Role of retinal pigment epithelium in age-related macular disease: a systematic review.

Age-related macular disease (AMD) is a major cause of blindness and there is little treatment currently available by which the progress of the basic disorder can be modulated. Histological and clinical studies show that the major tissues involved are the outer retina, retinal pigment epithelium, Bruch's membrane and choroid. Because of a wide variation of phenotype from one case to another, it has been suggested that accurate phenotyping would be necessary for assessment of the effectiveness of treatment that is tissue-directed. However, based on findings from the study of human donor material and animal models of disease and of cell culture, it is concluded that retinal pigment epithelial dysfunction plays a central role in the disease process in most, if not all, cases of early AMD. The metabolism of phagosomal material, particularly lipids, and energy generation are interdependent, and dysfunction of both appears to be important in the genesis of disease. Evidence exists to suggest that both can be modulated therapeutically. These metabolic functions are amenable to further investigation in both the normal state and in disease. Once fully characterised, it is likely that treatment could be directed towards a limited number of functions in single tissue, thus simplifying treatment strategies.

Dexamethasone delivery to the ocular posterior segment by sustained-release Laponite formulation.

This paper presents a novel nanoformulation for sustained-release delivery of dexamethasone (DEX) to the ocular posterior segment using a Laponite (LAP) carrier-DEX/LAP 1:10 w w-1 formulation; 10 mg ml-1. In vivo ocular feasibility and pharmacokinetics after intravitreal (IV) and suprachoroidal (SC) administration in rabbit eyes are compared against IV administration of a DEX solution (1 mg ml-1). Thirty rabbit eyes were injected with the DEX/LAP formulation (15 suprachoroid/15 intravitreous). Ophthalmological signs were monitored at day 1 and at weeks 1-4-12-24 post-administration. Three eyes per sample time point were used to quantify DEX concentration using high-performance liquid chromatography-mass spectrometry. The ocular tissues' pharmacokinetic parameters (lens, vitreous humour, choroid-retina unit and sclera) were studied. DEX/LAP was well tolerated under both administration methods. Peak intraocular DEX levels from the DEX/LAP were detected in the vitreous humour after both deliveries soon after administration. The vitreous area under the curve was significantly greater after both DEX/LAP deliveries (IV: 205 968.47; SC: 11 442.22 ng g-1 d-1) than after IV administration of the DEX solution (317.17 ng g-1 d-1). Intravitreal DEX/LAP delivery extended higher vitreous DEX levels up to week 24 (466.32 ± 311.15 ng g-1). With SC delivery, DEX levels were detectable in the choroid-retina unit (12.04 ± 20.85 ng g-1) and sclera (25.46 ± 44.09 ng g-1) up to week 24. This study demonstrated the intraocular feasibility of both SC and IV administration of the DEX/LAP formulation. The LAP increased the intraocular retention time of DEX when compared with conventional solutions. DEX/LAP could be considered a biocompatible and useful sustained-release formulation for treating posterior-pole eye diseases.

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.

Aging results in iron accumulations in the non-human primate choroid of the eye without an associated increase in zinc, copper or sulphur.

We present further analyses of a previous experiment published in 2016 where the distribution, concentration and correlation of iron, zinc, copper and sulphur in the choroid of the eye in young and aged old world primates (Macaca fascicularis) was studied with synchrotron X-ray fluorescence with a 2 µm resolution. The results indicate that iron accumulates in hotspots in the choroid with age with fluorescence intensity ranging from 2- to 7-fold (1002-3752 ppm) the mean level in the choroidal stroma (500 ppm) and maximum iron levels in blood vessel lumina. Iron hotspots with iron ppm > 1000 preferentially contained Fe3+ as demonstrated by Perls staining. There was a strong spatial co-localisation and correlation between copper and zinc (Pearson's correlation coefficient 0.97), and both elements with sulphur in the choroid of young animals. However, these are reduced in the choroid of aged animals and lost in the iron hotspots. The lack of proportional co-distribution suggests that iron accumulation does not induce a concomitant increase in zinc, copper or zinc-, copper-metalloproteins. It is possible that the iron hotspots are ferritin or hemosiderin molecules loaded with Fe3+ in stable, insoluble, non-toxic complexes without a significant oxidative environment.

Role of Fibulins 2 and 5 in Retinal Development and Maintenance.

Fibulins 2 and 5 are part of a seven-member family of proteins integral to the retinal extracellular matrix. Our study aimed to further explore the roles of both fibulins in retinal function. We obtained knockout mouse models of both fibulins and performed immunohistochemistry, electroretinography, and histology to investigate the outcome of eliminating these proteins. Immunohistochemical analysis showed that both fibulins are localized to the RPE, choroid, and Bruch's membrane. Functional testing showed a significantly reduced scotopic A response at 1 month of age, when compared to their wild-type counterpart. This functional reduction remained constant throughout the age of the animal and only declined as a result of normal aging. The functional decline was associated with reduced number of photoreceptor cells. The results presented clearly demonstrate that fibulins 2 and 5, as extracellular proteins, are necessary for normal retinal development.

Ocular drug delivery targeted by iontophoresis in the suprachoroidal space using a microneedle.

Treatment of many posterior-segment ocular indications would benefit from improved targeting of drug delivery to the back of the eye. Here, we propose the use of iontophoresis to direct delivery of negatively charged nanoparticles through the suprachoroidal space (SCS) toward the posterior pole of the eye. Injection of nanoparticles into the SCS of the rabbit eye ex vivo without iontophoresis led to a nanoparticle distribution mostly localized at the site of injection near the limbus and <15% of nanoparticles delivered to the most posterior region of SCS (>9 mm from the limbus). Iontophoresis using a novel microneedle-based device increased posterior targeting with >30% of nanoparticles in the most posterior region of SCS. Posterior targeting increased with increasing iontophoresis current and increasing application time up to 3 min, but further increasing to 5 min was not better, probably due to the observed collapse of the SCS within 5 min after injection ex vivo. Reversing the direction of iontophoretic flow inhibited posterior targeting, with just ~5% of nanoparticles reaching the most posterior region of SCS. In the rabbit eye in vivo, iontophoresis at 0.14 mA for 3 min after injection of a 100 µL suspension of nanoparticles resulted in ~30% of nanoparticles delivered to the most posterior region of the SCS, which was consistent with ex vivo findings. The procedure was well tolerated, with only mild, transient tissue effects at the site of injection. We conclude that iontophoresis in the SCS using a microneedle has promise as a method to target ocular drug delivery within the eye, especially toward the posterior pole.

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies.

Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. STATEMENT OF SIGNIFICANCE: Age-related macular degeneration (AMD) is a devastating disease affecting more than 600 million people worldwide. Endothelial cells of the choriocapillaris (CECs) are among the first cell types lost in early AMD, and cell replacement therapy is currently the most promising option for restoring vision in patients with advanced AMD. In order to study CEC replacement strategies we have generated a 3D choroid scaffold using a novel decellularization method in human RPE/choroid tissue. To our knowledge, this is the first report describing decellularization of human RPE/choroid, as well as recellularization of a choroid scaffold with CECs. This work will aid in our development and optimization of cell replacement strategies using a tissue scaffold that is similar to the in vivo environment.

Identification of Apolipoprotein A-I as a Retinoic Acid-binding Protein in the Eye.

All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-(3)H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent Mr of 27,000 Da. Following purification and mass spectrometry, the Mr 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive Mr 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly up-regulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth.

CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP. METHODS: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR. RESULTS: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01). CONCLUSIONS: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.

[Alterations of retinal tissue induced by amyloid ß(1- 42) subretinal injection in mice].

OBJECTIVE: To investigate the alterations of retinal tissue induced by OAß(1-42) subretinal injection in normal C57BL/6N mice. METHODS: Experimental study. One hundred and twenty C57BL/6N mice aged 8 weeks participated in this study. Among them, 60 eyes received subretinal injection of OAß(1-42); another 60 received double distilled water as control. The concentration of OAß(1-42) was 0.312 5 mmol/L. We took fundus photograph and autofluorescence, electroretinogram(ERG), haematoxylineosin staining of retinal paraffin sections, ß-galactosidase staining of neural retinal flatmounts, immunofluorescence of senescence marker P16INK4a of RPE/choroidal flatmounts, Western blot and RT-PCR of senescence relative cytokines IL-6, TGF-ß1 before and after the injection. The data were analyzed by independent sample t-test. RESULTS: Comparing with controls, 1 week post injection, the experimental group eyes had atrophied area with high autofluorescence site, and showed evident decrease in amplitudes of a wave (39.94±7.75)µV comparing with the controls (225.27±28.94)µV(t=12.45, P<0.001) in scotopia ERG, so did the amplitude of b wave (185.55±4.62)µV comparing with the controls (873.78±43.80)µV(t=27.06, P<0.001), retinal structures appeared significant loss of retinal thickness (t=75.13, P<0.001) along with hypo- or hyperpigmentation; the experimental eyes had high frequency of blue-green deposits in retinal ß-gal staining; the green fluorescence sites of RPE/choroidal flatmounts were clear and bright, indicating higher expression of P16INK4a in the nucleus; these results were the same till 8 weeks post operation. Western blot and RT-PCR vertified conspicuous increases in expression of TGF-ß1, IL-6 in retinas of experimental eyes compared with the control ones 1 week post injection. CONCLUSIONS: OAß(1-42) subretinal injection could induce visual function deficiency and retinal tissue senescence. All the manifestations were AMD-like retinopathy, which implied that Aß(1-42) might be an essential clue in human AMD pathology.

Immunohistochemical expression of CD117 and vascular endothelial growth factor in retinoblastoma: possible targets of new therapies.

CD117 (C-kit) is thought to play an important role in tumourigenesis. There are limited data in the literature concerning C-kit expression in retinoblastoma. To date, no immunohistochemical studies have been performed to assess the possible association of C-kit with vascular endothelial growth factor (VEGF) in retinoblastoma. This study was designed to investigate C-kit and VEGF immunoexpression in retinoblastoma, their relationship with prognostic parameters as well as the correlation between them. A prospective immunohistochemical study was conducted on 56 retinoblastoma cases. Patients who had received preoperative chemotherapy were excluded. Positive C-kit and VEGF immunoreactivity was observed in 48.2% and 76.8% of retinoblastoma cases respectively. No C-kit immunostaining was seen in the adjacent uninvolved retina. However, VEGF expression was detected within its vasculature. Retinoblastomas with combined pattern of tumour growth revealed a highly significant positive C-kit expression (P = 0.002) compared to cases with endophytic or exophytic growths. Also, positive C-kit expression was statistically higher in cases with optic nerve invasion (P = 0.001) and choroidal invasion (P ≤ 0.01) compared to negative cases. A highly significant positive VEGF expression was detected in cases with optic nerve invasion (P = 0.013) compared to negative cases. Moreover, a highly significant positive correlation was detected between C-kit and VEGF expression (P = 0.006). C-kit is a feature of more aggressive retinoblastomas, with increased expression in tumours spreading beyond the retina. Moreover, VEGF is vastly expressed in retinoblastoma and is associated with optic nerve invasion. Both C-kit and VEGF may represent potential therapeutic targets for retinoblastomas.

Effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits.

The aim of the present study was to evaluate the effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits. New Zealand male albino rabbits (n 21) were divided into two groups: group 1 (G1; n 11), fed a hypercholesterolaemic diet, and group 2 (G2; n 10), fed a hypercholesterolaemic diet enriched with flaxseed flour. The serum concentrations of total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, TAG and fasting blood glucose were determined at the start of the experiment and on the day of killing (8th week). Choroid and sclera samples were subjected to haematoxylin-eosin (HE) staining and histomorphometric and immunohistochemical analyses with the anti-oxidised LDL antibody. Sensory retina samples were subjected to an immunohistochemical analysis with the primary monoclonal nitrotyrosine antibody. At the end of the experiment, a significant increase was observed in TC and LDL-C concentrations in G1 rabbits when compared with G2 rabbits (P= 0·008 and P= 0·02, respectively). HE staining revealed a significant increase in choroid-sclera complex thickness in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of choroid and sclera samples with the anti-oxidised LDL marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of sensory retina samples with the anti-nitrotyrosine marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P= 0·002). Flaxseed reduced the choroid-sclera complex thickness of diet-induced hypercholesterolaemic rabbits and the expression of oxidised LDL in the choroid-sclera complex as well as the expression of nitrotyrosine in the sensory retina.

The proteomics of drusen.

The formation of extracellular deposits known as drusen below the macular region of the retina correlates with increased risk of severe visual loss from age-related macular degeneration (AMD). Inflammation and complement dysregulation contribute to AMD progression; however, disease mechanisms remain incompletely defined. Multiple genetic and environmental factors influence AMD pathology, and although immune system processes play a central role, multiple molecular mechanisms appear to be involved. Drusen proteomics, including the analyses of constituent proteins, oxidative protein modifications, and pattern recognition receptors, provide a foundation for deciphering mechanisms of drusen biogenesis and AMD pathology.

Detection of choroid- and retina-antigen reactive CD8(+) and CD4(+) T lymphocytes in the vitreous fluid of patients with birdshot chorioretinopathy.

Birdshot chorioretinopathy (BSCR), a progressive form of non-infectious uveitis, is the strongest HLA-associated disease described to date, with >95% of the patients displaying HLA-A29. Since indirect evidence indicates the involvement of T cells in the etiopathology of the disease, we now isolated, cultured and analyzed the vitreous fluid-infiltrating T cells from two BSCR patients with respect to their phenotype, cytokine profile, clonal distribution and antigen specificity. Phenotypic analyses revealed the predominant presence of both CD4(+) and CD8(+) T cells in vitreous fluid. Further analyses on short term expanded and cloned T cells suggested that eye-infiltrating T cells generally displayed a Th1 like cytokine profile with secretion of high levels of IFN-γ and TNF-α. In one patient an oligoclonal CD4(+) and CD8(+) T cell infiltration, with a moderate to strongly skewed TCR Vß usage was suggestive for an antigen driven infiltration/expansion. Indeed, a number of intraocular CD4(+) and CD8(+) T cells responded to crude retinal and choroidal lysates. These results, which demonstrate for the first time the existence of eye-antigen-specific T cells in the vitreous fluid of BSCR patients, substantiate the current view on the role of eye-antigen specific T cells in the etiopathology of BSCR.

Elastic properties of human posterior eye.

This study examines the elastic properties of the human posterior retina, choroid, and sclera. Twenty-four human eyes from 30- to 74-year-old donors were obtained from an eye bank. Vertically and horizontally oriented tissue strips of the retina, choroid, and sclera (ideally n = 12 in each group) were harvested from the posterior eyes. Their thicknesses were estimated optically. The samples were stretched at 1 mm/s in 37°C saline. Stress and strain were obtained from the mechanical tests, and then the transition stress, transition strain, toe modulus, and heel modulus were calculated. Statistical analysis was performed for comparison between groups. Linear regression analyses were used to explore the relationship between the mechanical parameters and age. We found that the stress-strain relationship of the retina, choroid, and sclera were nonlinear. Except for the retinal transition strain (p = 0.0124), no statistical difference was found between the vertical and horizontal meridian in the mechanical parameters (p > 0.05). Furthermore, weak relationship was observed between some of the mechanical parameters and the donors' age. Our results suggest that there is significant anisotropy in the retina, and mechanical properties of each layer may change with age.

Interleukin-17 production and T helper 17 cells in peripheral blood mononuclear cells in response to ocular lysate in patients with birdshot chorioretinopathy.

PURPOSE: To determine the cytokine response to ocular lysates of peripheral blood mononuclear cells (PBMCs) from patients with birdshot chorioretinopathy (BSCR). METHODS: In the PBMCs of 19 patients with BSCR, T cell cytokine production in response to human retina and choroid lysates was analyzed with flow cytometry and compared to the responses against skin lysates. Five patients had active disease and had not yet been treated (naïve to systemic therapy); 14 patients had either immunomodulatory therapy (IMT) or inactive disease (referred as inactive/IMT). The PBMCs of 11 HLA-A29-positive healthy individuals were used as controls. RESULTS: The levels of interleukin-17 (IL-17) in supernatant of cultures stimulated with retina lysate were higher in patients with active BSCR compared to the HLA-A29 positive controls. The levels of other T cell cytokines (IL-10 and interferon-γ [IFN-γ]) in PBMC cultures did not change significantly after stimulation with ocular lysate. The frequency of CD4(+) IL-17(+) (T helper 17 [Th17]) T cells but not of CD4(+) IFN-γ (Th1) T cells was elevated in the PBMCs of patients with active BSCR stimulated by retina lysates compared to skin lysates. CONCLUSIONS: Our data demonstrate that PBMCs exhibit an IL-17-mediated immune response to retina lysate in patients with active disease naïve to systemic therapy. This is accompanied by the enrichment of IL-17-producing CD4(+) T cells. These findings support the current concept of chronic Th17-cell mediated inflammation and provide evidence that links the Th17 signatures to ocular-specific immune responses in BSCR.

Mapping the differential distribution of proteoglycan core proteins in the adult human retina, choroid, and sclera.

PURPOSE: To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera. METHODS: Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections. RESULTS: The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera. CONCLUSIONS: A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease.

Intraocular distribution of melanin in human, monkey, rabbit, minipig and dog eyes.

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity.

A low zinc diet leads to loss of Zn in melanosomes of the RPE but not in melanosomes of the choroidal melanocytes.

Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.06 at% (choroid), respectively. For ZD-rats the Zn mole fraction of these granules was almost unchanged in the choroid but decreased and was below the detection limit (0.02 at%) in the RPE. ZD-rats produced also giant melanosomes (diameter 1-3 µm) in the choroid and greatly increased amounts of RPE lipofuscin. The giant melanosomes contained about six times as much Cu as control melanosomes. Lipofuscin granules were identified by AEM and could be clearly distinguished from melanosomes by their chemical composition. Thus, changes of the ultrastructure and transition metal storage of melanosomes due to a ZD can be directly traced using AEM.

Quantitative chemical analysis of ocular melanosomes in stained and non-stained tissues.

Energy-filtered Analytical Electron Microscopy (AEM) was used to image the ultrastructure and determine quantitatively the chemical composition of rat melanosomes of the choroid and the Retinal Pigment Epithelium (RPE). For the first time, the effect of staining in elemental analysis of melanosomes was investigated. Detection limits and accuracies of the applied methods were determined. Compared to previous work applying only quantitative Energy Dispersive X-ray microanalysis (EDX) in the TEM (Eibl, O., et al., 2006. Micron 37, 262), here we present a combined quantitative EDX and Electron Energy Loss Spectroscopy (EELS) analysis, including N. This yields the fraction of eumelanin and pheomelanin in melanosomes by the S/N mole fraction ratio. Melanosomes of the sepia ink sac, used as eumelanin standard, showed an S/N mole fraction ratio of <0.004. Thus, they consist primarily of eumelanin as reported by degradation analysis. In contrast, melanosomes of the rats contained mixed melanin with significant amounts of pheomelanin (S/N 0.02) in the RPE and the choroid. Consistent with the previous publication, it was shown that oxygen mole fractions are especially large in melanosomes (7-10 at.%) compared to other cell compartments, e.g. 2-4 at.% oxygen in the cytoplasm. In the melanosomes of non-stained tissue, the oxygen mole fraction clearly correlated with the Ca mole fraction. EDX spectra used for quantitative analysis had about 15,000 net counts under the oxygen peak, which is necessary to obtain (i) a small statistical error for oxygen and (ii) optimum minimum detectable mole fractions for S, Ca and transition metals. The precise determination of the oxygen mole fraction in melanosomes is important for understanding metabolism. Therefore, a detailed analysis was carried out on the possible errors affecting quantification. While O, S, and N mole fractions yielded similar results in stained and non-stained ocular melanosomes of rats, transition metals can only be determined reliably in non-stained tissues. High-precision EDX analysis of melanosomes yielded minimum detectable mole fractions of less than 0.04 at.% for Cu and Zn, these elements were present in melanosomes with mole fractions of about 0.3 at.% and 0.1at.%, respectively. Zn is of great importance for metabolism and for age related macular degeneration. Its mole fraction in melanosomes of rats is large enough to be detected and to be quantitatively analyzed by EDX spectroscopy. Ultrastructural information can now be correlated to the elemental composition. This is important to better understand the physical and chemical properties of melanosomal metabolism and turnover.