bluuebunny/crossref_metadata_2025 huggingface dataset:
Sustained neuroprotection after a single intravitreal injection of PGJ2 in a rodent model of NAION
Authors: TOUITOU V , JOHNSON M , MILLER NR , GUO Y , BERNSTEIN SL
Published: November 2010

Purpose Prostaglandin‐J2 (PGJ2) has been proposed as a potential neuroprotective agent. We wanted to evaluate the toxicity/efficacy of a single intravitreal (IVT) injection of PGJ2 in a rodent model of nonarteritic anterior ischemic optic neuropathy (NAION).

Methods We used the laser‐activated rose Bengal induction method to produce AION in Long‐Evans rats. We evaluated IVT‐PGJ2 retinal and ON toxicity. Following induction, PGJ2 was IVT‐injected in the treatment‐group. IVT phosphate‐buffered‐saline (PBS) was used as control. Functional studies (VEP) were performed at baseline and at 7days post‐treatment. Structural studies included immunohistochemical (IHC), electron microscopic (EM) analysis of the optic nerve (ON), and stereologic analysis of retinal ganglion cell (RGC) numbers at30 day 30.

Results Toxicity: IVT PGJ2 (5 eyes) did not induce any significant functional/structural changes in the retina or ON of treated animals compared with animals injected with PBS (5 eyes) 30 days post‐injection. Efficacy: After a single IVT‐injection, day7 VEPs in the PGJ2‐treatment group (n=7) had amplitudes 103.6% of baseline, whereas the PBS‐treated group (n=6) had VEPs that were 42.4% of the baseline. 30days post‐stroke, EM of ON from the treatment‐group demonstrated significant preservation of axons and decreased demyelination. Stereological RGCcounts confirmed significant (p<0.04) RGC preservation in PGJ2‐treated animals (1462.6 cells/µm2) compared w the stroke+PBS group (1156.5 cells/µm2).

Conclusion A single IVT of PGJ2 preserves RGCs and their axons, and provides sustained neuroprotection for at least 1 month following initial ischemic event.
Src family tyrosine kinase activation is required for endothelin‐1 to inhibit Na,K‐ATPase in porcine lens epithelium
Authors: DELAMERE NA , SHAHIDULLAH M , BEIMGRABEN C , MANDAL A
Published: November 2010

Purpose Earlier studies point to the involvement of Src family tyrosine kinases (SFKs) in the stimulation of of Na,K‐ATPase activity by purinergic receptor agonists ATP and UTP. Src itself was activated (Tamiya, et al. 2007, Am. J. Physiol. 293: C790‐6). Here, we examined the role of SFKs in the response to endothelin‐1 (ET‐1), an ET receptor agonist that causes Na,K‐ATPase inhibition.

Methods Porcine lenses were incubated 30 min in Krebs’ solution with ET‐1 or other test agents. The epithelium was removed, homogenized and analyzed by western blot or Na,K‐ATPase activity assay.

Results Exposure of the intact lens to ET‐1 caused a reduction in Na,K‐ATPase activity. The Na,K‐ATPase response was not observed when lenses were pretreated with 10 uM PP2, a selective inhibitor of SFKs. ET‐1 caused SFK activation evident from an increase in Y416 phosphorylation and decrease in Y527 phosphorylation of a ~61kDa SFK. The SFK inhibitor PP2 abolished the SFK phosphorylation response. Since SFKs Fyn, Src, Hck and Yes may contribute to the observed 61kDa band, these SFKs were isolated by immunoprecipitation and analyzed separately. Based on Y416 phosphorylation, ET‐1 appeared to activate Fyn, while Src and Hck were inhibited. Yes activity was unaltered.

Conclusion ET‐1, which causes Na,K‐ATPase inhibition, elicits a different pattern of SFK activation from that reported earlier for purinergic agonists which stimulate Na,K‐ATPase activity. Previously, Na,K‐ATPase stimulation was observed when Src was activated. The ET‐1 response points to Na,K‐ATPase inhibition when Fyn kinase is activated.
High glucose sensitizes human retinal endothelial cells for IFN‐g‐mediated apoptosis
Authors: NAGARAJ R , MAILANKOT M
Published: November 2010

Purpose The biochemical mechanisms by which inflammatory cytokines cause damage in the diabetic retina are poorly understood. Indoelamine 2, 3‐dioxygenase (IDO) is an inducible by IFN‐γ enzyme and is the first enzyme of the kynurenine pathway, which produces cytotoxic kynurenines. In this study we have investigated the role of IDO in apoptosis of human retinal capillary endothelial cells (HREC) under hyperglycemic conditions.

Methods HREC were cultured in medium containing high glucose (25 mM) or low glucose (7.5 mM) and incubated with 1‐100 U/ml of IFN‐γ. IDO activity was measured by an HPLC assay. Expression of IFN‐γ receptor 1, and activation of the JAK‐STAT signaling pathway along with activation of PKC‐δ was assessed by Western blotting. HREC apoptosis was measured by Hoechst staining. The role of IDO in HREC apoptosis was evaluated in the presence specific chemical inhibitors of the kynurenine pathway.

Results IFN‐γ dose‐dependently activated JAK‐STAT signaling and PKC‐δ, and upregulated IDO. The IDO‐mediated tryptophan oxidation led to formation of kynurenines, which was followed by chemical modification of proteins by kynurenines in HREC. These changes were accompanied by production of reactive oxygen species (ROS) and depletion of protein‐free thiols. IFN‐γ inhibited cell cycle at low concentrations and caused caspase‐3‐mediated apoptosis and at higher concentrations, and those effects were amplified in the presence of high glucose in HREC. We found that IFN‐γ mediated cytotoxicity in HREC was primarily due to ROS generated by 3‐hydroxykynurenine.

Conclusion Our results suggest that high glucose sensitizes HREC to deleterious effects IFN‐γ and provide a novel mechanistic pathway for retinal capillary endothelial cell death in diabetes.
Retinal and cortical functions in adult mice lacking cannabinoid receptors
Authors: CASANOVA C , ZABOURI N , FARISHTA A , ROBERT C , VANNI M , BELANGER S , MINVILLE K , BOUCHARD JF
Published: November 2010

Purpose Cannabinoid receptor type 1 (CB1R) has been localized in the adult retina of rodents. It is expressed in cones, horizontal, bipolar, some amacrine and ganglion cells. The expression of the cannabinoid receptor type 2 (CB2R) mRNA in the retina of adult rats was also reported. The goal of the present study was to investigate the functional roles of CB1R and CB2R in the retina by comparing retinal electrophysiological responses and cortical optical signals in normal and genetically modified mice.

Methods Experiments were conducted on four different groups of C57BL/6 mice: CB1R wild type (WT), CB1R knockout (KO), CB2R WT and KO. Scotopic electroretinograms (ERG) luminance‐response functions and photopic ERGs were recorded. In a subset of CB1 groups, intrinsic signals acquired by optical brain imaging were used to determine spatial frequency, contrast sensitivity and retinotopic maps in the visual cortex.

Results The CB1R KO retina showed a stronger photopic response. No differences were observed for scotopic responses. For the CB2R groups, the scotopic b‐wave response was stronger in the KO mice. No differences could be seen between visual cortices maps with respect to SF and contrast sensitivity. Retinotopic maps differed only along the azimuth. Significant differences were observed between hemodynamic response functions.

Conclusion These results indicate that CB receptors can play a regulatory effect on the neurovascular coupling at the retinal and cortical levels and on the functional organization of the mice visual cortex along the azimuth Axis.(NSERC)
Primary cell culture of mengiothelial cells ‐ a new model for future studies of optic nerve disease
Authors: MEYER P , XIN X , KILLER HE , FLAMMER J
Published: November 2010

Purpose Our previous study revealed that the occurrence and amount of meningothelial cell nests in the subarachnoid space were significantly increased in glaucoma histologic examination. For further investigation of the possible role of meningothelial cells in the pathogenesis of optic nerve diseases in vitro, pure meningothelial cells are needed. Accordingly, we developed a culture method of meningothelial cells harvested from the arachnoid layer covering the optic nerve.

Methods Menigiothelial cells were prepared from porcine optic nerve sheaths by scraping cells from the arachnoid layer into cell culture media. After outgrowth of cells, contaminating fibroblasts were eliminated using MACS. The remaining cells were characterized using immunofluorescence staining of fibroblast and meningothelial cell markers.

Results Primary cells grew within 1‐3 weeks and formed a monolayer composed of two different cell types, flattened cells with round nuclei and spindle shaped cells. Cells were separated by automatic fibroblast microbeads. Positive cells labelled with anti‐fibroblast microbeads showed a characteristic elongated fibroblast‐like cytoplasm whereas negative cells against fibroblast bead presented a flattened cell type. We used GFAP as well as keratan sulphate antibodies as markers for identifying meningothelial cells. Cells not

Conclusion Our method for primary meningothelial cell culture combines harvesting of superficial arachnoid cells from the optic nerve sheath with a purification process based on magnetic beads and is effective in providing pure and rich meningothelial cells.
Celastrol regulates innate immunity response via NF‐kB and HSP70 in ARPE‐19 cells
Authors: PAIMELA T
Published: November 2010

Purpose Chronic inflammation participates in the pathology of age‐related macular degeneration (AMD). Recent studies indicate that celastrol, a novel triterpene compound, modulates inflammatory responses, but its effect on the human retinal pigment epithelial cells is poorly understood. In this study, we investigated the potential anti‐inflammatory role of celastrol and its effect on nuclear factor kappa B (NF‐kB) activity 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 concentrations of celastrol and the secretion of IL‐6 cytokine was analyzed by ELISA. The effect of celastrol exposure on heat shock protein 70 (HSP70) expression was analyzed by western blotting. In response to celastrol and modulated HSP70 levels NF‐kB activity was examined by ELISA.

Results Celastrol suppressed the LPS‐induced IL‐6 expression levels via NF‐kB transcription factor in ARPE‐19 cells. Celastrol evoked elevated HSP70 levels without cytotoxicity. Interestingly, celastrols capability to inhibit NF‐kB activity was diminished when HSP70 response was suppressed by siRNA. This reveals that celastrol has potent anti‐inflammatory capacity in ARPE‐19 cells, and its effect is modulated through NF‐kB and HSP70.

Conclusion Our findings reveal that celastrol is a novel compound to suppress innate immunity response in human retinal pigment epithelial cells.
p62/sequestosome 1 as a regulator of proteasome inhibitor‐induced autophagy in human retinal pigment epithelial cells
Authors: KAARNIRANTA K
Published: November 2010

Purpose The pathogenesis of age‐related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress –induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates.The functional roles of p62 and HSP70 were evaluated in conjunction with protesome inhibitor ‐induced autophagy in human RPE cells (ARPE‐19).

Methods The p62, HSP70 and ubiquitin protein levels and localization were analyzed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay.

Results Proteasome inhibition evoked the accumulation of p62 and HSP70 that strongly co‐localized with each other in perinuclear aggregates. The p62 accumulation was time and concentration dependent after MG‐132 proteasome inhibitor loading. Interestingly, autophagy induction was p62 and Hsp70 independent. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently we showed that hsp70 mRNA depletion increased cell death in ARPE‐19 cells. Here we now demonstrate that p62 mRNA silencing has similar effects on cellular viability.

Conclusion The p62 and HSP70 are central molecules in the regulation of protein turnover in human retinal pigment epithelial cells in proteasome inhibitor‐ induced autophagy.
OCT features of the optic disk parameters and peripapillary nervous fibres layer thickness in children with acquired myopia
Authors: BOYCHUK IM , GORBATYUK TL
Published: November 2010

Purpose To reveal morphological and structural peculiarities of retinal nerve fiber layer thickness (RNFLT) and optic nerve parameters in schoolchildren with acquired myopia in dependence of myopia degree.

Methods 135 children (270 eyes) of 10‐14 y.o. with bilateral light (86 eyes), moderate (50) and high degree of myopia (42) as well as control group of healthy children (40) were observed. Morphostructural indices (PRNFLTh) in four segments (nasal, temporal, superior, and inferior) and optic disk parameters were investigated by optical coherent tomography SOCT Copernicus, OPTOPOL Tech.Sp.z o.o. applying standard protocols – Retina analysis and Disk analysis. Evaluation of data and comparisons in groups were performed with the help of STATISTICA for Windows 98, a Newman‐Kales criterion was applied.

Results Significantly less (p=0,03) peripapillary RNFLT values were found in nasal segments in children with the high degree of myopia as compared to children in control group and with the light degree of myopia. We founded that it depended on Rim Area values. Disk Area (2,08 – 2,16) mm2 was found in 82,7% and above 3,0 mm2 in 17,3% of myopic patients. Disk Area 2,07 ‐ 4,25 mm2 was revealed in myopic patients and 2,08 – 3,11 mm2 in control group. Cup Area was 0,23‐0,54 mm 2 in myopic and 0,38‐1,13 mm2 in healthy children Rim Area 1,2‐3,2 mm2 in myopic and 1,66 – 2,63 mm2 in control group. Cup/Disk ratio 0,19‐1,18 was established in myopic and 0,19 – 1,18 in control group.

Conclusion Axial myopia is often accompanied with cone near the optic disk. It is suggested that disk parameters such as – Disk Area and Rim Area can be identified as parameters which can be considered as the signs of myopia progression.
Fitting of challenging corneas with a special design of rigid gas permeable (RGP) lens
Authors: ABOU SAMRA W
Published: November 2010

Purpose To study the safety and efficacy of a new design of RGP lens namely Rose K irregular cornea (IC) in management of corneas with high degree of irregular astigmatism.

Methods The study was made in 66 eyes of 40 patients with irregular astigmatism.The selection criterion was to obtain a sample population with irregular astigmatism that was unlikely to be corrected with spectacles or conventional contact lens.A complete ophthalmologic exploration which included topography was made. All eyes were fitted with Rose K IC after measuring precisely lens parameters according to a special trial set of this design.Refraction, visual acuity before and after fitting,maximum wearing time, contact lens related problems and patient satisfaction were analyzed.

Results The studied eyes included eyes with high degree of irregular astigmatism due to keratoconus in 42 eyes (63.6%),corneal injuries in 6 eyes (9.1%), PMD in 12 eyes (18.2%), and post Lasik ectasia in 6 eyes (9.1%).After fitting of the eyes with the RGP lens, there was a significant improvement in the visual acuity (VA)in all fitted eyes. The mean VA improved from 0.16±0.25 to 0.85± 0.13(p<0.001).Study of spherical and astigmatic errors showed also a highly significant improvement. The mean spherical error was ‐4.6 ± 2.4 and improved to 0.50 ± 0.20 after final fitting (p<0.001).The mean astigmatic error of studied eyes was ‐6.2 ± 2.3 while after fitting was ‐0.75±0.32(p<0.001).Statistical analysis of the subjective responses indicated a strong acceptance of the lens by all subjects.

Conclusion Rose K IC proved to be effective in correcting high degree of corneal astigmatism caused by a variable number of challenging corneal conditions with high visual performance and good patient comfortability.
Ulrastructural features of keratoconus cornea after cross‐linking by riboflavin/UVA
Authors: AKHTAR S , ALMUBRAD TM , PALADINI I , MENCUCCI R
Published: November 2010

Purpose In the present studies we assess the effects of collagen cross‐linking on ultrastructure organisation of the corneal stroma of keratoconus human corneas.

Methods One normal, one keratoconus (KC) and three cross‐linked keratoconus corneas were analysed. One was treated with standard cross linking (SXL) and two with trans‐epithelial collagen cross linking (TEXL). Penetrating keratoplasty was performed three months after treatment. All samples were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy.

Results The structure of SXL corneas was very similar to normal corneas in their hemidesmosomes, basement membrane (BM), Bowman’s layer (BW) and stromal lamellae that were not undulated. The architecture of TEXL corneas presented some differences. The BM was thick with degenerated hemidesmosomes. Bowman’s layer was disorganised at some places and replaced by thin filaments forming pannus. There were thin undulating lamellae in anterior, middle and posterior stroma. The keratocytes were embedded between undulating lamellae. Large amounts of abnormal PGs were attached around collagen fibrils. The parallel running lamellae were very thin. In some parts of the anterior stroma collagen fibrils were oriented (running) in random directions instead of running parallel. There were some parts of the stroma which showed a normal appearance.

Conclusion The present studies demonstrate that corneal cross‐linking leads to modifications in keratocytes and in the organisation of collagen fibril. The morphological changes might be correlated to the process of increase in biomechanical stability although there are differences between stromal structures treated by standard and trans
Corneal keratocyte population after laser subepithelial keratectomy (LASEK) with mitomycin C (MMC): 3 months vs 15 months comparison
Authors: CANADAS SUAREZ P , DRAKE P , HERNÁNDEZ‐VERDEJO JL , DE BENITO‐LLOPIS L , TEUS MA
Published: November 2010

Purpose To study corneal keratocyte population after LASEK with mitomycin C (MMC) 15 months vs 3 months after surgery

Methods Sixty eyes were included in this study, all of them treated with LASEK with intraoperative 0.02% MMC, divided into 2 groups. 32 eyes had LASEK performed 3 months before, versus 28 in the 15 months post‐op group. Keratocyte density was examined by using confocal microscopy in the anterior, mid and posterior stroma in both groups, and the mean density was compared with a control group of healthy eyes

Results Keratocyte density in the anterior stroma was 16993,75+ 8001,7 cells/mm3 and 18529,1 + 2917,7 cells/mm3(p=0,3) in the 3 months and in the 15 months groups respectively, in the mid stroma it was 30783,3+ 9300 cells/mm3 in the 3 months and 20754,7 + 3615,3 cells/mm3 in the 15 months groups (p= 0,0001) and 30286,75+ 8321 cells/mm3and 19994+ 3085,9 cells/mm3 (p= 0,0001) in the posterior stroma in the 3 months and in the 15 months groups respectively. The comparison between the average densities through the whole cornea showed statistically significant difference between the 3 months vs 15 months group and 3 months vs normals, but it was not statistically significant different between 15 months vs normals

Conclusion The changes in the corneal keratocyte population after LASEK + MMC seen in the early post‐op, seem to change towards the normal values 15 months after the procedure.
LASEK with MMC vs femtosecond sub‐Bowmann keratomileusis to correct myopic astigmatism: a pilot study
Authors: TEUS MA , IGLESIAS MLUZ , GIMÉNEZ C , SANCHEZ‐PINA JM , CANADAS‐SUAREZ P
Published: November 2010

Purpose To evaluate the safety and efficacy of LASEK and Femtosecond sub‐Bowmann Keratomileusis (FSBK) in the correction of myopic astigmatism

Methods Prospective, masked study of consecutive eyes, with myopic astigmatism higher than 1.5D. MMC was used in LASEK eyes if ablation > 50 microns. Visual results were evaluated three months post‐op

Results 252 eyes were analyzed, 125 LASEK and 127 FSBK. Preoperative sphere and cylinder was similar in both groups (p=0.1). Safety and Efficacy indexes were 1.01 vs 1.02 and 0.90 vs 0.88 in LASEK and FSBK groups, respectively (p=0.8 and 0.3)

Conclusion LASEK and FSBK achieve comparable visual results when used to correct myopic astigmatism at 3 months follow‐up
Neuroprotective effect of intravitreal cell‐based glucagon‐like peptide‐1 production in the optic nerve crush model
Authors: JONAS JB , ZHANG R , ZHANG H , XU L , MA KE , WALLRAPP C
Published: November 2010

Purpose To examine the effect of intraocularly produced glucagon‐like peptide‐1 (GLP‐1) on the survival rate of retinal ganglion cells in an optic nerve crush model.

Methods Forty‐one Sprague‐Dawley rats were divided into a study group (21 animals) in which 4 beads with 3000 genetically modified cells to produce GLP‐1 were intravitreally implanted into the right eye; a saline control group (n=12) with intravitreal saline injection; and a GLP‐1 negative bead control group (n=8) in which 4 beads with 3000 cells without GLP‐1 production were intravitreally implanted. The right optic nerves of all animals were crushed in a standardized manner. After labeling the retinal ganglion cells by injecting 3% fluorogold into the superior colliculus, the animals were sacrificed, and the ganglion cells were counted on retinal flat mounts.

Results The retinal ganglion cell density of the right eyes was significantly higher in the study group (2030±470 cells/mm2) than in the GLP‐1 bead negative control group (1390±367 cells/mm2;P=0.001) and than in the saline control group (1734±424 cells/mm2;P=0.07). Correspondingly, the survival rate (ratio of retinal ganglion cell density of right eye / left eye) was significantly higher in the study group (71±19%) than in the GLP‐1 bead negative control group (49±14%; P=0.003) and than in the saline control group (57±16%;P=0.03).

Conclusion Glucagon‐like peptide‐1 produced by intravitreally implanted cell beads was associated with a higher survival rate of retinal ganglion cells after an experimental optic nerve crush in rats.

Commercial interest
Interaction of vascular and biomechanical aspects of glaucoma
Authors: LESK M
Published: November 2010

Purpose In an attempt to understand some of the reasons why some optic nerves appear to be sensitive to IOP and others not, I will review studies describing the interaction between vascular and biomechanical factors in open angle glaucoma.

Methods Studies using biomechanical modelling, epidemiologic data, measurements of ocular or systemic blood flow, measurment of peripheral vasospasticity, and measurement of ocular biomechanical parameters will be reviewed. Hypotheses will be presented regarding the interpretation of these data.

Results Studies suggest that the optic nerves of vasospasctic patients may be more IOP‐sensitive than those of non‐vasospastic patients. Non‐invasive measurements of ocular blood flow suggest that this pressure‐sensitivity may be related to IOP‐sensitive optic nerve blood flow. Biomechanical modelling suggests that scleral and lamina cribrosa elasticity, axial length, and eye wall thickness contribute to optic nerve head stress and strain. Cross‐sectional clinical data supports the role of increased ocular elasticity in the susceptibilty of the optic nerve to glaucoma damage, especially in vasospastic patients. Some promising new avenues for research in this area will be presented.

Conclusion There is increasing evidence that biomechanical and vascular ocular factors interact leading to an elevated susceptibilty of the optic nerve to glaucomatous optic nerve damage.

Commercial interest
Oxidative stress and glaucoma
Authors: FLAMMER J
Published: November 2010

Abstract not provided
Development of a next‐generation sequencing platform for retinal dystrophies, with LCA and RP as proof of concept
Authors: COPPIETERS F
Published: November 2010

Purpose Retinal dystrophies represent an emerging group of hereditary disorders that lead to degeneration of the photoreceptors and/or the retinal pigment epithelium, resulting in irreversible blindness. They are genetically complex, with over 200 disease loci identified so far. Current genetic screening consists of microarray analysis (Asper Ophthalmics) for the most recurrent mutations, and subsequent Sanger sequencing. However, the high cost and low throughput of the latter technology limits testing to only the most recurrent genes. This project aims to develop a high throughput and cost‐effective platform for screening of all known disease genes for Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP), using the next‐generation sequencing (NGS) technology.

Methods A NGS panel will be developed for all 16 and 47 known LCA and RP genes, respectively, including coding and untranslated regions, regulatory regions and microRNA binding sites. The protocol will consist of the following steps: 1) high throughput primerdesign and qPCR, 2) ligation, 3) shearing and 4) sequencing on the Illumina Genome Analyser IIx (GAIIx). This innovative protocol overcomes the need for short amplicons in order to render short‐read sequences by the GAIIx. This sequencing instrument was chosen because of its high capacity, low cost per base and the absence of interpretation problems at homopolymeric regions. Analysis of the variants will be performed using in‐house developed and commercial software, which ranks all variants according to their pathogenic potential.

Conclusion Using the proposed protocol, comprehensive screening for all known disease genes for LCA and RP will be available for the first time.
Identification of novel disease gene for primary congenital glaucoma (PCG) through homozygosity mapping and next‐generation sequencing strategies in a large consanguineous pedigree
Authors: VERDIN H
Published: November 2010

Purpose Primary congenital glaucoma (PCG) is caused by developmental anomalies of the trabecular meshwork and the anterior chamber angle resulting in an increased ocular pressure (IOP) and optic nerve damage. In general PCG displays an autosomal recessive inheritance pattern and is genetically heterogeneous. To date, three PCG loci are known, namely GLC3A, GLC3B and GLC3C, and two causal genes have been identified, CYP1B1 located in the GLC3A locus and LTPB2 located at 1.3 MB proximal to the GLC3C locus. The purpose of the current study is to identify the causal disease gene in a large consanguineous family with PCG, originating from Jordany. CYP1B1 mutations and linkage to the LTBP2, GLCB3 and GLCC3 locus were previously excluded.

Methods In a first step, DNA from members from the consanguineous family will be genotyped by 250K GeneChip Mapping Affymetrix arrays. Homozygosity mapping will be applied to identify potential disease loci, using a homemade Perl script. Next, microsatellite analysis will be performed in order to confirm findings and to narrow down candidate regions. Subsequently, candidate regions of interest will be captured (Agilent) and sequenced on the Illumina Genome Analyser IIx (GAIIx). Gene and variant prioritization will be done using in‐house developed software, followed by segregation analysis and screening in control individuals. At last, a cohort of 30 molecularly unsolved PCG patients will be screened for mutations in the newly identified disease.

Conclusion The identification of a new disease gene for PCG may lead to better insights into the molecular pathogenesis of glaucoma, and might uncover novel therapeutic strategies.
Role of placental growth factor (PIGF) in wound healing after glaucoma filtration surgery
Authors: VAN BERGEN T
Published: November 2010

Purpose Failing filtering surgery due to excessive wound healing is a considerable challenge in ophthalmology, and largely contributes to progressive vision loss in glaucoma patients. Anti‐VEGF therapy helps to prevent post‐surgical scarring by inhibiting angiogenesis and collagen deposition, but does not influence inflammation (which is also an important player in postoperative wound healing). We will check the hypothesis that placental growth factor (PlGF) plays a role in scar formation after glaucoma filtration surgery, and that it may be a(n) (additional) target for improvement of the outcome of this surgery through its known anti‐angiogenic and anti‐inflammatory, and possibly anti‐fibrotic properties.

Methods The effect of an anti‐PlGF antibody (ThromboGenics) will be investigated in vitro on the proliferation of endothelial cells (HUVEC), inflammatory cells (Jurkat cells) and of Tenon fibroblasts (TF). The effect of the antibody will also be investigated in vivo in a rabbit model for glaucoma surgery by measuring intra‐ocular pressure (IOP), filtration bleb function and survival, and by (immuno‐)histological analysis of angiogenesis (CD31), inflammation (CD45) and fibrosis (Sirius Red).

Conclusion Our proposed research project will elucidate the potential role of PlGF‐inhibition in the improvement of filtration surgery outcome, and will highlight any angiostatic, anti‐inflammatory, and/or anti‐fibrotic effects. PlGF‐inhibition as an adjuvant anti‐inflammatory therapy to anti‐VEGF treatment in glaucoma surgery might open new perspectives for more efficient surgery. In conclusion, our project opens exciting perspectives for the treatment of the blinding condition of glaucoma, and thus might improve the visual prognosis of glaucoma patients.
Tear film biomarkers as prognostic indicators for recurrent pterygium
Authors: ZAKARIA N
Published: November 2010

Purpose The aim of this project is to establish the use of IL‐6, IL‐8 and VEGF as biomarkers in the tear film for early detection of recurrent pterygium.

Methods Cytometric Bead Array (CBA) kits will be used to perform multicytokine assays in the tear samples of eyes having pterygium before and after surgical resection of the pterygia. This technique utilizes flow cytometry to determine the concentrations of multiple analytes namely IL‐6, IL‐8 and VEGF (proposed biomarkers) present in small volumes of tear fluid.Patients with pterygia showing corneal encroachment and requiring surgical excision will be recruited in this study along with a second population of control subjects consisting of individuals with no history of eye diseases or contact lens wear. After instilling a local anesthetic, 3 drops of normal saline will be applied and the at least 50µl of the diluted epithelial secretions collected and stored at ‐80°C for CBA analysis. From the results we can determine the baseline levels of IL‐6, IL‐8 and VEGF present in normal epithelial secretions and correlate it with potentially higher levels in the eyes of patients with pterygia. By collecting post op epithelial secretions at different time points along with regular ocular surface photographs and grading of any recurrent pterygia we will be able to ascertain the role of these cytokines and growth factors as biomarkers for recurrent pterygia.

Conclusion By establishing higher tear film levels of IL‐6, IL‐8 and VEGF in eyes with pterygia compared to normal eyes and the return to baseline levels post excision we can begin to ascertain the role of these key players in the pathogenesis of pterygia.
Cellular genic therapy: evaluation of mesenchymal stem cells engraftment transduced by an Ecdysone inductible lentiviral vector expressing luciferase gene
Authors: NOCHEZ Y , COLLIN C , BELLICAUD D , CARDON A , GICQUEL JJ , PISELLA PJ , GABISON E , PAGES JC
Published: November 2010

Purpose Gene therapy might offer an opportunity to improve new treatments in ophthalmology. Several studies, using limbal stem cells or mesenchymal stem cells (MCSs), have confirmed the usefulness of these cells to repair epithelial and stromal corneal tissue.

Methods We designed a strategy aimed at expressing Luciferase gene. We have tested the viability of mesenchymal stem cells modified by a lentiviral vector delivery system. To allow an adaptative treatment, the target genes would be transcriptionally controlled by the use of an Ecdysone inductible promoter.

Results Construction of a lentiviral plasmid with the transgene (luciferase) and inductible promoter: Ecdysone by recombination. Construction of a lentiviral plasmid with the RXR gene and VgEcR gene and a constitutive enhancer. Transfection of 293 FT cells for lentiviral production. MSCs Transduction by “V/R” lentivirus and by “E/Transgene” lentirus. We have tested induction of Ecdysone system and transcription of the transgene (luciferase) in MSCs adding Ponasterone.

Conclusion The expression of luciferase, using conditionnal expression vectors, could be applied after an engraftment of mesenchymal stem cells for pathological wound healing in Sprague‐Dawley rat cornea.
Body height and ocular dimensions in the adult population in rural Central India. The Central India eye and medical study
Authors: JONAS JB , NANGIA V , MATIN A , KULKARNI M , SINHA A , GUPTA R
Published: November 2010

Purpose To investigate associations between anthropomorphic parameters and ocular dimensions in a typical rural society untouched by the effects of urbanization.

Methods The Central India Eye and Medical Study performed in rural Central India included 4711 participants aged 30 or more years. The participants underwent a detailed ophthalmic and medical examination.

Results After controlling for age, gender, level of education, and body mass index (BMI), taller subjects were more likely to have larger eyes with a longer axial length, lower corneal refractive power, deeper anterior chambers, and longer vitreous cavity . Central corneal thickness (P=0.97) and lens thickness (P=0.08) were not significantly associated with body height. Subjects with a higher BMI had shorter globes, flatter corneas and thicker corneas, thicker lenses and longer vitreous cavities. Body height as compared with the BMI had a stronger influence on the ocular biometric data. After correcting for age, gender, level of education and axial length, for each increase in body height by 10 cm or for each increase in BMI by one unit, the refractive error significantly (P<0.001) increased by 0.23 diopters and by 0.40 diopters (P<0.001), respectively.

Conclusion In the rural population of Central India without urbanization associated myopization, body height and size of the eye was associated with each other. Subjects with a higher body mass index had shorter eyes, flatter and thicker corneas, and thicker lenses. Since the occurrence of some ocular diseases depends of eye size and refractive error, the results may be helpful for screening examinations and for elucidating pathogenic associations.
The utility of a rabbit model of adenovirus ocular infection
Authors: ROMANOWSKI E
Published: November 2010

Acute adenovirus ocular infections are the most common ocular viral infections worldwide and are associated with community and medical facility epidemics. While not permanently blinding, ocular adenoviral infections are associated with significant patient morbidity, including symptomatic distress with visual disturbances which can last years, and loss of time from school or work. Currently, the treatment of these acute infections is symptomatic due to the lack of an approved antiviral. This symptomatic treatment presents a controversy of whether to treat these viral infections with immunomodulating agents (e.g. topical corticosteroids, NSAIDs, cyclosporine A), which are contraindicated for use in the treatment of the other major external ocular viral infection, HSV‐1 epithelial keratitis. The adenovirus type 5 (Ad5)/New Zealand White (NZW) rabbit ocular model has provided data for potential clinical guidelines for the use of immunomodulating agents in the treatment of adenovirus ocular infections. Moreover, this model has been used extensively to evaluate promising candidate antivirals for adenovirus. Multiple candidate antivirals have been evaluated in vivo using this model, and several have proceeded to human clinical trials. The current presentation will discuss the potential clinical guidelines for the use of immunomodulating agents, present data on potential new topical antiviral agents, discuss the potential combination therapy of an antiviral and immunomodulatory agent in the treatment of adenovirus ocular infections as well as the limitations of the model.

Commercial interest
Animal models for vaccinia virus keratitis
Authors: BRANDT C
Published: November 2010

Purpose Studies on pathogenic mechanisms involved in viral ocular infections and testing of potential therapies requires well established quantitative animal models. We present a new model of Vaccinia virus keratitis.

Methods Rabbits were infected with the Dryvax strain of Vaccinia virus and disease was scored using a modified MacDonald‐Shadduck scoring system. The model will be described and examples of the use of the model for studies of pathogenesis and testing therapeutic approaches will be presented.

Results The optimum dose of virus was determined (10e5 PFU). Infiltration of neutrophils followed predominantly by CD4+ cells occurred in the cornea and the optimum therapy was determined to be topical viroptic 9 times per day for 10 days. Steroid use should be avoided.

Conclusion A new model of Vaccinia keratitis has been developed that is useful for pathogenesis and therapeutic studies.

Commercial interest
Cytomegalovirus infections
Authors: ATHERTON S , ZHANG M , MARSHALL B
Published: November 2010

Even in the era of highly‐active retroviral therapy (HAART), some HIV‐infected patients continue to develop cytomegalovirus retinitis. The purpose of this SIS presentation is to discuss how results from a mouse model of CMV retinitis using murine CMV (MCMV) may help us understand the pathogenesis of CMV infection of the retina in immunosuppressed human patients. One interesting aspect of MCMV retinitis in the mouse that has also been described in human patients is the striking juxtaposition of uninfected retinal cells that are apoptotic with cytomegalovirus‐infected retinal cells that are not apoptotic. Using in vivo approaches (using MCMV infected BALB/c mice) as well as complementary in vitro studies (using virus infected cells), our laboratory has been attempting to decipher the virus and host cells processes that contribute to this interesting pattern of pathology. In addition to consideration of the most likely virus and cell modulators that mediate this process, the possible role of this juxtaposition of infected and uninfected retinal cells in preventing panretinal infection and/or in reducing spread of the virus in the retina during cytomegalovirus infection will be discussed.
Correlation of SNPs in exon 1 of loxl‐1 gene with pseudoexfoliation syndrome in patiens from Western Greece
Authors: PANOUTSOPOULOS AA , GARTAGANIS VS , GIANNAKOPOULOS MP , PANTELI VS , GARTAGANIS SP
Published: November 2010

Purpose Pseudoexfoliation (PEX) syndrome is a clinically important systemic disorder of the extracellular matrix characterized by pathologic accumulation of an abnormal fibrillar material in various ocular tissues. LOXL1 is a cross‐linking enzyme in extracellular matrix metabolism and is required for elastic fiber formation. Single nucleotide polymorphisms (SNPs) in exon 1 of the lysyl oxidaselike 1 (LOXL1) gene have been recently identified as strong genetic risk factors for PEX syndrome. The purpose of this project is to correlate certain Single Nucleotide Polymorphisms (SNPs) in exon 1 of the gene loxl‐1 with patients from the area of Western Greece who present PEX syndrome

Methods After isolating genomic DNA from the blood of PEX syndrome and cataract patients from Western Greece, exon 1 of the loxl‐1 gene was generated by Polymerase Chain Reaction (PCR) with specific primers. The DNA samples were sent for sequencing and the haplotypes that were sequenced for the areas of the loxl‐1 exon1 SNPs were statistically processed.

Results In more than 50% of the PEX syndrome patients the SNPs rs3825942 and rs1048661 that are previously associated with Exfoliation Glaucoma (XFG), were shown in our population. Moreover, a significant number of cataract patients with no PEX syndrome phenotype had the above haplotype for the loxl‐1 gene.

Conclusion We have performed an analysis of LOXL1 and PEX syndrome in a Western Greece patient population and have confirmed the strong association previously reported for Icelandic and Swedish samples. Preliminary results from 50 patients show that there is strong correlation for SNPs rs3825942 and rs1048661 with patients with PEX syndrome.
Gases and the ocular vasculature
Authors: SCHMETTERER L
Published: November 2010

Purpose The regulation of ocular blood flow and oxygenation is largely controlled by metabolic factors. Among them gases like oxygen (O2), carbon dioxide (CO2), carbon monoxide (CO) and nitroc oxide (NO) play a key role.

Methods In a series of clinical trials we focussed on the role of O2, CO2, CO and NO in the control of ocular blood flow. These studies were done on healthy subjects and employed a number of techniques including laser Doppler flowmetry, color Doppler imaging and pulsatile ocular blood flow techniques.

Results O2 is a very potent vasoconstrictor in the retina and optic nerve head, but not in the choroid. The mechanisms underlying this effect is not entirely clear, but appears to involve endothelin A receptors. CO and CO2 are potent vasodilators in retina, choroid and optic nerve head. NO plays a major role in the control of basal vascular tone in all ocular vessels, but is also involved in many agonist‐induced vasodilator effects.

Conclusion Gases play a key role in controlling ocular blood flow. An imblanace of the production of these mediators is associated with ischemia, hypoxia and endothelial dysfunction.
Hydrogen sulphide: a new CNS mediator
Authors: OSBORNE N
Published: November 2010

The recent discovery that hydrogen sulphide (H2S) is an endogenously produced gaseous secondary messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential role of H2S as a retinal neuroprotective agent. In the current study we use dithiolethiones (kindly provided by Dr. Piero Del Soldato, Milan, Italy) as H2S donors and show that such substances attenuate the effect of retinal ischemia as well as oxidative and light‐induced injury to a transformed line of cells (RGC‐5 cells) in culture. Ischemia was delivered to rats by elevation of the intraocular pressure above the systolic blood pressure. Partial damage to the retina after seven days was determined by a combination of procedures which included analysis of electroretinograms, immunohistochemistry and changes in the retinal content of proteins and mRNAs known to be associated with ganglion cell function and apoptosis. Most of the changes caused by ischemia were significantly attenuated by intravitreal injection of a H2S donor directly after ischemia. Both light (400‐700nm, intensity 1000 lux) and hydrogen peroxide caused death to RGC‐5 cells in culture over a period of 24‐48 hours in a time and dose‐dependent manner, respectively. Light and hydrogen peroxide‐induced RGC‐5 cell death is by different forms of apoptosis but they are both attenuated by the H2S donor, ACS1. These initial findings demonstrate that donors of H2S may be value in the treatment of various retinal dysfunctions where oxidative stress, light or ischemia is implicated as causative fact
Carbon monoxide as a mediator in the retina
Authors: BUCOLO C , DRAGO F
Published: November 2010

Purpose Carbonic monoxide (CO) is organic gas ubiquitously synthesized in mammalian tissues by enzyme that has constitutive and inducible forms. This gas is produced as metabolic end‐product in specific cell life phases, and may acts as atypical neuronal messenger. Evidence has recently accumulated suggesting that CO may be cytoprotective because its bioactions, including inhibition of apoptosis, platelet aggregation, complement activation, and inflammatory cytokine production. CO appears to be important to counteract the cytotoxicity caused by excessive production of reactive oxygen (ROS) and nitrogen (RNS) species.

Methods In vitro and in vivo models.

Results Induction of heme oxygenase (HO)‐1 by hemin has been found to prevent retinal cell death after ischemia provoked by ocular hypertension in rats. The LPS‐induced expression of pro‐inflammatory cytokines, in rat eye, is also inhibited by CO. Interestingly, drugs active as inhibitors of iNOS block CO‐induced increases in cGMP in the retina.Drugs inhibiting NO formation by acting on iNOS activity have been found to potently reduce intraocular pressure. Studies from our lab showed that an increase of CO availability by hemin or carbon monoxide‐releasing molecules lower the intraocular pressure, suggesting a suppress action of iNOS‐derived NO production.

Conclusion A better understanding of CO regulation may lead to new therapeutic options that are safer and more efficacious than currently available treatments for various sight‐threatening eye diseases, such as retinal degenerations.
Oxygenation of the human retina
Authors: STEFANSSON E
Published: November 2010

Purpose Partial pressure of oxygen in the optic nerve and retina is regulated by the intraocular pressure and systemic blood pressure, the resistance in the blood vessels and oxygen consumption of the tissue. The PO2 is autoregulated and moderate changes in intraocular pressure, blood pressure or tissue oxygen consumption do not affect the retinal and optic nerve oxygen tension.

Methods If the intraocular pressure is increased above 40 mmHg or the ocular perfusion pressure decreased below 50 mmHg the autoregulation is overwhelmed and the optic nerve becomes hypoxic. The levels of perfusion pressure that lead to optic nerve hypoxia in the laboratory correspond remarkably well to the levels that increase the risk of glaucomatous optic nerve atrophy in human glaucoma patients. Medical intervention can affect optic nerve PO2. Lowering the intraocular pressure tends to increase the optic nerve PO2, even though this effect may be masked by the autoregulation when the optic nerve PO2 and perfusion pressure is in the normal range.

Results Carbonic anhydrase inhibitors increase retinal PO2 through a mechanism of vasodilatation and lowering of the intraocular pressure. Carbonic anhydrase inhibition reduces the removal of CO2 from the tissue and the CO2 accumulation induces vasodilatation resulting in increased blood flow and improved oxygen supply. This effect is inhibited by indomethacin but not other cyclo‐oxygenase inhibitors.

Conclusion Carbonic anhydrase inhibitors increase retinal blood flow and increase oxygen delivery. Glaucoma drugs and glaucoma surgery lower intraocular pressure, increase ocular perfusion pressure and blood flow. Demand of oxygen by retinal cells may be reduced through apoptosis and tissue atrophy, as well as active destruction of tissue by laser photocoagulation.