Optometry and Vision Sciences - Research Publications
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ItemNo Preview AvailableAge-related deficits in retinal autophagy following intraocular pressure elevation in autophagy reporter mouse model(ELSEVIER SCIENCE INC, 2023-11)This study quantified age-related changes to retinal autophagy using the CAG-RFP-EGFP-LC3 autophagy reporter mice and considered how aging impacts autophagic responses to acute intraocular pressure (IOP) stress. IOP was elevated to 50 mm Hg for 30 minutes in 3-month-old and 12-month-old CAG-RFP-EGFP-LC3 (n = 7 per age group) and Thy1-YFPh transgenic mice (n = 3 per age group). Compared with younger eyes, older eyes showed diminished basal autophagy in the outer retina, while the inner retina was unaffected. Autophagic flux (red:yellow puncta ratio) was elevated in the inner plexiform layer. Three days following IOP elevation, older eyes showed poorer functional recovery, most notably in ganglion cell responses compared to younger eyes (12 months old: -33.4 ± 5.3% vs. 3 months mice: -13.4 ± 4.5%). This paralleled a reduced capacity to upregulate autophagic puncta volume in the inner retina in older eyes, a response that was seen in younger eyes. Age-related decline in basal and stress-induced autophagy in the retina is associated with greater retinal ganglion cells' susceptibility to IOP elevation.
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ItemRetinal Dysfunction in a Mouse Model of HCN1 Genetic Epilepsy(SOC NEUROSCIENCE, 2023-03-22)Pathogenic variants in HCN1 are associated with a range of epilepsy syndromes including a developmental and epileptic encephalopathy. The recurrent de novo HCN1 pathogenic variant (M305L) results in a cation leak, allowing the flux of excitatory ions at potentials where the wild-type channels are closed. The Hcn1M294L mouse recapitulates patient seizure and behavioral phenotypes. As HCN1 channels are highly expressed in rod and cone photoreceptor inner segments, where they shape the light response, mutated channels are likely to impact visual function. Electroretinogram (ERG) recordings from male and female mice Hcn1M294L mice revealed a significant decrease in the photoreceptor sensitivity to light, as well as attenuated bipolar cell (P2) and retinal ganglion cell responses. Hcn1M294L mice also showed attenuated ERG responses to flickering lights. ERG abnormalities are consistent with the response recorded from a single female human subject. There was no impact of the variant on the structure or expression of the Hcn1 protein in the retina. In silico modeling of photoreceptors revealed that the mutated HCN1 channel dramatically reduced light-induced hyperpolarization, resulting in more Ca2+ flux during the response when compared with the wild-type situation. We propose that the light-induced change in glutamate release from photoreceptors during a stimulus will be diminished, significantly blunting the dynamic range of this response. Our data highlight the importance of HCN1 channels to retinal function and suggest that patients with HCN1 pathogenic variants are likely to have a dramatically reduced sensitivity to light and a limited ability to process temporal information.SIGNIFICANCE STATEMENT Pathogenic variants in HCN1 are emerging as an important cause of catastrophic epilepsy. HCN1 channels are ubiquitously expressed throughout the body, including the retina. Electroretinogram recordings from a mouse model of HCN1 genetic epilepsy showed a marked decrease in the photoreceptor sensitivity to light and a reduced ability to respond to high rates of light flicker. No morphologic deficits were noted. Simulation data suggest that the mutated HCN1 channel blunts light-induced hyperpolarization and consequently limits the dynamic range of this response. Our results provide insights into the role HCN1 channels play in retinal function as well as highlighting the need to consider retinal dysfunction in disease caused by HCN1 variants. The characteristic changes in the electroretinogram open the possibility of using this tool as a biomarker for this HCN1 epilepsy variant and to facilitate development of treatments.
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ItemNo Preview AvailableRetinal alpha-synuclein accumulation correlates with retinal dysfunction and structural thinning in the A53T mouse model of Parkinson's disease(FRONTIERS MEDIA SA, 2023-05-05)Abnormal alpha-synuclein (α-SYN) protein deposition has long been recognized as one of the pathological hallmarks of Parkinson's disease's (PD). This study considers the potential utility of PD retinal biomarkers by investigating retinal changes in a well characterized PD model of α-SYN overexpression and how these correspond to the presence of retinal α-SYN. Transgenic A53T homozygous (HOM) mice overexpressing human α-SYN and wildtype (WT) control littermates were assessed at 4, 6, and 14 months of age (male and female, n = 15-29 per group). In vivo retinal function (electroretinography, ERG) and structure (optical coherence tomography, OCT) were recorded, and retinal immunohistochemistry and western blot assays were performed to examine retinal α-SYN and tyrosine hydroxylase. Compared to WT controls, A53T mice exhibited reduced light-adapted (cone photoreceptor and bipolar cell amplitude, p < 0.0001) ERG responses and outer retinal thinning (outer plexiform layer, outer nuclear layer, p < 0.0001) which correlated with elevated levels of α-SYN. These retinal signatures provide a high throughput means to study α-SYN induced neurodegeneration and may be useful in vivo endpoints for PD drug discovery.
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ItemTargeted delivery of LM22A-4 by cubosomes protects retinal ganglion cells in an experimental glaucoma model(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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ItemEffect of hydroxychloroquine or chloroquine and short wavelength light on in vivo retinal function and structure in mouse eyes(TAYLOR & FRANCIS LTD, 2023-07-04)CLINICAL RELEVANCE: The use of chloroquine or hydroxychloroquine can lead to both acute and chronic changes to both retinal structure and function. BACKGROUND: Chloroquine (CQ) and hydroxychloroquine (HCQ) have the potential for retina toxicity. The acute impact of short-term drug exposure (2-4 weeks) on in vivo retinal structure and function and assess whether short wavelength light exposure further exacerbates any structural and functional changes was assessed in a murine model. METHODS: Adult C57BL/6 J mice received intraperitoneal injection of vehicle or hydroxychloroquine (10 mg/kg) 3 times per week for 2 or 4 weeks, or chloroquine for 4 weeks (10 mg/kg). Over this period, animals were exposed to room light (8 hours) or short-wavelength light 4 hours per day (4 hours of normal room light) for 5 days each week. Retinal changes were assessed using electroretinography (ERG), in vivo optical coherence tomography (OCT) imaging. RESULTS: Short-term low-dose HCQ and CQ treatment led to RPE thickening and elongation of photoreceptors. These structural changes were associated with a no dysfunction in the case of HCQ treatments and widespread functional changes (photoreceptor sensitivity, bipolar cell amplitude and oscillatory potential amplitude) in the case of CQ treatment. Exposure to low intensity short-wavelength light does not appear to alter the effect of HCQ or CQ. CONCLUSIONS: HCQ and CQ treatment has acute effects on both retinal structure and function, effects that were not exacerbated by short wavelength light exposure. Whether chronic short wavelength light exposure exacerbates these changes require further study.
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ItemCharacterization of retinal function and structure in the MPTP murine model of Parkinson's disease(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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ItemThe Effect of Aging on Retinal Function and Retinal Ganglion Cell Morphology Following Intraocular Pressure Elevation(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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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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ItemNo Preview AvailableFractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy( 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.