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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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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ItemNo Preview AvailableResponse of the trilaminar retinal vessel network to intraocular pressure elevation in rat eyes(Association for Research in Vision and Ophthalmology, 2018-07-01)Purpose : It is known that inner retinal blood flow is autoregulated to compensate for changes in ocular perfusion pressure (OPP). However, studies have focused on the superficial vessels. Here we test the hypothesis that the superficial, intermediate and deep retinal vascular plexus show different responses to intraocular pressure (IOP) elevation. Methods : Anesthetized (60:5mg/kg ketamine:xylazine) adult Long Evans rats (n=14) were imaged using optical coherence tomography angiography (OCTA) at baseline (IOP 10mmHg) and in follow up mode to examine the vasculature during IOP elevation (10mmHg steps to 110mmHg, each lasting 3 min). We imaged a 20 x 10-degree field starting at one disc diameter from the optic disc margin. Capillary area (i.e. diameter) within a 2D projection image was determined (% region of interest) for three layers based on segmentation of the structural OCT: superficial vascular complex (SVC), intermediate capillary plexus (ICP) and deep capillary plexus (DVP). Increases and decreases in this parameter can be interpreted as functional “vasodilation” and “vasoconstriction”, respectively, of the column of blood flowing above threshold. Comparisons were made between layers (2-way repeated measures ANOVA, layer vs IOP) following normalisation to baseline (% relative to 10mmHg). Results : Group mean arterial blood pressure at baseline was 125±5 mmHg, thus for the IOPs examined OPP spanned 115±5 to -9±4 mmHg. For OPPs from 115±5 to 77±4 mmHg capillaries within the DCP (9±8%, p<0.05) and ICP (11±10%, p<0.05) showed significant “vasodilation”, whereas those in the SVC showed constriction (-14±6%, p<0.05). For OPPs between 63±4 and 38±4 mmHg, capillary diameter was maintained, by for OPPs below 38mmHg, all layers showed linear attenuation. Significant compression in tissue thickness (retinal nerve fibre, ganglion cell and inner plexiform layers and total retinal thickness) for the same regions were not found until OPP fell below 38mmHg. Conclusions : These data show that the intermediate and deep vascular plexus in the rat retina have a greater capacity for autoregulation against IOP elevation. This might reflect a redistribution of blood flow to the deeper layers during stress. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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ItemNo Preview AvailableReversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.(Association for Research in Vision and Ophthalmology, 2018-07-01)Purpose : To determine the duration of chronic IOP elevation beyond which ganglion cell function can no longer recover using the mouse circumlimbal suture model. Methods : IOP elevation was induced in anaesthetized (isoflurane) adult male C57BL6/J mice by attaching a circumlimbal suture (nylon, 10/0) around the equator of one eye, with the contralateral eye serving as a control. The suture was left in place for 8, 12 and 16 weeks (n=27, 23 and 27), respectively, and animals underwent electroretinography and optical coherence tomography at these time points. In two other groups, the suture was removed after 8 and 12 weeks (n=26 and 28), and the capacity for recovery assessed 4 weeks later. IOP was measured weekly (Tonolab). Retinal ganglion cell (RGC) function (or integrity) was assessed with the positive scotopic threshold response (pSTR) and retinal nerve fibre layer (RNFL) thickness. Data (mean ± SEM) were compared using t-test (control vs. treatment) and one-way ANOVA (within groups). Results : IOP in sutured eyes was higher than control eyes (8wk: 17.1 ± 0.3 vs. 26.8 ± 0.6 mmHg, 12wk: 13.8 ± 0.3 vs. 19.5 ± 0.5 mmHg, 16wk: 17.1 ± 0.2 vs. 27.4 ± 0.6 mmHg; all P<0.001). After suture removal, IOP returned to levels comparable to control eyes (8+4wk: 16.9 ± 0.3 vs. 16.1 ± 0.3 mmHg; P=0.08, 12+4wk: 17.3 ± 0.2 vs. 17.1 ± 0.3 mmHg; P=0.5). With IOP elevation, RGC function declined to 75% ± 8% (8wk), 78% ± 7% (12wk) and 59% ± 4% (16wk, all P<0.001) of control eyes. RNFL thinning was also evident (8wk: 84% ± 4%, 12wk: 83% ± 5%; 16wk: 83% ± 3%; P<0.001) but no change in total retinal thickness was noted (P=0.33). Suture removal at week 8 facilitated full recovery of RGC function (97% ± 7%, P=0.9 vs. baseline) 4 weeks later. However, there was no recovery in RNFL thickness (87% ± 3%, P<0.001 vs. baseline). When the suture was removed at week 12, neither function (79% ± 9%, P<0.05) nor RNFL thickness recovered (89% ± 3%, P<0.01) 4 weeks later. Conclusions : RGC dysfunction can be recovered 4 weeks after an 8-week period of mild IOP elevation, but not after a 12-week period. Beyond 12 weeks, IOP reversal only served to prevent further functional decline. This identifies a critical chronic IOP duration that results in irreversible ganglion cell dysfunction. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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ItemNo Preview AvailableResponse of the Rat Optic Nerve to Acute Intraocular and Intracranial Pressure Changes(Springer Singapore, 2019)Glaucoma is a neurodegenerative disease, characterized by the progressive death of retinal ganglion cells. Elevated intraocular pressure (IOP) is known to be an important risk factor for glaucoma; however, it is not the only force acting on the optic nerve. Intracranial pressure (ICP) also exerts an effect on the optic nerve head, effectively opposing the force applied by IOP. Indeed, this balance of forces creates a pressure gradient (or the translaminar pressure gradient) across the optic nerve head [1]. Increasingly it is thought that the pressure difference between IOP and ICP, the translaminar pressure (TLP), may be critical for the integrity of the retina and optic nerve [2], and thus ICP may be an important risk factor for glaucoma [2–6].
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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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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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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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ItemNo Preview AvailableHow ganglion cell responses to IOP elevation are impacted by blood pressure and intracranial pressure(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : The extent to which blood pressure or intracranial pressure modifies ganglion cell responses to acute intraocular pressure (IOP) elevation incompletely understood. Using the electroretinogram (ERG) we measure ganglion cell mediated responses in rat retina, whilst acutely modifying IOP, BP and ICP in a systematic manner. We quantify the relationship between ganglion cell function and ocular perfusion pressure (BP - IOP) at low, normal and high ICP. Methods : Six groups of adult Long-Evans rats (n=7-11 eyes/group, total animals = 25) were anaesthetised (60:5mg/kg ketamine:xylazine) and underwent acute pressure modification. A femoral artery and vein were cannulated for blood pressure measurement and manipulation (sodium nitroprusside to lower and angiotensin II to elevate pressure). ICP was set to -5, 5 or 25 mmHg via a dual cannula (30G infusion needle inside a 23G measurement needle) placed into the lateral ventricle (-1.5mm from bregma, ±2mm from midline) on the ipsilateral side to the cannulated eye (30G, vitreal chamber). At each ICP (-5, 5 or 25 mmHg) and BP setting (normal or high), IOP was raised from 10 to 90 mmHg in 10 mmHg steps (3 min each). At each IOP level ganglion cell function was assessed using the scotopic threshold response (-5 log cd.s/m2, 20 repeats). Data were compared using one- and two-way ANOVA. Results : Average blood pressure at baseline was similar for the normal blood pressure groups (ICP-5 93±3; ICP5 99±5; ICP25 105±3mmHg, p=0.8). There was significant BP elevation in all the high blood pressure groups (ICP-5 160±3; ICP5 157±3; ICP25 157±5mmHg p<0.001). Compared with normal blood pressure groups (32.0±2.0μV), animals with high blood pressure (24.5±1.8μV) had significantly smaller baseline STR amplitudes (p<0.01). There was also a significant ICP effect (p<0.01), with larger baseline amplitudes in the 25mmHg ICP group (34.8±1.6μV) compared with normal (26.4±2.5μV) and low ICP groups (23.9±2.5μV). The ocular perfusion pressure (BP-IOP) relationship fully could not account for difference in ganglion cell function between ICP levels. Conclusions : Ganglion cell function is dependent on ocular perfusion pressure, excessive low or high perfusion attenuates function. Higher intracranial pressure appears to protect against acute ocular perfusion stress.