Immunopathological aspects of age-related macular degeneration

Age-related macular degeneration (AMD) represents a leading cause of blindness worldwide. While the clinical and histopathological aspects of AMD are well characterized, its etiology and pathogenesis remain unclear. Recent findings suggest a role for immunologic processes in AMD pathogenesis, including the age-related generation of extracellular deposits inside the Brusch membrane and beneath the retinal pigment epithelium, recruitment of macrophages for clearance of these deposits, complement activation, recruitment of tissue-destructive macrophages, microglial activation and accumulation, and proinflammatory effects of chronic inflammation by Chlamydia pneumoniae. This review discusses the evidence for the role of inflammation in human AMD and in animal models of AMD.     

Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration

This work presents a combined light and electron microscopical approach to investigate the initial breakdown of the retinal pigment epithelium (RPE) and choriocapillaris (CC) in age-related macular degeneration (AMD). Perimacular sections of 12 dry and wet AMD eyes (82 ± 15 years) and 7 age-matched controls (75 ± 10 years) without retinal pathology were investigated. Disease progression was classified into 5 stages of retinal degeneration to investigate the concurrent CC breakdown. Special emphasis was laid on transitions where intact CC–RPE–retina complexes went over into highly atrophied areas. AMD sections showed elevated loss of photoreceptors, RPE and CC (p < 0.01), and thickened Bruch's membrane with increased basal laminar and linear deposits compared with controls. Up to 27% of the CC was lost in controls although RPE and retina were still intact. This primary loss of CC further increased with AMD (up to 100%). The data implicate that CC breakdown already occurs during normal aging and precedes degeneration of the RPE and retina with AMD, defining AMD as a vascular disease. Particular attention should be given to the investigation of early AMD stages and transitional stages to the late stage that reveal a possible sequence of degenerative steps with aging and AMD.     

Lipofuscin can be eliminated from the retinal pigment epithelium of monkeys

Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruch's membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to remove lipofuscin from RPE cells, which may have implications for the treatment of age-related macular degeneration in which lipofuscin accumulation in cells is a causative factor.     

VEGF-binding aptides and the inhibition of choroidal and retinal neovascularization

Age-related macular degeneration and diabetic retinopathy are leading causes of blindness. Vascular endothelial growth factor (VEGF) is known to be the main factor that induces pathological angiogenesis in these diseases. In this study, we investigate the therapeutic potential and safety profiles of high-affinity peptides targeting VEGF which are identified using an ‘aptide’ technology. We show that two VEGF-binding aptides, APT_VEGF1 and APT_VEGF2, demonstrate high binding affinity and specificity to VEGF. Furthermore, they suppress VEGF-induced activation of VEGF receptor-2, in vitro angiogenesis, and in vivo pathological choroidal and retinal neovascularization. Despite potent anti-angiogenic effects, both VEGF-binding aptides do not induce any definite toxicity at the level of cellular viability, histological integrity, and gene expression. Our data show the therapeutic potential of VEGF-binding peptides for the treatment of choroidal and retinal neovascularization.     

Retinal blood vessels are damaged in a rat model of NMDA-induced retinal degeneration

Retinal ischemia–reperfusion causes capillary degeneration but the mechanisms of damage are not well understood. The NMDA receptor plays an important role in neuronal damage after ischemia–reperfusion. Therefore, we determined whether retinal blood vessels are damaged structurally and functionally in a rat model of retinal degeneration induced by NMDA. At 7 days after a single intravitreal injection of NMDA (200 nmol) into the eye, loss of retinal ganglion cells and thinning of the inner plexiform layer were observed. Endothelial cells disappeared in some regressing vessels and empty basement membrane sleeves were left as remnants of the vessels. The number of basement membrane sleeves was increased in the NMDA-treated retina and non-perfused vessels were found in the injured retina. These results indicate that retinal blood vessels are damaged in the NMDA-induced retinal degeneration model. Neuronal cells may play a role in maintaining normal structure and function of the vasculature in the retina.     

Phototransduction and retinal degeneration in Drosophila

Drosophila visual transduction is the fastest known G-protein-coupled signaling cascade and has therefore served as a genetically tractable animal model for characterizing rapid responses to sensory stimulation. Mutations in over 30 genes have been identified, which affect activation, adaptation, or termination of the photoresponse. Based on analyses of these genes, a model for phototransduction has emerged, which involves phosphoinoside signaling and culminates with opening of the TRP and TRPL cation channels. Many of the proteins that function in phototransduction are coupled to the PDZ containing scaffold protein INAD and form a supramolecular signaling complex, the signalplex. Arrestin, TRPL, and Gα_q undergo dynamic light-dependent trafficking, and these movements function in long-term adaptation. Other proteins play important roles either in the formation or maturation of rhodopsin, or in regeneration of phosphatidylinositol 4,5-bisphosphate (PIP₂), which is required for the photoresponse. Mutation of nearly any gene that functions in the photoresponse results in retinal degeneration. The underlying bases of photoreceptor cell death are diverse and involve mechanisms such as excessive endocytosis of rhodopsin due to stable rhodopsin/arrestin complexes and abnormally low or high levels of Ca²⁺. Drosophila visual transduction appears to have particular relevance to the cascade in the intrinsically photosensitive retinal ganglion cells in mammals, as the photoresponse in these latter cells appears to operate through a remarkably similar mechanism.     

MicroRNAs in the regulation of autophagy and their possible use in age-related macular degeneration therapy

Age-related macular degeneration (AMD) is a progressive sight-impairing disease of the elderly. The pathogenic mechanisms of AMD are not well understood although both genetic and many environmental factors have been associated with the development of AMD. One clinical hallmark of AMD is the detrimental aggregation of damaged proteins. Recently, it has been suggested that the weakening of autophagy clearance is an important mechanism in the pathogenesis of AMD. Autophagy is important in the removal of damaged or no longer needed cellular material and its recycling. A considerable number of autophagy-targeting microRNAs (miRNAs), small RNA molecules and epigenetic regulators have been found to be either up- or down-regulated in AMD patients and experimental models. The important role of autophagy-targeting miRNAs is supported by several studies and can open the prospect of the use of these miRNAs in the therapy for AMD.     

The complement system in age-related macular degeneration: A review of rare genetic variants and implications for personalized treatment

Age-related macular degeneration (AMD) is a progressive retinal disease and the major cause of irreversible vision loss in the elderly. Numerous studies have found both common and rare genetic variants in the complement pathway to play a role in the pathogenesis of AMD. In this review we provide an overview of rare variants identified in AMD patients, and summarize the functional consequences of rare genetic variation in complement genes on the complement system. Finally, we discuss the relevance of this work in light of ongoing clinical trials that study the effectiveness of complement inhibitors against AMD.     

Significant photoreceptor rescue by treatment with a combination of antioxidants in an animal model for retinal degeneration

The purpose of this study was to investigate the presence of oxidative DNA damage in the photoreceptors of the rd1 mouse, an animal model for retinitis pigmentosa, and to determine if antioxidants could delay the progress of photoreceptor cell death. Retinas of rd1 mice and congenic wild type controls were examined for DNA oxidation and fragmentation. To study the rescue effect of antioxidants on retinal degeneration, rd1 retinas were studied in vitro and in vivo using lutein, zeaxanthin, alpha lipoic acid and reduced l-glutathione. For the in vitro studies, antioxidants were added to the culture medium. For the in vivo studies, postnatal day (PN3) pups of rd1 mice were fed antioxidants either individually or in combination and control rd1 animals received vehicle alone. Histological evaluation was performed using hematoxylin/eosin and avidin staining, as well as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Many of the rd1 rod photoreceptors at PN11 displayed oxidative DNA damage and TUNEL positive reaction which co-localized in a subset of rod photoreceptors. Avidin-labeled rod photoreceptors were more abundant than the TUNEL positive photoreceptors of the rd1 mouse, indicating that oxidative DNA damage precedes fragmentation. The number of TUNEL positive and avidin positive cells was considerably decreased upon treatment with the combination of the antioxidants. Rescue of rd1 photoreceptors was significant at PN18 and PN17, respectively, in the in vitro and in vivo studies. In conclusion individual antioxidants had no significant rescue effect but the combination slowed down the rd1 rod photoreceptor degeneration, indicating an additive or synergistic effect.     

IL-17A promotes microglial activation and neuroinflammation in mouse models of intracerebral haemorrhage

Microglial activation is an important contributor to neuroinflammation in intracerebral haemorrhage (ICH). IL-17A has been demonstrated to be involved in neuroinflammatory diseases such as multiple sclerosis. However, the exact mechanism of IL-17A mediated microglial activation in ICH has not been well identified. The purpose of this experiment is to investigate the role of IL-17A in ICH induced microglial activation and neuroinflammation. ICH mice were made by injection of autologous blood model. IL-17A expression and inflammatory factors in perihematomal region, and neurological function of mice were examined after ICH. In addition, IL-17A-neutralizing antibody was utilized to potentially prevent microglial activation and neuroinflammation in ICH mice. The expression of IL-17A, inflammatory factors and microglial activation in perihematomal region were significantly increased, and neurological function of mice was impaired after ICH. In addition, IL-17A Ab prevented ICH-induced cytokine expression, including TNF-α, IL-1β and IL-6, and downstream signaling molecules, including MyD88, TRIF, IκBα, and NF-κBp65 expression, and attenuated microglial activation. IL-17A Ab significantly reduced brain water content and improved neurological function of ICH mice. In conclusion, our results demonstrated that IL-17A was involved in ICH-induced microglial activation and neuroinflammation. IL-17A Ab might also provide a promising therapeutic strategy in ICH.     

Automatic segmentation of age-related macular degeneration in retinal fundus images

Every year an increasing number of people are affected by age-related macular degeneration (ARMD). Consequently, vast amount of information is accumulated in medical databases and manual classification of this information is becoming more and more difficult. Therefore, there is an increasing interest in developing automated evaluation methods to follow up the diseases. In this paper, we have presented an automatic method for segmenting the ARMD in retinal fundus images. Previously used direct segmentation techniques, generating unsatisfactory results in some cases, are more complex and costly than our inverse method. This is because of the fact that the texture of unhealthy areas of macula is quite irregular and varies from eye to eye. Therefore, a simple inverse segmentation method is proposed to exploit the homogeneity of healthy areas of the macula rather than unhealthy areas. This method first extracts healthy areas of the macula by employing a simple region growing method. Then, blood vessels are also extracted and classified as healthy regions. In order to produce the final segmented image, the inverse image of the segmented image is generated as unhealthy region of the macula. The performance of the method is examined on various qualities of retinal fundus images. The segmentation method without any user involvement provides over 90% segmentation accuracy. Segmented images with reference invariants are also compared with consecutive images of the same patient to follow up the changes in the disease.     

Genetically induced retinal degeneration leads to changes in metabotropic glutamate receptor expression

In the retina, neurotransmission from photoreceptors to ON-cone and rod bipolar cells is sign reversing and mediated by the metabotropic glutamate receptor mGluR6, which converts the light-evoked hyperpolarization of the photoreceptors into depolarization of ON bipolar cells. The Royal College of Surgeons (RCS) rat retina undergoes progressive photoreceptor loss due to a genetic defect in the pigment epithelium cells. The consequences of photoreceptor loss and the concomitant loss of glutamatergic input to second-order retinal neurons on the expression of the metabotropic glutamate receptor was investigated in the RCS rat retina from early stages of photoreceptor degeneration (P17) up to several months after complete rod and cone degeneration (P120). The expression of the gene encoding mGluR6 was studied by in situ hybridization in the retina, using an [(35)S]dATP-labeled oligonucleotide probe. In congenic control and RCS retina, we found mRNA expression of mGluR6 receptor only in the outer half of the inner nuclear layer (INL) on emulsion-coated retinal sections. Quantitative analysis of the hybridization signal obtained from the autoradiographic films revealed decreased expression levels of the mGluR6 mRNA at early stages of photoreceptor degeneration (P17). On the contrary, increased expression levels were observed at late stages of degeneration (P60 and P120) in RCS compared to congenic control retina. In conclusion, our data demonstrate that the metabotropic glutamate receptor-6 mRNA levels are altered in the young and adult RCS rat retina and suggest that the genetically induced degeneration of photoreceptors affects the expression of this receptor by the INL retinal neurons.     

A2E accumulation influences retinal microglial activation and complement regulation

Age-related macular degeneration is an outer retinal disease that involves aging and immune dysfunction. In the aging retina, microglia aggregate in the outer retina and acquire intracellular autofluorescent lipofuscin deposits. In this study, we investigated whether accumulation of A2E, a key bisretinoid constituent of ocular lipofuscin, alters the physiology of retinal microglia in pathologically relevant ways. Our findings show that sublethal accumulations of intracellular A2E in cultured retinal microglia increased microglial activation and decreased microglial neuroprotection of photoreceptors. Increased A2E accumulation also lowered microglial expression of chemokine receptors and suppressed microglial chemotaxis, suggesting that lipofuscin accumulation may potentiate subretinal microglial accumulation. Significantly, A2E accumulation altered microglial complement regulation by increasing complement factor B and decreasing complement factor H expression, favoring increased complement activation and deposition in the outer retina. Taken together, our findings highlight the role of microglia in the local control of complement activation in the retina and present the age-related accumulation of ocular lipofuscin in subretinal microglia as a cellular mechanism capable of driving outer retinal immune dysregulation in age-related macular degeneration pathogenesis.     

黑视素基因转染给光双极细胞部分恢复MNU诱导的视网膜感光细胞变性小鼠的视觉

 目的： 探索定向转染内源性光感受蛋白黑视素（melanopsin/opsin 4,Opn4）基因进入给光型双极细胞后,视网膜变性小鼠模型中视网膜神经元的光反应以及整体视觉行为的改变。方法：选用由甲基亚硝基脲（N-methyl-N-nitrosourea,MNU）诱导的成年CD1小鼠作为视网膜变性模型。于P0~P1 CD1乳鼠视网膜底注射Grm6-Opn4-GFP质粒,Grm6-GFP作为阴性对照。通过电转进行基因转染。术后5周对基因转染小鼠腹腔注射MNU诱导视网膜感光细胞变性,对照组注射生理盐水,共设计5个实验组：正常对照组（normal）、生理盐水Grm6-Opn4-GFP对照组（NS+melanopsin）、MNU诱导模型Grm6-Opn4-GFP治疗组（MNU+melanopsin）、MNU诱导模型Grm6-GFP对照组（MNU+GFP）和MNU诱导组（MNU）。诱导后连续7 d进行明暗箱测试,统计动物在暗箱中的活动时间比。随后进行视网膜电图（electroretinogram,ERG）测试,计算体现给光双极细胞光反应的b波峰值、潜伏期和反映视网膜神经节细胞（retinal ganglion cells,RGCs）光反应的明视负波反应（photopic negative response,PhNR）。利用免疫荧光法检测动物视网膜黑视素基因转导效果。结果：黑视素可以被定向转染进入视网膜给光双极细胞。明暗箱实验显示MNU诱导7 d后Grm6-Opn4-GFP转染的CD1小鼠滞留在黑箱的时间显著长于未转染组（P<0.05）,ERG测试显示Grm6-Opn4-GFP转染的CD1小鼠的b波也有明显恢复（P<0.05）。结论：定向转染内源性光感受蛋白黑视素基因进入给光型双极细胞可部分恢复视网膜变性模型动物视觉。     

rd1小鼠视网膜退变中期节细胞功能的变化

 目的:探讨视网膜色素变性模型rd1小鼠视网膜退变中期各类视网膜节细胞(retinal ganglion cells, RGCs)功能的变化情况。方法:运用多电极阵列(multi-electrode arrays,MEA)记录方法,记录视网膜退变中期(出生后20 d,P20)的rd1小鼠或正常对照小鼠视网膜中多个节细胞动作电位的发放,并比较自发发放和光反应特征等指标,评价幸存的节细胞功能变化。另外,采用免疫组化染色方法验证视网膜感光细胞的退化情况。结果:免疫组化的结果表明rd1小鼠视网膜感光层的厚度显著低于正常小鼠。根据节细胞光反应特性的不同,可以将其分成6类:ON sustained、ON transient、ON-OFF sustained、ON-OFF transient、OFF sustained和OFF transient RGCs,但OFF sustained RGCs所占比重极小(1.0%~3.1%)。rd1小鼠视网膜中保持光反应的节细胞比例显著低于正常小鼠。rd1小鼠节细胞的自发发放显著高于正常小鼠,而不同类型的节细胞变化情况有所不同。rd1小鼠视网膜各类节细胞的光反应强度及光敏感度均显著低于正常小鼠。结论:在rd1小鼠退变的中期,视网膜感光层明显退变;rd1小鼠退变中期的视网膜节细胞发生明显的功能退变,而且不同类型的节细胞变化情况有所不同。     

CFH基因R1210C突变与中国人老年黄斑变性的相关性研究

目的 补体因子H(complement factor H,CFH)基因R1210C突变与高加索人老年黄斑变性(age-related macular degeneration,AMD)显著相关.我们通过对中国大陆汉族人群进行病例-对照研究,评估CFH基因R1210C突变与中国人老年黄斑变性的相关性.方法 收集258例中国汉族人湿性AMD患者作为病例组,同时收集来自同一地区426名正常中国汉族人作为对照,应用直接测序法检测CFH基因中R1210C的突变情况,并比较病例组和对照组的差异.结果 258例中国人AMD患者和426名正常中国人的CFH基因中未发现R1210C突变.结论 中国人老年黄斑变性与CFH基因R1210C突变可能无关.     

Association between vascular endothelial growth factor gene polymorphisms and age-related macular degeneration in a Polish population

The pathogenesis of age-related macular degeneration (AMD) is thought to be determined by an array of environmental and genetic factors. The association of increased expression of vascular endothelial growth factor (VEGF) with AMD, especially the wet form of AMD, was reported in several studies. The VEGF gene is highly polymorphic and some of its polymorphisms may affect its expression. In our work, we searched for an association between the −460C> (rs833061) and −634G>C (rs2010963) polymorphisms of the VEGF gene and the occurrence of AMD and its dry and wet forms. We have chosen these polymorphisms because they were shown to be significant in other studies and we previously showed their association with diabetic retinopathy. A total of 401 individuals were enrolled in this study: 136 controls, and 88 patients with dry and 177 with wet AMD. The polymorphisms were determined with DNA from peripheral blood lymphocytes by allele-specific and restriction fragment length polymorphism polymerase chain reaction. The significance of the polymorphisms was assessed by multiple logistic regression, producing odds ratios (ORs) and 95% confidence intervals (CIs). We observed a weak association (OR 2.90) between AMD occurrence and the C/T genotype of the −460C>T polymorphism. An association (OR 3.77) between the C/T genotype of the −460C>T polymorphism and the occurrence of dry AMD was observed. The T/T genotype considerably lowered the risk of dry AMD (OR 0.19). Dry AMD was associated with the C/C genotype of the −634G>C polymorphism (OR 3.68). Another weak association (OR 2.63) was found between the C/T genotype of the −460C>T polymorphism and the occurrence of wet AMD. The occurrence of AMD was correlated with the presence of the combined C/T–G/G genotype of both polymorphisms (OR 2.41), whereas the T/T–G/G and T/T–G/C genotypes exerted a protective effect against the disease (OR 0.22 and 0.48, respectively). The presence of the C/T–G/G and C/T–C/C combined genotypes increased the risk of dry AMD (OR 2.08 and 3.77, respectively), whereas the presence of the T/T–G/G and T/T–G/C genotypes decreased the risk (OR 0.15 and 0.28, respectively). In the wet form of AMD, the combined genotype C/T–G/G slightly favored the disease (OR 2.61) and the T/T–G/G genotype had a protective effect (OR 0.25). Analysis of haplotypes of both polymorphisms yielded similar results for AMD in general as well as for the dry and wet forms of the disease: the CG haplotype favored both forms of AMD, whereas the TG haplotype protected against both forms of AMD. The results obtained indicate that the −460C>T and −634G>C polymorphisms of the VEGF gene may be associated with the dry and wet forms of AMD in a Polish population.     

Age-related macular degeneration and 5-year incidence of impaired activities of daily living

Objectives

We aimed to assess the prospective association between age-related macular degeneration (AMD) and impaired activities of daily living (ADL) among a large cohort of older adults.

Study design

Functional status was determined by the Older Americans Resources and Services ADL scale from 2002–2004 to 2007–2009 among 761 participants aged 60+ years. AMD was assessed from retinal photographs.

Results

After adjusting for age, sex, living status, self-rated poor health, smoking, body mass index, visual impairment, hypertension, diabetes, hospital admissions in the past year, walking disability, probable depression, mini-mental state examination scores, having any AMD or late AMD increased the risk of incident impaired total ADL 5 years later, odds ratio, OR 2.87 (95% confidence intervals, CI 1.44–5.71) and OR 12.95 (95% CI 3.78–44.35), respectively. Having any AMD increased the risk of developing instrumental ADL disability over the 5 years, multivariable-adjusted OR 2.06 (95% CI 1.11–3.83).

Conclusions

This study shows that the presence of AMD could independently signal an increased risk of functional disability, particularly in performing instrumental ADL tasks.     

杞菊地黄汤防治MNU诱导大鼠视网膜变性的早期效应

目的:观察杞菊地黄汤在N-甲基-N-亚硝脲(MNU)诱导的SD大鼠视网膜变性早期的拮抗效应。 方法:生后46 d的雌性SD大鼠30只随机分为3组(n=10)：药物组以杞菊地黄汤灌胃，正常组和模型组以等量蒸馏水灌胃，每日1次，连续4 d。用药后第5 d，药物组和模型组大鼠皮下注射MNU 40 mg/kg，正常组皮下注射等量生理盐水做对照。注射后12 h，处死大鼠，每组大鼠的左眼（10眼）用于透射电镜观察；右眼（10眼）用于苏木素-伊红(hematoxylin eosin,HE)染色观察并测量分析视网膜全层及外核层厚度，部分眼球行TUNEL法染色并计算细胞凋亡百分率。 结果:光镜下各组大鼠视网膜组织学未见明显改变，视网膜全层及外核层厚度组间比较均无显著差异（P>0.05）。TUNEL法染色见模型组和药物组外核层存在细胞凋亡，正常组未见。电镜下正常组和药物组光感受器细胞大小和核染色质分布均匀；模型组光感受器细胞开始变小，核染色质开始浓缩, 外节膜盘疏松，膜型不完整。 结论:杞菊地黄汤通过抑制细胞凋亡等选择性拮抗MNU对大鼠视网膜光感受器细胞的早期损伤。     

Genetic deficiency of complement factor H in a patient with age-related macular degeneration and membranoproliferative glomerulonephritis

Age-related macular degeneration (AMD) and membranoproliferative glomerulonephritis type II (MPGN2) are dense deposit diseases that share a genetic association with complement genes and have complement proteins as important components of the dense deposits. Here, we present the case of a 64-year-old smoker male who developed both AMD and MPGN2 in his late 50s. The patient presented persistent low plasma levels of C3, factor H levels in the lower part of the normal range and C3NeF traces. Genetic analyses of the CFH, CFB, C3, CFHR1-CFHR3 and LOC387715/HTRA1 genes revealed that the patient was heterozygote for a novel missense mutation in exon 9 of CFH (c.1292 G > A) that results in a Cys431Tyr substitution in SCR7 of the factor H protein. In addition, he was homozygote for the His402 CFH allele, heterozygote for the Ser69 LOC387715 allele, homozygote for the Arg32 (BFS) CFB allele, heterozygote for the Gly102 (C3F) C3 allele and carried no deletion of the CFHR1/CFHR3 genes. Proteomic and functional analyses indicate absence in plasma of the factor H allele carrying the Cys431Tyr mutation. As a whole, these data recapitulate a prototypical complement genetic profile, including a partial factor H deficiency and the presence of major risk factors for AMD and MPGN2, which support the hypothesis that these dense deposit diseases have a common pathogenic mechanism involving dysregulation of the alternative pathway of complement activation.