Thrombospondin-1-deficient (TSP-1) mice are used as an animal model of Sjögren's Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren's Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1 and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1 compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1 mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1 compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1 mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1 mice age.

Purpose: To compare the diagnostic capability of three-dimensional (3D) macular parameters against traditional two-dimensional (2D) retinal nerve fiber layer (RNFL) thickness using spectral domain optical coherence tomography. To determine if manual correction and interpolation of B-scans improve the ability of 3D macular parameters to diagnose glaucoma. Methods: A total of 101 open angle glaucoma patients (29 with early glaucoma) and 57 healthy subjects had peripapillary 2D RNFL thickness and 3D macular volume scans. Four parameters were calculated for six different-sized annuli: total macular thickness (M-thickness), total macular volume (M-volume), ganglion cell complex (GCC) thickness, and GCC volume of the innermost 3 macular layers (retinal nerve fiber layer + ganglion cell layer + inner plexiform layer). All macular parameters were calculated with and without correction and interpolation of frames with artifacts. The areas under the receiver operating characteristic curves (AUROC) were calculated for all the parameters. Results: The 3D macular parameter with the best diagnostic performance was GCC-volume-34, with an inner diameter of 3 mm and an outer of 4 mm. The AUROC for RNFL thickness and GCC-volume-34 were statistically similar for all regions (global: RNFL thickness 0.956, GCC-volume-34 0.939, P value = 0.3827), except for the temporal GCC-volume-34, which was significantly better than temporal RNFL thickness (P value = 0.0067). Correction of artifacts did not significantly change the AUROC of macular parameters (P values between 0.8452 and 1.0000). Conclusions: The diagnostic performance of best macular parameters (GCC-volume-34 and GCC-thickness-34) were similar to or better than 2D RNFL thickness. Manual correction of artifacts with data interpolation is unnecessary in the clinical setting.

Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Purpose: Proper control of eye movements is critical to vision, but relatively little is known about the molecular mechanisms that regulate development and axon guidance in the ocular motor system or cause the abnormal innervation patterns (oculomotor synkinesis) seen in developmental disorders and after oculomotor nerve palsy. We developed an ex vivo slice assay that allows for live imaging and molecular manipulation of the growing oculomotor nerve, which we used to identify axon guidance cues that affect the oculomotor nerve. Methods: Ex vivo slices were generated from E10.5 IslMN-GFP embryos and grown for 24 to 72 hours. To assess for CXCR4 function, the specific inhibitor AMD3100 was added to the culture media. Cxcr4cko/cko:Isl-Cre:ISLMN-GFP and Cxcl12KO/KO:ISLMN-GFP embryos were cleared and imaged on a confocal microscope. Results: When AMD3100 was added to the slice cultures, oculomotor axons grew dorsally (away from the eye) rather than ventrally (toward the eye). Axons that had already exited the midbrain continued toward the eye. Loss of Cxcr4 or Cxcl12 in vivo caused misrouting of the oculomotor nerve dorsally and motor axons from the trigeminal motor nerve, which normally innervate the muscles of mastication, aberrantly innervated extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis. Conclusions: CXCR4/CXCL12 signaling is critical for the initial pathfinding decisions of oculomotor axons and their proper exit from the midbrain. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles lacking innervation may secrete cues that attract motor axons.

PURPOSE: To describe a case of a central retinal vein occlusion in a young patient with a history of eosinophilic pneumonia. METHODS: A retrospective case report of a 45-year-old woman with acute painless vision loss for 9 days after multiple episodes of eosinophilic pneumonia and thalamic stroke. Fluorescein angiography, spectral domain optical coherence tomography, and clinical examination were performed. She was then treated with intravitreal bevacizumab and pan-retinal photocoagulations. RESULTS: Retinal examination revealed tortuosity and dilatation of all branches of the central retinal vein and flame-shaped hemorrhages in all four quadrants of the right eye associated with cystoid macular edema, optic disc edema, and cotton wool spots. The left eye had mild venous dilatation and tortuosity with a few dot retinal hemorrhages in the far temporal periphery. The cystoid macular edema resolved after one intravitreal injection of bevacizumab and remained resolved at the most recent follow-up. Fluorescein angiography at the most recent follow-up revealed vasculitis in the far periphery of the nontreated eye. CONCLUSION: Central retinal vein occlusion in young patients is a rare condition often presenting as a manifestation of an underlying inflammatory or hematological disorder. Combined anti-vascular endothelial growth factor treatment and pan-retinal photocoagulation may have resolved the associated cystoid macular edema in this case, although continued observation is necessary.

Breast milk (BM) hormones have been hypothesised as a nutritional link between maternal and infant metabolic health. This study aimed to evaluate hormone concentrations in BM of women with and without gestational diabetes mellitus (GDM), and the relationship between maternal factors, BM hormones and infant growth. We studied ninety-six nulliparous women with (n 48) and without GDM and their exclusively breastfed term singletons. Women with GDM received dietary therapy or insulin injection for euglycaemia during pregnancy. Hormone concentrations in BM, maternal BMI and infant growth were longitudinally evaluated on postnatal days 3, 42 and 90. Mothers with GDM had decreased concentrations of adiponectin (P colostrum<0·001; P mature-milk=0·009) and ghrelin (P colostrum=0·011; P mature-milk<0·001) and increased concentration of insulin in BM (P colostrum=0·047; P mature-milk=0·021). Maternal BMI was positively associated with adiponectin (β=0·06; 95 % CI 0·02, 0·1; P=0·001), leptin (β=0·16; 95 % CI 0·12, 0·2; P<0·001) and insulin concentrations (β=0·06; 95 % CI 0·02, 0·1; P<0·001), and inversely associated with ghrelin concentration in BM (β=-0·08; 95 % CI -0·1, -0·06; P<0·001). Among the four hormones, adiponectin was inversely associated with infant growth in both the GDM (β weight-for-height=-2·49; 95 % CI -3·83, -1·15; P<0·001; β head-circumference=-0·39; 95 % CI -0·65, -0·13; P=0·003) and healthy groups (β weight-for-height=-1·42; 95 % CI -2·38, -0·46; P=0·003; β head-circumference=-0·15; 95 % CI -0·27, -0·03; P=0·007). Maternal BMI and GDM are important determinants of BM hormone concentrations. Milk-borne adiponectin is determined by maternal metabolic status and plays an independent down-regulating role in early infant growth.

Woundhealing disorders characterized by impaired or delayed re-epithelialization are a serious medical problem that is painful and difficult to treat. Gelsolin (GSN), a known actin modulator, supports epithelial cell regeneration and apoptosis. The aim of this study was to estimate the potential of recombinant gelsolin (rhu-pGSN) for ocular surface regeneration to establish a novel therapy for delayed or complicated wound healing. We analyzed the influence of gelsolin on cell proliferation and wound healing in vitro, in vivo/ex vivo and by gene knockdown. Gelsolin is expressed in all tested tissues of the ocular system as shown by molecular analysis. The concentration of GSN is significantly increased in tear fluid samples of patients with dry eye disease. rhu-pGSN induces cell proliferation and faster wound healing in vitro as well as in vivo/ex vivo. TGF-β dependent transcription of SMA is significantly decreased after GSN gene knockdown. Gelsolin is an inherent protein of the ocular system and is secreted into the tear fluid. Our results show a positive effect on corneal cell proliferation and wound healing. Furthermore, GSN regulates the synthesis of SMA in myofibroblasts, which establishes GSN as a key protein of TGF-β dependent cell differentiation.

The meibomian gland (MG) is a sebaceous gland that secretes through a holocrine process. Because such secretion requires the destruction of MG acinar epithelial cells, they need constant renewal and differentiation. The processes that promote these regenerative events in the human MG are unknown, nor is it known how to distinguish MG progenitor and differentiated cells. We discovered that Lrig1 and DNase2 serve as biomarkers for human MG progenitor and differentiated cells, respectively. Lrig1 is expressed in MG basal epithelial cells in the acinar periphery, a location where progenitor cells originate in sebaceous glands. DNase2 is expressed in the differentiated epithelial cells of the MG central acinus. Furthermore, proliferation stimulates, and differentiation suppresses, Lrig1 expression in human MG epithelial cells. The opposite is true for DNase2 expression. Our biomarker identification may have significant value in clinical efforts to restore MG function and to regenerate MGs after disease-induced dropout. Stem Cells Translational Medicine 2018;7:887-892.

In visual search tasks, observers can guide their attention towards items in the visual field that share features with the target item. In this series of studies, we examined the time course of guidance toward a subset of items that have the same color as the target item. Landolt Cs were placed on 16 colored disks. Fifteen distractor Cs had gaps facing up or down while one target C had a gap facing left or right. Observers searched for the target C and reported which side contained the gap as quickly as possible. In the absence of other information, observers must search at random through the Cs. However, during the trial, the disks changed colors. Twelve disks were now of one color and four disks were of another color. Observers knew that the target C would always be in the smaller color set. The experimental question was how quickly observers could guide their attention to the smaller color set. Results indicate that observers could not make instantaneous use of color information to guide the search, even when they knew which two colors would be appearing on every trial. In each study, it took participants 200-300 ms to fully utilize the color information once presented. Control studies replicated the finding with more saturated colors and with colored C stimuli (rather than Cs on colored disks). We conclude that segregation of a display by color for the purposes of guidance takes 200-300 ms to fully develop.