Pre-corneal tear film thickness in humans measured with a novel technique.

PurposeThe purpose of this work was to gather preliminary data in normals and dry eye subjects, using a new, non-invasive imaging platform to measure the thickness of pre-corneal tear film.MethodsHuman subjects were screened for dry eye and classified as dry or normal. Tear film thickness over the inferior paracentral cornea was measured using laser illumination and a complementary metal-oxide-semiconductor (CMOS) camera. A previously developed mathematical model was used to calculate the thickness of the tear film by applying the principle of spatial auto-correlation function (ACF).ResultsMean tear film thickness values (±SD) were 3.05 μm (0.20) and 2.48 μm (0.32) on the initial visit for normals (n=18) and dry eye subjects (n=22), respectively, and were significantly different (p<0.001, 2-sample t-test). Repeatability was good between visit 1 and 2 for normals (intraclass correlation coefficient [ICC]=0.935) and dry eye subjects (ICC=0.950). Tear film thickness increased above baseline for the dry eye subjects following viscous drop instillation and remained significantly elevated for up to approximately 32 min (n=20; p<0.05 until 32 min; general linear mixed model and Dunnett's tests).ConclusionsThis technique for imaging the ocular surface appears to provide tear thickness values in agreement with other non-invasive methods. Moreover, the technique can differentiate between normal and dry eye patient types

Nonlinear Optical Corneal Crosslinking, Mechanical Stiffening, and Corneal Flattening Using Amplified Femtosecond Pulses.

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 < 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

Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD)

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

Evaluation of topical cysteamine therapy in the CTNS−/− knockout mouse using in vivo confocal microscopy

PurposeThe purpose of this study was to assess the ability of quantitative in vivo confocal microscopy (CM) to detect changes in cystine crystal volume in the cystinosisn (Ctns−/−)mouse cornea following topical cysteamine therapy.MethodsFifteen Ctns−/− mice were sequentially followed using in vivo CM from 3 to 10 months of age. In a second experiment, five mice receiving topical cysteamine eyedrops (0.55%) for 4 weeks were compared to five untreated mice. The volume of corneal cystine crystals was determined by thresholding and counting high intensity pixels in the in vivo CM scans and dividing by the stromal volume to calculate a crystal volume index (CVI).ResultsCorneal crystals progressively increased in density with age, reaching a peak density at 6–8 months and showing a 70 fold increase in CVI. Eyes treated with cysteamine drops showed significantly less crystal accumulation compared to control eyes (p<0.001) with only a 15% increase in treated eyes (p=ns) compared to 173% increase (p<0.04) for untreated eyes.ConclusionsMeasurement of CVI shows that there is a progressive increase in cystine crystal volume up to 8 months of age and that cysteamine eyedrops significantly inhibits progression in the Ctns−/− mouse. These findings are similar to those seen clinically in patients with cystinosis, and suggest that measurement of CVI in the Ctns−/− mouse may be used as a model to develop novel therapeutic strategies for treating corneal cystinosis

Epithelial Migration and Non-adhesive Periderm Are Required for Digit Separation during Mammalian Development.

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) when 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

Recapitulation of normal collagen architecture in embryonic wounded corneas

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

Herpes simplex virus type 1 ICP0 localizes in the stromal layer of infected rabbit corneas and resides predominantly in the cytoplasm and/or perinuclear region of rabbit keratocytes

Herpes stromal keratitis (HSK) results from the reactivation of herpes simplex virus type-1 (HSV-1) in the cornea. The subsequent corneal inflammation and neovascularization may lead to scarring and visual loss. The cellular and molecular mechanisms underlying HSK remain unknown. The presence of stromal HSV-1 viral proteins or antigens in the HSK cornea remains a subject of debate. It was recently reported that HSV-1 ICP0 rapidly diffuses out of infected rabbit corneas. To investigate further the presence of HSV-1 ICP0 in the infected cornea, particularly in the corneal stroma, ex vivo confocal microscopy was used to scan rabbit corneas infected with the virus ICP0–EYFP, an HSV-1 derivative (strain 17+) that expresses ICP0 fused to the enhanced yellow fluorescent protein (EYFP). These results demonstrate that ICP0 is expressed in the corneal epithelium and stromal cells (keratocytes) of infected rabbit corneas throughout acute infection. Furthermore, expression of ICP0–EYFP appears localized to punctate, granular deposits within stromal keratocytes, showing both a cytoplasmic and perinuclear localization. These findings provide new data demonstrating that anterior corneal keratocytes become infected and express ICP0 during acute HSV-1 infection

The development of meibomian glands in mice

PurposeThe purpose of this study was to characterize the natural history of meibomian gland morphogenesis in the mouse.MethodsEmbryonic (E) and post natal (P) C57Bl/6 mouse pups were obtained at E18.5, P0, P1, P3, P5, P8, P15, and P60. Eyelids were fixed and processed for en bloc staining with Phalloidin/DAPI to identify gland morphogenesis, or frozen for immunohistochemistry staining with Oil red O (ORO) to identify lipid and antibodies specific against peroxisome proliferator-activated receptor gamma (PPARγ) to identify meibocyte differentiation. Samples were then evaluated using a Zeiss 510 Meta laser scanning confocal microscope or Nikon epi-fluorescent microscope. Tissues from adult mice (2 month-old) were also collected for western blotting.ResultsMeibomian gland morphogenesis was first detected at E18.5 with the formation of an epithelial placode within the fused eyelid margin. Invagination of the epithelium into the eyelid was detected at P0. From P1 to P3 there was continued extension of the epithelium into the eyelid. ORO and PPARγ staining was first detected at P3, localized to the central core of the epithelial cord thus forming the presumptive ductal lumen. Ductal branching was first detected at P5 associated with acinar differentiation identified by ORO and PPARγ staining. Adult meibomian glands were observed by P15. Western blotting of meibomian gland proteins identified a 50 kDa and a 72 kDa band that stained with antibodies specific to PPARγ.ConclusionsWe have demonstrated that meibomian gland development bears distinct similarities to hair development with the formation of an epithelial placode and expression of PPARγ co-incident with lipid synthesis and meibocyte differentiation