This review compared the clinical results of transepithelial corneal crosslinking (CXL) to epithelium-off (epi-off) CXL in progressive corneal ectasia using a metaanalysis. The Cochrane databases and Medline were searched for randomized controlled trials (RCTs). Seven RCTs involving 505 eyes that met the eligibility criteria were identified. The epi-off CXL group showed significantly better outcomes in postoperative changes in maximum keratometry (K) during 1-year observation periods. Transepithelial CXL resulted in significantly greater post-treatment central corneal thickness and best spectacle-corrected visual acuity (BSCVA). The presence of a postoperative demarcation line was significantly more frequent after epi-off CXL than that after transepithelial CXL. No statistically significant difference was found between other parameters. Although patients in the transepithelial CXL group demonstrated a greater improvement in BSCVA compared with patients in the epi-off CXL group at the 1 year follow-up, transepithelial CXL had less impact on halting progressive corneal ectasia in terms of maximum K than epi-off CXL.

PURPOSE: To investigate systemic and ocular determinants of peripapillary retinal nerve fiber layer thickness (pRNFLT) in the European population. DESIGN: Cross-sectional meta-analysis. PARTICIPANTS: A total of 16 084 European adults from 8 cohort studies (mean age range, 56.9±12.3-82.1±4.2 years) of the European Eye Epidemiology (E3) consortium. METHODS: We examined associations with pRNFLT measured by spectral-domain OCT in each study using multivariable linear regression and pooled results using random effects meta-analysis. MAIN OUTCOME MEASURES: Determinants of pRNFLT. RESULTS: Mean pRNFLT ranged from 86.8±21.4 μm in the Rotterdam Study I to 104.7±12.5 μm in the Rotterdam Study III. We found the following factors to be associated with reduced pRNFLT: Older age (β = -0.38 μm/year; 95% confidence interval [CI], -0.57 to -0.18), higher intraocular pressure (IOP) (β = -0.36 μm/mmHg; 95% CI, -0.56 to -0.15), visual impairment (β = -5.50 μm; 95% CI, -9.37 to -1.64), and history of systemic hypertension (β = -0.54 μm; 95% CI, -1.01 to -0.07) and stroke (β = -1.94 μm; 95% CI, -3.17 to -0.72). A suggestive, albeit nonsignificant, association was observed for dementia (β = -3.11 μm; 95% CI, -6.22 to 0.01). Higher pRNFLT was associated with more hyperopic spherical equivalent (β = 1.39 μm/diopter; 95% CI, 1.19-1.59) and smoking (β = 1.53 μm; 95% CI, 1.00-2.06 for current smokers compared with never-smokers). CONCLUSIONS: In addition to previously described determinants such as age and refraction, we found that systemic vascular and neurovascular diseases were associated with reduced pRNFLT. These may be of clinical relevance, especially in glaucoma monitoring of patients with newly occurring vascular comorbidities.

In 1955, Human adenovirus type 14 (HAdV-B14p) was firstly identified in a military trainee diagnosed as acute respiratory disease (ARD) in the Netherlands. Fifty years later, a genomic variant, HAdV-B14p1, re-emerged in the U.S. and caused large and fatal ARD outbreaks. Subsequently, more and more ARD outbreaks occurred in Canada, the UK, Ireland, and China, in both military and civil settings. To generate a tool for the efficient characterization of this new genomic variant, a full-length infectious genomic clone of HAdV-B14 was successfully constructed using one-step Gibson Assembly method in this study. Firstly, the full genome of HAdV-B14p1 strain GZ01, the first HAdV-B14 isolate in China, was assembled into pBR322 plasmid by Gibson Assembly. The pBRAdV14 plasmid, generated by Gibson Assembly, was analyzed and verified by PCR, restriction enzymes digestion and the sequencing. Secondly, viruses were rescued from pBRAdV14-transfected A549 cells. The integrity of the rescued viruses was identified by restriction enzyme analysis. The complete sequence of the infectious clone was further sequenced. No mutation was found in the infectious clone during the construction when compared with the parental virus and pBR322 sequences. The direct immunofluorescence assay indicated the expression of the hexon protein. Finally, typical virions were observed; the one-step growth curves further showed that the DNA replication and viral reproduction efficiency of pBRAd14 derived viruses was similar with that of wild-type HAdV-B14 strain. The successful construction of the replication-competent infectious clone of pBRAdV14 facilitates the development of vaccine and antiviral drugs against HAdV-B14, as well as provides a novel strategy for rapid construction of infectious viral clones for other large-genome DNA viruses.

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals HO (via VEGFR2/PI3K/AKT signaling) and N-methyl-D-aspartate (via reduced Ca influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.

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.