bluuebunny/crossref_metadata_2025 huggingface dataset:
Form‐deprivation myopia in the guinea pig: scleral myofibroblasts and biomechanics
Authors: PHILLIPS JR , BACKHOUSE S
Published: September 2008

Purpose To investigate the effect of induced myopia on cell populations and biomechanical properties of the sclera.

Methods Week‐old guinea pigs (n = 29) were monocularly deprived (MD) of form vision for 14 days. Total cells and myofibroblast (contractile) cells were visualized using DAPI and α‐smooth muscle actin (α‐SMA) antibodies respectively in 20 µm thick scleral sections. Ocular biomechanics were investigated by raising the IOP to 50 mmHg for one hour in anaesthetized animals. Ultrasound biometry measures were made before, during (every 10 min) and after raising the IOP.

Results As expected, MD eyes developed significant axial myopia. Generally, MD had no effect on total cell number or myofibroblast number compared to control or normal eyes. However, in MD eyes a significant decrease in total cell number was observed between the optic nerve and 10° nasal (equivalent to human temporal myopic crescent). Overall, myofibroblasts accounted for 63.67 ± 1.65% of scleral cells (mean ± SEM: three groups combined; n = 30 eyes). On increasing IOP, there was significant elastic expansion of the vitreous chamber depth (VCD) in deprived and control eyes but not in normal eyes. The VCD creep response to increased IOP stabilized at equivalent lengths to MD and control eyes.

Conclusion A high proportion of scleral cells are myofibroblasts, but cell number is unaffected by MD. The biomechanical response of MD eyes differs from normal eyes, suggesting that something other than myofibroblast cell number controls axial length.
Contribution of Müller cells to blue light injury
Authors: WIEDEMANN P , BRINGMANN A
Published: September 2008

Purpose Excessive light causes both damage to the photoreceptors and pigment epithelium, and degenerative alterations in the inner retina, for example apoptotic death of ganglion cells and a decrease in the thickness of the inner plexiform layer. We investigated whether gliotic alterations of Müller cells may contribute to the light‐evoked degenerative alterations in the inner retina.

Methods Retinas of adult rats were exposed to blue light for 30 minutes. At various time periods after treatment, retinal slices were immunostained against potassium and water channel proteins. Recordings of potassium currents were made in isolated Müller cells, and the swelling of Müller cell bodies was recorded in retinal slices.

Results After blue light treatment, Müller cells displayed hypertrophy and an increase in glial fibrillary acidic protein. The immunostaining of the glial water channel aquaporin‐4 was increased in the outer retina while the immunostaining of the photoreceptor water channel aquaporin‐1 disappeared. Blue light treatment resulted in a decrease and mislocation of the glial Kir4.1 protein in the whole retinal tissue, and a decrease in the potassium conductance of Müller cells. Hypoosmotic stress evoked a swelling of Müller cell bodies in blue light‐treated retinas that was not observed in control tissues.

Conclusion Gliosis of Müller cells associated with a loss of functional Kir4.1 channels will result in a disturbance of the retinal potassium and water homeostasis contributing to the degenerative alterations of the inner retina after exposure to blue light.
Localization of cathepsin x in the mouse retina
Authors: BERGER ST , STAHL A , GROSS NJ , SEVENICH L , MARTIN G , REINHECKEL T , AGOSTINI HT
Published: September 2008

Purpose Cathepsin X is a recently identified member of the papain‐like cysteine protease family that is expressed by many cell types. The in vivo functions of Cathepsin X are unknown. Hence, the aims of this study are to localize cathepsin X expression in the mouse retina and to compare the retinal morphology and ERG function of cathepsin X knock‐out mice (Ctsx‐/‐) to congenic wild type C57Bl/6 mice (Ctsx+/+).

Methods Left eyes of adult mice were prepared for paraffin‐ and right eyes for cryo‐sections. Histology was accomplished by Hämalaun‐Eosin and PAS staining after depigmentation. Expression of cathepsin X and the proliferation marker Ki67 were investigated by immunohistochemistry. Retinal function was tested by ERG.

Results Cathepsin X expression in WT mice accumulates along the external limiting membrane. Additionally, a netlike pattern of cathepsin X was found around the neuronal cells in the inner nuclear and ganglion cell layer. There is no significant difference between cathepsin X knock‐out and WT mice in retinal morphology, cell proliferation or ERG readings.

Conclusion We found prominent expression of cathepsin X in WT mice retina. Normal retinal morphology and ERG function in cathepsin X knock‐out mice, however, suggest that other cathepsins might compensate for the loss of retinal cathepsin X.
Cx43 internalization is mediated by the ubiquitin‐binding adaptor protein Eps15 in retinal endothelial cells
Authors: GIRAO H , CATARINO S , LOPES C , PEREIRA P
Published: September 2008

Purpose Intercellular communication through gap junctions (GJIC) is important in maintaining the integrity of the blood‐retinal barrier. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. The purpose of this study is to investigate the molecular mechanisms involved in the regulation of connexin 43 (Cx43) stability at plasma membrane by eps15 in retinal endothelial cells.

Methods To investigate the interaction of Cx43 with eps15, Cx43 was selectively immunoprecipitated (IP), followed by western blot (WB) and probing with antibodies against eps15. To investigate the role of eps15 in Cx43 stability and subcellular distribution cells were transfected with a dominant negative form of eps15 or siRNA targeted against eps15, cell extracts were WB and probed with antibodies against Cx43. Alternatively, cells were fixed and simultaneously stained with antibodies against Cx43 and eps15, and imaged by confocal microscopy.

Results Data obtained in this study indicates that eps15 interacts with Cx43 mediating its internalization from the plasma membrane. When the normal function of eps15 is disrupted by the presence of a dominant negative form of the protein or through silencing of the eps15 gene with siRNA, Cx43 accumulates at the plasma membrane.

Conclusion Data suggests a novel protein interaction between Cx43 and eps15 that mediates the plasma membrane stability of Cx43. Disruption of this interaction may impact on GJIC, contributing to endothelial cell dysfunction associated with the breakdown of the blood‐retinal barrier, as observed in diabetic retinopathy. Supported by POCI/SAU‐MMO/57216/2004
Receptor retinoid‐binding protein (IRBP) binds cone outer segments differentially in light and dark
Authors: GONZALEZ‐FERNANDEZ F , GARLIPP MA , KEATING AM
Published: September 2008

Purpose Vision requires the continuous exchanges of visual cycle retinoids through the interphotoreceptor matrix (IPM). IRBP, which is synthesized by the rods and cones, is the major soluble protein component of the IPM. In the matrix, IRBP protects and targets retinoids trafficking between the photoreceptors, RPE and Muller cells.

Methods Organ culture of isolated Xenopus retina was used to characterize the cellular binding of recombinant full‐length IRBP protein free of fusion tags. Xenopus was selected for these studies because of their large photoreceptors which are easily detached in both light and dark‐adapted states. xIRBP was covalently bound to the fluorescent dye Alexa‐647 at 1:1 molar ratio. Labeling had no effect on the retinoid ligand‐binding properties of the protein. Xenopus retinas were detached and incubated with xIRBP‐647 for 1 hour at room temperature in light and dark. The distribution of xIRBP‐647 was studied by confocal microscopy in retinal flat whole mounts and cryosections.

Results In light, xIRBP‐647 bound to cone outer segments with lighter labeling of the rod outer segments. Retinas incubated with the xIRBP‐647 in the presence of a 50 fold excess of unlabeled protein showed reduced cone and rod outer segment labeling. In dark, outer segment binding was not appreciated. Positive staining near the level of the external‐limiting membrane was noted in dark but not light.

Conclusion The binding of xIRBP‐647 to cells of the isolated retina was different in light versus dark. Our data suggests that the direct interaction of IRBP with the outer segments and Muller cells plays an important role in retinoid trafficking in the the visual cycle.
Selective estrogen receptor modulators regulate IL‐6 inflammatory response in ARPE‐19 cells
Authors: PAIMELA T , RYHÄNEN T , VIIRI J , HYTTINEN J , UNKILA M , UUSITALO HMT , SALMINEN A , KAARNIRANTA K
Published: September 2008

Purpose Recent findings reveal that Toll‐like receptors (TLRs) and innate immunity participate in the pathology of age‐related macular degeneration (AMD). Many studies indicate that estrogens and selective estrogen modulators (SERMs) modulate inflammatory responses, but their effect on the development of AMD is weakly understood. In this study, we investigated the regulatory role of various SERMs (provided by Hormos Medical Ltd.) on IL‐6 expression in human retinal pigment epithelial cells (ARPE‐19).

Methods ARPE‐19 cells were exposed to lipopolysaccharide (LPS; TLR 4 agonist) with simultaneous exposure to various SERMs and the secretion of IL‐6 cytokine was analyzed by ELISA. The estrogen receptor alpha and beta were qualitatively measured by RT‐PCR. To study the effect of various SERM treatments of estrogen response element (ERE) ‐mediated transcription, the ARPE‐19 cells were transiently transfected with ERE‐luciferase vector. The activity of ERE was measured by Luciferase assay.

Results Simultaneous exposures to LPS and SERM‐320 reduced the IL‐6 expression levels in ARPE‐19 cells compared to LPS exposure alone. The RT‐PCR analysis showed that ARPE‐19 cells expressed estrogen receptor alpha but not beta proteins. Interestingly, SERM‐320 did not increase the activity of ERE in ARPE cells. This reveals that SERM‐320 is implicated in regulation of IL‐6 expression, but is not mediated through estrogen response element.

Conclusion Our findings reveal that SERM‐320 is a novel compound to suppress innate immunity response in human retinal pigment epithelial cells.
Activation of autophagy induced by 7‐Ketocholesterol and dysfunction of RPE in AMD
Authors: OLIVEIRA FERREIRA J , GIRAO H , PEREIRA P
Published: September 2008

Purpose Retinal pigment epithelial (RPE) cell death is a hallmark of age‐related macular degeneration (AMD). Lysosomal impairment in RPE cells is one of the traits present in the pathology of AMD. Authophagy, a process whereby proteins and subcellular organelles are degraded in the lysosome is activated by several stress stimuli. RPEs internalize large amounts of oxidized lipids including 7‐ketocholesterol, the most cytotoxic of oxysterols. The purpose of this study is to determine if exposing RPE to 7‐ketocholesterol can act as a stimulus to induce autophagy and thus account to the dysfunction of the RPE.

Methods RPE cells where incubated with 7‐ketocholesterol (20 ug/ml) for 6 or 12 h, or deprived from nutrients for 12h. To assess Macroautophagy cells were transfected with EGFP‐LC3 plasmid. Formation of autophagic vesicles was assessed by confocal microscopy. LC3 processing was measured by western blot with antibodies against GFP. Chaperone mediated autophagy (CMA) was assessed by determining if chaperones interacted with Lamp2, performing Lamp2 immunoprecipitation.

Results 7‐ketocholesterol induces the formation of autophagic vesicles and the processing of the LC3‐I into LC3‐II, both hallmarks of Macroautophagy, Moreover treatment with 7‐ketocholesterol increases the interaction between Lamp2 and Hsc70, Hsp90 and Hsp40, suggesting that this cholesterol oxide also stimulates CMA.

Conclusion RPE cells undergo Macroautophagy and CMA activation when exposed to 7‐ketocholesterol. At early stages both may act as a cell survival mechanism whereas prolonged exposure may result in macroautophagic cell death.
New developments in OCT evaluations
Authors: BERNARDES R , CUNHA‐VAZ J
Published: September 2008

Diabetic macular edema (DME) is one of the most common causes for visual loss (Ciulla et al., Diabetes Care, 2003). DME is characterized by an abnormal increase of the retinal thickness (RT) due to the accumulation of liquids in the retinal tissue, consequence of the first response to the inflammatory process occurring in diabetic retinopathy (DR). It is therefore considered one of the most important indicators of DR progression. Other indicators, such as microaneurysms and retinal leakage, were also observed and quantified in a prospective longitudinal 3‐year follow‐up study involving type 2 diabetic patients with nonproliferative DR. Patterns of progression were established based on these ophthalmic indicators (Lobo et al., Arch Opthalmol., 2004). OCT plays an important role in the assessment and quantification of DME. Stratus OCT version established a 9 area map for RT, being the central area of 1000µm in diameter. However, there is a lack of detailed information when studying DME. This problem was addressed by the use of an atlas of the human macula in a recent work from our group, where the merging of radial and circular scans was made possible (Bernardes et al., IOVS, 2008). Recently, Carl Zeiss introduced Cirrus HD‐OCT, a Fourier domain OCT, allowing to achieve more detailed information of the macular area, performing either 512×128 or 200×200 B‐scans, and allowing to export an eye fundus image for reference. It is now possible to assess local RT and correlate it with any imaging modalities available.
Role of inflammation in blood‐retinal barrier breakdown: animal models
Authors: AMBROSIO AF , LEAL EC , MANIVANNAN A , CUNHA‐VAZ J , FORRESTER JV
Published: September 2008

Purpose Animal models are valuable tools to study the pathogenic mechanisms of many diseases. Diabetic retinopathy is a low‐grade chronic inflammatory disease. Nitric oxide (NO) is involved in leukostasis and blood‐retinal barrier (BRB) breakdown in the early stages of the disease. However, the role of the different NO synthase (NOS) isoforms was not elucidated. We aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO‐mediated leukostasis and BRB breakdown, by using an experimental model of diabetes and iNOS KO mice.

Methods Diabetes was induced by streptozotocin in normal and KO mice (2 weeks of diabetes). Normal mice were treated with L‐NAME (NOS inhibitor). Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat‐mounted retinas and in vivo. ICAM‐1, occludin, ZO‐1 and nitrotyrosine levels were assessed by Western blotting or immunohistochemistry.

Results Diabetes increased the permeability of BRB and leukostasis, which were reduced by L‐NAME. Similar effects were observed in diabetic iNOS KO mice. In diabetic mice, the immunoreactivity of tight junction proteins, occludin and ZO‐1, decreased, whereas ICAM‐1 protein levels increased. Those effects were prevented by L‐NAME and in diabetic iNOS KO mice. Diabetes also upregulated all NOS isoforms and increased nitrotyrosine levels in normal mice, but did not significantly increase eNOS and nNOS, and nitrotyrosine levels, in iNOS KO mice.

Conclusion These data demonstrate that iNOS plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM‐1 upregulation and tight junction proteins downregulation. Support: FCT, Portugal
The role of FGF2 in the in vitro proliferation and angiogenesis of human choroidal endothelial cells
Authors: STEWART EA , AMOAKU WMK
Published: September 2008

Purpose Age‐related macular degeneration (AMD) is the most common cause of irreversible visual loss in elderly populations in the western world. The mechanism of this disease is not yet fully understood, although vascular endothelial growth factor (VEGF) is thought to be the most significant growth factor in AMD, other growth factors, including FGF2 also stimulates the proliferation of human choroidal endothelial cells (hCEC). These factors may explain the only partial success of current anti‐VEGF AMD treatments.

Methods hCEC were isolated from cadaver eyes and cultured in EBM2‐MV medium as previously published. hCEC proliferation after exposure to VEGF and FGF2 was measured using the WST‐1 assay and angiogenesis assessed using a double layer Matrigel technique. Western blotting was used to examine the intracellular signalling stimulated by FGF2 and VEGF.

Results hCEC proliferation was increased in response to both FGF2 and VEGF. This effect was cumulative, indicating that these work through different intracellular signalling pathways. Western blotting identified common and differential intracellular signalling between the two growth factors.

Conclusion Currently VEGF is thought to be the most important growth factor which stimulates neovascularisation leading to AMD and treatments developed reflect this. However, these results show that FGF2 also stimulates neovascularisation and may play a role in AMD development. A treatment which targets downstream signalling common to both growth factors may provide a more targeted and therefore successful treatment.
Form deprivation amblyopia ‐ a treatable cause of blindness
Authors: SJÖSTRAND JB
Published: September 2008

Purpose Form deprivation amblyopia is associated with the obstruction or deformation of the passage of light causing degraded images on the retina. This lack of normal visual experience delays or blocks the visually driven development. If the obstacle is removed within the critical period the developing visual system has the capacity to regain visual function. Left untreated the deprivation and blocked visual experience may cause severe visual impairment and blindness.

Methods To review our current understanding of the pathophysiology and risk factors in form deprivation amblyopia and to evaluate the effectiveness of treatment. The main conditions obstructing light passage during early development of vision will be described. Form deprivation amblyopia and its functional consequences are defined. Factors of importance for a satisfactory long term visual outcome are evaluated.

Results Valid experimental and clinical models of form deprivation amblyopia are reviewed with focus on the role of visual deprivation per se and unequal visual input to the visual cortex, the primary site of functional change. Recent clinical observations of the visual outcome following early surgery of congenital dense bilateral and unilateral cataracts are used to evaluate the factors involved.

Conclusion Form deprivation amblyopia is a complex condition with a hallmark of impaired visual acuity. For a satisfactory visual outcome early detection and intervention is important.
Development of immunostaining of cell cycle related proteins in flat mounted corneal endothelium
Authors: HE Z , THURET G , PIPPARELLI A , PISELLI S , DUMOLLARD JM , PEOC'H M , GAIN P
Published: September 2008

Purpose Immunostaining of cell cycle proteins is necessary for the study of the proliferative status of corneal endothelial cells (EC). Most studies use cross sections, which offer direct access to intracellular antigens but allow visualization of only few cells, without giving a global view of an intact endothelium. We developed protocols for immunostaining of ECs of flat mounted corneas

Methods Studied proteins: 4 proteins with a known expression pattern (intra‐membranous ZO‐1, cytoplasmic α actin, nuclear hnRNP and H3 histone) and 8 others, already described within EC: 3 cell cycle inhibitors (P27, P21, P16); 3 proliferation markers (PCNA, MCM2, Ki67), cyclins D1, and E. Fresh and organ cultured (OC) human corneas were used. Fixation/permeabilization: paraformaldehyde (PFA), acetone, methanol, ethanol, Hg, Zn, acetic acid, triton, sodium docecyl sulfate, alone or in combination. Antigen retrieval: heating and biochemical or chemical agents. Patterns of expression were also compared between endothelium and epithelium

Results There was no universal protocol. Most of the time 0.5% PFA gave the best results, but for p21, and cyclins, specific protocols were necessary. Homogeneous staining was always obtained with a clear subcellular localization (nuclear or cytoplasmic). OC did not globally modify expression within EC whereas several nuclear or cytoplasmic translocations were observed within the epithelium

Conclusion The use of protocols specifically designed for the endothelium of whole intact flat mounted corneas would allow a better localization of cell cycle proteins. They will be especially useful during attempts to alter the cell cycle and trigger EC proliferation. Grant: Fondation de l’Avenir 2007 ET7‐468
Inflammation in AMD pathology
Authors: NOWAK JZ
Published: September 2008

Age‐related macular degeneration (AMD) is a progressive retinal disease that leads to substantial irreversible vision loss in elderly patients. Two clinical categories of AMD are distinguished: the “dry” atrophic form and the exudative neovascular or “wet” form. There is neither a preventive therapy nor a cure for both forms, although recent efforts succeeded in a more effective treatment of the wet AMD with PDT and anti‐VEGF drugs. AMD is a multifactorial pathology which involves complex interaction of metabolic, genetic and environmental factors, with major biochemical‐clinical abnormalities seen in four functionally interrelated tissues: photoreceptors, retinal pigment epithelium, Bruch’s membrane and choriocapilaries. Four processes specifically contribute to the development of AMD pathology: lipofuscinogenesis (in RPE cells), drusogenesis (with drusen located between RPE and Bruch’s membrane), inflammation (local) and choroidal neovascularization (in wet form). Although the role of immune system and inflammation has been implicated in AMD pathogenesis for many years, an impetus to intensify the research in this direction gave a recent discovery of polymorphisms in genes that encode for elements of the complement system, including factor H (CFH; Y402H), factor B, and complement component 2. An increased activity of the complement alternative pathway due to the lack of or insufficient control by CFH appears to contribute to AMD progression via immunologic mechanism which drives inflammatory response. An arising question is whether blockade of overactive complement system will be a therapeutic strategy safe for patients and effective to prevent or slowing down the macula‐devastating and vision‐threatening disease. Supported by grant no. 503‐1023‐1 from Medical University of Lodz.
Complement factor H and factor B expression in RPE cells
Authors: XU H , CHEN M , FORRESTER JV
Published: September 2008

Purpose Age‐related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface.

Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT‐PCR.

Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch’s membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF‐alpha and IL‐6, whereas the expression of CFB was positively regulated by TNF‐alpha and IFN‐gamma. Short‐term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long‐term incubation of RPE cells with POS significantly down‐regulated CFH expression but up‐regulated CFB expression.

Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS.
Inflammatory gene defects in AMD
Authors: IMMONEN I
Published: September 2008

Inflammatory markers have been raported in AMD patients at the serum, gene an tissue levels. Of the inflammatory cytokines especially IL‐6, IL‐8 and IL‐10 have been associated with AMD. Inflammatory cytokines are multifunctional molecules potentially stimulating macrophages and other inflammatory cells and choroidal neovascularization (CNV) in AMD lesions. The cytokines a modulate the expression of other inflammatory markers and are associated e.g with elevated levels of CRP in AMD. Targeting these pathways with non‐steroidal anti‐inflammatory agents may be used in future pharmacologic treatments for AMD before and after CNV formation. This presentation reviews current knowledge of inflammatory cytokines in relation to the RPE and AMD in general.
Cell cycle genes expression in human corneal endothelium: study by microarray and qRT‐PCR
Authors: PIPPARELLI A , THURET G , PISELLI S , PEOC'H M , ACQUART S , GARRAUD O , GAIN P
Published: September 2008

Purpose To use microarray and qRT‐PCR to identify changes in cell cycle genes expression in human corneal endothelial cells (CEC) from “ in vivo”, post mortem, and organ cultured (OC) corneas, and also in confluent primary culture and immortalized CEC

Methods Total RNAs were extracted. “in vivo” corneas were obtained during penetrating grafts for keratoconus, immediately after trephination, post mortem corneas (body donation to Science) within 24H after death. OC corneas were stored for 15D. Descemet was peeled off to avoid contamination with other cell types. Biotin‐labeled cRNA probe was synthesized from RNAs extract and hybridized to the pretreated Oligo GEArray Human Cell Cycle Microarrays, which covered 112 key genes of cell cycle. Microarrays were performed in duplicate. Gene expression of 'in vivo' CEC served as a reference. A difference >1,5 fold of the transcriptional level was considered significant. qRT‐PCR were done on cyclin E1; cyclin D1; p16INK4a; p21CIP1 and p27Kip1 to confirm the microarrays

Results Compared to ‘in vivo’, CEC from OC corneas had 1 upregulated gene (CDK7) and 26 downregulated (CCNE1; CDKN1B; MCM2; RB1…), post mortem corneas had 2 up(CDC28; CDK7) genes and 16 downregulated (ATM; BAX; CDC16; CDKN1B…), primary culture had 1 up(CKS2) and 9 downregulated (BAX; CCNE1; CDK2; MCM2…), and the immortalized cell line had 16 up (ATM; CCNH; CKS2; MCM2…) and 2 downregulated genes (BAX; CDK5R2). qRT‐PCR of the 5 aforementioned genes validated the Microarrays data

Conclusion Microarray seems to be a powerful tool to better understand the proliferative status of human CEC. It will help to choose the targets we need to alter in order to trigger CEC proliferation. Grant: Fondationdel’Avenir2007 ET7‐468
TGFbeta signalling through Smad‐independent mechanisms in human lens cells
Authors: WORMSTONE IM , ELDRED JA , DAWES LJ , REDDAN JR , WANG L
Published: September 2008

Purpose Previous work has shown that TGFß can induce matrix contraction and is likely to be regulated by Smad‐independent pathways. The present study investigated the role of the classical MAPK signalling and Rho kinase signalling pathways in TGFß mediated matrix contraction.

Methods Cells from the human lens line FHL 124 were employed. Contraction was assessed using a patch assay, whereby the area covered by cells, was measured using imaging techniques. ERK activation was evaluated using western blot methods. To study SMAD2/3 distribution immunocytochemistry was used. Following a 24hr period of serum starvation, cells were maintained in the following conditions: Control medium +/– 10µM MEK inhibitor (U0126); 10ng/ml TGFß2 +/– U0126; Control medium +/– 10µM Rho kinase Inhibitor (Y–27632); 10ng/ml TGFß2 +/– Y–27632. The experimental duration for Smad2/3 staining was 2 hrs; western blots, 30mins & 24hr; patch contraction assays, 2‐3 days.

Results Addition of 10ng/ml TGFß2 lead to a significant elevation of P‐ERK levels following both 30 min and 24hr periods of exposure. Addition of 10ng/ml TGFß2 to patch assays caused a significant contractile event to take place. Maintenance of cultures in the presence of 10µM U0126 or Y–27632 had no significant effect on patch area when added to control medium; however, when added in the presence of TGFß2, contraction was inhibited. With respect to cell population all groups were similar. Immunocytochemistry for SMAD2/3 showed clear nuclear translocation in response to 10ng/ml TGFß2; this signalling event was unaffected by U0126 and Y–27632.

Conclusion MAPK and Rho kinase signalling play important roles in TGFß mediated matrix contraction of human lens cells.

Commercial interest
A novel cytocompatible thermoresponsive co‐polymer for corneal tissue engineering
Authors: HOPKINSON A , VARGHESE VM , KUMARY TV , DUA HS
Published: September 2008

Purpose Using thermoresponsive polymers as a substrate to expand healthy sheets of transplantable cells for the immediate cover of wounds is an attractive novel treatment for ocular surface reconstruction. However, the cytocompatibility of thermoresponsive polymer and the effect on cell expansion must be assessed. Changes in the expression of secretory proteins in response to different microenvironments can be used to determine a more precise suitability of polymers in cell culture at molecular level. We assess the specific cytocompatibility of a novel thermoresponsive co‐polymer with the future goal of its use in corneal reconstruction.

Methods Protein was extracted from conditioned medium of SV‐40 immortalised corneal cell line (CCL) cultured on both thermoresponsive polymer and tissue culture plastic. Proteins were separated by two‐dimensional gel electrophoresis and the resulting spot map profiles were differentially compared. Differences in the protein profiles were characterised by mass spectrometry and western blot analysis.

Results Differential analysis of whole cell extract, and conditioned medium from CCL on plastic identified over 50 potentially secreted proteins. Mass spec characterisation confirmed these proteins as secretory, but also suggested healthy cell expansion was occurring. Comparison of CCL on plastic and thermoresponsive polymer revealed few differences suggesting the polymer did not effect cell expansion.

Conclusion Thermoresponsive polymer is cytocompatible for CEC cell growth. This novel co‐polymer maybe a potential substrate for the future ex‐vivo engineering of transplantable cell sheets, for improved corneal reconstruction
Determining rates of visual field progression in glaucoma
Authors: CHAUHAN B
Published: September 2008

Purpose To provide practical guidelines on detecting rates of visual field progression in glaucoma

Methods Using a mixture of real patient data, computer simulation and statistical analysis, the frequency of visual field examinations for detecting various rates of visual field change were determined.

Results Our results show that the ability to detect rates of visual field change depends critically on the magnitude of the change we wish to detect and the variability of visual fields. They also show that performing only one visual field per year will lead to failure to detect very significant visual field loss. The statistical power to detect clinically meaningful rates increases with 2 or 3 examinations per year

Conclusion This study provides guidance to general ophthalmologists and glaucoma specialists on detecting rates of visual field progression in glaucoma.

Commercial interest
Myofibroblast and smooth muscle actin in scarred corneal stroma
Authors: AKHTAR S , ALMUBRAD TM , OGBUEHI KC , BRON AJ
Published: September 2008

Purpose Mohan et al, (2003) hypothesised that after corneal injury, the site of injury is invaded by myofibroblasts (MFBCs) derived from dividing keratocytes. We report here the distribution of myofibroblasts (MFBCs), and cellular location of α‐‐ smooth muscle actin (α‐‐SMA) in scarred cornea.

Methods Three penetrating keratoplasty buttons, two with post‐zoster scarring and one with hypertrophic dendriform epitheliopathy, were fixed in: 1) 2.5% glutaraldehyde containing cuprolinic blue for ultrastructural studies and 2) 4% paraformaldehyde for immunogold localisation. Primary α‐‐SMA and vimentin antibodies were localised by gold conjugates.

Results Large numbers of MFBCs were observed in the sub‐epithelial collagenous pannus, posterior stroma and posterior collagenous layer in these corneas. The MFBCs were very large in size, lay in layers and occasionally fused with each other. Labelling for α‐SMA was very strong in all MFBCs. Labelling of α‐SMA was also observed in basal epithelial cells. MFBCs contained very prominent rough endoplasmic reticulum, vimentin filaments and large nuclei. Significantly, large proteoglycans (nm) and collagen fibrils (nm) were present around these cells.

Conclusion It has been suggested that cytokynes released from the overlying epithelium act to maintain the myofibroblast phenotype (Jester et al 1999a, 1999b). We hypothesise that MFBCs synthesise large amounts of proteoglycan which disrupt the organisation of collagen fibrils and lead to the opacities of scarred cornea. The finding of labelling for α‐SMA in basal epithelial cells suggests that some epithelial cells may transform into MFBCs.
Gene transfer of disease regulated promoters during experimental autoimmune uveitis
Authors: CHTARTO A
Published: September 2008

Abstract not provided
Osmotic stress regulation of aquaporin 4 expression in a human retinal pigmented epithelial cell line
Authors: JANSSENS S
Published: September 2008

Abstract not provided
AAV mediated SOCS1 gene expression in ARPE cells in an immune rejection model of xeno‐graft for AMD diseases
Authors: KOCH P
Published: September 2008

Abstract not provided
Geometric characterization of anterior segment in the hen’s eye
Authors: ALIO SANZ JL , PINERO LLORENS DP , SIREROL B , BATAILLE L
Published: September 2008

Purpose To characterize the corneal geometry and the anatomical configuration of the anterior segment in the hen’s eye

Methods 6 eyes of 3 brown leghorn hens with a mean age of 6 months were analyzed. The animals were anesthesized prior to the beginning of the measurements. In all cases, corneal geometry and anatomical configuration of the anterior segment were evaluated by means of the CSO and Visante OCT systems, respectively

Results Mean corneal curvature in the 3‐mm central area was 69.35 ± 1.77 D, with a marked corneal astigmatism of a mean value of 3.00 ± 1.86 D. Mean corneal asphericity was ‐0.23 ± 0.41. Regarding central pachymetry, a mean value of 306.67 ± 46.76 microns was found. Anterior chamber had a mean diameter of 8.14 ± 0.13 mm and a mean associated sagittal depth of 2.33 ± 0.23 mm

Conclusion The hen’s eye has a thin cornea, slightly prolate and with significant steepness, and an anterior chamber with a reduced diameter
Two distinct populations of corneal epithelial cells with limbal stem cell characteristics in the mouse
Authors: HOLAN V , KRULOVA M , POKORNA K , LENCOVA A , ZAJICOVA A , FILIPEC M
Published: September 2008

Purpose Purpose. To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse.

Methods Methods. Limbal tissues from BALB/c mice were trypsin‐dissociated and separated on a Percoll gradient. Several fractions were isolated and characterized by Real‐time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells, by flow cytometry for the determination of the side‐population (SP) phenotype and for growth properties in vitro.

Results Results. Cells retained in the 40% Percoll fraction and in the 80% Percoll fraction of the gradient were both enriched for populations with a high expression of SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction was K12 negative and contained small non‐spontaneously proliferating cells, which instead were positive for p63.

Conclusion Conclusion. These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12‐ p63+ population being closer to the primitive limbal SC.
Depth profile study of molecular collagen structure in normal human cornea
Authors: KAMMA‐LORGER CS , BOOTE C , YOUNG RD , HAYES S , QUANTOCK AJ , MEEK KM
Published: September 2008

Purpose To investigate the structure and organisation of corneal collagen as a function of stromal depth.

Methods A strip extending from limbus to limbus along the superior to posterior meridian was cut from a normal human cornea and 100µm serial sections, parallel to the surface plane of the tissue, were obtained. Wide angle x‐ray diffraction experiments were performed in ID‐13 at the European Synchrotron Radiation Facility (ESRF) using a 25µm square beam. Each section was scanned at 450µm or 500µm steps. Additionally, another thin vertical strip (1mm thickness) was mounted in such a way that the beam was scanning edge‐on. Two scans were performed (1mm apart, each covering the depth of the cornea) from the anterior to the posterior surface at 25µm intervals. Data analysis provided information on the direction and degree of alignment of preferentially oriented collagen fibrils, as well as collagen intermolecular spacing (IMS).

Results IMS is relatively stable throughout the depth of the central cornea, but is decreased in the posterior peripheral cornea. The degree of preferential alignment of fibrillar collagen is lower in the peripheral anterior part. In addition, the previously well‐documented tangential or annular collagen structure at the limbus is mainly restricted to the most posterior 100µm of stroma. At more superficial layers collagen preferred orientation tends to obtain an “x” shaped pattern, possibly forming part of the "anchoring fibril" structure alluded to previously in full‐thickness averaged x‐ray studies.

Conclusion The preferred directions of the lamellae and IMS are depth‐dependent. The annulus at the limbus, which is thought to be implicated in the maintenance of corneal curvature, resides in the posterior limbus.
Leber´s stellate neuroretinitis. A case report
Authors: IDOIPE CORTA M , GIL ARRIBAS L , GARCIA‐MARTIN E , FERRER NOVELLA E
Published: September 2008

Purpose A 49 years old man was admitted in our hospital with a 4‐day history on sudden loss of vision in the right eye.

Methods Completed ocular examination, including visual acuity, anterior ocular segment, pupillary light reflex, intaocular pressure, ophthalmoscopic examination, visual fields, fluorescein angiography, system examination, including computed tomography and serologic test were done.

Results We present a 49 years old man with sudden loss of vision who presented unilateral disc swelling. Five days later, the fundus examination reveled a macular star, and peripapillary retinal detachment. The fundus findings were normalized in a few weeks, leaving behind only retinal pigment epithelial defects and an inferior visual field defect. Infectious aetiology and others causes were rejected.

Conclusion Leber´s idiopathic stellate neuroretinitis is un uncommon syndrome characterized by unilateral optic disc swelling followed by a macular star. It has a spontaneous resolution, and the aetiology is unknown.
European vision research ‐ An independent point of view
Authors: VON BONHORST C
Published: September 2008

Abstract not provided
International research funding advocacy: The US experience
Authors: ANGLE J
Published: September 2008

Abstract not provided
Unilateral optic neuritis as presenting feature of acute hemorrhagic leukoencephalitis
Authors: SKET KONTESTABILE A , SEGA S , SVIGELJ V , HAWLINA M
Published: September 2008

Purpose We report a case of 23 year‐old previously healthy female with acute hemorrhagic leukoencephalitis AHLE (Hürst disease) leading to lethal outcome within two weeks after presentation with unilateral visual loss and moderate upper respiratory tract infection with leukocytosis.

Methods At presentation, VA was 0.3 RE and 1.2 LE with centrocecal defect on right eye, positive RAPD and dyschromatopsia. Optic nerve on fundus examination was normal. Neurological examination revealed right sided hyperreflexia and diminished right plantar response. Initial MRI revealed few focal periventricular hyperintensive lesions consistent with demyelination.

Results Treatment started with 1g metilprednisolon i.v. for 3 days with good initial effect. Third day of treatment, VA in right eye was normal (1.2), RAPD was negative and dyschromatopsia improved. Three days later, altered consciousness with seizures developed, subsequently followed by right hemiparesis and coma. Cerebrospinal fluid pleocytosis was present. Follow‐ up MRI, obtained 4 days after initial one, revealed large areas of hemorrhagic necrosis in the left hemisphere.Despite continued high dose steroid treatment,antiviral prophylaxis and plasmapharesis, the disease progressed to death within days.

Conclusion Acute hemorrhagic leukoencephalitis AHLE (Hürst disease) is a rare fulminant demyelinating disorder characterized by a fulminant clinical course due to hemorrhagic necrosis of the white matter associated with high rate of mortality. We present a case of this severe disease in which optic neuritis was presenting focal neurological deficit.
Hypertensive retinopathy mimicking papilledema
Authors: LEE A
Published: September 2008

Abstract not provided
Acute painful ophthalmoplegia: infectious considerations
Authors: GOLNIK K
Published: September 2008

Acute painful ophthalmoplegia may be caused by a number of processes including inflammation (giant cell arteritis, sarcoidosis, thyroid eye disease, idiopathic), vascular (aneurysm, hemorrhage, thrombosis, ischemia, fistula), tumor, and migraine. However, infectious causes (fungal, bacterial) of ophthalmoplegia may be life‐threatening if the diagnosis and treatment is delayed. Presenting symptoms and signs, differential diagnosis, and the appropriate treatment of these infectious entities will be discussed.
Microarray study of limbal epithelial crypt demonstrates its putative limbal stem cell niche characteristics
Authors: KULKARNI B , FOSTER T , YEUNG AM , TIGHE P , HOPKINSON A , DUA HS
Published: September 2008

Purpose We have discovered a novel anatomical structure at the limbus, termed the Limbal Epithelial crypt (LEC). Previous anatomical and immunohistological studies suggest its potential as the limbal stem cell niche. We aim to characterise the differential gene expression of the LEC by microarrays with a view to finding putative stem cell marker(s).

Methods Frozen tissue blocks of corneoscleral buttons dissected from cadaver eyes were cryosectioned. These tissue sections from different ocular surface regions were laser microdissected. Extracted RNA was amplified & hybridized to microarray chips. Raw data obtained with Genepix Pro6 software was filtered, normalized & analysed on BASE, TMeV & Jexpresspro software. Unpaired T‐Test, Significance Analysis of Microarrays and k‐means clustering were performed on the data. Quantitative gene expression analysis (qPCR) was performed on the genes of interest.

Results 150 differentially expressed genes in the LEC were obtained(p ≤ 0.01). These genes were clustered according to stem cell related functions. qPCR was performed on the Integrin Beta 1 binding protein gene (ITGB1BP1). The protein expressed by this gene specifically binds to Beta 1 Integrin (ITGB1) which is a cell adhesion molecule, is predominantly an inhibitor of cell proliferation and has a role in maintaining stem cells in their niche. These genes were significantly expressed in the LEC (P=0.03).

Conclusion The constituents of the LEC are stem cells with neighbouring niche cells, hence representing a Limbal Epithelial Stem Cell niche.
Distribution of BIG‐H3 protein in primary and recurrent granular corneal dystrophy
Authors: AKHTAR S , ALMUBRAD TM , OGBUEHI KC , BRON AJ
Published: September 2008

Purpose Granular corneal dystrophy (GCD) is a hereditary disease involving the formation of opaque granular deposits in the corneal stroma. In the present project we investigated the distribution of big‐h3 protein in the cornea of primary GCD (PGCD), recurrent GCD (RGCD) and following epithelial debridement of RGCD.

Methods The distribution of big‐h3 protein was studied in 2 PGCDs, 2 RGCDs, and in epithelial debridement specimens from 2 RCGD corneas. The corneas were fixed in 4% paraformaldehyde at 4°C and embedded in LR white under UV light. The primary antibody big‐h3 was located by secondary, goat anti‐rabbit antibody conjugated with gold.

Results In normal cornea moderate distribution of big‐h3 was observed. In PGCD and RGCD corneas, strong labelling for big‐h3 was observed in deposits, within very thin microfilaments (nm) and at the inter‐lamellar junctions. Labelling was significantly higher in GCD Descemet’s membrane compared to normal Descemet’s membrane. Very strong labelling was observed in the deposits and in the cytoplasm of epithelial cells, of debridement specimens.

Conclusion Big‐h3 protein, of epithelial origin, is thought to diffuse into the stroma to form stromal deposits. The presence of aggregates of the protein around keratocytes suggests that it is also produced by these cells. The presence of granular deposits and big‐h3 protein suggested that the protein migrated from limbal epithelium during resurfacing of host epithelium.
Anterior segment imaging with fourier‐domain optical coherence tomography system compared to time‐domain OCT
Authors: NOWINSKA A , WYLEGALA E , MILKA M , MANKOWSKI W
Published: September 2008

Purpose To compare anterior segment measurements and morphology obtained from two anterior segment optical coherence tomography systems

Methods 30 eyes of 30 healthy volunteers were recruited for the study. Central corneal thickness (CCT), trabecular‐iris angle (TIA) and angle opening distance (AOD 500) at the nasal and temporal angles were measured three times during one visit with two systems: Visante OCT (Carl Zeiss Meditec, Inc, Dublin, California, USA) and RTVue FD‐OCT Corneal Anterior Module system (Optovue, Inc. Fremont, CA, USC). Anterior segment morphology was assessed and compared.

Results Mean values of CCT, TIA and AOD by Visante OCT and RTVue FD‐OCT were as follows: automatic CCT 535 ± 33,07 μm, 538 ± 31,82 μm; manual CCT 545 ± 30,91 μm, 542 ± 30,57 μm; nasal TIA 34,7± 9,5, 35,2 ± 8,9; temporal TIA 35,3 ± 8,5, 35,5 ± 9; nasal AOD 435 ± 95 μm, 444 ± 98 μm; temporal AOD 443 ± 103 μm, 452 ± 99 μm. There was no significant difference between mean values and they were highly correlated. Morphologic analysis of two OCT systems proved that Visante OCT has lower resolution, but in one anterior chamber image, all the anterior chamber structures are visible, while RTVue provides very precise information about small fragments of anterior chamber.

Conclusion RTVue FD‐OCT provides accurate anterior segment parameters measurements and shows agreement wit OCT Visante.