Animal Model Of Age-related Macular Degeneration Research Articles

Atorvastatin Alleviates Age-Related Macular Degeneration via AIM2-Regulated Pyroptosis.

The underlying causes of age-related macular degeneration (AMD) remain elusive and treatment options of it are limited, while atorvastatin (AT) is expected to improve AMD. Our study sought to uncover the specific mechanisms that initiate pyroptosis in AMD and elucidate whether AT ameliorates Aβ1-40-induced retinal damage by inhibiting pyroptosis. An animal model of AMD was triggered by Aβ1-40, and the therapeutic efficacy of AT was evaluated by hematoxylin and eosin staining (H&E), Optical Coherence Tomography (OCT), Electroretinogram (ERG) and other methods. Utilizing network pharmacology in conjunction with transcriptomics, we identified potential therapeutic pathways. we employed Western blotting (WB) and quantitative real-time PCR (qPCR) methodologies to evaluate the levels of pyroptosis. In vitro system of retinal pigment epithelium (RPE) cells injury was caused by Aβ1-40 and subsequently treated with AT or JC2-11. The extent of pyroptosis was quantified using enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining and WB. Cell morphological changes were examined using light microscopy and scanning electron microscopy. Network pharmacology and transcriptomics identified AIM2/Caspase-1/GSDMD as the key pathway. AT improved the retinal morphological and functional damage caused by Aβ1-40, and decreased the production of AIM2, Asc, Caspase-1, GSDMD-N, Cleaved Caspase-1 and cytokines to exert an anti-inflammatory effect. In addition, AT improved the ruptured membrane of RPE cells caused by Aβ1-40. The use of JC2-11 further demonstrated that AT inhibits pyroptosis of RPE via AIM2/Caspase-1/GSDMD pathway activated by Aβ1-40. These discoveries illuminate the retinal conservation role of AT by effectively hindering the progression of pyroptosis.

REGULATION OF RETINOGENESIS IN PRENATAL ONTOGENESIS AND ITS DISRUPTION IN AGE-RELATED MACULAR DEGENERATION

One of the main causes of blindness in the elderly in developed countries is age-related macular degeneration (AMD) [1, 2, 3]. Active research is being conducted to identify putative molecular targets for the development of an effective strategy for conservative treatment of both atrophic and neovascular forms of this disease [4, 5, 6], but at the present stage, pharmacotherapy is effective only for some patients, and is insufficient to stop the progression of the disease or eliminate the damage that has already occurred. The most promising and promising potential treatment options are considered to be the introduction of cellular technologies with retinal cell replacement and targeted pharmacotherapy. The aim of this study was to investigate the role of the retinal development inducer, SIRT1 [7, 8, 9], in retinogenesis to justify pharmacological inhibition of this protein as a possible treatment option for neovascular AMD. Using immunohistochemistry and immunocytochemistry, we assessed the expression and subcellular localization of SIRT1 and its innate inhibitor DBC1 in the retina of fetuses, adults, and mice. We also studied SIRT1 in retinal progenitor cells derived from mice and humans, the former having been used in small interfering RNA studies. SIRT1 is widely expressed in the developing and adult retina and is a regulator of key genes in retinal development, namely PAX6, Nestin, and CRX [10, 11, 12]. Moreover, we found that photoreceptor progenitor cells were among the smallest cells in a heterogeneous mouse retinal progenitor cell population [13, 14, 15]. Collectively, these results provide a basis for manipulating SIRT1 expression in small retinal progenitors as a means to increase the yield of photoreceptors for transplantation in retinal degeneration models [16, 17]. Furthermore, SIRT1 was found to be highly expressed in human-derived choroidal neovascular membranes, allowing its activity to be pharmacologically inhibited by the drug nicotinamide as a potent regulator of angiogenic and hypoxic signaling in a human retinal pigment epithelial cell line at both the protein level using angiogenesis arrays and at the RNA level using whole genome microarrays [18, 19]. These results point to the SIRT1 inhibitor, Nicotinamide, as a possible agent for treatment of neovascular AMD. Further studies of Nicotinamide are warranted in animal models of AMD. To the best of our knowledge, this is the first time that a detailed analysis of SIRT1 as a regulator of both retinal development and choroidal neovascularization has been reported.

Prevalence of Age-Related Macular Degeneration in Patients with Chronic Exposure to P2X7R Inhibitors.

P2X7 receptor (P2X7R) is a purinergic cation channel whose activation has been linked with age-related macular degeneration (ARMD). Several nucleoside reverse transcriptase inhibitors, zidovudine (AZT), lamivudine (3TC) and abacavir (ABC), have been shown to inhibit P2X7R and improve outcomes in animal models of ARMD. Our aim is to investigate the association between chronic AZT, 3TC, and ABC therapy and ARMD in a clinical setting. This is a retrospective cohort study comparing 445 patients with HIV and confirmed usage of AZT, 3TC, and ABC against 200 patients with HIV without usage of AZT, 3TC, and ABC and 445 non-HIV infected patients. Fundus examination and spectral domain optical coherence tomography (SD-ODT) were used to measure prevalence of early-intermediate stage ARMD, geographic atrophy, and exudative ARMD. There was no statistically significant difference in the prevalence of early-intermediate stage ARMD between the HIV infected patients with a history of AZT, 3TC, and ABC use and the HIV infected patients without AZT, 3TC, and ABC use (p = 0.887). There was also no statistically significant difference in the prevalence of geographical atrophy (p = 0.062) and exudative AMD (p > 0.999) between the HIV infected patients with a history of AZT, 3TC, and ABC use and non-HIV infected patients. We did not find an effect of P2X7R inhibiting antiretrovirals usage on early-intermediate stage ARMD, geographical atrophy, or exudative ARMD. Studies with larger cohort and more rigorous medication history are needed to assess the effects on geographical atrophy or exudative ARMD.

Extracellular vesicles as a potential therapeutic for age-related macular degeneration.

Age-related macular degeneration is a major global cause of central visual impairment and severe vision loss. With an aging population, the already immense economic burden of costly anti-vascular endothelial growth factor treatment is likely to increase. In addition, current conventional treatment is only available for the late neovascular stage of age-related macular degeneration, and injections can come with potentially devastating complications, introducing the need for more economical and risk-free treatment. In recent years, exosomes, which are nano-sized extracellular vesicles of an endocytic origin, have shown immense potential as diagnostic biomarkers and in the therapeutic application, as they are bestowed with characteristics including an expansive cargo that closely resembles their parent cell and exceptional ability of intercellular communication and targeting neighboring cells. Exosomes are currently undergoing clinical trials for various conditions such as type 1 diabetes and autoimmune diseases; however, exosomes as a potential therapy for several retinal diseases have just begun to undergo scrutinizing investigation with little literature on age-related macular degeneration specifically. This article will focus on the limited literature available on exosome transplantation treatment in age-related macular degeneration animal models and in vitro cell cultures, as well as briefly identify future research directions. Current literature on exosome therapy using age-related macular degeneration rodent models includes laser retinal injury, N-methyl-N-nitrosourea, and royal college of surgeon models, which mimic inflammatory and degenerative aspects of age-related macular degeneration. These have shown promising results in preserving retinal function and morphology, as well as protecting photoreceptors from apoptosis. Exosomes from their respective cellular origins may also act by regulating the expression of various inflammatory cytokines, mRNAs, and proteins involved in photoreceptor degeneration pathways to exert a therapeutic effect. Various findings have also opened exciting prospects for the involvement of cargo components in remedial effects on the damaged macula or retina.

Absence of Gut Microbiota Is Associated with RPE/Choroid Transcriptomic Changes Related to Age-Related Macular Degeneration Pathobiology and Decreased Choroidal Neovascularization.

Studies have begun to reveal significant connections between the gut microbiome and various retinal diseases, including age-related macular degeneration (AMD). As critical supporting tissues of the retina, the retinal pigment epithelium (RPE) and underlying choroid play a critical role in retinal homeostasis and degeneration. However, the relationship between the microbiome and RPE/choroid remains poorly understood, particularly in animal models of AMD. In order to better elucidate this role, we performed high-throughput RNA sequencing of RPE/choroid tissue in germ-free (GF) and specific pathogen-free (SPF) mice. Furthermore, utilizing a specialized laser-induced choroidal neovascularization (CNV) model that we developed, we compared CNV size and inflammatory response between GF and SPF mice. After correction of raw data, 660 differentially expressed genes (DEGs) were identified, including those involved in angiogenesis regulation, scavenger and cytokine receptor activity, and inflammatory response—all of which have been implicated in AMD pathogenesis. Among lasered mice, the GF group showed significantly decreased CNV lesion size and microglial infiltration around CNV compared to the SPF group. Together, these findings provide evidence for a potential gut–RPE/choroidal axis as well as a correlation with neovascular features of AMD.

Cytoprotective Potential of Fucoxanthin in Oxidative Stress-Induced Age-Related Macular Degeneration and Retinal Pigment Epithelial Cell Senescence In Vivo and In Vitro.

Oxidative stress is identified as a major inducer of retinal pigment epithelium (RPE) cell dysregulation and is associated with age-related macular degeneration (AMD). The protection of RPE disorders plays an essential role in the pathological progress of retinal degeneration diseases. The pharmacological functions of fucoxanthin, a characteristic carotenoid, including anti-inflammatory and antioxidant properties, may ameliorate an outstanding bioactivity against premature senescence and cellular dysfunction. This study demonstrates that fucoxanthin protects RPE cells from oxidative stress-induced premature senescence and decreased photoreceptor cell loss in a sodium iodate-induced AMD animal model. Similarly, oxidative stress induced by hydrogen peroxide, nuclear phosphorylated histone (γH2AX) deposition and premature senescence-associated β-galactosidase staining were inhibited by fucoxanthin pretreatment in a human RPE cell line, ARPE-19 cells. Results reveal that fucoxanthin treatment significantly inhibited reactive oxygen species (ROS) generation, reduced malondialdehyde (MDA) concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli was inhibited in cells treated with fucoxanthin after oxidative stress. During aging, the RPE undergoes well-characterized pathological changes, including amyloid beta (Aβ) deposition, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression and tight junction disruption, which were also reduced in fucoxanthin-treated groups by immunofluorescence. Altogether, pretreatment with fucoxanthin may protect against premature senescence and cellular dysfunction in retinal cells by oxidative stress in experimental AMD animal and human RPE cell models.

Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats’ eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

The Impact of Oxidative Stress on Blood-Retinal Barrier Physiology in Age-Related Macular Degeneration

The blood retinal barrier (BRB) is a fundamental eye component, whose function is to select the flow of molecules from the blood to the retina and vice-versa, and its integrity allows the maintenance of a finely regulated microenvironment. The outer BRB, composed by the choriocapillaris, the Bruch’s membrane, and the retinal pigment epithelium, undergoes structural and functional changes in age-related macular degeneration (AMD), the leading cause of blindness worldwide. BRB alterations lead to retinal dysfunction and neurodegeneration. Several risk factors have been associated with AMD onset in the past decades and oxidative stress is widely recognized as a key factor, even if the exact AMD pathophysiology has not been exactly elucidated yet. The present review describes the BRB physiology, the BRB changes occurring in AMD, the role of oxidative stress in AMD with a focus on the outer BRB structures. Moreover, we propose the use of cerium oxide nanoparticles as a new powerful anti-oxidant agent to combat AMD, based on the relevant existing data which demonstrated their beneficial effects in protecting the outer BRB in animal models of AMD.

Central Role of Oxidative Stress in Age-Related Macular Degeneration: Evidence from a Review of the Molecular Mechanisms and Animal Models.

Age-related macular degeneration (AMD) is a common cause of visual impairment in the elderly. There are very limited therapeutic options for AMD with the predominant therapies targeting vascular endothelial growth factor (VEGF) in the retina of patients afflicted with wet AMD. Hence, it is important to remind readers, especially those interested in AMD, about current studies that may help to develop novel therapies for other stages of AMD. This study, therefore, provides a comprehensive review of studies on human specimens as well as rodent models of the disease, to identify and analyze the molecular mechanisms behind AMD development and progression. The evaluation of this information highlights the central role that oxidative damage in the retina plays in contributing to major pathways, including inflammation and angiogenesis, found in the AMD phenotype. Following on the debate of oxidative stress as the earliest injury in the AMD pathogenesis, we demonstrated how the targeting of oxidative stress-associated pathways, such as autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, might be the futuristic direction to explore in the search of an effective treatment for AMD, as the dysregulation of these mechanisms is crucial to oxidative injury in the retina. In addition, animal models of AMD have been discussed in great detail, with their strengths and pitfalls included, to assist inform in the selection of suitable models for investigating any of the molecular mechanisms.

Therapeutic Effect of Garcinia cambogia Extract and Hydroxycitric Acid Inhibiting Hypoxia-Inducible Factor in a Murine Model of Age-Related Macular Degeneration.

Background: Age-related macular degeneration (AMD) is the leading cause of blindness and can be classified into two types called atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choriocapillaris, and photoreceptors. Wet AMD is characterized by the invasion of abnormal vessels from the choroid. Although anti-vascular endothelial growth factor (VEGF) therapy has a potent therapeutic effect against the disease, there is a possibility of chorio-retinal atrophy and adverse systemic events due to long-term robust VEGF antagonism. We focused on hypoxia-inducible factor (HIF) regulation of VEGF transcription, and report the suppressive effects of HIF inhibition against ocular phenotypes in animal models. Many of the known HIF inhibitors are categorized as anti-cancer drugs, and their systemic side effects are cause for concern in clinical use. In this study, we explored food ingredients that have HIF inhibitory effects and verified their effects in an animal model of AMD. Methods: Food ingredients were screened using a luciferase assay. C57BL6/J mice were administered the Garcinia cambogia extract (Garcinia extract) and hydroxycitric acid (HCA). Choroidal neovascularization (CNV) was induced by laser irradiation. Results: Garcinia extract and HCA showed inhibitory effects on HIF in the luciferase assay. The laser CNV model mice showed significant reduction of CNV volume by administering Garcinia extract and HCA. Conclusions: Garcinia extract and HCA showed therapeutic effects in a murine AMD model.

4-Hydroxy-7-oxo-5-heptenoic Acid Lactone Is a Potent Inducer of the Complement Pathway in Human Retinal Pigmented Epithelial Cells.

We previously discovered that oxidative cleavage of docosahexaenoate (DHA), which is especially abundant in the retinal photoreceptor rod outer segments and retinal pigmented endothelial (RPE) cells, generates 4-hydroxy-7-oxo-5-heptenoate (HOHA) lactone, and that HOHA lactone can enter RPE cells that metabolize it through conjugation with glutathione (GSH). The consequent depletion of GSH results in oxidative stress. We now find that HOHA lactone induces upregulation of the antioxidant transcription factor Nrf2 in ARPE-19 cells. This leads to expression of GCLM, HO1, and NQO1, three known Nrf2-responsive antioxidant genes. Besides this protective response, HOHA lactone also triggers a countervailing inflammatory activation of innate immunity. Evidence for a contribution of the complement pathway to age-related macular degeneration (AMD) pathology includes the presence of complement proteins in drusen and Bruch's membrane from AMD donor eyes, and the identification of genetic susceptibility loci for AMD in the complement pathway. In eye tissues from a mouse model of AMD, accumulation of complement protein in Bruch's membrane below the RPE suggested that the complement pathway targets this interface, where lesions occur in the RPE and photoreceptor rod outer segments. In animal models of AMD, intravenous injection of NaIO3 to induce oxidative injury selectively destroys the RPE and causes secretion of factor C3 from the RPE into areas directly adjacent to sites of RPE damage. However, a molecular-level link between oxidative injury and complement activation remained elusive. We now find that sub-micromolar concentrations of HOHA lactone foster expression of C3, CFB, and C5 in ARPE-19 cells and induce a countervailing upregulation of CD55, an inhibitor of C3 convertase production and complement cascade amplification. Ultimately, HOHA lactone causes membrane attack complex formation on the plasma membrane. Thus, HOHA lactone provides a molecular-level connection between free-radical-induced oxidative cleavage of DHA and activation of the complement pathway in AMD pathology.

48 - Lipid Radicals Cause Light Induced-Retinal Degeneration

Age-related macular degeneration (AMD) is a progressive degenerative disease of the macula, the centre of the retina. It is the most common cause of adult vision loss in developed countries, and the numbers of patients are increasing every year. Although the cause of AMD remains unknown, it is believed that chronic oxidative stresses, such as light, smoking, and radiation, are related to the progression of AMD, especially dry-type AMD. The retina consumes large amounts of oxygen per tissue, and has high oxygen tension. This leads to the production of excessive reactive oxygen species (ROS). In addition, polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and arachidonic acid, are abundantly present in the retina. Thus, in the retina, ROS can easily oxidize PUFAs and produce lipid radicals, which are key factors in the chain reaction during lipid peroxidation (LPO). The resulting lipid radicals produce many kinds of reactive aldehydes, including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). These reactive aldehydes bind to intracellular proteins and DNA, and induce inflammation and apoptosis. In fact, LPO end-products often accumulate in the retina of elderly people and AMD animal models. However, there are hundreds of LPO end-products with different functions, and it is challenging to detect and inhibit all of these products in vivo . Here, we focused on lipid-derived radicals as common compounds in the production of reactive aldehydes and most upstream compounds during the LPO chain reaction. As lipid radicals are too highly reactive for detection in cells and animals, we recently developed a lipid radical detection fluorescent probe, NBD-Pen ( Nat Chem Biol , 2016). In this report, we describe imaging of lipid radicals in the retina using light-induced retinal damage models as dry-type AMD animal models, and the effect of a newly-synthesized lipid radical inhibition molecule, CPC-Pen, on AMD with therapeutic relevance.

Hemorheological parameters and their correlations in OXYS rats: a new model of hyperviscosity syndrome.

Rheohaemapheresis aims to normalize major rheological parameters and is used to treat patients with dry age-related macular degeneration (AMD). While effective, this approach is invasive and requires specially trained personnel. Therefore, the search for novel effective compounds with hemorheological properties that can be taken orally to treat AMD is justified. The use of a robust rodent model of AMD with high blood viscosity is crucial to test the efficacy of potential hemorheological drugs to treat this disease. The objective of this study was to investigate whether OXYS rats, generally used as an animal model of AMD, have hyperviscosity syndrome. The results of this study show that blood viscosity in OXYS rats at low (3-10 s -1) and high (45-300 s -1) shear rates were 14-20% and 7-10% higher than in Wistar rats, while hematocrit and plasma viscosity were not different. Red blood cells (RBCs) in OXYS rats were more prone to aggregation as shown by 39% shorter half-time than in Wistar rats. RBCs were also more rigid in OXYS than in Wistar rats as shown by 21-33% lower index of elongation at the shear stress of 1-7 Pa. These data indicate that OXYS rats have hyperviscosity syndrome as the result of abnormal RBC deformability and aggregation. We propose to use OXYS rats as an animal model for preclinical studies to test compounds with hemorheological properties aimed to treat AMD.

Inhibition of micro‐fibrillar associated protein 4 as a potential therapy targeting choroidal neovascularisation in age related macular degeneration

PurposeTo evaluate inhibition of Micro‐Fibrillar Associated Protein 4 (MFAP4) on choroidal neovascularization (CNV) in a mouse model of age‐related macular degeneration (AMD).MethodsAll experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Female C57BL/6J mice were subjected to laser‐induced CNV, and intravitreally injected with either 1 µg ±MFAP4, 5 µg ±MFAP4, 1 µg mouse IgG or 1 µg ±VEGF‐A on day 0 and day 7. Fluorescein angiography (FA) was undertaken at day 7 and day 14, and choroids stained for inflammation (CD45) and vasculature (isolectin B4, IB4).ResultsFA showed that injection of αMFAP4 reduced average lesion size and density on day 7 compared to IgG (p < 0.01) and αVEGF‐A positive controls (p < 0.05) and both αMFAP4 and αVEGF‐A reduced average lesion size and density by day 14 compared to IgG (p < 0.001 and p < 0.05 respectively). IB4 staining indicated that both αMFAP4 and αVEGF‐A reduced lesion fluorescence intensity (p < 0.05). Both αMFAP4 and αVEGF‐A treatments also reduced infiltration of macrophages into the lesion site (p < 0.01 and p < 0.05).ConclusionsThese results show that inhibition of MFAP4 results in a significant decrease in neovascular lesions in an animal model of AMD. The reduction in macrophage infiltration suggests a potential mechanism of action for anti‐MFAP4 treatment. Together, this suggests that inhibition of MFAP4 could be a potential novel AMD therapeutic.

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Potential Sources and Roles of Adaptive Immunity in Age-Related Macular Degeneration: Shall We Rename AMD into Autoimmune Macular Disease?

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly throughout the industrialized world. Its most prominent pathologic features are lesions involving the retinal pigment epithelium (RPE) the Bruch's membrane, the degeneration of photoreceptors, and, in the most aggressive cases, choroidal neovascularization. Genetic associations between the risk of developing AMD and polymorphism within components of the complement system, as well as chemokine receptors expressed on microglial cells and macrophages, have linked retinal degeneration and choroidal neovascularization to innate immunity (inflammation). In addition to inflammation, players of the adaptive immunity including cytokines, chemokines, antibodies, and T cells have been detected in animal models of AMD and in patients suffering from this pathology. These observations suggest that adaptive immunity might play a role in different processes associated with AMD such as RPE atrophy, neovascularization, and retinal degeneration. To this date however, the exact roles (if any) of autoantibodies and T cells in AMD remain unknown. In this review we discuss the potential effects of adaptive immune responses in AMD pathogenesis.

Altered mRNA Expression of 'Ahr-Nrf2 Gene Batteries' in the Retinas of Senescence-Accelerated OXYS Rats during Development of AMDLike Retinopathy

Background: Etiology of age-related macular degeneration (AMD) is poorly understood, although oxidative stress and environmental risk factors have been implicated. Recently, AhR (arylhydrocarbon receptor) and Nrf2 (nuclear factor erythroid 2-related factor 2) were considered as AMD candidate genes. Transcription products of the AhR-Nrf2 “gene battery” are important in mediating cellular response to oxidative stress. The animal model for AMD (senescence accelerated OXYS rats) was used. Ophthalmoscopy revealed no pathologic changes in OXYS rats’ retinas at the age of 1 month; however, at the age of 3 months, the first signs of retinopathy were recorded in the eyes of all animals tested. The aim of the present study was to determine whether the balance between prooxidizing (AhR-dependent) and antioxidant (Nrf2-dependent) systems plays a crucial role in the onset and/or progression of age-related retinopathy. Methods: In the retina of 1-, 3- and 12- month-old OXYS and Wistar rats mRNA levels of CYP1A1, CYP1A2, CYP1B1, GSTA1, NQO1, ALDH3A1, UGT1A6, UGT1A9, AhR and Nrf2 genes were measured by qRT-PCR. Results: In the retinas of 1-month OXYS rats, the mRNA level of only AhR was reduced when compared with Wistar rats. At the age of 3 months, a decline in the mRNA levels was detected for CYP1A and CYP1A2, but not for CYP1B1 in OXYS rats. mRNA levels of Nrf2, were higher in OXYS rats when compared with Wistar rats. Levels of the genes that are regulated by AhR and Nrf2 (NQO1 and UGT1A6), were reduced when compared with Wistar rats, and GSTA1: mRNA level was increased. Conclusions: The data obtained allow us to conclude that the AhR-Nrf2 “gene battery” may be involved in the pathogenesis of retinopathy in the OXYS rats. One of the triggers for the starting of oxidative stress during the progression of retinopathy may be the inborn reduced level of AhR expression.

Potential of melatonin for prevention of age-related macular degeneration: Experimental study

Decline with age of the content of melatonin is considered as one of the leading mechanisms of aging and development of associated diseases, including age-related macular degeneration (AMD)--the disease, which becomes the most common cause of blindness and acuity of vision deterioration in elderly. The prospects of the use of melatonin in the prevention of AMD is being actively discussed, but as a rule on the basis of the results of the experiments on cells in retinal pigment epithelium (RPE). We showed previously that the senescence-accelerated OXYS rat is an adequate animal model of AMD, already used for identifying the relevant therapeutic targets. Here we have investigated the effect of Melatonin (Melaksen, 0,004 mg per kg--a dose equivalent to the recommended one for people) on the development of retinopathy similar to AMD in OXYS rats. Ophthalmoscopic examinations show that Melatonin supplementation decreased the incidence and severity of retinopathy and improved some (but not all) histological abnormalities associated with retinopathy. Thus, melatonin prevented the structural and functional changes in RPE cells, reduced the severity of microcirculatory disorders. Importantly, Melatonin prevented destruction of neurosensory cells, associative and gangliolar neurons in the retina. Taken together, our data suggest the therapeutic potential of Melatonin for treatment and prevention of AMD.

Keep Your Eyes Open: Challenges And Opportunities In Ophthalmic Therapeutics

The eyes are our windows to the world. Major ocular diseases, including cataracts, glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), dry eye syndrome and allergic conjunctivitis, can cause vision impairment and even blindness, which not only affect our daily life, but also have a significant socio-economic and psychological impact on our society. The prevalence of vision impairment was estimated to be 147 million people in 2010, and is projected to reach 191 million in the word in 2020 due to an increase in aging populations. The market for ophthalmic disease therapeutics is expected to reach $18.7 billion this year, and is predicted to grow steadily. Ophthalmic drug development has been in the forefront of therapeutic research partly because the eye is an accessible and immunologically isolated organ, which has the advantage of avoiding the complications of systemic drug delivery. With the recent advances in genetics, genomics and stem cell biology, the mechanisms of many ophthalmic diseases are being increasingly revealed. Accordingly, the field of ophthalmic therapeutics is facing unprecedented challenges and opportunities. Looking back in history, ophthalmic drug development has been driven by the scientific breakthroughs. For example, the discovery that vascular endothelial growth factor (VEGF) is a driving force of choroidal neovascularization in AMD has led to the successful development of anti-VEGF antibodies for wet AMD therapy. The discovery that prostaglandin mediates reduction in intraocular pressure (IOP) has led to the treatment of ocular hypertension in glaucoma using prostaglandin analogues. Research over the past decade has uncovered novel mechanisms for many ocular diseases, which will likely spawn new therapeutic solutions. But this field is not without challenges: (A) currently there is no cure for many ocular diseases, including dry AMD, DR and glaucoma; (B) current ophthalmic drug development is not based on the unique characteristics of ocular diseases, but mainly applies agents developed for non-ocular diseases to the eye; (C) most of the ocular diseases are multi-factorial diseases with both genetic and environmental (nutritional) risk factors, therefore many drugs only work for a subset of patients. For example, cyclosporine, acting as an immune-modulator for dry-eye disease, is only effective in about 15% of all patients; (D) ophthalmic drug delivery, especially to the posterior eye, remains a considerable challenge. These challenges present unique opportunities for researchers in the field of ophthalmology to further elucidate the mechanisms of and develop innovative therapeutics and delivery approaches for ocular diseases. Despite the challenges, significant progress has recently been made towards understanding the mechanisms of several ocular diseases, which may translate into future therapeutics. Dry AMD accounts for up to 90% of the AMD cases and is currently without cure. Polymorphisms in members of the complement system, especially complement factor H (CFH), have been associated with AMD. Moreover, CFH was recently shown to protect against oxidative stress-induced inflammation in animal model. Inhibitors of the complement system and the recombinant form of CFH are being tested preclinically for AMD treatment. Aβ amyloid, originally implicated in Alzheimer’s disease, was recently associated with pathogenesis of AMD. Anti-Aβ amyloid antibody has shown promise in animal models of AMD. Autophagy is currently being investigated for AMD involvement, and may represent a potential therapeutics for AMD (See review in this issue). Diabetic retinopathy is one of the leading causes of blindness in the working class. Besides laser photocoagulation and vitrectomy surgery, several therapeutic options, including protein kinase inhibitors, cyclooxygenase inhibitors, anti-VEGF and slow release steroid, are on clinical trials. Some of them may enter the market in the near future. As reviewed in this issue, pericytes and the angiopoietin (Ang)-Tie-2 signaling also play an important role in the development of DR, and may be potential therapeutic targets for DR. For glaucoma, the current mainstay for treatment is to lower IOP, therefore preventing further damage to the optic nerve. β-blocker, prostaglandin, carbonic anhydrase inhibitor, miotics, α-adrenergic agonist, or their combinations are routinely used for lowering IOP. Rho kinase inhibitors and actin depolymerization agents are currently tested in clinical trials for safely reducing IOP. Dominant mutations in the olfactomedin (OLF) domain of myocilin have been associated with several populations of familial glaucoma, and molecular studies suggest that the OLF domain of myocilin may be a bona fide target for future glaucoma therapeutics (reviewed in this issue). Looking into future, the explosion in basic research in ocular biology and disease will lead to revolutionarily innovative ophthalmic therapeutics. Numerous opportunities also present themselves to researchers working on medicinal chemistry, new biomaterials, and new delivery system (see reviews for these subjects in the issue). With the coordinated effort of all these groups, the future of ophthalmic therapeutics is bright.

Therapeutic Action of the Mitochondria-Targeted Antioxidant SkQ1 on Retinopathy in OXYS Rats Linked with Improvement of VEGF and PEDF Gene Expression

The incidence of age-related macular degeneration (AMD), the main cause of blindness in older patients in the developed countries, is increasing with the ageing population. At present there is no effective treatment for the prevailing geographic atrophy, dry AMD, whereas antiangiogenic therapies successful used in managing the wet form of AMD. Recently we showed that mitochondria-targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium (SkQ1) is able to prevent the development and moreover caused regression of pre-existing signs of the retinopathy in OXYS rats, an animal model of AMD. Here we examine the effects of SkQ1 on expression of key regulators of angiogenesis vascular endothelial growth factor A (VEGF) and its antagonist pigment epithelium-derived factor (PEDF) genes in the retina of OXYS rats as evidenced by real-time PCR and an ELISA test for VEGF using Wistar rats as control. Ophthalmoscopic examinations confirmed that SkQ1 supplementation (from 1.5 to 3 months of age, 250 nmol/kg) prevented development while eye drops SkQ1 (250 nM, from 9 to 12 months) caused some reduction of retinopathy signs in OXYS rats and did not reveal any negative effects on the control Wistar rat's retina. Prevention of premature retinopathy by SkQ1 was connected with an increase of VEGF mRNA and protein in OXYS rat's retina up to the levels corresponding to the Wistar rats, and did not involve changes in PEDF expression. In contrast the treatment with SkQ1 drops caused a decrease of VEGF mRNA and protein levels and an increase in the PEDF mRNA level in the middle-aged OXYS rats, but in Wistar rats the changes of gene expression were the opposite. Conclusions: The beneficial effects of SkQ1 on retinopathy connected with normalization of expression of VEGF and PEDF in the retina of OXYS rats and depended on age of the animals and the stage of retinopathy.