at the tips of extending cell processes. Cells on fibrin matrices also developed more prominent stress fibers than those on collagen matrices. Importantly, these spreading and migration patterns were consistently observed on both rigid and compliant substrates, thus differences in ECM mechanical stiffness were not the underlying cause. Overall, these results demonstrate for the first time that ECM protein composition alone (collagen vs. fibrin) can induce a switch from individual to collective fibroblast spreading and migration in 3-D culture. Similar processes may also influence cell behavior during wound healing, development, tumor invasion and repopulation of engineered tissues.

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice.Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3.Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. After 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro.The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.

Endothelial dysfunction remains the leading indication for corneal transplantation in the United States.1 Visual rehabilitation after penetrating keratoplasty (PKP) may be limited by delayed wound healing, surgically induced astigmatism, suture-related complications, and anisometropia.2,3 In contrast, endothelial keratoplasty in the form of deep lamellar endothelial keratoplasty (DLEK) or Descemet stripping automated endothelial keratoplasty (DSAEK) allows selective replacement of diseased host endothelium, more rapid visual rehabilitation, minimal induced astigmatism, and a predictable change in corneal power postoperatively.4–6 DSAEK employs stripping of the diseased host endothelium and replacement with a thin layer of donor stromal tissue and endothelium harvested with an automated microkeratome.7,8 The manual lamellar dissections required to prepare the recipient cornea and donor tissue in DLEK surgery, which are technically difficult and time consuming for the surgeon, have been eliminated in DSAEK, helping this procedure become the procedure of choice for endothelial dysfunction. Despite excellent postoperative results, the available data suggest that best-corrected visual acuity after DLEK or DSAEK surgeries is at times less than that of PKP patients.8–11 Persistent subepithelial haze and interface haze have been suggested to affect the best-corrected visual acuity after DSAEK surgery.12 The better visual outcomes after DSAEK in comparison to DLEK may be attributable to the smoother interface in DSAEK compared with the manually created DLEK interface.13 Despite several advantages of DSAEK over PKP, endothelial cell loss remains a concern with endothelial keratoplasty. Endothelial cell loss of 34% has been reported at the 6- month postoperative time period after DSAEK.14 Endothelial survival may be impacted by every step of the procedure including donor tissue preparation, folding of the donor disc, insertion site size and technique, intraoperative manipulation of the tissue to center and attach the donor disc, and the postoperative course. Donor cornea preparation requires the surgeon or the eye bank to create a deep lamellar dissection to create a thin posterior stromal lenticule with viable endothelium. “Precut” donor tissue by the eye bank has been shown to have no increased risk of tissue-related complications and may increase surgeon’s efficiency.15 In this masked study, we attempted to evaluate the quality of the stromal bed and the safety on the endothelium in the preparation of donor tissue for DSAEK using the Moria ALTK system with the mechanical CBm microkeratome, disposable Horizon mechanical microkeratome system, and Intralase FS60 femtosecond laser.