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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.

During corneal wound healing, keratocytes present within the corneal stroma become activated into a repair phenotype upon the release of growth factors, such as transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). The process of injury and repair can lead to changes in the mechanical properties of the tissue, and previous work has shown that the TGF-β1-mediated myofibroblast differentiation of corneal keratocytes depends on substratum stiffness. It is still unclear, however, if changes in stiffness can modulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in stiffness influence the proliferation and motility of primary corneal keratocytes treated with PDGF-BB. In the presence of PDGF-BB, cells on stiffer substrata exhibited a more elongated morphology and had higher rates of proliferation than cells in a more compliant microenvironment. Using a freeze-injury to assay cell motility, however, we did not observe any stiffness-dependent differences in the migration of keratocytes treated with PDGF-BB. Taken together, these data highlight the importance of biophysical cues during corneal wound healing and suggest that keratocytes respond differently to changes in ECM stiffness in the presence of different growth factors.

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.

Seventy percent of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the transcription factor 4 gene (Cross-sectional, single-center study.Fifty-two eyes of 29 subjects with a modified Krachmer grade of FECD severity from 1 to 6 were included in the study. Fifteen of the 29 subjects carried an expandedIn vivo confocal microscopy assessments of corneal nerve fiber length (CNFL), corneal nerve branch density, corneal nerve fiber density (CNFD), and anterior corneal stromal backscatter (haze); Scheimpflug tomography densitometry measurements of haze in anterior, central, and posterior corneal layers.Using confocal microscopy, we detected a negative correlation between FECD severity and both CNFL and CNFD in the eyes of RE+ subjects (Spearman ρ = -0.45,Loss of corneal nerves in FECD supports the classification of the

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.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

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.

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.

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 generation of cellular forces and the application of these physical forces to the ECM play a central role in mediating matrix patterning and remodeling during fundamental processes such as developmental morphogenesis and wound healing. In addition to growth factors and other biochemical factors that can modulate the keratocyte mechanical phenotype, another key player in the regulation of cell-induced ECM patterning is the mechanical state of the ECM itself. In this review we provide an overview of the biochemical and biophysical factors regulating the mechanical interactions between corneal keratocytes and the stromal ECM at the cellular level. We first provide an overview of how Rho GTPases regulate the sub-cellular pattern of force generation by corneal keratocytes, and the impact these forces have on the surrounding ECM. We next review how feedback from local matrix structural and mechanical properties can modulate keratocyte phenotype and mechanical activity. Throughout this review, we provide examples of how these biophysical interactions may contribute to clinical outcomes, with a focus on corneal wound healing.

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.

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p

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.

This paper reviews new developments in microscopy that combine gene transfer technology, multiphoton confocal fluorescence microscopy, live cell imaging and digital imaging techniques that provide unique insights into the complex physiological processes involved in tissue function at the cellular and subcellular level. The evolution of this novel, new technology is discussed with particular attention to earlier achievements in noninvasive ocular surface imaging. The practical basis of confocal microscopy, multiphoton confocal fluorescence microscopy, and the vital fluorescent labeling of cells in living tissues are also discussed. Additionally, one application using retroviral gene transfer to express enhanced green fluorescent protein in living wound healing fibroblasts is presented as an example of how living biology can be studied in situ in four dimensions (x, y, z, time).

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.

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.

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.