Optometry and Vision Sciences - Research Publications
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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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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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ItemAge-Specific Retinal and Cerebral Immunodetection of Amyloid-β Plaques and Oligomers in a Rodent Model of Alzheimer's Disease(IOS PRESS, 2020)BACKGROUND: Amyloid-β soluble oligomers (Aβo) are believed to be the cause of the pathophysiology underlying Alzheimer's disease (AD) and are normally detected some two decades before clinical onset of the disease. Retinal pathology associated with AD pathogenesis has previously been reported, including ganglion cell loss, accumulation of Aβ deposits in the retina, and reduction of nerve fiber layer thickness as well as abnormalities of the microvasculature. OBJECTIVE: This study's aim is to better understand the relationship between brain and retinal Aβo deposition and in particular to quantify levels of the toxic Aβo as a function of age in the retina of a rodent model of AD. METHODS: Retinas and brain tissue from 5×FAD mice were stained with Congo red, Thioflavin-T (Th-T), and Aβ plaque-specific and Aβo-specific antibodies. RESULTS: We show that retinas displayed an age-dependent increase of Th-T-specific amyloid fibrils. Staining with anti-Aβ antibody confirmed the presence of the Aβ plaques in all 5×FAD retinas tested. In contrast, staining with anti-Aβo antibody showed an age-dependent decrease of retinal Aβo. Of note, Aβo was observed mainly in the retinal nuclear layers. Finally, we confirmed the localization of Aβo to neurons, typically accumulating in late endosomes, indicating possible impairment of the endocytic pathway. CONCLUSION: Our results demonstrate the presence of intraneuronal Aβo in the retina and its accumulation inversely correlated with retinal Aβ plaque deposition, indicating an age-related conversion in this animal model. These results support the development of an early AD diagnostic test targeting Aβo in the eye.
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ItemGene Therapy Intervention in Neovascular Eye Disease: A Recent Update(CELL PRESS, 2020-10-07)Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.
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ItemReversibility of Retinal Ganglion Cell Dysfunction From Chronic IOP Elevation(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019-09)PURPOSE: To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. METHODS: IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. RESULTS: With IOP elevation (∼10 mm Hg above baseline), ganglion cell function declined to 75% ± 8% at 8 weeks and 59% ± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% ± 4%) and 16 weeks (83% ± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% ± 7%), but not at week 12 (73% ± 6%). Ganglion cell loss was found in all groups (-8% to -13%). CONCLUSIONS: In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.
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ItemTyro3 Contributes to Retinal Ganglion Cell Function, Survival and Dendritic Density in the Mouse Retina(FRONTIERS MEDIA SA, 2020-08-14)Retinal ganglion cells (RGCs) are the only output neurons of the vertebrate retina, integrating signals from other retinal neurons and transmitting information to the visual centers of the brain. The death of RGCs is a common outcome in many optic neuropathies, such as glaucoma, demyelinating optic neuritis and ischemic optic neuropathy, resulting in visual defects and blindness. There are currently no therapies in clinical use which can prevent RGC death in optic neuropathies; therefore, the identification of new targets for supporting RGC survival is crucial in the development of novel treatments for eye diseases. In this study we identify that the receptor tyrosine kinase, Tyro3, is critical for normal neuronal function in the adult mouse retina. The loss of Tyro3 results in a reduction in photoreceptor and RGC function as measured using electroretinography. The reduction in RGC function was associated with a thinner retinal nerve fiber layer and fewer RGCs. In the central retina, independent of the loss of RGCs, Tyro3 deficiency resulted in a dramatic reduction in the number of RGC dendrites in the inner plexiform layer. Our results show that Tyro3 has a novel, previously unidentified role in retinal function, RGC survival and RGC morphology. The Tyro3 pathway could therefore provide an alternative, targetable pathway for RGC protective therapeutics.
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ItemBlocking endothelial apoptosis revascularizes the retina in a model of ischemic retinopathy(AMER SOC CLINICAL INVESTIGATION INC, 2020-08-03)Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary nonperfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularization. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularization. Vessel reassembly was not limited by VEGFA neutralization, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralization of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue revascularization by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.
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ItemResponse of the Trilaminar Retinal Vessel Network to Intraocular Pressure Elevation in Rat Eyes(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2020-02)PURPOSE: The purpose of this study was to test the hypothesis that the superficial, intermediate, and deep retinal vascular plexus show different responses to intraocular pressure (IOP) elevation. METHODS: Anesthetized adult Long Evans rats (n = 14) were imaged using optical coherence tomography angiography (OCTA; Spectralis) at baseline (IOP 10 mm Hg) and in follow-up mode to examine the vasculature during IOP elevation (10 to 110 mm Hg, 10 mm Hg steps, each step 3 minutes). A 20° × 10° field was imaged. Vessel density within a 2D projection image was determined (%) for the superficial vascular complex (SVC), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). Comparisons were made between layers using 2-way repeated measures ANOVA (layer versus IOP) following normalization to baseline (% relative to 10 mm Hg). RESULTS: The three vascular layers responded differently to IOP elevation. For IOPs between 40 and 60 mm Hg, DCP and ICP capillaries were significantly more resistant to IOP elevation than those in the SVC. When IOP was elevated above 70 mm Hg, all layers showed reduced vessel density. IOP induced change in SVC vessel density closely followed reductions in thickness of the inner retinal layers (nerve fiber, ganglion cell, and inner plexiform layer). This close relationship between reductions in tissue thickness and vessel density was less apparent for the ICP and DCP. CONCLUSIONS: These data show that the intermediate and deep vascular plexus in the rat retina have a greater capacity for autoregulation against mild IOP elevation but are more affected at high IOP.
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ItemAge-related changes in the response of retinal structure, function and blood flow to pressure modification in rats(NATURE PORTFOLIO, 2018-02-13)Age-related changes to the balance between the pressure inside the eye (intraocular pressure, IOP) and the pressure inside the brain (intracranial pressure, ICP) can modify the risk of glaucoma. In this study, we consider whether the optic nerve in older rat eyes is more susceptible to acute IOP and ICP modification. We systematically manipulate both ICP and IOP and quantify their effects on ganglion cell function (electroretinography, ERG), optic nerve structure (optical coherence tomography, OCT) and retinal blood flow (Doppler OCT). We show that ganglion cell function in older eyes was more susceptible to a higher optic nerve pressure difference (ONPD = IOP - ICP). This age-related susceptibility could not be explained by poorer blood flow with elevated ONPD. Rather, as ONPD increased the retinal nerve fibre layer showed greater compression, and the retinal surface showed less deformation in older eyes. Our data suggest that age-related changes to connective tissues in and around the rat optic nerve make it less flexible, which may result in greater strain on ganglion cell axons. This may account for greater functional susceptibility to higher optic nerve pressure differences in older rat eyes. Further studies in a species with a well-developed lamina cribrosa are needed to determine the clinical importance of these observations.
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ItemCharacterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.(Frontiers Media SA, 2017)To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (-15 ± 4%), bipolar cell (-15 ± 4%) and ganglion cell responses (-19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior -12 ± 5%; temporal, -7% ± 2%; inferior -9 ± 4%; nasal -8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.