Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

Synthesis and Propagation of Complement C3 by Microglia/Monocytes in the Aging Retina

Introduction

Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging.

Methods

SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes.

Results

C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines.

Conclusions

Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.     

Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats

Objective

Royal College of Surgeons (RCS) rats develop vasculopathy as photoreceptors degenerate. The aim of this study was to examine the effect of erythropoietin (EPO) on retinopathy in RCS rats.

Methods

Fluorescein angiography was used to monitor retinal vascular changes over time. Changes in retinal glia and vasculature were studied by immunostaining. To study the effects of EPO on retinal pathology, EPO (5000 IU/kg) was injected intraperitoneally in 14 week old normal and RCS rats twice a week for 4 weeks. Changes in the retinal vasculature, glia and microglia, photoreceptor apoptosis, differential expression of p75 neurotrophin receptor (p75^NTR), pro-neurotrophin 3 (pro-NT3), tumour necrosis factor-α (TNFα), pigment epithelium derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A), the production of CD34⁺ cells and mobilization of CD34⁺/VEGF-R2⁺ cells as well as recruitment of CD34⁺ cells into the retina were examined after EPO treatment.

Results

RCS rats developed progressive capillary dropout and subretinal neovascularization which were accompanied by retinal gliosis. Systemic administration of EPO stabilized the retinal vasculature and inhibited the development of focal vascular lesions. Further studies showed that EPO modulated retinal gliosis, attenuated photoreceptor apoptosis and p75^NTR and pro-NT3 upregulation, promoted the infiltration of ramified microglia and stimulated VEGF-A expression but had little effect on TNFα and PEDF expression. EPO stimulated the production of red and white blood cells and CD34⁺ cells along with effective mobilization of CD34⁺/VEGF-R2⁺ cells. Immunofluorescence study demonstrated that EPO enhanced the recruitment of CD34⁺ cells into the retina.

Conclusions

Our results suggest that EPO has therapeutic potentials in treatment of neuronal and vascular pathology in retinal disease. The protective effects of EPO on photoreceptors and the retinal vasculature may involve multiple mechanisms including regulation of retinal glia and microglia, inhibition of p75^NTR-pro-NT3 signaling together with stimulation of production and mobilization of bone marrow derived cells.     

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

Background

Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).

Results

We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.

Conclusions

RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.     

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo

Background

Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin⁺ cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo.

Methodology/Principal Findings

For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin⁺ microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin⁺ microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU⁺ and nestin⁺, although no further local cell proliferation occurred. In addition, nestin⁺ microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin⁺NG2⁺ microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons.

Conclusions/Significance

The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed.     

A chick model of retinal detachment: cone rich and novel

Background

Development of retinal detachment models in small animals can be difficult and expensive. Here we create and characterize a novel, cone-rich retinal detachment (RD) model in the chick.

Methodology/Principal Findings

Retinal detachments were created in chicks between postnatal days 7 and 21 by subretinal injections of either saline (SA) or hyaluronic acid (HA). Injections were performed through a dilated pupil with observation via surgical microscope, using the fellow eye as a control. Immunohistochemical analyses were performed at days 1, 3, 7, 10 and 14 after retinal detachment to evaluate the cellular responses of photoreceptors, Müller glia, microglia and nonastrocytic inner retinal glia (NIRG). Cell proliferation was detected with bromodeoxyuridine (BrdU)-incorporation and by the expression of proliferating cell nuclear antigen (PCNA). Cell death was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). As in mammalian models of RD, there is shortening of photoreceptor outer segments and mis-trafficking of photoreceptor opsins in areas of RD. Photoreceptor cell death was maximal 1 day after RD, but continued until 14 days after RD. Müller glia up-regulated glial fibriliary acidic protein (GFAP), proliferated, showed interkinetic nuclear migration, and migrated to the subretinal space in areas of detachment. Microglia became reactive; they up-regulated CD45, acquired amoeboid morphology, and migrated toward outer retina in areas of RD. Reactive NIRG cells accumulated in detached areas.

Conclusions/Significance

Subretinal injections of SA or HA in the chick eye successfully produced retinal detachments and cellular responses similar to those seen in standard mammalian models. Given the relatively large eye size, and considering the low cost, the chick model of RD offers advantages for high-throughput studies.     

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

Background

Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.

Methodology/Principal Findings

We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.

Conclusion/Significance

In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.     

Orally active multi-functional antioxidants are neuroprotective in a rat model of light-induced retinal damage

Background

Progression of age-related macular degeneration has been linked to iron dysregulation and oxidative stress that induce apoptosis of neural retinal cells. Since both antioxidants and chelating agents have been reported to reduce the progression of retinal lesions associated with AMD in experimental animals, the present study evaluates the ability of multi-functional antioxidants containing functional groups that can independently chelate redox metals and quench free radicals to protect the retina against light-induced retinal degeneration, a rat model of dry atrophic AMD.

Methods/Results

Proof of concept studies were conducted to evaluate the ability of 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8) to reduce retinal damage in 2-week dark adapted Wistar rats exposed to 1000 lx of light for 3 hours. Assessment of the oxidative stress markers 4- hydroxynonenal and nitrotyrosine modified proteins and Thioredoxin by ELISA and Western blots indicated that these compounds reduced the oxidative insult caused by light exposure. The beneficial antioxidant effects of these compounds in providing significant functional and structural protection were confirmed by electroretinography and quantitative histology of the retina.

Conclusions/Significance

The present study suggests that multi-functional compounds may be effective candidates for preventive therapy of AMD.     

Alternative Complement Pathway Deficiency Ameliorates Chronic Smoke-Induced Functional and Morphological Ocular Injury

Background

Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required.

Methods

Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB^−/−) using molecular, histological, electrophysiological, and behavioral outcomes.

Results

CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch''s membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB^−/− mice were protected from developing these CE-mediated alterations.

Conclusions

Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.     

Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo

Background

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.

Principal Findings

MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

Conclusion

We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.     

Microglial morphology and dynamic behavior is regulated by ionotropic glutamatergic and GABAergic neurotransmission

Purpose

Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior.

Methods

We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia.

Results

Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels.

Conclusions

Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.     

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina

Purpose

To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology.

Methods

After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection.

Results

After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group.

Conclusions

Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.     

A Pilot Study of Morphometric Analysis of Choroidal Vasculature In Vivo,Using En Face Optical Coherence Tomography

Purpose

To study the ability of volumetric spectral domain optical coherence tomography (SD-OCT) to perform quantitative measurement of the choroidal vasculature in vivo.

Methods

Choroidal vascular density and vessel size were quantified using en face choroidal scans from various depths below the retinal pigment epithelium (RPE) in 58 eyes of 58 patients with either epiretinal membranes (ERM), early age-related macular degeneration (AMD), or reticular pseudo-drusen (RPD). For each patient, we used the macular volume scan (6×6 mm cube) for vessel quantification, while high-definition (HD) cross-section raster scans were used to qualitatively assess vascularity of the choroidal sub-layers, and measure choroidal thickness.

Results

Of the 58 patients, more were female (66% versus 34% male), of whom 14 (24%) had ERM, 11 (19%) early AMD, and 33 (57%) RPD. Compared to intact choriocapillaris in all ERM (100%), none of the RPD and only 5/11 (45%) early AMD eyes had visible choriocapillaris on either cross section or C-scans (p-value<0.001). When comparing select regions from the most superficial C-scans, early AMD group had lowest vascular density and RPD had highest (p-value 0.04). Qualitative evaluation of C-scans from all three groups revealed a more granular appearance of the choriocapillaris in ERM versus increased stroma and larger vessels in the RPD eyes.

Conclusions

SD-OCT can be used to qualitatively and quantitatively assess choroidal vascularity in vivo. Our findings correlate to previously reported histopathologic studies. Lack of choriocapillaris on HD cross-sections or C-scans in all RPD and about half of early AMD eyes suggests earlier choroidal involvement in AMD and specifically, RPD.     

The Association between Plasma 25-Hydroxyvitamin D and Subgroups in Age-Related Macular Degeneration: A Cross-Sectional Study

Objectives

To evaluate potential differences in plasma 25-hydroxyvitamin in subtypes of age-related macular degeneration (AMD), and in patients in Clinical Age-Related Maculopathy Staging (CARMS) group 5 with or without subretinal fibrosis.

Methods

This single-center cross-sectional study included 178 participants during a period of 20 months. Ninety-five patients belonged to CARMS 5; twelve belonged to CARMS 4; twenty-two belonged to CARMS 2 or 3; and 49 individuals did not have AMD (CARMS 1). Following a structured interview, a detailed bilateral retinal examination was performed and participants were allocated to their respective subgroups in accordance with the Clinical Age-Related Maculopathy Staging system. Plasma 25-hydroxyvitamin D2 and D3 were analyzed using liquid chromatography-tandem mass spectrometry. Genomic DNA was extracted from leukocytes and genotyped for single nucleotide polymorphisms (SNPs) in the vitamin D metabolism. Differences in plasma 25-hydroxyvitamin D were determined in the subgroups as well as between patients in CARMS 5 with or without subretinal fibrosis.

Results

Plasma 25-hydroxyvitamin D was comparable in patients across CARMS groups 1 to 5 (p = 0.83). In CARMS 5, the presence of subretinal fibrosis was associated with significantly lower concentrations of 25-hydroxyvitamin D as compared to the absence of subretinal fibrosis (47.2 versus 75.6 nmol/L, p<0.001). Patients in CARMS 5 with subretinal fibrosis were more likely to have insufficient levels of 25-hydroxyvitamin D compared to patients without subretinal fibrosis (p = 0.006). No association was found between the SNPs rs10877012, rs2228570, rs4588, or rs7041 and AMD subgroups or plasma 25-hydroxyvitamin levels.

Conclusions

This study suggests that the presence of subretinal fibrosis in patients belonging to CARMS 5 may be associated with a poor vitamin D status. Our observations warrant further investigation into the role of vitamin D in the development of subretinal fibrosis.     

Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

Background

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods

We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results

We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier.

Conclusion

These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.     

Retinal ganglion cell loss is accompanied by antibody depositions and increased levels of microglia after immunization with retinal antigens

Background

Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats.

Methodology/Principal Findings

Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3⁺retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP⁺ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1⁺ microglia cells (p = 0.003) was observed in retinas of RGA animals.

Conclusions/Significance

Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.