Showing papers in "Molecular Vision in 2008"

Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration.

Abstract: Purpose Our objective was to comprehensively assess the nature and chronology of neural remodeling in retinal degenerations triggered by light-induced retinal damage (LIRD) in adult albino rodents. Our primary hypothesis is that all complete photoreceptor degenerations devolve to extensive remodeling. An hypothesis emergent from data analysis is that the LIRD model closely mimics late-stage atrophic age relared macular degeneration (AMD).

Oxygen and blood flow: players in the pathogenesis of glaucoma

Abstract: The increase of IOP in POAG is due an increased resistance of aqueous outflow through the trabecular meshwork (TM). The exact mechanisms leading to the corresponding changes in the TM are not yet known. We know, however, that all risk factors for arteriosclerosis are also risk factors for an increase in IOP. RESULTS: The association between IOP increase and these factors is relatively weak but nevertheless significant. Similar to the pathogenesis of arteriosclerosis, oxidative stress plays a role in the development of TM damage. Even less is known about the pathogenesis of glaucomatous optic neuropathy (GON). Obviously the risk factors for arteriosclerosis play a role via increasing the IOP. When corrected for IOP, however, these factors only play a minor role. In contrast, factors associated with disturbed autoregulation, in particular a systemic primary vascular dysregulation (PVD), increase the risk for GON. This is best observed in normal tension glaucoma patients. An insufficient autoregulation increases the chance for an unstable ocular perfusion and thereby an unstable oxygen supply. This, in turn, leads to oxidative stress. The concentration of superoxide (O(2)(-)) within the axons of the optic nerve head increases. If neighboring astrocytes are activated, either by mechanical or by ischemic stress, in excess produced nitric oxide (NO) molecules diffuse also into the axons and fuse with oxygen. The resulting peroxynitrat (ONOO(-)) diffuses within the axons towards the retina and the lateral geniculate nucleus and induces apoptosis.

Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential.

Abstract: Purpose Safety and efficiency are critical for successful gene therapy. Adeno-associated viral (AAV) vectors are commonly used for gene transfer in both human and animal studies. However, administration of AAV vectors can lead to development of neutralizing antibodies against the vector capsid, thus decreasing the efficiency of therapeutic gene transfer and preventing effective vector readministration. We investigated immune responses to different routes of ocular administration and readministration of AAV vectors, and the effect of previous exposure of AAV vector in one eye on the transduction efficacy of subsequent intraocular AAV-mediated gene delivery to the partner eye.

Mesenchymal cells from limbal stroma of human eye.

Abstract: Purpose Mesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye. Methods Fresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco's Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT-PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium. Results Spindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-L(CD105, CD106, CD54, CD166, CD90, CD29, CD71, pax -6 +/ p75, SSEA1, Tra-1-61, Tra-1-81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -). The profile was further confirmed by RT-PCR. These cells also showed differentiation into adipocytes and osteocytes. Conclusions We demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.

Investigation of the human tear film proteome using multiple proteomic approaches

Abstract: Purpose The purpose of this work was to examine the tear film proteome using a combination of one-dimensional (1D) and two dimensional (2D) gel electrophoresis and mass spectrometry-based techniques and to explore the effect of the tear collection methods on the tear proteome.

Crystallin gene mutations in Indian families with inherited pediatric cataract

Abstract: Purpose: Pediatric cataract is the most common form of treatable childhood blindness and is both clinically and genetically heterogeneous. Autosomal dominant and recessive forms of cataract have been reported to be caused by mutations in 22 different genes so far. Of the cataract mutations reported to date, about half the mutations occur in crystallins, a quarter of the mutations in connexins, and the remainder is evenly divided between intrinsic membrane proteins, intermediate filament proteins, and transcription factors. This study is aimed at identification of the spectrum and frequency of crystallin gene mutations in cataractous patients in an Indian population. Methods: Genetic analysis was extended to screen the entire coding region of the CRYAA, CRYAB, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, and CRYGS genes using single stranded conformational polymorphism (SSCP) analysis as a screening technique followed by direct sequencing of all subjects that displayed an electrophoretic shift. Results: This report describes the first simultaneous mutation analysis of 10 crystallin genes in the same population, represented by 60 south Indian families. The analysis allowed the identification of causative mutations in 10 of the families (three novel and six reported). This includes six missense mutations (CRYAA-R12C, R21W, R54C, CRYAB- A171T, CRYGC-R168W, CRYGS- S39C), two nonsense mutations (CRYBB2- Q155X, CRYGD- R140X), and one splice mutation, which was identified in two families (CRYBA1-IVS3+1G>A). Conclusions: Crystallin mutations are responsible for 16.6% of the inherited pediatric cataract in this population. As causative mutations have not been found in many of the families analyzed, this study suggests the presence of further novel genes or sequence elements involved in the pathogenesis of cataract in these families.

The EPHA2 gene is associated with cataracts linked to chromosome 1p.

Abstract: PURPOSE Cataracts are a clinically and genetically heterogeneous disorder affecting the ocular lens, and the leading cause of treatable vision loss and blindness worldwide. Here we identify a novel gene linked with a rare autosomal dominant form of childhood cataracts segregating in a four generation pedigree, and further show that this gene is likely associated with much more common forms of age-related cataracts in a case-control cohort. METHODS Genomic DNA was prepared from blood leukocytes, and genotyping was performed by means of single nucleotide polymorphism (SNP) markers, and short tandem repeat (STR) markers. Linkage analyses were performed with the GeneHunter and MLINK programs, and association analyses were performed with the Haploview and Exemplar programs. Mutation detection was achieved by PCR amplification of exons and di-deoxy cycle-sequencing. RESULTS Genome-wide linkage analysis with SNP markers, identified a likely disease-haplotype interval on chromosome 1p (rs707455-[approximately 10 Mb]-rs477558). Linkage to chromosome 1p was confirmed using STR markers D1S2672 (LOD score [Z]=3.56, recombination distance [theta]=0), and D1S2697 (Z=2.92, theta=0). Mutation profiling of positional-candidate genes detected a heterozygous transversion (c.2842G>T) in exon 17 of the gene coding for Eph-receptor type-A2 (EPHA2) that cosegregated with the disease. This missense change was predicted to result in the non-conservative substitution of a tryptophan residue for a phylogenetically conserved glycine residue at codon 948 (p.G948W), within a conserved cytoplasmic domain of the receptor. Candidate gene association analysis further identified SNPs in the EPHA2 region of chromosome 1p that were suggestively associated with age-related cataracts (p=0.007 for cortical cataracts, and p=0.01 for cortical and/or nuclear cataracts). CONCLUSIONS These data provide the first evidence that EPHA2, which functions in the Eph-ephrin bidirectional signaling pathway of mammalian cells, plays a vital role in maintaining lens transparency.

Effect of circulation on the disposition and ocular tissue distribution of 20 nm nanoparticles after periocular administration

Abstract: Purpose Our previous studies indicated that while 20 nm particles are rapidly cleared from the periocular space of the rat following posterior subconjunctival injection, 200 nm particles persisted for at least two months. To understand faster clearance of 20 nm particles, the purpose of this study was to determine transscleral permeability and in vivo disposition in the presence and absence of circulation. Further, it was the purpose of this study to simulate sustained retinal drug delivery after periocular administration of rapidly cleared and slowly cleared nanoparticles.

The application of in vivo laser confocal microscopy to the diagnosis and evaluation of meibomian gland dysfunction

Abstract: Purpose: To evaluate the morphological changes of the meibomian glands (MG) in patients with meibomian gland dysfunction (MGD) compared to normal subjects by in vivo confocal microscopy and to investigate the relation of these changes to the clinical ocular surface findings and tear functions. Methods: Twenty MGD patients and 15 normal subjects were recruited into this prospective study. Patients and controls underwent slit lamp examinations, tear film break-up time (BUT) measurements, fluorescein and Rose-Bengal stainings, Schirmer test I without anesthesia, tear evaporation rate assessment (TEROS), tear film lipid layer interferometry (DR-1), transillumination of the lids (meibography), MG expressibility test, and in vivo laser confocal microscopy of the lids (HRTII-RCM). Results: The BUT, DR-1 tear film lipid layer interferometry grades, fluorescein and Rose-Bengal staining scores, MG drop out grade in meibography, and MG expressibility grades were significantly worse in MGD patients compared to normal controls (p<0.05). The severity of both MG dropout and MG expressibility related significantly with the BUT, DR-1 grades, and TEROS (p<0.05). The mean density of acinar units of MGs as measured by HRTII-RCM was significantly lower in MGD patients (47.6±26.6/mm 2 ) than in control subjects (101.3±33.8/mm 2 ; p<0.05). The mean acinar unit diameter as determined by HRTII-RCM was significantly larger in MGD patients (98.2±53.3 μm) than in controls (41.6±11.9 μm; p<0.05). Both the density and diameter of MG acinar units related significantly with the severity of MG dropout and MG expression grades (p<0.05). Conclusions: In vivo confocal microscopy can effectively demonstrate the morphological changes of the MG in patients with MGD. Glandular acinar density and acinar unit diameter seemed to be promising new parameters of in vivo confocal microscopy, which is significantly related to the clinical ocular surface and tear function findings of MGD. Meibomian glands (MG) are holocrine lipid excreting glands embedded in the tarsal plate of the upper and lower lids. Each MG comprises multiple acini connected by a long common central duct running throughout the entire length of the gland. Epithelial cells comprising the acini synthesize and release lipids into the central ducts, which are then excreted onto the ocular surface. Lipids excreted by MGs form the superficial layer of the tear film and are thought to retard tear evaporation and function as lubricants for the eyelids during blinking [1].

Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid.

Abstract: Purpose: In the central nervous system (CNS), increased mitochondrial DNA (mtDNA) damage is associated with aging and may underlie, contribute to, or increase the susceptibility to neurodegenerative diseases. Because of the focus on the retinal pigment epithelium (RPE) and choroid as tissue relevant to age-related macular degeneration (AMD), we examined young and aged RPE and choroid, harvested from rodent eyes, for DNA damage and for changes in selected DNA repair enzymes. Methods: Immunohistochemical labeling and quantitative ELISA for the oxidative DNA damage marker, 8-hydroxy-2’deoxy-guanosine (8-OHdG), were measured in young and aged rodent RPE and choroid. mtDNA and nuclear DNA (nDNA) damage was determined by quantitative polymerase chain reaction (PCR) by comparing the relative amplification of small and large DNA fragments. Expression of several DNA repair enzymes was measured using real-time quantitative reverse transcription -PCR (qRT–PCR) and immunoblot. Results: Immunohistochemical labeling for 8-OHdG increased in aged rodent RPE and choroid. Quantitative ELISA confirmed increased levels of 8-OHdG. Measurements of nDNA and mtDNA lesions indicated that DNA damage is primarily in mtDNA in aged RPE and choroid. Using qRT–PCR, we found that gene expression of DNA repair enzymes, 8-oxoguanine-DNA glycosylase 1 (OGG1), mutY homolog (MYH), and thymine DNA glycosylase were decreased in an age-dependent pattern in RPE and choroid. However, endonuclease III homolog 1 was not significantly changed in aged RPE and choroid. Using immunoblots, we found that protein levels of OGG1 and MYH were decreased in aged RPE and choroid. Conclusions: Our results show that there is increased mtDNA damage in aged RPE and choroid, which is likely due to decreased DNA repair capability. mtDNA damage in the RPE and choroid may be a susceptibility factor that underlies the development of AMD.

Epigallocatechin gallate protects against oxidative stress-induced mitochondria-dependent apoptosis in human lens epithelial cells.

Abstract: Purpose: Oxidative stress has long been recognized as an important mediator of apoptosis in lens epithelial cells and also plays an important role in the pathogenesis of cataracts (-)-Epigallocatechin gallate (EGCG), the most abundant component in green tea, has potent antioxidant activity The goals of this study were to determine the protective effect of EGCG against H 2 O 2 -induced apoptotic death and the possible mechanisms involved in human lens epithelial (HLE) cells Methods: HLEB-3, a human lens epithelial cell line, was exposed to various concentrations of H 2 O 2 and EGCG and subsequently monitored for cell death by the MTT assay and flow cytometric analysis using Annexin V and PI The effect of EGCG in protecting HLE cells from cell death was determined by various assays after the cells were exposed to H 2 O 2 The ability of EGCG to block the accumulation of intracellular reactive oxygen species and the loss of mitochondrial membrane potential (∆ψm) induced by H 2 O 2 was examined with dichlorofluorescein (DCF) fluorescence and 5,5',6,6'tetrachloro-1,1',3,3'-tetrathylbenzimidazol carbocyanine iodide (JC-1) The expression of cytochrome c, caspase-9, caspase3, and Bcl-2 family proteins was measured by western blotting The changed expression of the mitogen activated protein kinase (MAPK) and Akt pathways was also detected by western blot Results: In the present study, EGCG protected against cell death caused by H 2 O 2 in HLEB-3 cells EGCG reduced the H 2 O 2 -induced generation of reactive oxygen species (ROS), the loss of mitochondrial membrane potential ( ∆ψm), and the release of cytochrome c from the mitochondria into the cytosol EGCG inhibited the H 2 O 2 -stimulated increase of caspase9 and caspase-3 expression and the decrease of the Bcl-2/Bax ratio Moreover, EGCG attenuated the reduced activation and expression of ERK, p38 MAPK, and Akt induced by H 2 O 2 Conclusions: These findings suggest that EGCG protects HLE cells from the mitochondria-mediated apoptosis induced by H 2 O 2 through the modulation of caspases, the Bcl-2 family, and the MAPK and Akt pathways

Multiplex bead analysis of vitreous and serum concentrations of inflammatory and proangiogenic factors in diabetic patients.

Abstract: Purpose: To investigate the role of inflammatory and angiogenic factors in the pathogenesis of diabetic retinopathy, we determined, in diabetic patients and controls, vitreous and serum concentrations of interferon-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated upon activation, normal T-expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF). Methods: We recruited 36 probands with type 2 diabetes mellitus (15 noninsulin-dependent and 21 insulin-dependent) and 69 normal controls. Using Cytometric Bead Array Technology, we measured vitreous and serum concentrations of IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, and VEGF. Results: In diabetic patients the mean vitreous levels of IP-10, MCP-1 and VEGF were significantly higher compared normal controls. [IP-10 (pg/mL) 254.84 +/−311.67 versus 78.90 +/− 67.94 (p<0.001); MCP-1 (pg/mL) 1127.14 +/− 738.91 versus 700.80 +/−419.21 (p=0.002); VEGF (pg/mL) 954.98 +/− 2315.09 versus 37.90 +/− 28.51(p<0.001)]. Vitreous levels of VEGF correlated with vitreous levels of both IP-10 and MCP-1 (p<0.05). MIP-1β, RANTES, and VEGF mean serum levels were significantly raised in diabetic probands while IP-10, MCP-1, and MIP-1α serum levels showed no significant elevation compared to controls [IP-10 (pg/mL) 346.20 +/− 287.36 versus 328.74 +/−352.35 (p=0.88); MCP-1(pg/mL) 133.10 +/− 89.10 versus 141.47 +/− 222.15 (p=0.50); MIP-1β (pg/mL) 184.40 +/− 100.20 versus 139.56 +/− 151.38 (p=0.003); RANTES (pg/mL) 51336.23 +/− 19940.31 versus 33629.2 +/− 33301.0 (p=0.002); VEGF (pg/mL) 304.88 +/− 257.52 versus 154.45 +/− 114.78 (p<0.001)]. Conclusions: Our results suggest that in diabetics, there is an upregulation of IP-10, MCP-1, and VEGF in the vitreous and an upregulation of MIP-1β, RANTES, and VEGF in the serum. These findings support the concept of an angiogenic and inflammatory element in the development of diabetic retinopathy.

Topographical characterization of cone photoreceptors and the area centralis of the canine retina.

Abstract: Purpose: The canine is an important large animal model of human retinal genetic disorders. Studies of ganglion cell distribution in the canine retina have identified a visual streak of high density superior to the optic disc with a temporal area of peak density known as the area centralis. The topography of cone photoreceptors in the canine retina has not been characterized in detail, and in contrast to the macula in humans, the position of the area centralis in dogs is not apparent on clinical funduscopic examination. The purpose of this study was to define the location of the area centralis in the dog and to characterize in detail the topography of rod and cone photoreceptors within the area centralis. This will facilitate the investigation and treatment of retinal disease in the canine.Methods: We used peanut agglutinin, which labels cone matrix sheaths and antibodies against long/medium wavelength (L/M)- and short wavelength (S)-cone opsins, to stain retinal cryosections and flatmounts from beagle dogs. Retinas were imaged using differential interference contrast imaging, fluorescence, and confocal microscopy. Within the area centralis, rod and cone size and density were quantified, and the proportion of cones expressing each cone opsin subtype was calculated. Using a grid pattern of sampling in 9 retinal flatmounts, we investigated the distribution of cones throughout the retina to predict the location of the area centralis.Results: We identified the area centralis as the site of maximal density of rod and cone photoreceptor cells, which have a smaller inner segment cross-sectional area in this region. L/M opsin was expressed by the majority of cones in the retina, both within the area centralis and in the peripheral retina. Using the mean of cone density distribution from 9 retinas, we calculated that the area centralis is likely to be centered at a point 1.5 mm temporal and 0.6 mm superior to the optic disc. For clinical funduscopic examination, this represents 1.2 disc diameters temporal and 0.4 disc diameters superior to the optic disc.Conclusions: We have described the distribution of rods and cone subtypes within the canine retina and calculated a predictable location for the area centralis. These findings will facilitate the characterization and treatment of cone photoreceptor dystrophies in the dog.

γ-Synuclein as a marker of retinal ganglion cells

Abstract: PURPOSE Previous studies have described gamma-synuclein as a protein highly expressed in retinal ganglion cells (RGCs), and a loss of RGCs correlates with a downregulation of gamma-synuclein gene expression in glaucoma. Here we asked whether gamma-synuclein expression in the retina can be considered a specific marker of RGCs. METHODS gamma-Synuclein expression was examined with immunohistochemistry in retinal sections from normal and glaucomatous human eyes. Primary cultures of RGCs from Sprague-Dawley rats purified by sequential immunopanning using a monoclonal antibody to Thy1-1, cultures of A7 immortalized optic nerve astrocytes from newborn rats, and the immortalized RGC-5 cell line were studied using immunofluorescence and quantitative RT-PCR. RESULTS gamma-Synuclein was highly expressed in RGCs in the human retina and was localized in cytoplasm adjacent to the RGC nuclear marker, Brn-3a. Axons of RGCs were immunopositive for gamma-synuclein in the nerve fiber layer (NFL), the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings were likewise immunopositive, whereas in the retina of patients with retinoblastoma, NFL staining appeared reduced. In primary rat RGCs and in immortalized RGC-5 cultures, gamma-synuclein was localized predominantly in the perinuclear area and in cell processes. Among rat retinal cells in culture, all Brn-3a positive cells were stained with a gamma-synuclein antibody; rare gamma-synuclein-positive cells were not stained by the Brn-3a antibody. CONCLUSIONS gamma-Synuclein is selectively and abundantly expressed in human RGCs in vivo, primary rat RGCs in vitro, and immortalized RGC-5 cells. In pathology, gamma-synuclein abundance may vary between RGC somas and axons. Coincident Brn-3a and gamma-synuclein expression suggests that strong gamma-synuclein expression can be considered a marker of RGCs. Future translational approaches might include using a gamma-synuclein promoter for the specific delivery of siRNA or therapeutic proteins to RGCs.

Analysis of LOXL1 polymorphisms in a United States population with pseudoexfoliation glaucoma.

Abstract: Purpose To identify if recently described LOXL1 (lysyl oxidase-like 1) polymorphisms are associated with pseudoexfoliation glaucoma (XFG) in a United States (U.S.) Caucasian patient population. Methods Individuals with XFG were identified using standard clinical examination techniques. TaqMan allelic discrimination assays were used to genotype 13 single nucleotide polymorphisms (SNPs) that tag LOXL1 in Caucasian individuals. The coding region of exon 1 that includes the previously associated SNP, rs1048661, was sequenced. Allele and genotype frequencies were compared between cases and unrelated controls. Results Fifty affected individuals and 235 control individuals were recruited into this study. We replicated the previously reported association of three SNPs (rs1048661, rs2165241, and rs3825942) in our independent XFG population (single SNP p-values were 0.001-0.02). The risk alleles at these three and several other intragenic SNPs are part of an extended XFG-associated LOXL1 haplotype with a frequency of 32.0% in XFG patients and 21.6% in controls. Conclusions We have performed an analysis of LOXL1 and XFG in a United States patient population and have confirmed the strong association previously reported for Icelandic and Swedish samples. However, due to the high frequency of risk alleles in non-XFG individuals, this association should not form the basis of a diagnostic test for XFG. It is likely that additional genetic or environmental factors modulate the penetrance of LOXL1 susceptibility alleles.

Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush.

Abstract: PURPOSE: A time-course analysis of gene regulation in the adult rat retina after intraorbital nerve crush (IONC) and intraorbital nerve transection (IONT). METHODS: RNA was extracted from adult rat ...

Association of non-synonymous single nucleotide polymorphisms in the LOXL1 gene with pseudoexfoliation syndrome in India

Abstract: Pseudoexfoliation syndrome (XFS) is an age-related,systemic, elastic microfibrillopathy affecting both intraocularand extraocular tissues [1]. In the eye, XFS is characterizedby the presence of fibrogranular extracellular debris in theanterior segment, which is composed of a complexglycoprotein–proteoglycan structure having epitopes of abasement membrane, elastic fiber system, and components ofelastic microfibrils [ 2]. The average worldwide prevalence ofXFS is 10%–20% of the general population over the age of 60years. However, studies have shown much higher prevalencein certain populations like Scandinavian countries and Greece[3-5].Glaucoma, the second most common cause of blindness,is a heterogeneous group of disorders characterized by opticnerve damage [ 6]. XFS is the most common identifiable causeof secondary glaucoma such as pseudoexfoliation glaucoma(XFG), which is due to the accumulation of the exfoliativematerial [ 7]. The risk of XFS converting to XFG over a periodof 15 years is about 60% [8,9].Linkage studies on a large Finnish family showing anautosomal dominant mode of inheritance for XFS traitidentified a locus, 18q12.1–21.33, with a two point LOD scoreof 3.45 and a multipoint LOD score of 4.2 and few other loci

Recombinant anti-vascular endothelial growth factor fusion protein efficiently suppresses choridal neovasularization in monkeys.

Abstract: Purpose KH902 is a fusion protein which combines ligand binding elements taken from the extracellular domains of vascular endothelial growth factor (VEGF) receptors 1 and 2 and the Fc portion of IgG1. This study is designed to examine the inhibitory effect of KH902 in the choroidal neovascularization (CNV) monkey model.

Proteomic analysis of aqueous humor from patients with myopia.

Abstract: Purpose: The mechanism of axial elongation in the myopic eyeball remains to be elucidated. It is known that the expression profile for some proteins in the aqueous humor (AH) changes in some diseases. Accordingly, determinations of these AH proteins may serve to understand their potential role in this pathogenesis. To identify the possible mechanism in myopia development, a proteomic analysis of the AH composition from high myopic eyes (patients) was performed and compared with that of the AH composition from non-myopic cataract eyes (controls). Methods: Total protein concentration in AH was determined by the Bradford method, and separation profiles were analyzed by two-dimensional (2D) gel electrophoresis. Protein in gel was determined by silver stain, and the separation profiles were analyzed to assess spot density changes between myopia and non-myopia patients. These spots in gel were isolated and identified by mass spectrometry. Results: The total protein concentration in AH with high myopia was significantly greater than that of non-myopia. A total of six spots were significantly increased in 2D gels from high myopia. The spots were derived from albumin, transthyretin, and a vitamin D-binding protein. Conclusions: The protein composition in AH was significantly different between myopia and non-myopia. The identified proteins could be a potential biomarker for high myopia development and may play a role in the mechanisms of myopia ocular axial elongation. Myopia is the most common eye disease in the world with substantial social, educational, and economic impact [1]. The prevalence of high myopia (usually defined as eyes with ≥6 diopters [D] of myopia or ≥26 mm in length) in world populations has been estimated to be between 0.3% and 9.6%. However, this percentage has recently been shown to be as high as 16% in certain young Asian populations, and evidence suggests that this prevalence is increasing [2]. It is well documented that individuals with high myopia have a greatly increased risk of ocular pathology such as retinal detachment, macular degeneration, and glaucoma [3,4]. However, the pathogenesis of high myopia is not well understood. Previous studies have focused upon investigating changes in the sclera, retina, and choroid in high myopia, but few studies have been directed toward assessing the aqueous humor (AH) in these patients. It was reported that the pigment epithelium–derived factor (PEDF) is elevated in patients with high myopia [5]. Such findings suggest that some proteins in AH might be involved in the development of high myopia.

Evaluation of LOXL1 gene polymorphisms in exfoliation syndrome and exfoliation glaucoma.

Abstract: Purpose To evaluate genetic susceptibility of lysyl oxidase-like 1 (LOXL1) gene polymorphisms to exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) in a case-control cohort of American and European patients.

Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.

Abstract: Purpose To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.

Association of CFH, LOC387715, and HTRA1 polymorphisms with exudative age-related macular degeneration in a northern Chinese population.

Abstract: Purpose: Variants in complement factor H (CFH), the hypothetical LOC387715, and the high-temperature requirement A-1 (HTRA1) genes have been reported to be associated with age-related macular degeneration (AMD). The purpose of this study was to investigate the association of reported common single-nucleotide polymorphisms (SNPs) in CFH, LOC387715, and HTRA1 with exudative AMD in a northern Chinese population. Methods: A cohort of 121 unrelated patients with exudative AMD and 132 control subjects were enrolled in this study. Genomic DNA was extracted from blood leukocytes. Genotyping for SNPs rs1061170:T>C in CFH (Y402H), rs10490924:G>T in LOC387715 (A69S), and rs11200638:G>A in the promoter of HTRA1 was performed using a polymerase chain reaction (PCR) method followed by allele-specific restriction enzyme digestion and direct sequencing. Results: The Y402H variant in CFH was not associated with exudative AMD in our study population. Frequencies of Y402H was 10.3% in AMD cases and 8.0% in controls (p=0.353). Significant associations were detected for exudative AMD with SNPs rs10490924:G>T in LOC387715 (A69S), and rs11200638:G>A in the promoter of HTRA1. The risk Tallele frequency of rs10490924 in LOC387715 was 64.9% in cases versus 43.2% in controls (p<0.001). The odds ratio for risk of AMD was 1.56 (95% CI; 0.80–3.03) for the GT genotype and 5.45 (95% CI; 2.59–11.49) for the TT genotype. The A allele frequency of rs11200638 in the HTRA1 promoter was 67.8% in cases versus 42.4% in controls (p<0.001). The odds ratio was 2.75 (95% CI; 1.34–5.64) for the GA genotype and 7.90 (95% CI; 3.61–17.26) for the AA genotype. An odds ratio of 7.94 (95% CI; 3.49–18.04) was obtained for carriers with both TT genotype in LOC387715 and AA genotype in the HTRA1 promoter. Conclusions: Our data suggest that the LOC387715 and HTRA1 polymorphisms are associated with a higher risk of exudative AMD in northern Chinese. We found no association of CFH Y402H with exudative AMD. The low frequency of CFH Y402H variant was further confirmed in this study population.

Lowered cortistatin expression is an early event in the human diabetic retina and is associated with apoptosis and glial activation

Abstract: Purpose: Cortistatin (CST), a neuropeptide with strong structural and functional similarities to somatostatin, is abundant in the vitreous fluid, and it is decreased in patients with proliferative diabetic retinopathy. The aims of the present study were to explore whether the retina produces CST, and to compare its expression between diabetic and nondiabetic donors. Retinal neurodegeneration was assessed by measuring glial fibrilar acidic protein (GFAP) by confocal laser microscopy and counting the apoptotic TUNEL positive cells in which nuclear fragmentation as well as condensation were present. Methods: Human postmortem eyes (10) from five diabetic donors were compared with 10 eyes from five nondiabetic donors, matched by age. CST mRNA (RT–PCR) and CST (confocal laser microscopy) were measured separately in both the neuroretina and retinal pigment epithelium (RPE). Retinal neurodegeneration was assessed by measuring glial fibrillar acidic protein (GFAP) by confocal laser microscopy and counting the apoptotic cells by TUNEL. Results: CST was found to be produced by the human retina, and higher levels of CST mRNA were found in RPE than in the neuroretina. CST mRNA levels in diabetic donors were significantly lower in both the RPE (p=0.001) and the neuroretina (p=0.03) in comparison with nondiabetic donors. CST immunofluorescence was in agreement with mRNA expression, but the differences were only significant when comparing neuroretinas (p=0.03). Increased GFAP and a higher degree of apoptosis were observed in diabetic retinas in comparison with nondiabetic retinas. These changes were inversely related with CST levels. Conclusions: CST is expressed in the human retina. There is more CST in the RPE than in the neuroretina. A lower expression of CST exists in diabetic retinas and it is associated with retinal neurodegeneration.

Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells.

Abstract: Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 18 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM)

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina

Abstract: Purpose To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Local and genetic determinants of vascular endothelial growth factor expression in advanced proliferative diabetic retinopathy

Abstract: Purpose: In proliferative diabetic retinopathy (PDR) and other angiogenesis-associated diseases, increased levels of cytokines, inflammatory cells, growth factors, and angiogenic factors are present. Vascular endothelial growth factor (VEGF) appears to play a central role in mediating microvascular pathology in PDR. The purpose of the present study was to search for the association between the –634 C/G polymorphism of the VEGF gene and PDR. Moreover, it was hoped to determine whether serum and vitreous levels of VEGF are affected by genetic factors. Methods: This cross-sectional case-control study enrolled 349 unrelated Slovene subjects (Caucasians) with type 2 diabetes mellitus. The case group consisted of 206 patients with an advanced form of PDR and for whom vitrectomy was performed, and the control group had 143 patients who had no clinical signs of diabetic retinopathy but did have type 2 diabetes of more than 10 years duration. To analyze the genotype distribution we had to compare the genotype frequencies in diabetics with PDR (cases, n=206) and diabetics without diabetic retinopathy (control group, n=143). Additionally, to evaluate the effect of diabetes on the VEGF serum levels 2 groups, diabetics and non diabetics, were compared. First group were diabetics (diabetics with PDR, n=104), and second group were 29 subjects without diabetes. Results: The –634 C/G VEGF polymorphism was not associated with PDR. Mean serum and vitreous levels of VEGF were statistically significantly higher in PDR in comparison to the control group. Moreover, significantly higher serum and vitreous levels of VEGF were demonstrated in diabetics with the CC genotype compared to those with the other (CG + GG) genotypes. Conclusions: VEGF is an important cytokine in PDR. Despite the effect of the –634 C/G VEGF polymorphism on serum and vitreous levels of VEGF in PDR, it failed to contribute to the genetic susceptibility to PDR.

LOXL1 genetic polymorphisms are associated with exfoliation glaucoma in the Japanese population

Abstract: PURPOSE We performed genetic association studies using a native Japanese population to examine the reproducibility of results of lysyl oxidase-like 1 (LOXL1) genetic association studies for exfoliation glaucoma (XFG) beyond the differences of ethnicity. We also quantified LOXL1 mRNA expression in the human lens capsule to examine the possible correlation between LOXL1 expression and XFG pathogenesis. METHODS We performed a case-control study using 95 Japanese XFG patients and 190 controls. Real-time polymerase chain reaction (PCR) analysis was performed using lens capsules obtained during surgery. RESULTS The TT genotype in the single nucleotide polymorphism (SNP) rs1048661 and the GG genotype in the SNP rs3825942 in exon 1 of LOXL1 were significantly associated with an increased risk of XFG under recessive models (chi(2) test, p=5.34 x 10(-34) and p=2.1 x 10(-8), respectively). Quantification of LOXL1 mRNA expression demonstrated no significant difference between XFG and senile cataract samples. CONCLUSIONS Although the functional effects of the LOXL1 SNP appear to be qualitative rather than quantitative, the amino acid substitution (R141L) caused by SNP rs1048661 is not a simple decisive factor for XFG due to the inverted allele frequency between Japanese XFG and Caucasian XFG patients. Further genetic and functional studies are essential for clarifying XFG pathogenesis.

Neutralizing antibody to VEGF reduces intravitreous neovascularization and may not interfere with ongoing intraretinal vascularization in a rat model of retinopathy of prematurity

Abstract: Purpose: To study the effects of a neutralizing antibody to vascular endothelial growth factor (VEGF), given as an intravitreous injection, on intravitreous neovascularization (IVNV) and ongoing vascular development of avascular retina in a rat model relevant to human retinopathy of prematurity. Methods: Newborn Sprague-Dawley rats were exposed to oxygen fluctuations alternating between 50% O2 and 10% O2 every 24 h. At postnatal day (p)12, rat pups received intravitreous injections of a neutralizing antibody to VEGF or control nonimmune rat IgG in one eye and were returned to oxygen cycling until p14, at which time they were placed into room air. At p18 (time of maximal IVNV) or p25 (time point in regression), animals were sacrificed. Their retinas were dissected, flat mounted, and stained with Alexa-isolectin for fluorescence microscopy. IVNV was measured as number of clock hours involved in injected VEGF antibody and control eyes. Mean clock hours of IVNV, avascular/total retinal areas and capillary densities within vascularized retinas were determined in injected eyes of control and treatment groups. Mean clock hours of IVNV in fellow noninjected eyes from control and treatment groups were analyzed by Student’s ttests to assess possible crossover effects from systemic absorption of antibody. Eyes from p13 rat pups were sectioned for immunohistochemistry or analyzed for VEGF receptor 2 (VEGFR2) phosphorylation by western blot. Free retinal VEGF at p13, one day following injections, was measured by ELISA. Results: Neutralizing antibody to VEGF at 25 ng and 50 ng caused a modest but significant inhibition of IVNV compared to IgG injected controls at p18, but only the 50 ng dose decreased IVNV compared to control at p25 (one-way ANOVA p=0.003; posthoc Bonferroni t-test p=0.003). Neither dose caused a significant difference in avascular/total retinal area at p18 compared to control. However, at p25, the 50 ng dose caused a significant reduction in avascular/total retinal area compared to the 25 ng dose (ANOVA p=0.038; posthoc Student’s t-test p=0.038). There was no difference in avascular/ total retinal area between IgG and the 25 ng dose. At p13, qualitative analysis of immunohistochemical sections of retina showed the 50 ng dose of VEGF antibody reduced VEGFR2 phosphorylation within the retina and around blood vessels. Also at p13, there was a significant increase in free intraretinal VEGF protein in eyes that had been treated with 50 ng dose of VEGF antibody compared to IgG injected control (Student’s t-test p=0.042). There were no differences in capillary densities in the vascularized retinas between eyes injected with the 50 ng dose of VEGF antibody and IgG control. There was also no difference in weight gain between treated and control groups. Conclusions: Neutralizing antibody to VEGF at a 50 ng dose caused a significant and sustained reduction in IVNV without interfering with ongoing retinal vascularization in a rat model of ROP, whereas a lower dose of antibody did not. These data also suggest that compensatory regulatory mechanisms may lead to increased VEGF concentration after intravitreous injection of a neutralizing antibody to VEGF. Further study is necessary for safety and for determination of drug dose of VEGF antibody, since dose of treatment appears important and may vary among infants with severe ROP. In this study, survival of already developed retinal capillaries did not appear affected. Neutralizing VEGF by an intravitreous injection of antibody may offer a treatment consideration for severe ROP, which fails current standard of care management.

Neuroprotective effects of cannabidiol in endotoxin-induced uveitis: critical role of p38 MAPK activation.

Abstract: Purpose Degenerative retinal diseases are characterized by inflammation and microglial activation The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma However, the cellular and molecular mechanisms are largely unknown We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes

Reconstruction of damaged cornea by autologous transplantation of epidermal adult stem cells.

Abstract: PURPOSE It is crucial for the treatment of severe ocular surface diseases such as Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP) to find strategies that avoid the risks of allograft rejection and immunosuppression. Here, we report a new strategy for reconstructing the damaged corneal surface in a goat model of total limbal stem cell deficiency (LSCD) by autologous transplantation of epidermal adult stem cells (EpiASC). METHODS EpiASC derived from adult goat ear skin by explant culture were purified by selecting single cell-derived clones. These EpiASC were cultivated on denuded human amniotic membrane (HAM) and transplanted onto goat eyes with total LSCD. The characteristics of both EpiASC and reconstructed corneal epithelium were identified by histology and immunohistochemistry. The clinical characteristic of reconstructed corneal surface was observed by digital camera. RESULTS Ten LSCD goats (10 eyes) were treated with EpiASC transplantation, leading to the restoration of corneal transparency and improvement of postoperative visual acuity to varying degrees in 80.00% (8/10) of the experimental eyes. The corneal epithelium of control groups either with HAM transplantation only or without any transplantation showed irregular surfaces, diffuse vascularization, and pannus on the entire cornea. The reconstructed corneal epithelium (RCE) expressed CK3, CK12, and PAX-6 and had the function of secreting glycocalyx-like material (AB-PAS positive). During the follow-up period, all corneal surfaces remained transparent and there were no serious complications. We also observed that the REC expressed CK1/10 weakly at six months after operation but not at 12 months after operation, suggesting that the REC was derived from grafted EpiASC. CONCLUSIONS Our results showed that EpiASC repaired the damaged cornea of goats with total LSCD and demonstrated that EpiASC can be induced to differentiate into corneal epithelial cell types in vivo, which at least in part correlated with down-regulation of CK1/10 and upregulation of PAX-6.