Yael Nisgav

Gucy2f zebrafish knockdown - a model for Gucy2d-related leber congenital amaurosis

Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible.

Enhancement of Angiogenic Potential of Endothelial Cells by Contact with Retinal Pigment Epithelial Cells in a Model Simulating Pathological Conditions

PURPOSE Choroidal neovascularization (CNV) is the leading cause of vision loss in chorioretinal diseases involving contact between retinal pigment epithelial (RPE) and endothelial cells (ECs). The aim of this study was to investigate changes in the angiogenic potential of ECs induced by RPE-EC interaction in two models of RPE-EC coculture. METHODS RPE and ECs were grown in contact or noncontact coculture. Selection of ECs was achieved using magnetic beads coated with antibodies specific for EC surface proteins. Angiogenesis was assessed by analyzing the expression of EC genes involved in angiogenesis by RT-PCR. Tube formation on Matrigel was used as a functional angiogenesis assay. Expression and activity of matrix metalloproteases (MMPs) were examined by RT-PCR and zymography, respectively. RESULTS Coculture of ECs with RPE in the contact model under normoxic conditions induced markedly upregulated EC mRNA expression of 16 genes involved in positive regulation of angiogenesis. Solo ECs subjected to hypoxia demonstrated upregulated expression of the same 16 genes, including VEGF and HIF1. The EC VEGF level was not affected by coculture with RPE in the noncontact model. ECs demonstrated enhanced tube formation on Matrigel after contact coculture with RPE. EC MMP2 mRNA and activity levels were elevated in contact, but not in noncontact, coculture. CONCLUSIONS Coculture of ECs with RPE under conditions enabling direct EC-RPE contact enhances the proangiogenic potential of ECs under normoxia, to an extent similar to that induced by hypoxia, suggesting that ECs in direct contact with RPE cells might be more prone to pathologic angiogenesis involved in CNV formation.

Efficacy of Subconjunctival Aflibercept Versus Bevacizumab for Prevention of Corneal Neovascularization in a Rat Model.

Purpose: We aimed to evaluate the efficacy of subconjunctival aflibercept, a vascular endothelial growth factor trap compound, for the treatment of corneal neovascularization in a rat model. Methods: Chemical burn was produced in the central cornea of 31 male Sprague–Dawley rats. Animals were randomized to receive treatment with subconjunctival injection of 0.08 mL aflibercept (25 mg/mL), 0.05 mL bevacizumab (25 mg/mL), or 0.05 mL physiologic saline. Corneal neovascularization was evaluated on postinjury days 1, 3, 7, 9, and 13 by corneal photographs. The rats were killed on day 21 and samples were collected for histological and flat-mount immunofluorescence analyses. Results: In all rats, vascular sprouting began on day 3, reached maximum density on days 7–9, and spontaneously regressed thereafter. Mean burn area in the central cornea comprised ∼15% of the total corneal area. The aflibercept group had a significantly smaller relative area of neovascularization than both control group (P < 0.05, 12.27 ± 9.91, 29.66 ± 9.96 days 7) and bevacizumab group (P < 0.05, 12.27 ± 9.91, 21.27 ± 8.19 days 7 and 15.5 ± 10.25, 32.38 ± 9.44 days 9; Mann–Whitney test). On histological study, hematoxylin and eosin staining revealed blood vessels extending to the central cornea in the control and bevacizumab groups and limited to the periphery in the aflibercept group. Immunofluorescence study with an endothelial marker revealed a smaller area of staining in the aflibercept group. Conclusions: Aflibercept effectively inhibits corneal neovascularization in a rat model of chemical burn–induced neovascularization and warrants further study for potential use in humans.

The effects of the apoE4 genotype on the developing mouse retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimers disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of VEGF.

Knockdown of unc119c results in visual impairment and early-onset retinal dystrophy in zebrafish.

PURPOSE UNC119 proteins are involved in G protein trafficking in mouse retinal photoreceptors and Caenorhabditis elegans olfactory neurons. An Unc119 null allele is associated with cone-rod dystrophy in mouse, but the mechanism leading to disease is not understood. We studied the role of Unc119 paralogs and Arl3l2 in zebrafish vision and retinal organization resulting from unc119c and arl3l2 knockdown. METHODS Zebrafish unc119c was amplified by PCR from retina and pineal gland cDNA. Its expression pattern in the eye and pineal gland was determined by whole-mount in-situ hybridization. unc119c and arl3l2 were knocked down using morpholino-modified oligonucleotides (MO). Their visual function was assessed with a quantitative optomotor assay on 6 days post-fertilization larvae. Retinal morphology was analyzed using immunohistochemistry with anti-cone arrestin (zpr-1) and anti-cone transducin-α (GNAT2) antibodies. RESULTS The zebrafish genome contains four genes encoding unc119 paralogs located on different chromosomes. The exon/intron arrangements of these genes are identical. Three Unc119 paralogs are expressed in the zebrafish retina, termed Unc119a-c. Based on sequence similarity, Unc119a and Unc119b are orthologs of mammalian UNC119a and UNC119b, respectively. A third, Unc119c, is unique and not present in mammals. Whole mount in-situ hybridization revealed that unc119a and unc119b RNA are ubiquitously expressed in the CNS, and unc119c is specifically expressed in photoreceptive tissues (pineal gland and retina). A Unc119 interactant, Arl3l2 also localizes to the pineal gland and the retina. As measured by the optomotor response, unc119c and arl3l2 knockdown resulted in significantly lower vision compared to wild-type zebrafish larvae and control morpholino (MO). Immunohistological analysis with anti-cone transducin and anti-cone arrestin (zpr-1) indicates that knockdown of unc119c leads to photoreceptor degeneration mostly affecting cones. CONCLUSIONS Our results suggest that Unc119c is the only Unc119 paralog that is highly specific to the retina in zebrafish. Unc119c and Arl3l2 proteins are important for the function of cones.

Mobile Laser Indirect Ophthalmoscope: For the Induction of Choroidal Neovascularization in a Mouse Model

ABSTRACT Purpose: This study aims to evaluate and standardize the reliability of a mobile laser indirect ophthalmoscope in the induction of choroidal neovascularization (CNV) in a mouse model. Materials & Methods: A diode laser indirect ophthalmoscope was used to induce CNV in pigmented male C57BL/6J mice. Standardization of spot size and laser intensity was determined using different aspheric lenses with increasing laser intensities applied around the optic disc. Development of CNV was evaluated 1, 5, and 14 days post laser application using fluorescein angiography (FA), histology, and choroidal flat mounts stained for the endothelial marker CD31 and FITC-dextran. Correlation between the number of laser hits to the number and size of developed CNV lesions was determined using flat mount choroid staining. The ability of intravitreally injected anti-human and anti-mouse VEGF antibodies to inhibit CNV induced by the mobile laser was evaluated. Results: Laser parameters were standardized on 350 mW for 100 msec, using the 90 diopter lens to accomplish the highest incidence of Bruch’s membrane rupture. CNV lesions’ formation was validated on days 5 and 14 post laser injury, though FA showed leakage on as early as day 1. The number of laser hits was significantly correlated with the CNV area. CNV growth was successfully inhibited by both anti-human and mouse VEGF antibodies. Conclusion: The mobile laser indirect ophthalmoscope can serve as a feasible and a reliable alternative method for the CNV induction in a mouse model.

Correlation between Interleukin-6 and Thrombin–Antithrombin III Complex Levels in Retinal Diseases

ABSTRACT Purpose: This study aims to evaluate and correlate the levels of interleukin-6 (IL-6) and thrombin–antithrombin III complex (TAT) in the vitreous of patients with different vitreoretinal pathologies. Methods: Vitreous samples were collected from 78 patients scheduled for pars plana vitrectomy at a tertiary medical center. Patients were divided by the underlying vitreoretinal pathophysiology, as follows: macular hole (MH)/epiretinal membrane (ERM) (n = 26); rhegmatogenous retinal detachment (RRD) (n = 32); and proliferative diabetic retinopathy (PDR) (n = 20). Levels of IL-6 and TAT were measured by enzyme-linked immunosorbent assay and compared among the groups. Results: A significant difference was found in the vitreal IL-6 and TAT levels between the MH/ERM group and both the PDR and RRD groups (P < 0.001 for all). Diabetes was associated with higher IL-6 levels in the RRD group. Different relationships between the IL-6 and TAT levels were revealed in patients with different ocular pathologies. Conclusion: Our results imply that variations in vitreal TAT level may be attributable not only to an inflammatory reaction or blood–retinal barrier breakdown, but also to intraocular tissue-dependent regulation of thrombin.

Retinal Penetration of Intravitreally Injected Tissue Plasminogen Activator: A Rat Model Study

Purpose: To determine whether intravitreal unconjugated tissue plasminogen activator (tPA) (alteplase) can penetrate the intact neural retina and reach the subretinal space in an experimental model. Methods: This study was performed in 24 Sprague-Dawley rats aged 12 weeks. Under general anesthesia, the right eye was injected with either 0.75 μg of 3 μL tPA (14 rats; study group) or saline (10 rats, control group) into the vitreous. Animals were euthanized at 3, 24, and 48 h. The eyes were enucleated, and cryosections were prepared for immunofluorescence staining. Goat anti-tPA antibody was used to detect tPA. Results: In the study group, staining for tPA was detected in the deep retinal layers in all eyes. The staining was deeper and more intense at 3 and 24 h than at 48 h. There was no tPA staining in the retina of eyes injected with saline. Conclusions: This experimental study shows that unconjugated tPA administered into the vitreous is capable of penetrating the deep retinal layers and the subretinal space. These findings suggest that further clinical research is warranted on the benefits of intravitreal tPA in the treatment of submacular hemorrhage.

Remote manipulation of posterior lamellar corneal grafts using a magnetic field.

Purpose: In posterior lamellar keratoplasty procedures such as Descemet stripping endothelial keratoplasty and Descemet membrane endothelial keratoplasty, the lamellar graft is manipulated directly or by injecting an air bubble. This preliminary study sought to evaluate the feasibility of guiding lamellar corneal grafts by generating a magnetic field. Methods: Rabbit and porcine Descemet stripping endothelial keratoplasty and Descemet membrane endothelial keratoplasty grafts were manually produced and immersed in a ferromagnetic solution containing nanomagnetic particles conjugated to streptavidin or in gadoteric acid. For the feasibility study, grafts were transferred to an artificial anterior chamber or plastic test tube and a magnetic field was generated with a handheld NdFeB disc magnet. The presence and the sustainability of graft motion were documented under various conditions. For the semiquantitative study, whole or partial grafts were transferred to a plastic test tube after immersion, and the amount of tissue retraction induced by the remote magnet was graded. Results: The grafts were successfully manipulated in all directions by the magnet, from a distance of up to 7 mm. They remained ferromagnetic more than 24 hours after immersion in the ferromagnetic solutions. The degree of retraction was affected by graft size, immersion time, time from immersion, and immersion solution. Conclusions: Posterior lamellar corneal grafts may be made ferromagnetic and remotely manipulated by creation of a magnetic field. The ferromagnetic properties are adjustable. This technique holds promise in attaching and repositioning grafts during keratoplasty. Further research is needed to assess the possible effects of ferromagnetic solutions on corneal endothelial cells and on lamellar graft clarity.