Migraine-type photophobia, most commonly described as exacerbation of headache by light, affects nearly 90% of the patients. It is the most bothersome symptom accompanying an attack. Using subjective psychophysical assessments, we showed that migraine patients are more sensitive to all colors of light during ictal than during interictal phase and that control subjects do not experience pain when exposed to different colors of light. Based on these findings, we suggested that color preference is unique to migraineurs (as it was not found in control subjects) rather than migraine phase (as it was found in both phases). To identify the origin of this photophobia in migraineurs, we compared the electrical waveforms that were generated in the retina and visual cortex of 46 interictal migraineurs to those generated in 42 healthy controls using color-based electroretinography and visual-evoked potential paradigms. Unexpectedly, it was the amplitude of the retinal rod-driven b wave, which was consistently larger (by 14%-19% in the light-adapted and 18%-34% in the dark-adapted flash ERG) in the migraineurs than in the controls, rather than the retinal cone-driven a wave or the visual-evoked potentials that differs most strikingly between the 2 groups. Mechanistically, these findings suggest that the inherent hypersensitivity to light among migraine patients may originate in the retinal rods rather than retinal cones or the visual cortex. Clinically, the findings may explain why migraineurs complain that the light is too bright even when it is dim to the extent that nonmigraineurs feel as if they are in a cave.

Primary open angle glaucoma (POAG) is a complex disease with a major genetic contribution. Its prevalence varies greatly among ethnic groups, and is up to five times more frequent in black African populations compared to Europeans. So far, worldwide efforts to elucidate the genetic complexity of POAG in African populations has been limited. We conducted a genome-wide association study in 1113 POAG cases and 1826 controls from Tanzanian, South African and African American study samples. Apart from confirming evidence of association at TXNRD2 (rs16984299; OR 1.20; P = 0.003), we found that a genetic risk score combining the effects of the 15 previously reported POAG loci was significantly associated with POAG in our samples (OR 1.56; 95% CI 1.26-1.93; P = 4.79 × 10). By genome-wide association testing we identified a novel candidate locus, rs141186647, harboring EXOC4 (OR 0.48; P = 3.75 × 10), a gene transcribing a component of the exocyst complex involved in vesicle transport. The low frequency and high degree of genetic heterogeneity at this region hampered validation of this finding in predominantly West-African replication sets. Our results suggest that established genetic risk factors play a role in African POAG, however, they do not explain the higher disease load. The high heterogeneity within Africans remains a challenge to identify the genetic commonalities for POAG in this ethnicity, and demands studies of extremely large size.

PURPOSE: To ascertain the incidence of unexpected management changes at the postoperative week 1 visit in asymptomatic patients who have had an uncomplicated cataract surgery and a routine postoperative day 1 examination. DESIGN: Retrospective observational study. METHODS: A retrospective chart review was conducted of all cases of cataract extraction by phacoemulsification with intraocular lens insertion performed by the Comprehensive Ophthalmology Service at Massachusetts Eye and Ear between January 1, 2014 and December 31, 2014. The preoperative consultation, operative report, and postoperative day 1 and week 1 (postoperative days 5-14) visits were reviewed. Cases with intraoperative complications, as well as clinical findings at postoperative day 1 requiring close follow-up, were excluded. The main outcome measure was incidence of unexpected management changes at the postoperative week 1 visit after cataract surgery, defined as an unanticipated change in postoperative drops, additional procedures, or urgent referral to a specialty service. RESULTS: Overall, 1938 surgical cases of 1471 patients were reviewed, and 1510 cases (77.9%) underwent uncomplicated phacoemulsification with intraocular lens implantation with a routine postoperative day 1 examination. Of these 1510 cases, 238 (15.8%) reported symptoms at the postoperative week 1 visit, including flashes, floaters, redness, pain, or decreased vision, which warranted an examination. In total, 1272 cases were asymptomatic, and only 11 of these cases (0.9%) had an unexpected management change at postoperative week 1. Eight of 11 patients were asymptomatic steroid responders requiring alteration of their postoperative drops. Two of these patients had an intraocular pressure >30 mm Hg. CONCLUSIONS: Unexpected management changes at the postoperative week 1 timepoint after cataract surgery are rare in asymptomatic patients who have had uncomplicated cataract surgery and a routine postoperative day 1 examination. Limited data are available to outline an optimal postoperative regimen after cataract surgery. The results of this study suggest that postoperative week 1 examinations could potentially be performed on an as-needed basis in the appropriate subgroup of patients after cataract surgery.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Retinopathy of prematurity (ROP) is characterized by abnormal retinal neovascularization in response to vessel loss. Platelets regulate angiogenesis and may influence ROP progression. In preterm infants, we assessed ROP and correlated with longitudinal postnatal platelet counts (n = 202). Any episode of thrombocytopenia (<100 × 109/l) at ≥30 weeks postmenstrual age (at onset of ROP) was independently associated with severe ROP, requiring treatment. Infants with severe ROP also had a lower weekly median platelet count compared with infants with less severe ROP. In a mouse oxygen-induced retinopathy model of ROP, platelet counts were lower at P17 (peak neovascularization) versus controls. Platelet transfusions at P15 and P16 suppressed neovascularization, and platelet depletion increased neovascularization. Platelet transfusion decreased retinal of vascular endothelial growth factor A (VEGFA) mRNA and protein expression; platelet depletion increased retinal VEGFA mRNA and protein expression. Resting platelets with intact granules reduced neovascularization, while thrombin-activated degranulated platelets did not. These data suggest that platelet releasate has a local antiangiogenic effect on endothelial cells to exert a downstream suppression of VEGFA in neural retina. Low platelet counts during the neovascularization phase in ROP is significantly associated with the development of severe ROP in preterm infants. In a murine model of retinopathy, platelet transfusion during the period of neovascularization suppressed retinopathy.

Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (, ) and other potentially disease-relevant genes (, ) were upregulated in male lacrimal glands. Expression of and , in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, -deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.

PURPOSE: To test the effects of an encapsulated cell-based delivery of a neuroprotective agent, ciliary neurotrophic factor (CNTF), on progression of macular telangiectasia type 2, a neurodegenerative disease with no proven effective therapy. DESIGN: Randomized sham-controlled clinical trial. PARTICIPANTS: Ninety-nine study eyes of 67 eligible participants were enrolled. METHODS: Single-masked randomized clinical trial of 24 months' duration conducted from May 2014 through April 2017 in 11 clinical centers of retinal specialists in the United States and Australia. Participants were randomized 1:1 to surgical implantation of intravitreal sustained delivery of human CNTF versus a sham procedure. MAIN OUTCOME MEASURES: The primary outcome was the difference in the area of neurodegeneration as measured in the area of the ellipsoid zone disruption (or photoreceptor loss) measured on spectral-domain (SD) OCT images at 24 months from baseline between the treated and untreated groups. Secondary outcomes included comparison of visual function changes between treatment groups. RESULTS: Among the 67 participants who were randomized (mean age, 62±8.9 years; 41 women [61%]; 58 white persons [86%]), 65 (97%) completed the study. Two participants (3 study eyes) died and 3 participants (4 eyes) were found ineligible. The eyes receiving sham treatment had 31% greater progression of neurodegeneration than the CNTF-treated eyes. The difference in mean area of photoreceptor loss was 0.05±0.03 mm (P = 0.04) at 24 months. Retinal sensitivity changes, measured using microperimetry, were correlated highly with the changes in the area of photoreceptor loss (r = 0.86; P < 0.0001). The mean retinal sensitivity loss of the sham group was 45% greater than that of the treated group (decrease, 15.81±8.93 dB; P = 0.07). Reading speed deteriorated in the sham group (-13.9 words per minute) with no loss in the treated group (P = 0.02). Serious adverse ocular effects were found in 2 of 51 persons (4%) in the sham group and 2 of 48 persons (4%) in the treated group. CONCLUSIONS: In participants with macular telangiectasia type 2, a surgical implant that released CNTF into the vitreous cavity, compared with a sham procedure, slowed the progression of retinal degeneration. Further research is needed to assess longer-term clinical outcomes and safety.

BACKGROUND: Cerebrovascular disease (CVD) is highly comorbid with Alzheimer's disease (AD), yet its role is not entirely understood. Nailfold video capillaroscopy (NVC) is a noninvasive method of live imaging the capillaries near the fingernail's cuticle and may help to describe further vascular contributions to AD. OBJECTIVE: To examine finger nailfold capillary morphology using NVC in subjects with AD dementia, mild cognitive impairment (MCI), and normal cognition (NC). METHODS: We evaluated nailfold capillary hemorrhages, avascular zones ≥100 microns, and degree of tortuosity in 28 NC, 15 MCI, and 18 AD dementia subjects using NVC. Tortuosity was measured with a semi-quantitative rating scale. To assess the relation between nailfold capillary morphological features and diagnostic grouping, univariate and multivariable logistic regression models were fit to the data. RESULTS: 56% of subjects with AD dementia compared to 14% with NC and 13% with MCI displayed moderate to severe tortuosity. Greater severity of tortuosity was associated with 10.6-fold (95% confidence interval [CI]: 2.4, 46.2; p = 0.0018) and 7.4-fold (95% CI: 1.3, 41.3; p = 0.023) increased odds of AD dementia relative to NC and MCI, respectively, after adjusting for multiple covariates. CONCLUSION: Greater nailfold capillary tortuosity was found in participants with AD dementia compared to those with MCI or NC. These data provide preliminary evidence of a systemic microvasculopathy in AD that may be noninvasively and inexpensively evaluated through NVC.

Background: It is unknown whether dietary quality modifies genetic association with body mass index (BMI). Objective: This study examined whether dietary quality modifies genetic association with BMI. Design: We calculated 3 diet quality scores including the Alternative Healthy Eating Index 2010 (AHEI-2010), the Alternative Mediterranean Diet score (AMED), and the Dietary Approach to Stop Hypertension (DASH) diet score. We examined the interactions of a genetic risk score (GRS) based on 97 BMI-associated variants with the 3 diet quality scores on BMI in 30,904 participants from 3 large cohorts. Results: We found significant interactions between total GRS and all 3 diet scores on BMI assessed after 2-3 y, with an attenuated genetic effect observed in individuals with healthier diets (AHEI: P-interaction = 0.003; AMED: P = 0.001; DASH: P = 0.004). For example, the difference in BMI (kg/m2) per 10-unit increment of the GRS was smaller among participants in the highest tertile of AHEI score compared with those in the lowest tertile (0.84; 95% CI: 0.72, 0.96 compared with 1.14; 95% CI: 0.99, 1.29). Results were consistent across the 3 cohorts with no significant heterogeneity. The interactions with diet scores on BMI appeared more significant for central nervous system GRSs (P < 0.01 for 3 diet scores) than for non-central nervous system GRSs (P > 0.05 for 3 diet scores). Conclusions: A higher diet quality attenuated genetic predisposition to obesity. These findings underscore the importance of maintaining a healthful diet for the prevention of obesity, particularly for those individuals with a strong genetic predisposition to obesity. This trial was registered with the Clinical Trial Registry as NCT03577639.

Motion in a distorted virtual 3D space may cause visually induced motion sickness. Geometric distortions in stereoscopic 3D can result from mismatches among image capture, display, and viewing parameters. Three pairs of potential mismatches are considered, including 1) camera separation vs. eye separation, 2) camera field of view (FOV) vs. screen FOV, and 3) camera convergence distance (i.e., distance from the cameras to the point where the convergence axes intersect) vs. screen distance from the observer. The effect of the viewer's head positions (i.e., head lateral offset from the screen center) is also considered. The geometric model is expressed as a function of camera convergence distance, the ratios of the three parameter-pairs, and the offset of the head position. We analyze the impacts of these five variables separately and their interactions on geometric distortions. This model facilitates insights into the various distortions and leads to methods whereby the user can minimize geometric distortions caused by some parameter-pair mismatches through adjusting of other parameter pairs. For example, in postproduction, viewers can correct for a mismatch between camera separation and eye separation by adjusting their distance from the real screen and changing the effective camera convergence distance.

Seven unrelated individuals (four pediatric, three adults) with the TUBB3 E410K syndrome, harboring identical de novo heterozygous TUBB3 c.1228 G>A mutations, underwent neuropsychological testing and neuroimaging. Despite the absence of cortical malformations, they have intellectual and social disabilities. To search for potential etiologies for these deficits, we compared their brain's structural and white matter organization to 22 controls using structural and diffusion magnetic resonance imaging. Diffusion images were processed to calculate fractional anisotropy (FA) and perform tract reconstructions. Cortical parcellation-based network analysis and gyral topology-based FA analyses were performed. Major interhemispheric, projection and intrahemispheric tracts were manually segmented. Subjects had decreased corpus callosum volume and decreased network efficiency. While only pediatric subjects had diffuse decreases in FA predominantly affecting mid- and long-range tracts, only adult subjects had white matter volume loss associated with decreased cortical surface area. All subjects showed aberrant corticospinal tract trajectory and bilateral absence of the dorsal language network long segment. Furthermore, pediatric subjects had more tracts with decreased FA compared with controls than did adult subjects. These findings define a TUBB3 E410K neuroimaging endophenotype and lead to the hypothesis that the age-related changes are due to microscopic intrahemispheric misguided axons that are pruned during maturation.

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.

PURPOSE: To compare visual acuity (VA) improvement in children aged 7 to 12 years with amblyopia treated with a binocular iPad game plus continued spectacle correction vs. continued spectacle correction alone. DESIGN: Multicenter randomized clinical trial. PARTICIPANTS: One hundred thirty-eight participants aged 7 to 12 years with amblyopia (33-72 letters, i.e., approximately 20/200 to 20/40) resulting from strabismus, anisometropia, or both. Participants were required to have at least 16 weeks of optical treatment in spectacles if needed or demonstrate no improvement in amblyopic-eye visual acuity (VA) for at least 8 weeks prior to enrollment. METHODS: Eligible participants (mean age 9.6 years, mean baseline VA of 59.6 letters, history of prior amblyopia treatment other than spectacles in 96%) were randomly assigned to treatment for 8 weeks with the dichoptic binocular Dig Rush iPad game (prescribed for 1 hour per day 5 days per week) plus spectacle wear if needed (n = 69) or continued spectacle correction alone if needed (n = 69). MAIN OUTCOME MEASURES: Change in amblyopic-eye VA from baseline to 4 weeks, assessed by a masked examiner. RESULTS: At 4 weeks, mean amblyopic-eye VA letter score improved from baseline by 1.3 (2-sided 95% confidence interval [CI]: 0.1-2.6; 0.026 logMAR) with binocular treatment and by 1.7 (2-sided 95% CI: 0.4-3.0; 0.034 logMAR) with continued spectacle correction alone. After adjusment for baseline VA, the letter score difference between groups (binocular minus control) was -0.3 (95% CI: -2.2 to 1.5, P = 0.71, difference of -0.006 logMAR). No difference in letter scores was observed between groups when the analysis was repeated after 8 weeks of treatment (adjusted mean: -0.1, 98.3% CI: -2.4 to 2.1). For the binocular group, adherence data from the iPad indicated that slightly more than half of the participants (58% and 56%) completed >75% of prescribed treatment by the 4- and 8-week visits, respectively. CONCLUSIONS: In children aged 7 to 12 years who have received previous treatment for amblyopia other than spectacles, there was no benefit to VA or stereoacuity from 4 or 8 weeks of treatment with the dichoptic binocular Dig Rush iPad game.

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.

Severe, chronic eye allergy is an understudied, vision-threatening condition. Treatments remain limited. We used a mouse model of severe allergic eye disease (AED) to determine whether topical application of the pro-resolution mediator Resolvin D1 (RvD1) terminates the response. AED was induced by injection of ovalbumin (OVA) followed by topical challenge of OVA daily. RvD1 was applied topically prior to OVA. Clinical symptoms were scored. Eye washes were assayed for MUC5AC. After 7 days, eyes were removed and the number of goblet cells, T helper cell responses and presence of immune cells in draining lymph nodes and conjunctiva determined. Topical RvD1 treatment significantly reduced symptoms of AED. RvD1 did not alter the systemic type 2 immune response in the lymph nodes. AED increased the total amount of goblet cell mucin secretion, but not the number of goblet cells. RvD1 prevented this increase, but did not alter goblet cell number. Absolute numbers of CD4 + T cells, total CD11b + myeloid cells, eosinophils, neutrophils, and monocytes, but not macrophages increased in AED versus RvD1-treated mice. We conclude that topical application of RvD1 reduced the ocular allergic response by local actions in conjunctival immune response and a decrease in goblet cell mucin secretion.

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.