Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

Purpose: Ocular infection by human adenovirus species D type 37 (HAdV-D37) causes epidemic keratoconjunctivitis, a severe, hyperacute condition. The corneal component of epidemic keratoconjunctivitis begins upon infection of corneal epithelium, and the mechanism of viral entry dictates subsequent proinflammatory gene expression. Therefore, it is important to understand the specific pathways of adenoviral entry in these cells. Methods: Transmission electron microscopy of primary and tert-immortalized human corneal epithelial cells infected with HAdV-D37 was performed to identify the means of viral entry. Confocal microscopy was used to determine intracellular trafficking. The results of targeted small interfering RNA and specific chemical inhibitors were analyzed by quantitative PCR, and Western blot. Results: By transmission electron microscopy, HAdV-D37 was seen to enter by both clathrin-coated pits and macropinocytosis; however, entry was both pH and dynamin 2 independent. Small interfering RNA against clathrin, AP2A1, and lysosome-associated membrane protein 1, but not early endosome antigen 1, decreased early viral gene expression. Ethyl-isopropyl amiloride, which blocks micropinocytosis, did not affect HAdV-D37 entry, but IPA, an inhibitor of p21-activated kinase, and important to actin polymerization, decreased viral entry in a dose-dependent manner. Conclusions: HAdV-D37 enters human corneal epithelial cells by a noncanonical clathrin-mediated pathway involving lysosome-associated membrane protein 1 and PAK1, independent of pH, dynamin, and early endosome antigen 1. We showed earlier that HAdV-D37 enters human keratocytes through caveolae. Therefore, epidemic keratoconjunctivitis-associated viruses enter different corneal cell types via disparate pathways, which could account for a relative paucity of proinflammatory gene expression upon infection of corneal epithelial cells compared with keratocytes, as seen in prior studies.

The coordination of metabolic signals among different cellular components in pathological retinal angiogenesis is poorly understood. Here, we showed that in the pathological angiogenic vascular niche, retinal myeloid cells, particularly macrophages/microglia that are spatially adjacent to endothelial cells (ECs), are highly glycolytic. We refer to these macrophages/microglia that exhibit a unique angiogenic phenotype with increased expression of both M1 and M2 markers and enhanced production of both proinflammatory and proangiogenic cytokines as pathological retinal angiogenesis-associated glycolytic macrophages/microglia (PRAGMs). The phenotype of PRAGMs was recapitulated in bone marrow-derived macrophages or retinal microglia stimulated by lactate that was produced by hypoxic retinal ECs. Knockout of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (; for rodents), a glycolytic activator in myeloid cells, impaired the ability of macrophages/microglia to acquire an angiogenic phenotype, rendering them unable to promote EC proliferation and sprouting and pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Mechanistically, hyperglycolytic macrophages/microglia produced large amount of acetyl-coenzyme A, leading to histone acetylation and PRAGM-related gene induction, thus reprogramming macrophages/microglia into an angiogenic phenotype. These findings reveal a critical role of glycolytic metabolites as initiators of reciprocal activation of macrophages/microglia and ECs in the retinal angiogenic niche and suggest that strategies targeting the metabolic communication between these cell types may be efficacious in the treatment of pathological retinal angiogenesis.

Purpose To evaluate the parameters of the Fixed-Luminance and Multi-Luminance flicker electroretinography protocol among patients with early active birdshot chorioretinopathy. Methods Fixed-Luminance magnitude, Fixed-Luminance phase, Multi-Luminance magnitude area under the curve, and Multi-Luminance phase area under the curve parameters were compared between early active birdshot chorioretinopathy patients and an age-matched control group. Results There was no statistically significant difference between the Fixed-Luminance flicker magnitude ( = .6), the Fixed-Luminance flicker phase ( = .9), and the Multi-Luminance flicker phase area under the curve ( = .55) when each was compared to the normal population; however, the difference between the mean Multi-Luminance flicker magnitude area under the curve in our patients and the healthy control group was statistically significant. ( = .003) Conclusions Multi-Luminance flicker magnitude area under the curve has been shown to be significantly different from the normal population in the early active course of the disease. Abbreviations BSCR: birdshot chorioretinopathy; cd: Cadmium; ERG: Electroretinography; FA: Fluorescein angiography; FL-: Fixed-luminance; HVF: Humphrey visual field; Hz: Hertz; ICG: Indocyanine green; m: Square meter; ML-: Multi-luminance; ms: millisecond; SITA: Swedish interactive thresholding algorithm; SWAP: Short wave-length automated perimetry.

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

Macular telangiectasia type 2 (MacTel), a late-onset macular degeneration, has been linked to a loss in the retina of Müller glial cells and the amino acid serine, synthesized by the Müller cells. The disease is confined mainly to a central retinal region called the MacTel zone. We have used electron microscopic connectomics techniques, optimized for disease analysis, to study the retina from a 48-y-old woman suffering from MacTel. The major observations made were specific changes in mitochondrial structure within and outside the MacTel zone that were present in all retinal cell types. We also identified an abrupt boundary of the MacTel zone that coincides with the loss of Müller cells and macular pigment. Since Müller cells synthesize retinal serine, we propose that a deficiency of serine, required for mitochondrial maintenance, causes mitochondrial changes that underlie MacTel development.

PURPOSE: To examine disease profile of tubercular uveitis (TBU) in Paediatric population. METHODS: Among 945 patients of the retrospective multinational study by the Collaborative Ocular Tuberculosis Study (COTS)-1, 29 Paediatric patients diagnosed with TBU were analyzed. RESULTS: Mean age of disease presentation was 12.8 (range 4-18 years), with predominance of males (n = 14/20; 70.0%) and Asian ethnicity (n = 25/29; 86.2%). Posterior uveitis (n = 14/28; 50%) was the most frequent uveitis phenotype, with choroidal involvement occurring in 64.7% (n = 11/17). Incidence of optic disc edema and macular edema was higher in children (n = 8/18; 44.4% and n = 5/18; 27.8%, respectively) than in adults (n = 160/942; 16.9% and n = 135/942; 14.3%, respectively). Comparison of optic disc edema between subgroups showed a significant difference (). All patients received oral corticosteroids, most of them with antitubercular therapy. Treatment failure developed in 4.8% (n = 1/21). CONCLUSIONS: Children have a more severe inflammatory response to the disease, and an intensive anti-inflammatory therapeutic regimen is required to achieve a positive treatment outcome.

A normally sighted person can see a grating of 30 cycles per degree or higher, but spatial frequencies needed for motion perception are much lower than that. It is unknown for natural images with a wide spectrum how all the visible spatial frequencies contribute to motion speed perception. In this work, we studied the effect of spatial frequency content on motion speed estimation for sequences of natural and stochastic pixel images by simulating different visual conditions, including normal vision, low vision (low-pass filtering), and complementary vision (high-pass filtering at the same cutoff frequencies of the corresponding low-vision conditions) conditions. Speed was computed using a biological motion energy-based computational model. In natural sequences, there was no difference in speed estimation error between normal vision and low vision conditions, but it was significantly higher for complementary vision conditions (containing only high-frequency components) at higher speeds. In stochastic sequences that had a flat frequency distribution, the error in normal vision condition was significantly larger compared with low vision conditions at high speeds. On the contrary, such a detrimental effect on speed estimation accuracy was not found for low spatial frequencies. The simulation results were consistent with the motion direction detection task performed by human observers viewing stochastic sequences. Together, these results (i) reiterate the importance of low frequencies in motion perception, and (ii) indicate that high frequencies may be detrimental for speed estimation when low frequency content is weak or not present.