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Accumulation of 7-ketocholesterol (7KC) occurs in age-related macular degeneration (AMD) and was found previously to promote fibrosis, an untreatable cause of vision loss, partly through induction of endothelial-mesenchymal transition. To address the hypothesis that 7KC causes mesenchymal transition of retinal pigment epithelial cells (RPE), we exposed human primary RPE (hRPE) to 7KC or a control. 7KC-treated hRPE did not manifest increased mesenchymal markers, but instead maintained RPE-specific proteins and exhibited signs of senescence with increased serine phosphorylation of histone H3, serine/threonine phosphorylation of mammalian target of rapamycin (p-mTOR), p16 and p21, β-galactosidase labeling, and reduced LaminB1, suggesting senescence. The cells also developed senescence-associated secretory phenotype (SASP) determined by increased IL-1β, IL-6, and VEGF through mTOR-mediated NF-κB signaling, and reduced barrier integrity that was restored by the mTOR inhibitor, rapamycin. 7KC-induced p21, VEGF, and IL-1β were inhibited by an inhibitor of protein kinase C. The kinase regulates IQGAP1 serine phosphorylation. Furthermore, after 7KC injection and laser-induced injury, mice with an IQGAP1 serine 1441-point mutation had significantly reduced fibrosis compared to littermate control mice. Our results provide evidence that age-related accumulation of 7KC in drusen mediates senescence and SASP in RPE, and IQGAP1 serine phosphorylation is important in causing fibrosis in AMD.

Background Recent clinical studies suggest that preeclampsia, characterized by uteroplacental insufficiency (UPI) and infant intrauterine growth restriction (IUGR), may be protective against retinopathy of prematurity (ROP) in preterm infants. Experimental models of UPI/IUGR have found an association of erythropoietin (EPO) with less severe oxygen-induced retinopathy (OIR); however, it is unclear if EPO/EPO receptor (EPOR) signaling was involved. We hypothesized that maternal UPI and resultant infant IUGR would protect against features of ROP through EPO/EPOR signaling. Methods We compared transgenic mice with hypoactive EPOR signaling (hWtEPOR) to littermate wild-type mice (mWtEpoR) in a novel combined model of IUGR and ROP. Thromboxane A2 (TXA2) was infused into pregnant C57Bl/6J dams to produce UPI/IUGR; postnatal pups and their foster dams were subjected to a murine OIR model. Results Following hyperoxia, hematocrits were similar between littermate wild-type (mWtEpoR) TXA2/OIR and vehicle/OIR pups. mWtEpoR TXA2/OIR had increased serum EPO, retinal EPO and VEGF, and decreased avascular retinal area (AVA) compared to vehicle/OIR pups. In comparison to the mWtEpoR TXA2/OIR pups, AVA was not reduced in hWtEPOR TXA2/OIR pups. Conclusion Our findings provide biologic evidence that UPI/OIR-induced endogenous EPOR signaling confers protection against hyperoxia-induced vascular damage that may be related to pathophysiology in ROP. Impact Maternal preeclampsia and infant growth restriction confer retinovascular protection against high oxygen-induced damage through endogenous erythropoietin signaling.

The investigation of erythropoietin (EPO) has expanded to include potential nonhematopoietic roles in neural and retinal diseases, including diabetic retinopathy. However, it remains unclear how EPO functions to support the neural retina. Transgenic mice with hypoactive EPO receptor (EPOR) signaling (hWtEPOR) were compared with littermate control mice (WT) to test the role of EPOR signaling under normal conditions and after vascular injury and regrowth into the retina. Although retinal function tested with OptoMotry and electroretinography was comparable to adult (8-week–old) littermate WT mice, hWtEPOR mice had thinner inner and outer plexiform layers and a greater number of amacrine cells. Injury and repair caused by the oxygen-induced retinopathy model reduced visual acuity thresholds, reduced electroretinography amplitudes, and thinned the outer plexiform and inner nuclear layers of both WT and hWtEPOR 8-week–old mice. In hWtEPOR compared with WT mice, scotopic a-wave amplitudes were reduced by injury, despite no change in outer nuclear layer thickness; and peripheral rod, but not cone number, was reduced. Scotopic b-waves were reduced in injured hWtEPOR mice compared with WT, and rod bipolar cell ectopic neurites were increased in both genotypes after injury, suggesting a potential reparative process to preserve connectivity and the b-wave. Normal EPOR signaling appeared important because ectopic neurites and b-waves were lower in the hWtEPOR than WT injured mice.

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide. Blindness can occur from retinal detachment caused by pathologic retinal angiogenesis into the vitreous, termed intravitreal neovascularization (IVNV). Although agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) are now used to treat IVNV, concerns exist regarding the identification of optimal doses of anti-VEGF for individual infants and the effect of broad VEGF inhibition on physiologic angiogenesis in external organs or in the retina of a preterm infant. Therefore, it is important to understand VEGF signaling in both physiologic and pathologic angiogenesis in the retina. In this manuscript, we review the role of receptors that interact with VEGF in oxygen-induced retinopathy (OIR) models that represent features of ROP pathology. Specifically, we discuss our work regarding the regulation of VEGFR2 signaling in retinal endothelial cells to not only reduce severe ROP but also facilitate physiologic retinal vascular and neuronal development.

Aniket Ramshekar,1,2 Joshua Heczko,1 Ashlie Bernhisel,1 William Barlow Jnr,1 Brian Zaugg,1 Randall Olson,1 Jeff Pettey1 1Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, UT, 84132, USA; 2University of Utah School of Medicine, Salt Lake City, UT, 84132, USACorrespondence: Jeff PetteyDepartment of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, 65 Mario Capecchi Drive, Salt Lake City, UT, 84132, USATel +1 801-581-2352Fax +1 801-581-3357Email jeff.pettey@hsc.utah.eduPurpose: We assessed the effect of two lens cube sizes, three tip sizes, and two ultrasound (US) approaches on phacoemulsification efficiency and chatter.Methods: After porcine lens nuclei were soaked in formalin, we divided them into cubes measuring 2.0 mm or 3.0 mm. We collected efficiency and chatter data for 30-degree bent 19 G, 20 G, and 21 G tips with a continuous torsional US system; and for straight 19 G, 20 G, and 21 G tips with a micropulse longitudinal US system.Results: The average time needed for removal was always higher for the 3.0 mm lens cube than for the 2.0 mm lens cube. Statistically significant differences were observed between the 19 G and 21 G tips with micropulse longitudinal US using a 2.0 mm cube and a 3.0 mm cube, and with continuous transversal US using a 2.0 mm lens cube and a 3.0 mm cube. We did not observe significant differences between 19 G and 20 G tips with either cube size in either US system. However, we noted identical trends for both cube sizes with both US approaches; 19 G tips performed better than 20 G and 21 G tips.Conclusion: Regardless of the lens size, 19 G needles were the most efficient, and had both the fewest outliers and the smallest standard deviations.Keywords: cataract, chatter, efficiency, tip diameter, vacuum

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision-threatening CNV. IQGAP1 also binds the GTPase, Rap1, which when activated reduces Rac1 activation in CECs and CNV. In this study, we tested the hypothesis that active Rap1 binding to IQGAP1 is necessary and sufficient to reduce Rac1 activation in CECs, and CNV. We found that pharmacologic activation of Rap1 or adenoviral transduction of constitutively active Rap1a reduced VEGF-mediated Rac1 activation, migration, and tube formation in CECs. Following pharmacologic activation of Rap1, VEGF-mediated Rac1 activation was reduced in CECs transfected with an IQGAP1 construct that increased active Rap1-IQGAP1 binding but not in CECs transfected with an IQGAP1 construct lacking the Rap1 binding domain. Specific knockout of IQGAP1 in endothelial cells reduced laser-induced CNV and Rac1 activation in CNV lesions, but pharmacologic activation of Rap1 did not further reduce CNV compared to littermate controls. Taken together, our findings provide evidence that active Rap1 binding to the IQ domain of IQGAP1 is sufficient to interfere with active Rac1-mediated CEC activation and CNV formation.

Oxidized cholesterols and lipids accumulate in Bruch’s membrane in age-related macular degeneration (AMD). It remains unknown what causal relationship exists between these substances and AMD pathophysiology. We addressed the hypothesis that a prevalent form, 7-ketocholesterol (7KC), promotes choroidal endothelial cell (CEC) migration and macular neovascularization in AMD. Compared to control, 7KC injection caused 40% larger lectin-stained lesions, but 70% larger lesions measured by optical coherence tomography one week after laser-injury. At two weeks, 7KC-injected eyes had 86% larger alpha smooth muscle actin (αSMA)-labeled lesions and more collagen-labeling than control. There was no difference in cell death. 7KC-treated RPE/choroids had increased αSMA but decreased VE-cadherin. Compared to control-treated CECs, 7KC unexpectedly reduced endothelial VE-cadherin, CD31 and VEGFR2 and increased αSMA, fibroblast activation protein (FAP) and transforming growth factor beta (TGFβ). Inhibition of TGFβ receptor-mediated signaling by SB431542 abrogated 7KC-induced loss of endothelial and increase in mesenchymal proteins in association with decreased transcription factor, SMAD3. Knockdown of SMAD3 partially inhibited 7KC-mediated loss of endothelial proteins and increase in αSMA and FAP. Compared to control, 7KC-treatment of CECs increased Rac1GTP and migration, and both were inhibited by the Rac1 inhibitor; however, CECs treated with 7KC for 72 hours had reduced tube formation. These findings suggest that 7KC, which increases in AMD and with age, induces mesenchymal transition in CECs making them invasive and migratory, and causing fibrosis in macular neovascularization. Further studies to interfere with this process may reduce fibrosis and improve responses to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1(+/+), Iqgap1(−/−) mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration and tube formation. Our findings indicate that in CECs IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

Objectives To validate concurrent utility of within-scanner encoding and delayed recognition-memory paradigms to ascertain hippocampal activations during task-based memory fMRI. Methods Memory paradigms were designed for faces, word-pairs and abstract designs. A deep-encoding task was designed comprising of a total of 9 cycles run within a 1.5T MRI scanner. A recall session was performed after 1 h within the scanner using an event-related design. Group analysis was done with ‘correct-incorrect' responses applied as parametric modulators in Statistical Parametric Mapping version 8 using boot-strap method to enable estimation of laterality indices (LI) using custom anatomical masks involving the medio-basal temporal structures. Results Twenty seven subjects with drug-resistant mesial temporal lobe epilepsy due to hippocampal sclerosis (MTLE-HS) [17 patients of left-MTLE and 10 patients of right-MTLE] and 21 right handed age-matched healthy controls (HC) were recruited. For the encoding paradigm blood oxygen level dependent (BOLD) responses in HC demonstrated right laterality for faces, left laterality for word pairs, and bilaterality for design encoding over the regions of interest. Both right and left MTLE-HS groups revealed left lateralisation for word-pair encoding, bilateral activation for face encoding, with design encoding in right MTLE-HS demonstrating a left shift. As opposed to lateralization shown in controls, group analysis of cued-recall BOLD signals acquired within scanner in left MTLE-HS demonstrated right lateralization for word-pairs with bilaterality for faces and designs. The right MTLE-HS group demonstrated bilateral activations for faces, word-pairs and designs. Conclusion Recall-based fMRI paradigms indicate hippocampal plasticity in MTLE-HS, maximal for word-pair associate recall tasks.

Purpose Exogenous erythropoietin (EPO) is being considered for tissue protection and angiogenesis in retinal vascular diseases. However, studies are limited by insufficient tools to address signaling effects through the EPO receptor (EPOR). We used a humanized mouse model of hypoactive EPOR signaling to test the hypothesis that EPOR signaling supports angiogenesis in retinovascular diseases. Methods Humanized Knockin EPOR mice (hWtEPOR) with hypoactive EPOR signaling were compared to littermate wild-type mice (WT). Postnatal day (p)7 mice of each genotype were exposed to 75% oxygen for five days, followed by 21% oxygen in the oxygen-induced retinopathy model (OIR) and compared to room-air (RA)-raised pups. At time points after OIR, pups were sacrificed, and flat-mounted, lectin-stained retinas were analyzed for central avascular area or intravitreal neovascular area (IVNV). Flash-frozen retinas were analyzed for angiogenic protein (Epo, VEGF, p-Stat3) and gene (Vegfa, Kdr, Epo, Hif1α, Hif2α) expression levels. Results In OIR, hWtEPOR mice had increased AVA compared with WT at p8, p12, and p17, but there was no difference in IVNV between hWtEPOR and WT mice at p17. Although VEGF and p-STAT3 proteins were increased in WT at p17 OIR, there were no differences in retinal angiogenic factor expression levels between hWtEPOR and WT OIR at p17 despite similar areas of IVNV. Conclusions Our data support the hypothesis that EPOR signaling was associated with regrowth of vascularization following oxygen-induced capillary dropout and played a role in intravitreal angiogenesis. Additional study of EPOR signaling regulation on other angiogenic factor pathways may be considered.

Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner.

Status epilepticus (SE) is a neurological emergency resulting from prolonged Clinical or electroencephalographic seizure activity. Refractory SE refers to the persistence of seizure activity despite the initiation of first- and second-line anticonvulsant therapy. Sinister outcomes are often attributed to the etiology of SE. Despite randomized multicentre trials of established and promising therapeutic options, the management and prognostication in SE are fraught with challenges. Neither the duration of SE nor time-delay to initiation of therapy should discourage the aggressive approach to the management of SE. Neurointensive care of patients with SE consists of an algorithmic approach tailored to the etiology and systemic complications that arise as a consequence. This approach is also driven by the persistence of electrographic seizure activity, which is best followed with continuous EEG monitoring. The extent of patient support has to be augmentative to the degree of encephalopathy/coma and impairment of vital functions. Potential interactions of anticonvulsant drugs with other co-medications need to be considered during the course of treatment. This review discusses the existing literature on the epidemiological aspects, clinical approach, treatment and prognostication of SE.

Summary Introduction Failure to identify the association antiepileptic drug (AED)-resistant temporal lobe epilepsy (TLE) with idiopathic generalized epilepsy (IGE) can interfere with decision for anterior temporal lobectomy (ATL) and prediction of post-ATL seizure outcome. Methods Out of the 664 consecutive patients who underwent ATL between March 1995 and December 2007, 12 (1.8%) had coexisting IGE. The decision for ATL was made after a thorough discussion in the multidisciplinary patient management conference based upon the concordance between the clinical, electroencephalographic and magnetic resonance imaging data. All of them underwent epilepsy surgery for AED-resistant TLE. Results In seven of the 12 patients, IGE was not identified until post-ATL. During a median follow-up period of 8.5 years, 8 of our 12 patients were seizure-free; the remaining 4 patients only had infrequent myoclonus. In two them, AEDs were discontinued; others were on montherapy for IGE. Conclusions Our study highlights the rare association of IGE with TLE, the most common AED-resistant focal epilepsy syndrome. As the seizure outcome following ATL is similar in AED-resistant TLE patients with and without IGE, their co-existence is not a contraindication for ATL. Future studies should explore the molecular genetic basis of the rare association between these two epilepsy syndromes.

There is no uniform approach to assessment of an unprovoked seizure in an adult in India. Due to the huge socioeconomic differences among its vast population and the varied levels of available health care, the approach differs significantly across the country. Certain unique features of health care in India have direct bearing on any such approach. First, most of the health care costs are directly borne by the patient or caregivers. Secondly, the majority of the population live in rural areas and are cared for by primary care physicians or internists who have little training in the evaluation and management of seizures and do not have access to perform or expertise to interpret EEG or CT scans.1 On the positive side, phenytoin and phenobarbital are provided free of cost to all patients at primary and secondary level state-run hospitals. Similarly, the charges for various investigations including EEG, CT scan, and MRI are subsidized in the state-run tertiary care centers according to the economic status of the patient. Prevalence of neurocysticercosis within a region also influences this approach. The authors have worked in different regions of the country at various levels within the health care system and have noticed different approaches which are largely determined by the above-mentioned factors. Any approach is, however, applicable only to those patients who seek medical care. Higher levels of illiteracy, ignorance, perceived stigma, and belief in traditional practices prevent patients from seeking medical care, with a treatment gap as high as 50%–80%.2