Epithelial Membrane Protein 2 (EMP2) Blockade Attenuates Pathological Neovascularization in Murine Oxygen-Induced Retinopathy.

Purpose: Retinopathy of prematurity (ROP) results from postnatal hyperoxia exposure in premature infants and is characterized by aberrant neovascularization of retinal blood vessels. Epithelial membrane protein-2 (EMP2) regulates hypoxia-inducible factor (HIF)-induced vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line and genetic knock-out of Emp2 in a murine oxygen-induced retinopathy (OIR) model attenuates neovascularization. We hypothesize that EMP2 blockade via intravitreal injection protects against neovascularization. Methods: Ex vivo choroid sprouting assay was performed, comparing media and human IgG controls versus anti-EMP2 antibody (Ab) treatment. In vivo, eyes from wild-type (WT) mice exposed to hyperoxia from postnatal (P) days 7 to 12 were treated with P12 intravitreal injections of control IgG or anti-EMP2 Abs. Neovascularization was assessed at P17 by flat mount imaging. Local and systemic effects of anti-EMP2 Ab treatment were assessed. Results: Choroid sprouts treated with 30 µg/mL of anti-EMP2 Ab demonstrated a 48% reduction in vessel growth compared to control IgG-treated sprouts. Compared to IgG-treated controls, WT OIR mice treated with 4 µg/g of intravitreal anti-EMP2 Ab demonstrated a 42% reduction in neovascularization. They demonstrated down-regulation of retinal gene expression in pathways related to vasculature development and up-regulation in genes related to fatty acid oxidation and tricarboxylic acid cycle respiratory electron transport, compared to controls. Anti-EMP2 Ab-treated OIR mice did not exhibit gross retinal histologic abnormalities, vision transduction abnormalities, or weight loss. Conclusions: Our results suggest that EMP2 blockade could be a local and specific treatment modality for retinal neovascularization in oxygen-induced retinopathies, without systemic adverse effects.

Paraoxonase 2 Deficiency Causes Mitochondrial Dysfunction in Retinal Pigment Epithelial Cells and Retinal Degeneration in Mice.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO3 (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO3 treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

Oxidative Stress and Lipid Accumulation Augments Cell Death in LDLR-Deficient RPE Cells and Ldlr-/- Mice.

Lipid peroxidation from oxidative stress is considered a major contributor to age-related macular degeneration (AMD). The retina is abundant with circulating low-density lipoproteins (LDL), which are taken up by LDL receptor (LDLR) in the RPE and Müller cells. The purpose of this study is to investigate the role of LDLR in the NaIO3-induced model of dry AMD. Confluent primary human RPE (hRPE) and LDLR-silenced ARPE-19 cells were stressed with 150 µM tert-butyl hydroperoxide (tBH) and caspase 3/7 activation was determined. WT and Ldlr-/- mice were administered NaIO3 (20 mg/kg) intravenously. On day 7, fundus imaging, OCT, ERG, and retinal thickness were measured. Histology, TUNEL, cleaved caspase 3 and lipid accumulation were assessed. Treatment of hRPE with tBH markedly decreased LDLR expression. Caspase 3/7 activation was significantly increased in LDLR-silenced ARPE-19 cells treated with tBH. In Ldlr-/- mice, NaIO3 administration resulted in significant (a) retinal thinning, (b) compromised photoreceptor function, (c) increased percentage of cleaved caspase 3 positive and apoptotic cells, and (d) increased lipid droplet accumulation in the RPE, Bruch membrane, choroid, and sclera, compared to WT mice. Our findings imply that LDLR loss leads to lipid accumulation and impaired retinal function, which may contribute to the development of AMD.

Alleviation of extensive visual pathway dysfunction by a remyelinating drug in a chronic mouse model of multiple sclerosis.

Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor ß ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings relative to vehicle. Faster VEP conduction in IndCl-treated mice was associated with enhanced myelin basic protein signal in all visual system structures examined. IndCl preserved retinal ganglion cells (RGCs) and oligodendrocyte density in the prechiasmatic white matter, but similar retinal nerve fiber layer thinning by OCT was noted in vehicle and IndCl-treated mice. Although IndCl differentially attenuated leukocyte and astrocyte staining signal throughout the structures analyzed, axolemmal varicosities were observed in all visual fiber tracts of mice with EAE irrespective of treatment, suggesting impaired axonal energy homeostasis. These data support incomplete functional recovery of VEP amplitude with IndCl, as fiber tracts displayed persistent axon pathology despite remyelination-induced decreases in latencies, evidenced by reduced optic nerve g-ratio in IndCl-treated mice. Although additional studies are required, these findings demonstrate the dynamics of visual pathway dysfunction and disability during EAE, along with the importance of early treatment to mitigate EAE-induced axon damage.

LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.

A Novel HDL-Mimetic Peptide HM-10/10 Protects RPE and Photoreceptors in Murine Models of Retinal Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO3), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.

CENTRAL ELLIPSOID LOSS ASSOCIATED WITH CONE DYSTROPHY AND KCNV2 MUTATION.

PURPOSE: To report a case of central ellipsoid loss with supernormal rod electroretinogram and KCNV2 gene mutation. METHODS: Retrospective case report. PATIENT: Thirty-eight-year-old man. RESULTS: We report a patient with longstanding vision loss and photophobia who illustrated central atrophy of the inner segment ellipsoid zone band on spectral domain optical coherence tomography. Fundus autofluorescence displayed mild perifoveal mottled autofluorescence. Electroretinography demonstrated a diminished rod-isolated response with delayed timing but a normal dark-adapted maximal response to bright flashes. Cone-mediated responses under light-adapted conditions were abnormal with evidence of selective loss of the b wave and a normal a wave consistent with cone dystrophy with supernormal rod electroretinogram. Genetic testing demonstrated a frameshift mutation in the KCNV2 gene. CONCLUSION: Cone dystrophy with supernormal rod electroretinogram is believed to be a monogenic disease due to KCNV2 gene mutations that affect a transmembrane potassium channel found in rod and cone photoreceptors. We report the multimodal retinal findings associated with a signature electroretinogram in this disorder. Clinicians should consider this rare condition when evaluating patients with central ellipsoid loss and associated cone dystrophy.

NOVEL CONE DYSTROPHY WITH CENTRAL ELLIPSOID ZONE LOSS ASSOCIATED WITH HUMAN RETINAL FASCIN GENE (FSCN2) MUTATION.

PURPOSE: To report the first case of a cone dystrophy with central ellipsoid zone loss associated with a mutation in the human retinal fascin gene (FSCN2). METHODS: Multimodal retinal imaging findings including spectral domain optical coherence tomography and fundus autofluorescence are presented. The results of functional testing and mutational analysis are also discussed. PATIENTS: Single patient with a diagnosis of cone dystrophy. RESULTS: Spectral domain optical coherence tomography imaging illustrated central loss of the ellipsoid zone band in each eye. Full-field and multifocal electroretinogram testing confirmed a diagnosis of cone dystrophy in a 35-year-old male patient. Subsequent cone dystrophy genetic panel identified a novel mutation (p.Pro406Leu:c.1217C>T) in the FSCN2 gene. CONCLUSION: This is the first case report of a patient diagnosed with cone dystrophy associated with a novel mutation in the FSCN2 gene. FSCN2 genetic testing should be considered in patients with central ellipsoid loss and cone dystrophy, especially as specific gene therapy treatments emerge in the future.

Peripheral Sensory Neurons Expressing Melanopsin Respond to Light.

The ability of light to cause pain is paradoxical. The retina detects light but is devoid of nociceptors while the trigeminal sensory ganglia (TG) contain nociceptors but not photoreceptors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to mediate light-induced pain but recent evidence raises the possibility of an alternative light responsive pathway independent of the retina and optic nerve. Here, we show that melanopsin is expressed in both human and mouse TG neurons. In mice, they represent 3% of small TG neurons that are preferentially localized in the ophthalmic branch of the trigeminal nerve and are likely nociceptive C fibers and high-threshold mechanoreceptor Aδ fibers based on a strong size-function association. These isolated neurons respond to blue light stimuli with a delayed onset and sustained firing, similar to the melanopsin-dependent intrinsic photosensitivity observed in ipRGCs. Mice with severe bilateral optic nerve crush exhibit no light-induced responses including behavioral light aversion until treated with nitroglycerin, an inducer of migraine in people and migraine-like symptoms in mice. With nitroglycerin, these same mice with optic nerve crush exhibit significant light aversion. Furthermore, this retained light aversion remains dependent on melanopsin-expressing neurons. Our results demonstrate a novel light-responsive neural function independent of the optic nerve that may originate in the peripheral nervous system to provide the first direct mechanism for an alternative light detection pathway that influences motivated behavior.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

Light aversion and corneal mechanical sensitivity are altered by intrinscally photosensitive retinal ganglion cells in a mouse model of corneal surface damage.

Animal models of corneal surface damage reliably exhibit altered tear quality and quantity, apoptosis, nerve degeneration, immune responses and many other symptoms of dry eye disease. An important clinical symptom of dry eye disease is photoallodynia (photophobia), which can be modeled in mice using behavioral light aversion as a surrogate. Intrinsically photosensitive retinal ganglion cells (ipRGCs) function as irradiance detectors. They have been shown to mediate innate light aversion and are ideal candidates to initiate or modulate light aversion in disease or dysfunctional states. This study addresses the relationship between light aversion, corneal mechanical sensitivity and corneal surface damage in a preclinical mouse model using bilateral topical application of benzalkonium chloride (BAC). Corneal application of BAC resulted in similar levels of corneal surface damage by fluorescein staining in both wild type mice and mice lacking ipRGCs. Light aversion was an early symptom of corneal surface damage, was proportional to the level of corneal damage and dependent on melanopsin-expressing cells. A decrease in both corneal mechanosensitivity and light aversion was observed in mice lacking melanopsin-expressing cells, suggesting a connection in the neural circuits mediating the two most common symptoms of corneal surface damage.

Biocompatibility of a Synthetic Biopolymer for the Treatment of Rhegmatogenous Retinal Detachment.

OBJECTIVE: The aim of this study is to evaluate the retinal safety and toxicity of a novel synthetic biopolymer to be used as a patch to treat rhegmatogenous retinal detachment. METHODS: Thirty one adult wild type albino mice were divided in 2 groups. In Group A (n=9) 0.2 µl balanced salt solution (BSS) and in Group B (n=22), 0.2 µl biopolymer was injected in the subretinal space. Trans-scleral subretinal injection was performed in one eye and the fellow eye was used as control. In both groups, in vivo color fundus photography, electroretinogram (ERG), spectral domain optical coherence tomography (SD-OCT) were performed before injection and at days 7 and 14 post-intervention. Histological analysis was performed following euthanization at days 1, 7 and 21 post-injection. RESULTS: The biopolymer was visualized in the subretinal space in vivo by SD-OCT and post-life by histology up to 1 week after the injection. There were no significant differences in ERG parameters between the two groups at 1 and 2 weeks post-injection. Minimal inflammatory response and loss of photoreceptor cells was only observed in the immediate proximity of the site of scleral perforation, which was similar in both groups. Overall integrity of the outer, inner retina and retinal pigment epithelial (RPE) layers was unaffected by the presence of the biopolymer in the subretinal space. CONCLUSIONS: Functional and histological evaluation suggests that the synthetic biopolymer is non-inflammatory and non-toxic to the eye. It may represent a safe therapeutic agent in the future, for the treatment of rhegmatogenous retinal detachment.

Whole exome sequencing detects homozygosity for ABCA4 p.Arg602Trp missense mutation in a pediatric patient with rapidly progressive retinal dystrophy.

BACKGROUND: A pediatric patient presented with rapidly progressive vision loss, nyctalopia and retinal dystrophy. This is the first report of homozygosity for the p.Arg602Trp mutation in the ABCA4 gene. The child became legally blind within a period of 2 years. CASE PRESENTATION: An eight year-old Hispanic female presented with bilateral decreased vision following a febrile gastrointestinal illness with nausea and vomiting. Extensive workup involved pediatric infectious disease and rheumatology consultations.Initial visual acuity was 20/60 at distance and 20/30 at near in both eyes. Rapidly progressive vision loss occurred during a 2-year period resulting in visual acuities of 20/200 at distance in both eyes. Fundus exam disclosed attenuated vessels and multiple subretinal blister-like elevations. Optical coherence tomography showed far more lesions than were clinically evident with different levels of elevation. Autofluorescence imagery showed dramatic and widespread geographic areas of atrophy. The deposits that appeared drusen-like on clinical exam were hyperfluorescent, consistent with lipofuscin deposits containing A2e (N-retinylidene-N-retinylethanolamine) indicative of RPE cell dysfunction. Electroretinography was consistent with cone dystrophy, with relative preservation of rod function. Blood analysis and rheumatology evaluation found no evidence of a diffuse post-infectious/inflammatory process. The unique and rapid progression of her subretinal blister-like lesions was documented by fluorescein angiography, optical coherence tomography, autofluorescence imagery, and fundus photography. Family pedigree history disclosed consanguinity, her parents being first cousins. DNA analysis by whole exomic sequencing revealed homozygosity of p.Arg602Trp in the ABCA4 gene. CONCLUSION: The pediatric patient presented with a striking clinical appearance and dramatic rate of progression that was clinically more characteristic of an infectious or inflammatory process. This case expands the diverse range of phenotypes attributed to ABCA4 mutations and further supports the role of whole exome sequencing as a powerful new tool available to aid clinicians in establishing diagnosis for challenging cases.

Endophenotypes for Age-Related Macular Degeneration: Extending Our Reach into the Preclinical Stages of Disease.

The key to reducing the individual and societal burden of age-related macular degeneration (AMD)-related vision loss, is to be able to initiate therapies that slow or halt the progression at a point that will yield the maximum benefit while minimizing personal risk and cost. There is a critical need to find clinical markers that, when combined with the specificity of genetic testing, will identify individuals at the earliest stages of AMD who would benefit from preventive therapies. These clinical markers are endophenotypes for AMD, present in those who are likely to develop AMD, as well as in those who have clinical evidence of AMD. Clinical characteristics associated with AMD may also be possible endophenotypes if they can be detected before or at the earliest stages of the condition, but we and others have shown that this may not always be valid. Several studies have suggested that dynamic changes in rhodopsin regeneration (dark adaptation kinetics and/or critical flicker fusion frequencies) may be more subtle indicators of AMD-associated early retinal dysfunction. One can test for the relevance of these measures using genetic risk profiles based on known genetic risk variants. These functional measures may improve the sensitivity and specificity of predictive models for AMD and may also serve to delineate clinical subtypes of AMD that may differ with respect to prognosis and treatment.

CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in multiple retinal degeneration animal models. Recently, CNTF has been evaluated in clinical trials for the inherited degenerative disease retinitis pigmentosa (RP) and for dry age-related macular degeneration (AMD). Despite its potential as a broad-spectrum therapeutic treatment for blinding diseases, the target cells of exogenous CNTF and its mechanism of action remain poorly understood. We have shown previously that constitutive expression of CNTF prevents photoreceptor death but alters the retinal transcriptome and suppresses visual function. Here, we use a lentivirus to deliver the same secreted human CNTF used in clinical trials to a mouse model of RP. We found that low levels of CNTF halt photoreceptor death, improve photoreceptor morphology, and correct opsin mislocalization. However, we did not detect corresponding improvement of retinal function as measured by the electroretinogram. Disruption of the cytokine receptor gp130 gene in Müller glia reduces CNTF-dependent photoreceptor survival and prevents phosphorylation of STAT3 and ERK in Müller glia and the rest of the retina. Targeted deletion of gp130 in rods also demolishes neuroprotection by CNTF and prevents further activation of Müller glia. Moreover, CNTF elevates the expression of LIF and endothelin 2, thus positively promoting Müller and photoreceptor interactions. We propose that exogenous CNTF initially targets Müller glia, and subsequently induces cytokines acting through gp130 in photoreceptors to promote neuronal survival. These results elucidate a cellular mechanism for exogenous CNTF-triggered neuroprotection and provide insight into the complex cellular responses induced by CNTF in diseased retinas.

Functional burden of strabismus: decreased binocular summation and binocular inhibition.

IMPORTANCE: Binocular summation (BiS) is defined as the superiority of visual function for binocular over monocular viewing. Binocular summation decreases with age and large interocular differences in visual acuity. To our knowledge, BiS has not heretofore been well studied as a functional measure of binocularity in strabismus. OBJECTIVE: To evaluate the effect of strabismus on BiS using a battery of psychophysical tasks that are clinically relevant and easy to use and to determine whether strabismus is associated with binocular inhibition in extreme cases. DESIGN: Case-control study. SETTING: University-based eye institute. PARTICIPANTS: Strabismic patients recruited during 2010 to 2012 from a preoperative clinic and control participants with no history of eye disease other than refractive error. INTERVENTION: A battery of psychophysical and electrophysiological tests including Early Treatment Diabetic Retinopathy Study visual acuity, Sloan low-contrast acuity (LCA) (2.5% and 1.25%), Pelli-Robson contrast sensitivity, and sweep visual evoked potential contrast sensitivity. MAIN OUTCOME AND MEASURE: Binocular summation was calculated as the ratio between binocular and better-eye individual scores. RESULTS: Sixty strabismic and 80 control participants were prospectively examined (age range, 8-60 years). Mean BiS was significantly lower in the strabismic patients than controls for LCA (2.5% and 1.25%, P = .005 and <.001, respectively). For 1.25% LCA, strabismic patients had a mean BiS score less than 1, indicating binocular inhibition (ie, the binocular score was less than that of the better eye's monocular score). There was no significant difference in BiS for contrast thresholds on Early Treatment Diabetic Retinopathy Study visual acuity, Pelli-Robson contrast sensitivity, or sweep visual evoked potential contrast sensitivity. Regression analysis revealed a significant worsening of BiS with strabismus for 2.5% (P = .009) and 1.25% (P = .002) LCA, after accounting for age. CONCLUSIONS AND RELEVANCE: Strabismic patients demonstrate subnormal BiS and even binocular inhibition for LCA, suggesting that strabismus impairs visual function more than previously appreciated. This may explain why strabismic patients who are not diplopic close 1 eye in visually demanding situations. This finding clarifies the visual deficits impacting quality of life in strabismic patients and may represent a novel measure by which to evaluate and monitor function in strabismus.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

Comparative regional pupillography as a noninvasive biosensor screening method for diabetic retinopathy.

PURPOSE: We describe infrared regional pupillometry as an objective comparative assessment of midperipheral to central retinal sensitivity and to correlate with midperipheral retinal ischemia in diabetic subjects. METHODS: We tested 12 normal and 17 diabetic subjects using bilateral infrared pupillometry. The diabetic cohort included seven subjects without, five with mild, three with moderate, and two with severe non-proliferative diabetic retinopathy (NPDR). Central and annular stimuli of varying intensity were presented to one eye, and pupillary amplitude and constriction velocity were measured from both eyes. Light stimulus of increasing intensity was presented as 20 consecutive trials (stimulus duration of 300 ms with 3000 ms intervals). The ratio of central to peripheral responses (Q values) was calculated for each stimulus configuration. Average responses with respect to the stimulus strength were regressed with Gompertz sigmoid function. RESULTS: Control and moderate/severe NPDR cases comparison showed statistically significant differences in amplitude (Q(A)) and constriction velocity (Q(CV)) (Wilcoxon rank sum test P = 0.002, respectively). Age difference for these groups was not statistically significant (Wilcoxon rank sum test P = 0.15). The comparison of control and diabetic subjects without NPDR/mild NPDR was statistically significant for Q(A) and Q(CV) (Wilcoxon rank sum test P = 0.0002 and P = 0.001, respectively). Q(A) and Q(CV) differences were statistically significant between moderate/severe NPDR cases and subjects without or mild NPDR cases (Wilcoxon rank sum test P = 0.013). CONCLUSIONS: Q(A) and Q(CV) values correlated highly with the severity of diabetic retinopathy, but not with the duration of diabetes.

Intrinsically photosensitive retinal ganglion cells are the primary but not exclusive circuit for light aversion.

Photoallodynia (photophobia) occurs when normal levels of light cause pain ranging from uncomfortable to debilitating. The only current treatment for photoallodynia is light avoidance. The first step to understanding the mechanisms of photoallodynia is to develop reliable animal behavioral tests of light aversion and identify the photoreceptors required to initiate this response. A reliable light/dark box behavioral assay was developed that measures light aversion independently from anxiety, allowing direct testing of one endophenotype of photoallodynia in mice. Mice lacking intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit reduced aversion to bright light, suggesting these cells are the primary circuit for light aversion. Mice treated with exogenous µ opiate receptor agonists exhibited dramatically enhanced light aversion, which was not dependent on ipRGCs, suggesting an alternative pathway for light is engaged. Morphine enhances retinal electrophysiological responses to light but only at low levels. This suggests that for the dramatic light aversion observed, opiates also sensitize central brain regions of photoallodynia. Taken together, our results suggest that light aversion has at least two dissociable mechanisms by which light causes specific allodynia behaviors: a primary ipRGC-based circuit, and a secondary ipRGC-independent circuit that is unmasked by morphine sensitization. These models will be useful in delineating upstream light sensory pathways and downstream avoidance pathways that apply to photoallodynia.

Molecular diagnosis of putative Stargardt Disease probands by exome sequencing.

BACKGROUND: The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. METHODS: We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. RESULTS: Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. CONCLUSIONS: Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.