Optometry and Vision Sciences - Research Publications
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ItemImplantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats(JOURNAL OF VISUALIZED EXPERIMENTS, 2016-06-01)The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.
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ItemAAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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ItemAAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization(SPRINGER, 2018-02)Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.
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ItemA Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice(Journal of Visualized Experiments, 2018)The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.
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ItemTowards better estimates of uncorrected presbyopia(WORLD HEALTH ORGANIZATION, 2015-10)
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ItemUtility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina(MARY ANN LIEBERT, INC, 2019-11-01)Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.
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ItemElectroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode(MyJove Corporation, 2019-03-27)The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.
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ItemNo Preview AvailableOptical coherence tomography: seeing the unseen(TAYLOR & FRANCIS LTD, 2019-05-01)
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ItemHypercapnia Impairs Vasoreactivity to Changes in Blood Pressure and Intraocular Pressure in Rat Retina(LIPPINCOTT WILLIAMS & WILKINS, 2019-07)SIGNIFICANCE: The balance between oxygen and carbon dioxide sets the resting tone (or diameter) of retinal blood vessels. Eyes that are hypercapnic use up their "vasodilatory reserve" and therefore fail to respond adequately to changes in intraocular or blood pressure. PURPOSE: Retinal vessels are regulated by both myogenic and metabolic mechanisms. We considered whether alteration of metabolic status would modify the vascular response to ocular perfusion pressure (OPP) lowering in rat retina. METHODS: In pentobarbital anesthetized adult Brown-Norway rats, normocapnia or hypercapnia was achieved by artificially ventilating animals with air or 5% carbon dioxide in ~30% oxygen, respectively. Ocular perfusion pressure was gradually reduced to ~20 mmHg by either lowering blood pressure (slowly drawing blood from a femoral artery/vein) or manometrically increasing intraocular pressure under normocapnic or hypercapnic conditions. In all four groups (n = 7 eyes for each), a confocal scanning laser ophthalmoscope was used to acquire image sequences centered on the optic nerve throughout pressure modification. The diameter of arterioles and venules at various OPP levels was measured and expressed as percentage relative to their own baseline. The response of arterioles and venules to OPP lowering was compared between normocapnic and hypercapnic groups. RESULTS: Average arterial carbon dioxide partial pressures were 36.9 ± 2.6 mmHg in normocapnic and 64.1 ± 5.9 mmHg in hypercapnic (P < .001) animals. In the normocapnic groups, blood pressure lowering and intraocular pressure elevation resulted in significant vasodilation of both arterioles and venules (P < .0001). In the hypercapnic groups, OPP lowering-induced vasodilation was significantly attenuated compared with the corresponding normocapnic groups (P < .0001 for both, two-way analysis of variance). CONCLUSION: Hypercapnia significantly modified myogenic vascular autoregulation in response to OPP reduction.
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ItemCorrespondence Between Behavioral, Physiological, and Anatomical Measurements of Visual Function in Inhibitory Neuron Ablated Zebrafish(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019-11)PURPOSE: To compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels. METHODS: Inhibitory neurons were ablated in larval zebrafish retina. The Ptf1a gene, which determines inhibitory neuron fate in developing vertebrates, was used to express nitroreductase. By exposing larvae to the prodrug metronidazole, cytotoxicity was selectively induced in inhibitory neurons. Visual phenotypes were characterized at behavioral, physiological, and anatomical levels using an optomotor response (OMR) assay, electroretinography (ERG), and routine histology, respectively. Nonvisual locomotion was also assessed to reveal any general behavioral effects due to ablation of other nonvisual neurons that also express Ptf1a. RESULTS: Injured larvae showed severely reduced OMR relative to controls. Locomotor assessment showed unaltered swimming ability, indicating that reduced OMR was due to visual deficits. For ERG, injured larvae manifested either reduced (type-I) or absent (type-II) b-wave signals originating from bipolar interneurons in the retina. Histologic analysis showed altered retinal morphology in injured larvae, with reductions in synaptic inner plexiform layer (IPL) thickness and synaptic density more pronounced in type-II than type-I larvae; type-II larvae also had smaller retinae overall. CONCLUSIONS: The consequences of inhibitory neuron ablation corresponded closely across behavioral, physiological, and anatomical levels. Inhibitory neuron loss likely increases the ratio of neural excitation to inhibition, leading to hyperexcitability. In addition to modulating visual signals, inhibitory neurons may be critical for maintaining retinal structure and organization. This study highlights the utility of a multidisciplinary approach and provides a template for characterizing other zebrafish models of neurological disease.