Optometry and Vision Sciences - Research Publications

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    Targeted delivery of LM22A-4 by cubosomes protects retinal ganglion cells in an experimental glaucoma model
    Ding, Y ; Chow, SH ; Chen, J ; Le Brun, AP ; Wu, C-M ; Duff, AP ; Wang, Y ; Song, J ; Wang, J-H ; Wong, VHY ; Zhao, D ; Nishimura, T ; Lee, T-H ; Conn, CE ; Hsu, H-Y ; Bui, B ; Liu, G-S ; Shen, H-H (ELSEVIER SCI LTD, 2021-05)
    Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. STATEMENT OF SIGNIFICANCE: Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.
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    Characterization of retinal function and structure in the MPTP murine model of Parkinson's disease
    Tran, KKN ; Wong, VHY ; Lim, JKH ; Shahandeh, A ; Anh, H ; Finkelstein, D ; Bui, B ; Nguyen, CTO (NATURE PORTFOLIO, 2022-05-09)
    In addition to well characterized motor symptoms, visual disturbances are increasingly recognized as an early manifestation in Parkinson's disease (PD). A better understanding of the mechanisms underlying these changes would facilitate the development of vision tests which can be used as preclinical biomarkers to support the development of novel therapeutics for PD. This study aims to characterize the retinal phenotype of a mouse model of dopaminergic dysfunction and to examine whether these changes are reversible with levodopa treatment. We use a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD to characterize the neurotoxic effects of MPTP on in vivo retinal function (electroretinography, ERG), retinal structure (optical coherence tomography, OCT) and retinal dopaminergic cell number (tyrosine hydroxylase immunohistochemistry, IHC) at two time points (21 and 45 days) post MPTP model induction. We also investigate the effect of levodopa (L-DOPA) as a proof-of-principle chronic intervention against MPTP-induced changes in the retina. We show that MPTP decreases dopaminergic amacrine cell number (9%, p < 0.05) and that a component of the ERG that involves these cells, in particular oscillatory potential (OP) peak timing, was significantly delayed at Day 45 (7-13%, p < 0.01). This functional deficit was paralleled by outer plexiform layer (OPL) thinning (p < 0.05). L-DOPA treatment ameliorated oscillatory potential deficits (7-13%, p < 0.001) in MPTP animals. Our data suggest that the MPTP toxin slows the timing of inner retinal feedback circuits related to retinal dopaminergic pathways which mirrors findings from humans with PD. It also indicates that the MPTP model causes structural thinning of the outer retinal layer on OCT imaging that is not ameliorated with L-DOPA treatment. Together, these non-invasive measures serve as effective biomarkers for PD diagnosis as well as for quantifying the effect of therapy.
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    The Effect of Aging on Retinal Function and Retinal Ganglion Cell Morphology Following Intraocular Pressure Elevation
    Lee, PY ; Zhao, D ; Wong, VHY ; Chrysostomou, V ; Crowston, JG ; Bui, BV (FRONTIERS MEDIA SA, 2022-05-12)
    Aging and elevated intraocular pressure (IOP) are two major risk factors for glaucomatous optic neuropathy; a condition characterized by the selective, progressive injury, and subsequent loss of retinal ganglion cells (RGCs). We examined how age modified the capacity for RGCs to functionally recover following a reproducible IOP elevation (50 mmHg for 30 min). We found that RGC functional recovery (measured using electroretinography) was complete by 7 days in 3-month-old mice but was delayed in 12-month-old mice until 14 days. At the 7-day recovery endpoint when RGC function had recovered in young but not older eyes, we examined RGC structural responses to IOP-related stress by analyzing RGC dendritic morphology. ON-RGC cell volume was attenuated following IOP elevation in both young and older mice. We also found that following IOP elevation OFF-RGC dendritic morphology became less complex per cell volume in young mice, an effect that was not observed in older eyes. Our data suggest that adaptations in OFF-RGCs in young eyes were associated with better functional recovery 7 days after IOP elevation. Loss of RGC cellular adaptations may account for delayed functional recovery in older eyes.
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    Age-Specific Retinal and Cerebral Immunodetection of Amyloid-β Plaques and Oligomers in a Rodent Model of Alzheimer's Disease
    Habiba, U ; Merlin, S ; Lim, JKH ; Wong, VHY ; Nguyen, CTO ; Morley, JW ; Bui, B ; Tayebi, M (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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    Targeted Delivery of Lm22a-4 by Cubosomes Protects Retinal Ganglion Cells in an Experimental Glaucoma Model
    Ding, Y ; Chow, SH ; Chen, J ; Le Brun, AP ; Wu, C-M ; Duff, AP ; Wang, Y ; Wong, VHY ; Zhao, D ; Lee, T-H ; Conn, CE ; Hsu, H-Y ; Bui, BV ; Liu, G-S ; Shen, H-H ( 2020-01-01)
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    Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy
    Mills, S ; Jobling, A ; Dixon, M ; Bui, B ; Vessey, K ; Phipps, J ; Greferath, U ; Venables, G ; Wong, VHY ; Wong, CHY ; He, Z ; Hui, F ; Young, J ; Tonc, J ; Ivanova, E ; Sagdullaev, B ; Fletcher, E ( 2020)
    Local blood flow control within the CNS is critical to proper function and is dependent on coordination between neurons, glia and blood vessels. Macroglia such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial involvement, since mice lacking Cx3cr1, exhibited no fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan, abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 weeks due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

    Significance Statement

    This work identifies a novel role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signalling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial-vessel association, loss of microglial-capillary regulation and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial-vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow.
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    Tyro3 is a key regulator of myelin structure and retinal function in the central nervous system
    Blades, F ; Wong, VHY ; Nguyen, CTON ; Bui, BV ; Kilpatrick, TJ ; Binder, MD (SAGE PUBLICATIONS LTD, 2020-03)
    Background: While it is evident how critical myelin is for neural signalling in the CNS, the biological processes underpinning myelination remain unknown. Previously, we have shown that the receptor tyrosine kinase, Tyro3, regulates developmental myelination and myelin thickness in the CNS. Objectives: The aim of this study was to extend on our previous data by assessing the role of Tyro3 in regulating other myelin structures such as the node of Ranvier; and to assess the effect of Tyro3 loss on axonal conductivity and retinal function. Methods: To investigate node of Ranvier microstructure, we used electron microscopy and manually assessed paranodal loops. We measured nodal and paranodal widths using fluorescent immunohistochemistry staining and ImageJ software. To investigate neural conduction velocities, we measured visual evoked potentials in vivo and compound action potentials in slice cultures. Lastly, full-field electroretinograms and optical coherence tomographies were performed to assess retinal function. All experiments were wild-type to constitutive Tyro3 KO comparisons. Results: We show that Tyro3 receptor loss results in wider nodes of Ranvier and dysregular paranodal loop attachment at nodes. KO mice did not show significant alterations to axonal conduction in visual evoked potentials but did have reduced response amplitudes in compound action potentials. Finally, we show that Tyro3 loss results in a decrease in signal output from photoreceptor, bipolar and particularly retinal ganglion cells. Conclusion: Signalling via Tyro3 is key for the attachment of paranodal loops at nodes of Ranvier. Tyro3 deficient myelin results in a decrease in neural response amplitude. Tyro3 is important for the function of photoreceptor, bipolar and retinal ganglion cells of the retina.
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    Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update
    Lin, F-L ; Wang, P-Y ; Chuang, Y-F ; Wang, J-H ; Wong, VHY ; Bui, B ; Liu, G-S (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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    Increased episcleral venous pressure in a mouse model of circumlimbal suture induced ocular hypertension
    Wong, VHY ; Zhao, D ; Bui, B ; Millar, CJ ; Nguyen, CTO (ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2021-01)
    PURPOSE: To investigate changes in aqueous humor dynamics during intraocular pressure (IOP) elevation induced by circumlimbal suture in mice. METHODS: Ocular hypertension (OHT) was induced by applying a circumlimbal suture behind the limbus in male adult C57BL6/J mice. In the OHT group, the suture was left in place for an average of 8 weeks (n = 10, OHT group). In the sham control group the suture was cut at 2 days (n = 9, sham group) and in the naïve control group (n = 5) no suture was implanted. IOP was measured at baseline across 3 days, 1 h post-suture implantation, and at the chronic endpoint. Anterior segments were assessed using optical coherence tomography (OCT). Episcleral venous pressure (EVP), total outflow facility (C), uveoscleral outflow (Fu) and aqueous humor flow rate (Fin) were determined using a constant-flow infusion model. RESULTS: All aqueous dynamic and chronic IOP outcome measures showed no difference between sham and naïve controls (p > 0.05) and thus these groups were combined into a single control group. IOP was elevated in OHT group compared with controls (p < 0.01). Chronic suture implantation did not change pupil size, anterior chamber depth or iridocorneal angles (p > 0.05). EVP was significantly higher in OHT eyes compared to control eyes (p < 0.01). There was no statistical difference in C, Fu and Fin between groups (p > 0.05). A significant linear correlation was found between IOP and EVP (R2 = 0.35, p = 0.001). CONCLUSIONS: Circumlimbal suture implantation in mouse eyes results in chronic IOP elevation without angle closure. Chronic IOP elevation is likely to reflect higher EVP.
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    A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization
    Chen, J ; Lin, F-L ; Leung, JYK ; Tu, L ; Wang, J-H ; Chuang, Y-F ; Li, F ; Shen, H-H ; Dusting, GJ ; Wong, VHY ; Lisowski, L ; Hewitt, AW ; Bui, BV ; Zhong, J ; Liu, G-S (SPRINGER, 2021-02)
    Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.