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Purpose Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. Methods Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70–80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. Results Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a −16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. Conclusions AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure.

Author(s): Mikula, Eric; Holland, Guy; Srass, Hadi; Suarez, Carlos; Jester, James V; Juhasz, Tibor | Abstract: PurposeThis study investigated the initial feasibility of using femtosecond laser trabeculotomy (FLT) to create open channels through the trabecular meshwork into Schlemm's canal to lower intraocular pressure (IOP) in a perfused anterior segment model.MethodsHuman anterior segments (12 eyes) were assigned to either treatment (n = 6) or sham treatment (n = 6) groups. Both groups were perfused until a baseline IOP was recorded upon which a direct FLT treatment or a sham treatment was administered. IOP was recorded before and after the treatment. Spectral domain optical coherence tomography and second harmonic generation imaging we used to investigate the FLT channels.ResultsIn the FLT group, there was a significant mean decrease in the IOP of 22% compared with the pre-FLT IOP (7.13 ± 2.95 mm Hg to 5.34 ± 1.62 mm Hg; P l 0.05). In the control group, the post-sham IOP remained relatively unchanged compared with the pre-sham IOP (6.39 ± 3.69 mm Hg to 6.67 ± 4.12 mm Hg).ConclusionsThe results of this study indicate that FLT treatment can significantly decrease the IOP in a perfusion model and may provide a potential noninvasive treatment option for primary open angle glaucoma.Translational relevanceInvestigating the use of femtosecond lasers for photodisrupting the trabecular meshwork can lead to a clinically relevant alternative to current glaucoma procedures.

Purpose PPARγ plays a critical role in the maturation of immortalized human meibomian gland epithelial cells (hMGEC). To further understand the molecular changes associated with meibocyte differentiation, we analyzed transcriptome profiles from hMGEC after PPARγ activation. Methods Three sets of cultivated hMGEC with or without exposure to PPARγ agonist, rosiglitazone were used for RNA-seq analysis. RNA was isolated and processed to generate 6 libraries. The libraries were then sequenced and mapped to the human reference genome, and the expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using DESeq2 and NOISeq. Gene ontology enrichment analysis (GOEA) was performed on gene sets that were upregulated or downregulated after rosiglitazone treatment. Five genes were selected for validation and differential expression was confirmed using quantitative PCR. The Differential expression of CK5 was evaluated using Western blotting. Results Expression data indicated that about 58,000 genes are expressed in hMGEC. DESeq2 and NOISeq indicated that 296 and 3436 genes were upregulated and 258 and 3592 genes were down regulated after rosiglitazone treatment, respectively. Of genes showing significant differences > 2 fold, GOEA indicated that cellular and metabolic processes were highly represented. Expression of ANGPTL4, PLIN2, SQSTM1, and DDIT3 were significantly upregulated and HHIP was downregulated by rosiglitazone. CK5 was downregulated by rosiglitazone. Conclusions The RNA-seq data suggested that PPARγ activation induced alterations in cell differentiation and metabolic process and affected multiple signaling pathways such as PPAR, autophagy, WNT, and Hedgehog.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm.\ud \ud However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions.

Purpose While mouse models of dry eye disease (DED) have been developed, studies evaluating the role of the meibomian glands limited by the inability to temporally document changes. In this report we describe the development of a novel mouse transillumination meibography device and assess the ability of this device to detect age-related changes in the meibomian glands of young and old mice. Methods The mouse meibography device was comprised of a 3 mm wide right angle prism attached to broad spectrum light source by an optical fiber. Eyelids were then pulled over the prism using double tooth forceps and imaged using a stereomicroscope and low light level camera. Meibomian glands from four young and four old male, BALB/c mice were then imaged and analyzed using ImageJ. Results In young mice, meibography documented the presence of 7–8 meibomian glands appearing as black and distinct eyelid structures with the length shorter in the lower eyelid compared to the upper eyelids. Eyelids of old mice showed apparent dropout of meibomian glands along with smaller and more irregularly shaped acini. The mean acini area of one meibomian gland was 0.088 ± 0.025 mm2 in young mice and 0.080 ± 0.020 mm2 in old mice (p = 0.564), but the Meibomian gland density was significantly lower in older mice (41.7 ± 6.4%, 27.3 ± 4.2%) (p = 0.021). Conclusion We have developed an in vivo meibography device that may prove useful in sequentially documenting changes during development of meibomian gland dysfunction and following treatment.

Author(s): Bradford, Samantha; Mikula, Eric; Xie, Yilu; Juhasz, Tibor; Brown, Donald J; Jester, James V | Abstract: PurposeThis study describes a femtosecond laser (FS) approach to machine corneal epithelial microchannels for enhancing riboflavin (Rf) penetration into the cornea prior to corneal crosslinking (CXL).MethodsUsing a 1030-nm FS laser with 5- to 10-µJ pulse energy, the corneal epithelium of slaughterhouse rabbit eyes was machined to create 2-µm-diameter by 25-µm-long microchannels at a density of 100 or 400 channels/mm2. Rf penetration through the microchannels was then determined by applying 1% Rf in phosphate-buffered saline for 30 minutes followed by removal of the cornea and extraction from the central stromal button. Stromal Rf concentrations were then compared to those obtained using standard epithelial debridement or 0.01% benzalkonium chloride (BAK) to disrupt the epithelial barrier.ResultsMicrochannels formed using a 5-µJ/pulse at a density of 400 channels/mm2 achieved a stromal Rf concentration that was 50% of that achieved by removal of the corneal epithelium and imbibing with 1% Rf. Stromal Rf levels were also equal to that of debrided corneas soaked with 0.5% Rf, threefold higher than those soaked with 0.1% Rf, and twofold higher than corneas soaked in BAK without epithelial debridement. Organ culture of treated corneas showed a normal corneal epithelium following FS machining while BAK-treated corneas showed extensive epithelial and stromal damage at 24 hours posttreatment.ConclusionsFS corneal epithelial machining can be used to enhance penetration of Rf into the stroma for corneal CXL.Translational relevanceThe creation of epithelial microchannels allows for stromal Rf concentrations high enough to perform true transepithelial crosslinking.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 μm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ± 3.4% and 47.7 ± 7.6% of the corneal thickness at one and three months. There was a significant (P

PURPOSE: To evaluate the role of PPARγ in regulating meibocyte differentiation and lipid synthesis in a human meibomian gland epithelial cell line (hMGEC). METHODS: HMGEC were exposed to the PPARγ agonist, Rosiglitazone, from 10–50 μM. Cultures were also exposed to specific PPARγ antagonist, T0070907, to block PPARγ receptor signaling. Cells were then stained with Ki-67 and LipidTox to determine the effects on cell cycling and lipid synthesis, respectively. Expression of meibocyte differentiation related proteins, ADFP, PPARγ, ELOVL4, and FABP4, were evaluated by quantitative PCR and western blotting. A human corneal epithelial cell line (hTCEpi) was used as a control. RESULT: Rosiglitazone significantly decreased Ki-67 staining within 2 days in a dose-dependent manner (P = 0.003) and increased lipid accumulation in hMGEC in a dose dependent manner. T0070907 suppressed both lipid droplet synthesis and cell cycle exit. Rosiglitazone significantly upregulated expression of ADFP, PPARγ, ELOVL4, and FABP4 by 9.6, 2.7, 2.6, and 3.3 fold on average (all P < 0.05 except for FABP4, P = 0.057) in hMGEC. T0070907 significantly abrogated rosiglitazone-induced upregulation of these genes when treated prior to rosiglitazone treatment (all P < 0.05). The observed lipogenic differentiation response was not duplicated in hTCEpi after exposure to rosiglitazone. CONCLUSION: Rosiglitazone induced cell cycle exit and upregulation of lipogenic gene expression leading to lipid accumulation in hMGEC. These effects were suppressed by PPARγ antagonist indicating that PPARγ signaling specifically directs lipogenesis in hMGEC. These findings suggest that PPARγ plays a critical role in meibocyte differentiation.

PURPOSE: While meibography has proven useful in identifying structural changes in the meibomian gland (MG), little is known regarding the MG spectral transmission and absorption characteristics. The purpose of this study was to measure the transmission/absorption spectra of the MG compared to other eyelid tissues. METHODS: Human and rabbit eyelids were fixed in paraformaldehyde, serial sectioned (50 μm) using a cryotome and imaged by brightfield and reflectance microscopy. Eyelid regions (MG, muscle, tarsus and dermis) were then illuminated by a 100 μm spot using a infrared enhanced white light source. Transmission spectra over a 550–950 nm range were then measured using a spectrometer and differences compared using two-way analysis of variance. RESULTS: Brightfield microscopy of both human and rabbit eyelid tissue showed a marked decrease in light transmission for MG acini compared to other eyelid tissues. In rabbit, the dermis showed 5 × and the muscle showed 2 × more light transmission compared to MG (P < .001 and P < .001, respectively). For human, the muscle showed 14 × and the tarsus showed 84 × more light transmission compared to MG (P < .01 and P < .001, respectively). No specific spectral region of light absorption could be detected in either rabbit or human MG. Loss of light transmission in MG was localized to acini containing small lipid droplets, averaging 2.7 ± 0.8 μm in diameter. CONCLUSIONS: The data suggest that light transmission is dramatically reduced in the acini due to light scattering by small lipid droplets, suggesting that Meibography detects active lipid synthesis in differentiating meibocytes.

We recently identified a group of chemicals that are misclassified by most, if not all, in vitro alternative ocular irritation tests, suggesting that nonanimal tests may not fully model the ocular environment in which these chemicals interact. To address this, we evaluated the composition of tears, the first defense against foreign substances, and identified the presence of antioxidants that could detoxify reactive chemicals that otherwise may be falsely identified as irritants in alternative irritation tests. In this study, we evaluated the effects of tear antioxidants on the ocular irritation scoring of commonly overclassified chemicals (false positives) using the OptiSafe™ ocular irritation test. Six tear-related antioxidants were individually added to the OptiSafe formulation, and the effects on test outcome were determined. Ascorbic acid, the most abundant water-soluble antioxidant in tears, specifically reduced the OptiSafe false-positive rate. Titration curves showed that this reduction occurs at in vivo concentrations and is specific to chemicals identified either as producing reactive oxygen species or as crosslinkers. Importantly, the addition of tear antioxidants did not impact the detection of true negatives, true positives, or other false positives unassociated with reactive oxygen species or crosslinking. These results suggest that the addition of tear antioxidants to in vitro alternative test systems may substantially reduce the false-positive rate and improve ocular irritant detection.

PurposeTo examine effects of alkali injury of the ocular surface on meibomian gland pathology in mice.MethodsThree μL of 1N NaOH were applied under general anesthesia to the right eye of 10-week-old BALB/c (n=54) mice to produce a total ocular surface alkali burn. The meibomian gland morphology was examined at days 1, 2, 5, 10, and 20 by stereomicroscopy and non-contact infrared meibography. Mice were then sacrificed and eyelids processed for histology with hematoxylin-eosin and immunohistochemistry for ELOVL4, PPARγ, myeloperoxidase (a neutrophil marker) and F4/80 macrophage antigen, as well as TUNEL staining. Another set of specimens was processed for cryosectioning and Oil red O staining.ResultsAlkali injury to the ocular surface produced cellular apoptosis, infiltration of neutrophils and macrophages, degeneration of the meibomian gland, and ductal dilation. Inflammation in and destruction of acunal stricture seemed more prominent in the lower eyelid, while duct dilation was more frequently observed in the upper eyelid during healing. Surviving acinar cells were labeled for ELOVL4 and PPARγ. Oil red O staining showed that the substance in the dilated duct contained predominantly neutral lipid.ConclusionsAlkali injury to the ocular surface results in damage and destruction of the eyelid meibomian glands. The pattern of the tissue damage differs between glands of the upper and lower eyelids.

Summary The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future., Graphical Abstract, Highlights • Multipotent stem cells differentiate into distal Sox10+ and proximal Sox10− lineages • Lineage-restricted progenitor cells sustain the long-term lacrimal gland maintenance • Label-retaining cells are localized in the intercalated ducts and excretory ducts • Some cell lineages in the adult lacrimal gland retain plasticity, Biological Sciences; Cell Biology; Stem Cells Research; Developmental Biology

The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa, human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.

Author(s): Bradford, Samantha; Mikula, Eric; Kim, Sun Woong; Xie, Yilu; Juhasz, Tibor; Brown, Donald J; Jester, James V | Abstract: Purpose:We have shown that nonlinear optical corneal crosslinking (NLO CXL) and stiffening can be achieved in ex vivo rabbit corneas using an 80-MHz, 760-nm femtosecond (FS) laser, however the required power was beyond the American National Standard Institute limit. The purpose of this study was to test the efficacy of amplified FS pulses to perform CXL to reduce power by increasing pulse energy. Methods:A variable numerical aperture laser scanning delivery system was coupled to a 1030-nm laser with a noncollinear optical parametric amplifier to generate 760 nm, 50 to 150 kHz amplified FS pulses with 79.5-μm axial and 2.9-μm lateral two-photon focal volume. Ex vivo rabbit corneas received NLO CXL, and effectiveness was assessed by measuring collagen autofluorescence (CAF) and mechanical stiffening. NLO CXL was also performed in 14 live rabbits, and changes in corneal topography were measured using an Orbscan. Results:Amplified pulses (0.3 μJ) generated significant CAF that increased logarithmically with decreasing scan speed; achieving equivalent CAF to UVA CXL at 15.5 mm/s. Indentation testing detected a 62% increase in stiffness compared to control, and corneal topography measurements revealed a significant decrease of 1.0 ± 0.8 diopter by 1 month (P l 0.05). Conclusions:These results show that NLO CXL using amplified pulses can produce corneal collagen CXL comparable to UVA CXL. Translational Relevance:NLO CXL using amplified pulses can produce corneal CXL comparable to UVA CXL, suggesting a potential clinical application in which NLO CXL can be used to perform personalized crosslinking for treatment of refractive errors and keratoconus.

PurposeTo examine the effects of volume and method on fluorescein tear breakup time (TBUT) values and to evaluate test efficacy in an independent sample free of selection bias.MethodsSubjects were assessed using a battery of dry eye tests (DETs). Efficacy study: Subjects were randomized to the DET, standard strip, and liquid NaFl on separate days. A masked examiner measured TBUTs from video recordings. Verification study: Subjects were investigated for efficacy using volumes of 5.0 and 2.0 μL mL of NaFl for TBUT.ResultsEfficacy study: 46 subjects completed the study. Log-transformed TBUTs were significantly different, normal subjects versus dry subjects, for all 3 methods (all P values < 0.001). Area under the curves (AUCs), cut-points, sensitivity, and specificity were 1) DET: 0.873, 4.4 seconds, 0.97, and 0.67, respectively; 2) 2.0 mL: 0.901, 3.22 seconds, 0.90, and 0.87, respectively; and 3) standard strip: 0.912, 3.42 seconds, 0.97, and 0.80, respectively. Verification study: Data splitting analysis for the 2.0 μL data (n = 174 dry subjects and 97 normal subjects) generated an AUC of 0.917 and a cut-point of 6.05 seconds for a sensitivity of 0.87 and a specificity of 0.81. The 5.0 μL sample yielded an AUC of 0.940, with a sensitivity and specificity of 0.92 and 0.83, respectively, at a cut-point of 5.5 seconds.ConclusionsLittle difference in TBUT was found using the 3 clinical methods with video recordings. Analysis using liquid NaFl suggests that the TBUT test has excellent diagnostic accuracy and that a cut-point of 5.3 to 6.0 seconds is the optimum to differentiate normals from persons with dry eye.

The testing and classification of chemicals to determine adverse ocular effects are routinely conducted to ensure that materials are appropriately classified, labeled, and meet regulatory and safety guidelines. We have performed a same-chemical analysis using publicly available validation study results and compared the performance between tests for the same chemicals. To normalize for chemical selection, we matched chemicals tested by pairs of tests so that each matched set compared performance for the exact same chemicals. Same-chemical accuracy comparisons demonstrate a chemical selection effect that results in a wide range of overlapping false-positive (FP) rates and accuracies for all test methods. In addition, the analysis suggests that a tiered-testing strategy with specific combinations of tests can reduce the FP rate for some combinations. However, reductions in the FP rates were typically accompanied by an increase in the false-negative rates, resulting in minimal advantage in terms of accuracy. In addition, actual improvements in the FP rate after retesting positives with a second test are not as good as the theoretical improvements because some chemicals and functional groups appear to be broadly misclassified by all test methods, which, to the extent the tests make the same-chemical misclassifications, reduces the advantage of using tiered-testing strategies.

Purpose To determine whether riboflavin-induced collagen crosslinking (CXL) could be precisely achieved in the corneal stroma of ex vivo rabbit eyes using nonlinear optical excitation with a low numerical aperture lens and enlarged focal volume. Setting Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, USA. Design Experimental study. Methods The corneal epithelium was removed and the corneas were soaked in 0.5% riboflavin solution. Using a 0.1 numerical aperture objective, a theoretical excitation volume of 150 μm × 3 μm was generated using 1 W of 760 nm femtosecond laser light and raster scanned with 4.4 μm line separation at varying effective speeds over a 4.50 mm × 2.25 mm area. Corneal sections were examined for collagen autofluorescence. Results Collagen autofluorescence was enhanced 2.9 times compared with ultraviolet-A (UVA) CXL. Also, increasing speed was linearly associated with decreasing autofluorescence intensity. The slowest speed of 2.69 mm/s showed a mean of 182.97 μm ± 52.35 (SD) long autofluorescent scan lines axially in the central cornea compared with 147.84 ± 4.35 μm for UVA CXL. Conclusions Decreasing dwell time was linearly associated with decreasing autofluorescence intensity, approaching that of UVA CXL at a speed of 8.9 mm/s. Using an effective speed of 8.9 mm/s, nonlinear optical CXL could be achieved over a 3.0 mm diameter area in fewer than 4 minutes. Further development of nonlinear optical CXL might result in safer, faster, and more effective CXL treatments. Financial Disclosure None of the authors has a financial or proprietary interest in any material or method mentioned.

A synergy between the polymer biomaterial and drug plays an important role in enhancing the therapeutic efficacy, improving the drug stability, and minimizing the local immune responses in the development of drug delivery systems. Particularly, in the case of ocular drug delivery, the need for the development of synergistic drug delivery system becomes more pronounced because of the wet ocular mucosal surface and highly innervated cornea, which elicit a strong inflammatory response to the instilled drug formulations. This article presents the development of a synergistic cysteamine delivery nanowafer to treat corneal cystinosis. Corneal cystinosis is a rare metabolic disease that causes the accumulation of cystine crystals in the cornea resulting in corneal opacity and loss of vision. It is treated with topical cysteamine (Cys) eye drops that need to be instilled 6-12 times a day throughout the patient's life, which causes side effects such as eye pain, redness, and ocular inflammation. As a result, compliance and treatment outcomes are severely compromised. To surmount these issues, we have developed a clinically translatable Cys nanowafer (Cys-NW) that can be simply applied on the eye with a fingertip. During the course of the drug release, Cys-NW slowly dissolves and fades away. The in vivo studies in cystinosin knockout mice demonstrated twice the therapeutic efficacy of Cys-NW containing 10 μg of Cys administered once a day, compared to 44 μg of Cys as topical eye drops administered twice a day. Furthermore, Cys-NW stabilizes Cys for up to four months at room temperature compared to topical Cys eye drops that need to be frozen or refrigerated and still remain active for only 1 week. The Cys-NW, because of its enhanced therapeutic efficacy, safety profile, and extended drug stability at room temperature, can be rapidly translated to the clinic for human trials.

Ultraviolet A (UVA) light-based photoactivation of riboflavin (Rf) to induce corneal crosslinking (CXL) and mechanical stiffening is now a well-known treatment for corneal ectasia and Keratoconus that is being used in a topographically guided photorefractive intrastromal CXL (PiXL) procedure to treat low degrees of refractive errors. Alternative approaches for non-invasive treatment of refractive errors have also been proposed that use femtosecond lasers (FS) that provide much faster, more precise, and safer results than UVA CXL. One such treatment, nonlinear optical crosslinking (NLO CXL), has been able to replicate the effects of UVA CXL, while producing a smaller area of cellular damage and requiring a shorter procedure time. Unlike UVA CXL, the treatment volume of NLO CXL only occurs within the focal volume of the laser, which can be placed at any depth and scanned into any pattern for true topographically guided refractive correction. This review presents our experience with using FS lasers to photoactivate Rf and perform highly controlled corneal CXL that leads to mechanical stiffening and changes in corneal shape.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3., Graphical abstract Image 1, Highlights • Highly-ordered connective tissue appears early in development of the avian cornea. • Cell-independent mechanisms may contribute to the organisation of collagen fibrils into an orthogonal array. • Matrix cords from epithelium into stroma contact invading neural crest cells.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including Van der Woude syndrome. A subset of Van der Woude cases is caused by dominant negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3) and Grhl3(−/−) mice have soft tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3(−/−) embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) as the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3(−/−) embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function.Statement of significanceThis work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

The collagen fiber organization plays an important role in controlling the mechanical strength of the extracellular matrix tissues and in the cornea is thought to control tissue shape and refractive power. In the case of the cornea, collagen fibers run in orthogonal directions, interweaving, branching and anastomosing, that can be imaged using second harmonic-generated signals. But the complexity of the structure makes it difficult for identifying tunnels using previous methods. In the proposed work, we apply an efficient graph based algorithm to find fundamental cycles, including both tunnels and non-tunnels. After tightening the cycle and decoupling composite cycles, we classify the resulting fundamental cycles based on their geometric properties and their relationship between each other. The utility of our tunnel computation and location algorithm is demonstrated on various example cornea structures from various animals.

PurposeNitrogen mustard (NM), which simulates the effects of sulfur mustard (SM), is a potent vesicant known to cause irreversible corneal damage. This study investigates the mechanisms by which NM induces corneal damage by examining the impact of NM exposure on the morphology and lipidome of the cornea.MethodsIntact exvivo rabbit eyes were placed in serum-free DMEM organ culture. NM (0, 1, 2.5, 5 or 10mg/ml) was applied to the central cornea for 5, 10 or 15min using a 5mm filter disk and subsequently rinsed with DMEM. Corneas were then cultured for 3, 24, or 48h before being fixed for morphological analysis or for 24h before being snap frozen for lipidomic analysis.ResultsNo morphological changes were detected 3h after NM exposure. Twenty-four h after exposure, 1mg/ml NM caused erosion of the corneal epithelium, but no damage to the underlying stroma. Damage caused by 2.5mg/ml NM extended almost two thirds through the corneal stroma, while 5mg/ml completely penetrated the corneal stroma. An altered lipid profile occurred 24h after corneas were exposed to NM. Specific sphingomyelins, ceramides, and diacylglycerols were increased up to 9-, 60- and 10-fold, respectively.ConclusionsNM induces concentration- and exposure time-dependent damage to the cornea that increases in severity over time. Alterations in the sphingomyelin-ceramide pathway may contribute to the damaging effects of NM exposure.

This paper reviews our current understanding of age-related meibomian gland dysfunction (MGD) and the role of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), in the regulation of meibomian gland function, meibocyte differentiation and lipid synthesis. The studies suggest that PPARγ is a master regulator of meibocyte differentiation and function, whose expression and nuclear signaling coupled with meibocyte renewal is altered during aging, potentially leading to atrophy of the meibomian gland as seen in clinical MGD. Study of meibomian gland stem cells also suggest that there is a limited number of precursor meibocytes that provide progeny to the acini, that may be susceptible to exhaustion as occurs during aging and other environmental factors. Further study of pathways regulating PPARγ expression and function as well as meibocyte stem cell maintenance may provide clues to establishing cellular and molecular mechanisms underlying MGD and the development of novel therapeutic strategies to treating this disease.

This chapter presents some of the most important results obtained in adaptive optics (AO) visual testing, together with some important aspects of the required technology. AO visual testing provides the most powerful tool for the design of new optical solutions to correct our vision from refractive problems. The larger tolerance of vision when retinal images are degraded in a particular direction might be due to a kind of adaptation of the visual system to its own aberrations. AO visual testing can help to optimize new solutions and aids to correct vision with no need of real patients either true manufacturing or implantations of optical corrections. AO visual testing allowed studying the problem without the necessity of implantation of any intraocular lens (IOL). The incorporation of spherical aberration correction in the IOL could render a better image in the best plane for distant vision, impairing vision at any other closer distance more severely than the natural phakic eye.

To investigate the role of collagen structure in corneal biomechanics, measurement of localized corneal elasticity with minimal destruction to the tissue is necessary. We adopted the recently developed acoustic radiation force elastic microscopy (ARFEM) technique to measure localize biomechanical properties of the human cornea. In ARFEM, a low-frequency, high-intensity acoustic force is used to displace a femtosecond laser-generated microbubble, while high-frequency, low-intensity ultrasound is used to monitor the position of the microbubble within the cornea. Two ex vivo human corneas from a single donor were dehydrated to physiologic thickness, embedded in gelatin and then evaluated using the ARFEM technique. In the direction perpendicular to the corneal surface, ARFEM measurements provided elasticity values of E = 1.39 ± 0.28 kPa for the central anterior cornea and E = 0.71 ± 0.21 kPa for the central posterior cornea in pilot studies. The increased value of corneal elasticity in the anterior cornea correlates with the higher density of interweaving lamellae in this region.

The mechanical behavior of the optic nerve head (ONH) and the surrounding sclera play important roles in the development of optic neuropathy. We assessed the indentation behavior of the ONH and the surrounding sclera in unfixed human autopsy eyes from individuals between 27 and 87 years of age. Our testing device utilized a round-tipped 250 µm diameter stainless steel probe to measure the force applied in discrete 50 µm steps from the surface of the tissues to a depth of 400 µm. Thirteen eyes from eight individuals were indented in various locations within and around the ONH and the posterior sclera. Tissue thickness, force and depth were recorded at each position tested. Data was analyzed by evaluating the recorded force as a function of the depth of indentation. The data showed that, for both the sclera and the ONH, the force rose exponentially with increased depth of indentation. Apparent stiffness values were estimated using 2 different equations that assess soft biologic tissues. Results showed that significantly higher levels of force were required with aging to indent the posterior sclera, with a corresponding increase in stiffness values. However, no significant increase in indentation force as a function of age was noted for the ONH. Interestingly, the data suggests that some individuals have relatively large differences in the stiffness between the ONH and the posterior sclera, while others show less difference regardless of age. This data supports the notion that some individuals have relatively softer ONH tissue, and as the sclera becomes stiffer with age, this difference is magnified. This difference may be important in understanding the biomechanical contribution to the individually different susceptibility to glaucoma.

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet's membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.

The purpose of this study was to develop Globally Harmonized System (GHS) and U.S. Environmental Protection Agency (EPA) prediction models for classifying irritant materials based on histopathologic in vitro depth of injury (DoI) measurements. Sixteen different materials were selected, representing all classes of toxicity, according to the GHS and EPA classification systems. Food-source rabbit eyes, similar to eyes used for the widely accepted Bovine Corneal Opacity and Permeability and Isolated Chicken Eye ocular irritation tests, were used. Tissues were exposed to test material for 1 min, and corneas were collected at 3-and 24-hours post-exposure. Tissues were then fixed and processed for live/dead biomarker fluorescent staining using phalloidin. DoI was then measured, and the percent DoI values for the epithelium and stroma were compared to the EPA and GHS classifications. Excluding surfactants, EPA nonclassified (category IV) materials showed no stromal and very slight epithelial damage (≤10%) to the cornea, whereas EPA corrosive (category I) materials showed significantly greater damage (P < 0.001), ranging from 39% to 100% of the stromal depth. Importantly, EPA reversible (categories II and III) materials showed significant damage to the epithelium (> 10%, P < 0.005) but significantly less severe damage to the corneal stroma (P < 0.001), ranging from 1% to 38% of the stromal depth. GHS nonclassified (category NC) irritants caused damage to the epithelium but not to the stroma. All GHS class 2 materials showed damage to the stroma (1–11%), whereas GHS corrosives caused significantly greater damage to the stroma (38–100%; P < 0.001). Additionally, one corrosive material, which produced a stromal DoI of 99% at 24 h, produced no apparent damage at 3-hours post-exposure. Based on these findings, histopathologic EPA and GHS prediction models are proposed that appear to separate and identify reversible irritants from other irritant classes. Furthermore, GHS classification appears to require stromal damage, whereas NC materials may or may not damage the corneal epithelium.

PurposePrevious reports suggest that age-related meibomian gland atrophy is associated with decreased expression of the lipid-sensitive nuclear receptor, PPARγ. The purpose of this study was to identify the role of PPARγ in modulating meibocyte lipid synthesis.MethodsCytoplasmic and nuclear fractions from meibomian glands of young (2M) and old (2Y) C57Bl6 mice were probed using antibodies specific for PPARγ. Mouse meibocytes were cultured, immortalized using a SV40 lentiviral vector, and evaluated for lipid synthesis using LipidTox staining and CARS/Raman microspectroscopy. Lipid synthesizing clones were tested for effects of PPARγ agonist, rosiglitazone, on lipid synthesis and PPARγ localization, post-translational modification and induction of PPARγ response genes.ResultsThe cytoplasmic fraction in young mice contained both 50 and 72kDa PPARγ bands that were absent or reduced by 75% in older mice, respectively. Cultured meibocytes produced neutral lipid containing equal amounts of wax and cholesterol esters, similar to mouse meibum. Addition of rosiglitazone (10-50μM) significantly increased lipid production (P

Cornea Measurement of an Elasticity Map in the Human Cornea Eric R. Mikula, 1 James V. Jester, 1,2 and Tibor Juhasz 1,2 1 Gavin Herbert Eye Institute, University of California, Irvine, California, United States Department of Biomedical Engineering, University of California, Irvine, California, United States Correspondence: Eric R. Mikula, Hewitt Hall, 843 Health Sciences Road, Building 843, 2nd Floor, Room 2121, Irvine, CA 92697, USA; emikula@uci.edu. Submitted: September 22, 2015 Accepted: February 13, 2016 Citation: Mikula ER, Jester JV, Juhasz T. Measurement of an elasticity map in the human cornea. Invest Ophthalmol Vis Sci. 2016;57:3282–3286. DOI:10.1167/iovs.15-18248 P URPOSE . The biomechanical properties of the cornea have an important role in determining the shape of the cornea and visual acuity. Since the cornea is a nonhomogeneous tissue, it is thought that the elastic properties vary throughout the cornea. We aim to measure a map of corneal elasticity across the cornea. M ETHODS . An acoustic radiation force elasticity microscope (ARFEM) was used to create a map of corneal elasticity in the human cornea. This ARFEM uses a low frequency, high intensity acoustic force to displace a femtosecond laser-generated microbubble, while using a high frequency, low intensity ultrasound to monitor the position of the microbubble within the cornea. From the displacement of the bubble and the magnitude of the acoustic radiation force, the local value of corneal elasticity is calculated in the direction of the displacement. Measurements were conducted at 6 locations, ranging from the central to peripheral cornea at anterior and posterior depths. R ESULTS . The mean anterior elastic moduli were 4.2 6 1.2, 3.4 6 0.7, and 1.9 6 0.7 kPa in the central, mid, and peripheral regions, respectively, while the posterior elastic moduli were 2.3 6 0.7, 1.6 6 0.3, and 2.9 6 1.2 kPa in the same radial locations. C ONCLUSIONS . We found that there is a unique distribution of elasticity axially and radially throughout the cornea. Keywords: elasticity, ARFEM, corneal biomechanics, ultrasound T he biomechanical properties of the cornea, along with the underlying corneal microstructure, help determine the shape of the corneal surface, and, thus, also determine visual acuity. Since the cornea provides nearly two-thirds of the refractive power of the eye, understanding corneal biome- chanics is useful when considering refractive errors in the eye, such as keratoconus and post-LASIK ectasia. Further- more, a better knowledge of corneal mechanical properties can provide insight into the corneal response to refractive surgery or ultraviolet A (UVA) cross-linking procedures. Numerous methods have been used to evaluate corneal elasticity, including strip testing, globe inflation, atomic force microscopy, and various ultrasonic methods. Earlier methods, such as globe inflation and strip testing, treated the cornea as a homogenous material, measuring a bulk material property and not localized elastic moduli. 1–8 However, the cornea is a complex, nonhomogenous tissue composed of different layers and varying microstructure radially. There is significant interweaving of stromal lamellae in the central anterior cornea, while this interweaving is absent in the posterior. 9–12 Additionally, the interweaving in the anterior cornea affects the biomechanical properties and interlamellar cohesive strength increases with lamellar interweaving. 13,14 Recently, atomic force microscopy was used to probe varying biomechanical properties through the thickness of the cornea. 15 It was found that Bowman’s layer was nearly three times stiffer than the anterior stroma. Similarly, the trans- verse shear modulus in the anterior cornea was significantly higher than central and posterior layers using torsional rheometry. 16 A study using needle indentation found that the elastic modulus in the anterior cornea was nearly twice that of the posterior. 17,18 Furthermore, this same study also revealed that collagen fiber branching-point density was four times higher in the anterior third of the cornea than in the posterior third. These studies have shown that there is some depth dependence in corneal elasticity. As the cornea approaches the limbus and transitions into sclera tissue, there also is a transition in the microstructure of the tissue. Furthermore, x-ray scattering data suggest that collagen fibril orientation shifts from a preferred orthogonal orientation in the central cornea to a preferred circumferential orientation near the limbus. 19 The nonhomogenous structure of the cornea, through its thickness and radially towards the sclera, suggests that the elastic modulus will vary throughout the cornea. To date, there still is no consensus on regional corneal elastic modulus distribution, especially in the radial direction. Recently, we used acoustic radiation force elastic micros- copy (ARFEM) to measure localized elastic moduli in the human cornea. 20 Acoustic radiation force elastic microscopy uses a low frequency, high intensity acoustic force to displace a femtosecond laser-generated microbubble within the cornea, while using a high frequency, low intensity ultrasound to track the position of the microbubble within the tissue. 21 Importantly, the elasticity measurement is localized to the exact position of the microbubble, which can be created at any position within the cornea. This feature makes ARFEM ideal for probing biomechanical properties throughout the cornea. In this study, ARFEM was used to measure elasticity at varying positions through- out the cornea, with the goal of creating a map of corneal elastic moduli. iovs.arvojournals.org j ISSN: 1552-5783 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Recent studies suggest that mouse meibomian glands (MG) undergo age-related atrophy that mimics changes seen in age-related human MG dysfunction (MGD). To better understand the structural/functional changes that occur during aging, this study developed an imaging approach to generate quantifiable volumetric reconstructions of the mouse MG and measure total gland, cell, and lipid volume. Mouse eyelids were fixed in 4% paraformaldehyde, embedded in LR White resin and serially sectioned. Sections were then scanned using a 20× objective and a series of tiled images (1.35 × 1.35 × 0.5 mm) with a pixel size of 0.44 μm lateral and 2 μm axial were collected using a Zeiss 510 Meta LSM and a femtosecond laser to simultaneously detect second harmonic generated (SHG) and two-photon excited fluorescence (TPEF) signals from the tissue sections. The SHG signal from collagen was used to outline and generate an MG mask to create surface renderings of the total gland and extract relevant MG TPEF signals that were later separated into the cellular and lipid compartments. Using this technique, three-dimensional reconstructions of the mouse MG were obtained and the total, cell, and lipid volume of the MG measured. Volumetric reconstructions of mouse MG showed loss of acini in old mice that were not detected by routine histology. Furthermore, older mouse MG had reduced total gland volume that is primarily associated with loss of the lipid volume. These findings suggest that mice MG undergo “dropout” of acini, similar to that which occurs in human age-related MGD. Anat Rec, 2011. © 2010 Wiley-Liss, Inc.

Tear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. We present a non-invasive technique to quantify the pre-corneal tear film thickness. A cMOS camera is used to record the interference pattern produced by the reflections from multiple layers of the tear film Principles of spatial autocorrelation are applied to extract the frequency of the periodic patterns in the images. A mathematical model is developed to obtain the thickness of the tear film from the spatial autocorrelation image. The technique is validated using micro-fabricated thin parylene films. We obtained repeatable and precise measurement on a live rabbit model (N = 6). We obtained an average value of 10.2µm and standard deviation of, SD = 0.3 (N = 4). We measured one rabbit infected with HSV-1 virus that had a baseline tear film thickness of 4.7µm.

Over the past forty years, contact lenses have gained increasing popularity for the correction of refractive errors of the eye. At this time, about 140 million patients wear contact lenses world wide, with some 38 million exposed to the device in the United States, and a similar 10–12% of the population of Great Britain (Richard Weisbarth, personal communication). Importantly, for several decades contact lens use has continued to grow at a variable annual rate of 5–15%; thus, sight threatening complications while uncommon, adversely afflict significantly increased numbers of patients each year (Richard Weisbarth, personal communication). If, as recent epidemiological studies continue to confirm, the rates of lens-related microbial keratitis in daily wear remain 1/2500 and 1/500 in overnight or extended wear, then there will be increasing tens of thousands of new lens wearers at risk world wide every year.1–4 Given this challenge, it is critical to identify the biological mechanisms that predispose the cornea to infection in lens use, and search for modifiable risks that could reduce this sight-threatening complication. The purpose of this Castroviejo Lecture is to review significant clinical and basic science publications as well as ongoing studies to achieve the safest possible contact lens wear.

Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW femtosecond laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 μm diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 hours after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.

The current paper reviews the genetic basis of diseases affecting the cornea, including corneal dystrophies and systemic diseases with corneal manifestations (e.g. the mucopolysaccharidoses), discusses the mechanisms by which corneal injury and disease can affect corneal transparency and discusses stem cell therapy as a potential therapy for corneal injury and disease. Several diseases of the cornea have been described, many of which are inheritable. The identification of the genetic mutations responsible for these diseases has led to a better understanding of the mechanisms underlying their corneal manifestations. Corneal disease and injury can affect corneal transparency. Stromal keratocytes play an important role in cellular and extracellular transparency of the cornea. They synthesize type I and V collagen and keratan sulfate proteoglycans that together regulate collagen fibril size and spacing important for extracellular matrix transparency. There are strong indications that cellular transparency of keratocytes relies on the presence of corneal crystallins, which provide a uniform density of proteins in the cytoplasm that increase the spatial order necessary for transparency. After injury, activated keratocytes show heterogeneity of cytoplasmic proteins, decreased spatial ordering and increased light scattering. Stem cell transplantation is regarded as a potential new therapy that can replace keratocytes and restore corneal transparency and normal structure after injury or corneal disease.

While DOI is a mechanistic correlate to the ocular irritation response, attempts to measure DOI in alternative tests have been limited to qualitative histopathologic assessment by veterinarian pathologists. The purpose of this study was to determine whether DOI could be measured objectively by fluorescent staining for biomarkers of cell death and viability using an ex vivo isolated rabbit eye (IRE) test. A panel of nine materials characterized by in vivo DOI were selected that caused slight (3% acetic acid and 5% SDS), mild (acetone, sodium hypochlorite and 10% acetic acid), moderate (cyclohexanol and parafluoroanaline) and severe (8% sodium hydroxide and 10% benzalkonium chloride) irritation. Materials were then tested using a modified IRE test with 3h recovery and then processed for cyrosectioning and staining using a TUNEL assay to detect cell death, phalloidin to detect intracellular f-actin and DAPI staining to detect nuclei. Control eyes treated with water showed intense phalloidin staining of the corneal epithelium and stromal keratocytes but no TUNEL labeling. In general, eyes treated with mild, moderate and severe irritants showed regions of TUNEL labeled epithelial and keratocyte nuclei with no phalloidin stain overlying phalloidin stained, undamaged cells. DOI measurements showed that slight irritants damaged40% of the epithelium, mild and moderate irritants damaged50% of the epithelium, extending at times into the anterior stroma (20%), and the severe irritant damaged50% of the stroma. Regression analysis between ex vivo and in vivo DOI showed a significant (p0.007) correlation (r=0.785). These data suggest that fluorescent staining of fixed and sectioned tissue using biomarkers can be used to objectively identify the depth of injury caused by ocular irritants.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in every organ in the body and leads to organ damage and dysfunction, including renal defects. Using the murine model for cystinosis, Ctns−/− mice, we performed syngeneic bone marrow cell (BMC), hematopoietic stem cell (HSC), and mesenchymal stem cell transplantation. Organ-specific cystine content was reduced by 57% to 94% in all organs tested in the BMC-treated mice. Confocal microscopy and quantitative polymerase chain reaction revealed a large quantity of transplanted BMC in all organs tested, from 5% to 19% of the total cells. Most of these cells were not from the lymphoid lineage but part of the intrinsic structure of the organ. The natural progression of renal dysfunction was prevented, and deposition of corneal cystine crystals was significantly improved in the BMC-treated mice. HSC had the same therapeutic effect as whole BMC. In contrast, mesenchymal stem cell did not integrate efficiently in any organ. This work is a proof of concept for using HSC transplantation as a therapy for cystinosis and highlights the efficiency of this strategy for a chronic, progressive degenerative disease.

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6–8-week-old rabbits and corneas of 1–2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.

To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas.Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced.In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia.Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.

Past studies have established that the cornea like the lens abundantly expresses a few water-soluble enzyme/proteins in a taxon specific fashion. Based on these similarities it has been proposed that the lens and the cornea form a structural unit, the 'refracton', that has co-evolved through gene sharing to maximize light transmission and refraction to the retina. Thus far, the analogy between corneal crystallins and lens crystallins has been limited to similarities in the abundant expression, with few reports concerning their structural function. This review covers recent studies that establish a clear relationship between expression of corneal crystallins and light scattering from corneal stromal cells, i.e. keratocytes, that support a structural role for corneal crystallins in the development of transparency similar to that of lens crystallins that would be consistent with the 'refracton' hypothesis.

Author(s): Jester, JV; Morishige, N; BenMohamed, L; Brown, DJ; Osorio, N; Hsiang, C; Perng, GC; Jones, C; Wechsler, SL | Abstract: Purpose: Using CJLAT, a chimeric herpes simplex virus (HSV-1) that produces a high incidence of herpes stromal keratitis (HSK) in latently infected rabbits, and in vivo confocal microscopy (CM), we characterized the cellular events that precede the development of HSK. Methods: Thirty days after infection, in vivo CM was performed daily for 10 days and then weekly for up to 80 days after infection. Results: We detected 3 types of subclinical corneal lesions before HSK was clinically apparent: (1) small epithelial erosions; (2) regenerating epithelium overlying small cell infiltrates within the basal epithelial cell layer; and (3) dendritic-like cells within the basal epithelial layer overlying stromal foci containing infiltrating cells. Sequential in vivo CM observations suggested that subclinical foci resolved over time but were larger and more abundant with CJLAT than with wild-type HSV-1 McKrae. Active HSK was observed only with CJLAT and was initially associated with a large epithelial lesion overlying stromal immune cell infiltrates. Conclusions: These results suggest that replication in the cornea of reactivated virus from the trigeminal ganglia produces epithelial lesions, which recruit immune cell infiltrates into the basal epithelial layer and anterior stroma. The virus is usually cleared rapidly eliminating viral antigens before the arrival of the immune cells, which disperse. However, if the virus is not cleared rapidly, or if an additional reactivation results in an additional round of virus at the same site before the immune cells disperse, then the immune cells are stimulated and may induce an immunopathological response leading to the development of HSK.

We studied the phenotype and distribution of “naturally” occurring CD4+CD25+T regulatory cells (CD4+CD25+nTregcells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (Teff) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45+pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4+CD25(Bright+)T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4+CD25(Bright+)T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4+CD25(Bright+)T cells, but not CD4+CD25(low)T cells, efficiently suppressed HSV-specific CD4+and CD8+Teffcells. The CD4+CD25(Bright+)T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating Teffcells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor β. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3+CD4+CD25(Bright+)T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3+CD4+CD25(Bright+)Tregcells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4+CD25+nTregcells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.