Search

BACKGROUND: Visual biomarkers of Parkinson's disease (PD) are attractive as the retina is an outpouching of the brain. Although inner retinal neurodegeneration in PD is well-established this has overlap with other neurodegenerative diseases and thus outer retinal (photoreceptor) measures warrant further investigation. OBJECTIVE: To examine in a cross-sectional study whether clinically implementable measures targeting outer retinal function and structure can differentiate PD from healthy ageing and whether these are sensitive to intraday levodopa (L-DOPA) dosing. METHODS: Centre-surround perceptual contrast suppression, macular visual field sensitivity, colour discrimination, light-adapted electroretinography and optical coherence tomography (OCT) were tested in PD participants (n = 16) and controls (n = 21). Electroretinography and OCT were conducted before and after midday L-DOPA in PD participants, or repeated after ∼2 hours in controls. RESULTS: PD participants had decreased center-surround contrast suppression (p < 0.01), reduced macular visual field sensitivity (p < 0.05), color vision impairment (p < 0.01) photoreceptor dysfunction (a-wave, p < 0.01) and photoreceptor neurodegeneration (outer nuclear layer thinning, p < 0.05), relative to controls. Effect size comparison between inner and outer retinal parameters showed that photoreceptor metrics were similarly robust in differentiating the PD group from age-matched controls as inner retinal changes. Electroretinography and OCT were unaffected by L-DOPA treatment or time. CONCLUSIONS: We show that outer retinal outcomes of photoreceptoral dysfunction (decreased cone function and impaired color vision) and degeneration (i.e., outer nuclear layer thinning) were equivalent to inner retinal metrics at differentiating PD from healthy age-matched adults. These findings suggest outer retinal metrics may serve as useful biomarkers for PD.

Age-related neuronal adaptations are known to help maintain function. This study aims to examine gross age-related in vivo retinal functional adaptations (using electroretinography) in young and middle aged C57BL/6J and Thy1-YFPh mice and to relate this to in vivo retinal structure (using optical coherence tomography). Electroretinography responses were generally larger in Thy1-YFPh mice than in C57BL/6J mice, with similar in vivo retinal layer thicknesses except for longer inner/outer photoreceptor segment in Thy1-YFPh mice. Relative to 3-month-old mice, 12-month-old mice showed reduced photoreceptor (C57BL/6J 84.0±2.5 %; Thy1-YFPh 80.2±5.2 %) and bipolar cell (C57BL/6J 75.6±2.3 %; Thy1-YFPh 68.1±5.5 %) function. There was relative preservation of ganglion cell function (C57BL/6J 79.7±3.7 %; Thy1-YFPh 91.7±5.0 %) with age, which was associated with increased b-wave (bipolar cell) sensitivities to light. Ganglion cell function was correlated with both b-wave amplitude and sensitivity. This study shows that there are normal age-related adaptations to preserve functional output. Different mouse strains may have varied age-related adaptation capacity and should be taken into consideration when examining age-related susceptibility to injury.

Myopia is predicted to impact approximately 5 billion people by 2050, necessitating mechanistic understanding of its development. Myopia results from dysregulated genetic mechanisms of emmetropization, caused by over-exposure to aberrant visual environments; however, these genetic mechanisms remain unclear. Recent human genome-wide association studies have identified a range of novel myopia-risk genes. To facilitate large-scale in vivo mechanistic examination of gene-environment interactions, this study aims to establish a myopia model platform that allows efficient environmental and genetic manipulations. We established an environmental zebrafish myopia model by dark-rearing. Ocular biometrics including relative ocular refraction were quantified using optical coherence tomography images. Spatial vision was assessed using optomotor response (OMR). Retinal function was analyzed via electroretinography (ERG). Myopia-associated molecular contents or distributions were examined using RT-qPCR or immunohistochemistry. Our model produces robust phenotypic changes, showing myopia after 2 weeks of dark-rearing, which were recoverable within 2 weeks after returning animals to normal lighting. 2-week dark-reared zebrafish have reduced spatial-frequency tuning function. ERG showed reduced photoreceptor and bipolar cell function (a- and b-waves) after only 2 days of dark-rearing, which worsened after 2 weeks of dark-rearing. We also found dark-rearing-induced changes to expression of myopia-risk genes, including egr1, vegfaa, vegfab, rbp3, gjd2a and gjd2b, inner retinal distribution of EFEMP1, TIMP2 and MMP2, as well as transiently reduced PSD95 density in the inner plexiform layer. Coupled with the gene editing tools available for zebrafish, our environmental myopia model provides an excellent platform for large-scale investigation of gene-environment interactions in myopia development.

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.

Vision loss causes a significant burden on individuals and communities on a financial, emotional and social level. Common causes include age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma and retinitis pigmentosa (RP; also known as 'rod-cone dystrophy'). As the population continues to grow and age globally, an increasing number of people will experience vision loss. Hence, there is an urgent need to develop therapies that can curb early pathological events. The broccoli-derived compound, sulforaphane (SFN), is reported to have multiple health benefits and modes of action. In this review, we outline the preclinical findings on SFN in ocular diseases and discuss the future clinical testing of this compound.

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.

Iron plays an important role in a wide range of metabolic pathways that are important for neuronal health. Excessive levels of iron, however, can promote toxicity and cell death. An example of an iron overload disorder is hemochromatosis (HH) which is a genetic disorder of iron metabolism in which the body's ability to regulate iron absorption is altered, resulting in iron build-up and injury in several organs. The retina was traditionally assumed to be protected from high levels of systemic iron overload by the blood-retina barrier. However, recent data shows that expression of genes that are associated with HH can disrupt retinal iron metabolism. Thus, the effects of iron overload on the retina have become an area of research interest, as excessively high levels of iron are implicated in several retinal disorders, most notably age-related macular degeneration. This review is an effort to highlight risk factors for excessive levels of systemic iron build-up in the retina and its potential impact on the eye health. Information is integrated across clinical and preclinical animal studies to provide insights into the effects of systemic iron loading on the retina.

Blue light exposure-induced retinal damage has been extensively studied. Although many in vitro studies have shown the benefits of blue light-blocking lenses (BBL) there have been few comprehensive in vivo studies to assess the effects of BBL. We investigated the influence of blue light exposure using light-emitting diodes on retinal histology and visual cortex neurons in rodents. We also considered whether retinal and cortical changes induced by blue light could be ameliorated with blue light-blocking lenses. A total of n = 24 (n = 6 in each group; control, light exposure without lenses, two different BBLs)) male Wistar rats were subjected to blue light exposure (LEDs, 450-500 lux) without or with BBLs (400-490 nm) for 28 days on a 12:12 h light-dark cycle. Histological analysis of retinae revealed apoptosis and necrosis of the retinal pigment epithelium (RPE), photoreceptors, and inner retina in the light exposure (LE) group, along with increase caspase-3 immunostaining in the ganglion cell layer (p < 0.001). BBL groups showed less caspase-3 immunostaining compared with the LE group (p < 0.001). V1-L5PNs (primary visual cortex layer 5 pyramidal neurons) demonstrated reduced branching and intersections points for apical (p < 0.001) and basal (p < 0.05) dendrites following blue light exposure. Blue light-blocking lenses significantly improved the number of basal branching points compared with the LE group. Our study shows that prolonged exposure to high levels of blue light pose a significant hazard to the visual system resulting in damage to the retina with the associated remodeling of visual cortex neurons. BBL may offer moderate protection against exposure to high levels of blue light.

PURPOSE: Rhegmatogenous retinal detachment repair by intraoperative sealing of the tear without a tamponade agent should enable faster restoration of vision and resumption of normal activities. It avoids the need for further surgery in the case of silicone oil endotamponade. This study evaluated the retinal thermofusion (RTF) retinopexy method of subretinal space dehydration before photocoagulation to create an instantaneous intraoperative retina reattachment in a preclinical model. DESIGN: Preclinical study. PARTICIPANTS: Twenty Dutch Belt, pigmented rabbits that underwent RTF repair after experimental retinal detachment. METHODS: This ex vivo model quantified adhesion force between the retina and underlying retinal pigment epithelium and choroid after treatment of 1 retinal edge using postmortem porcine or human retina (6 × 12 mm). We compared (1) control, (2) laser photocoagulation alone, (3) dehydration alone, and (4) dehydration followed by photocoagulation (RTF). Optimized parameters for RTF were then applied in the in vivo rabbit model of retinal detachment. Animals were followed up for 14 days. MAIN OUTCOME MEASURES: For this ex vivo model, we measured adhesion force and related this to tissue temperature. For the in vivo study, we assessed retinal attachment using funduscopy and histologic analysis. RESULTS: The ex vivo model showed that RTF repair produced significantly higher adhesion force than photocoagulation alone independent of dehydration method: warm (60° C) high airflow (50-70 ml/minute) or using laser wavelengths targeting water absorption peaks (1470 or 1940 nm) with coaxial low airflow (10-20 ml/minute). The latter approach produced a smaller footprint of dehydration. Application of RTF (1940-nm laser with coaxial airflow) in an in vivo retinal detachment model in rabbit eyes resulted in immediate retinal adhesion, achieving forces similar to those in the ex vivo experiments. Retinal thermofusion repair resulted in stable reattachment of the re

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.

There is increasing evidence for the vulnerability of specific retinal ganglion cell (RGC) types in those with glaucoma and in animal models. In addition, the P2X7-receptor (P2X7-R) has been suggested to contribute to RGC death following stimulation and elevated IOP, though its role in RGC dysfunction prior to death has not been examined. Therefore, we examined the effect of an acute, non-ischemic intraocular pressure (IOP) insult (50 mmHg for 30 min) on RGC function in wildtype mice and P2X7-R knockout (P2X7-KO) mice. We examined retinal function using electroretinogram recordings and individual RGC responses using multielectrode arrays, 3 days following acute IOP elevation. Immunohistochemistry was used to examine RGC cell death and P2X7-R expression in several RGC types. Acute intraocular pressure elevation produced pronounced dysfunction in RGCs; whilst other retinal neuronal responses showed lesser changes. Dysfunction at 3 days post-injury was not associated with RGC loss or changes in receptive field size. However, in wildtype animals, OFF-RGCs showed reduced spontaneous and light-elicited activity. In the P2X7-KO, both ON- and OFF-RGC light-elicited responses were reduced. Expression of P2X7-R in wildtype ON-RGC dendrites was higher than in other RGC types. In conclusion, OFF-RGCs were vulnerable to acute IOP elevation and their dysfunction was not rescued by genetic ablation of P2X7-R. Indeed, knockout of P2X7-R also caused ON-RGC dysfunction. These findings aid our understanding of how pressure affects RGC function and suggest treatments targeting the P2X7-R need to be carefully considered.

PURPOSE: Glia and their communication via connexin 43 (Cx43) gap junctions are known to mediate neurovascular coupling, a process driven by metabolic demand. However, it is unclear whether Cx43 mediated glial communication intermediates classical autoregulation. Here we used viral transfection and a glial fibrillary acidic protein (GFAP) promoter to downregulate glial Cx43 to evaluate its role in retinal vascular autoregulation to ocular perfusion pressure (OPP) reduction. METHODS: Adult rats were intravitreally injected with the viral active construct or a control. Three weeks after the injection, eyes were imaged using confocal scanning laser ophthalmoscopy before and during a period of OPP decrease induced by blood draw to lower blood pressure or by manometric IOP elevation. Vessel diameter responses to the OPP decrease were compared between Cx43-downregulated and control-injected eyes. The extent of Cx43 downregulation was evaluated by Western blot and immunohistochemistry. RESULTS: In control eyes, the OPP decrease induced dilatation of arterioles, but not venules. In Cx43-downregulated eyes, Cx43 expression in whole retina was decreased by approximately 40%. In these eyes, the resting diameter of the venules increased significantly, but there was no effect on arterioles. In Cx43-downregulated eyes, vasoreactivity evoked by blood pressure lowering was significantly compromised in both arterioles (P = 0.005) and venules (P = 0.001). Cx43 downregulation did not affect the arteriole responses to IOP elevation, whereas the responses of the venules showed a significantly greater decrease in diameter (P < 0.001). CONCLUSIONS: The downregulation of retinal Cx43 in GFAP-expressing cells compromises vasoreactivity of both arterioles and venules in response to an OPP decrease achieved via blood pressure lowering or IOP elevation. The results also suggest that Cx43-mediated glial communication actively regulates resting venular diameter.

Purpose: We aimed to image the optic nerve, subarachnoid space and optic nerve sheath in emmetropes and myopes ultra-high field (7-Tesla) magnetic resonance imaging (MRI). We targeted the retrobulbar distance of approximately 3 mm behind the eyeball, an area of clinical interest because of optic nerve sheath distensibility and pressure-related enlargement. Methods: Eleven emmetropes (+0.75 to −0.50D, aged 20–41 years) and 10 myopes (−4.5 to −12D, aged 21–37 years) participated. Cross-sectional area of the optic nerve, subarachnoid space and optic nerve sheath at approximately 3 mm behind the eye were measured from two-dimensional T2-weighted coronal oblique MRI images obtained through the left optic nerve. Axial length of the left eye was measured from T2-weighted axial MRI images. In nine emmetropes and seven myopes, the optic nerve head was imaged with optical coherence tomography to compare retrobulbar and intraocular measures. Results: Retrobulbar optic nerve, subarachnoid space and optic nerve sheath dimensions differed between myopes and emmetropes. Myopes tended to have smaller optic nerve and subarachnoid space. Longer MRI-derived axial length was associated with smaller optic nerve area (P = 0.03). Bruch's membrane opening area did not predict retrobulbar optic nerve area (P = 0.48). Conclusions: This study demonstrates the feasibility of using 7-Tesla MRI to measure optic nerve, subarachnoid space, and optic nerve sheath dimensions behind the eye. In healthy adults, the retrobulbar optic nerve and subarachnoid space size are influenced by the degree of myopia. Translational Relevance: ultra-high field MRI is a practical tool for assessing the morphometry of the optic nerve and surrounding anatomy behind the eye.

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.

The human PDZK1 gene is located in a genomic susceptibility region for neurodevelopmental disorders. A genome-wide association study (GWAS) identified links between PDZK1 polymorphisms and altered visual contrast sensitivity, an endophenotype for schizophrenia and autism spectrum disorder. The PDZK1 protein is implicated in neurological functioning, interacting with synaptic molecules including post-synaptic density 95 (PSD-95), N-methyl-D-aspartate receptors (NMDAR), corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptors. To elucidate the role of PDZK1, we generated pdzk1-knockout (pdzk1-KO) zebrafish using CRISPR/Cas-9 genome editing. Visual function of 7-day-old fish was assessed at behavioural and functional levels using the optomotor response (OMR) and scotopic electroretinogram (ERG). We also quantified retinal morphology and densities of PSD-95, NMDAR1, CRFR1 and serotonin in the synaptic inner plexiform layer at 7 days, 4 weeks and 8 weeks of age. Relative to wild-type, pdzk1-KO larvae showed spatial-frequency tuning functions with increased amplitude (likely due to abnormal gain control) and reduced ERG b-waves (suggestive of inner retinal dysfunction). However, these functional differences were not associated with gross synaptic or morphological retinal phenotypes. The findings corroborate a role for pdzk1 in visual function, and our model system provides a platform for investigating other genes associated with abnormal visual behaviour.

Iron is essential for normal cellular function, however, excessive accumulation of iron in neural tissue has been implicated in both cortical and retinal diseases. The exact role of iron in the pathogenesis of neurodegenerative disorders remains incompletely understood. However, iron-induced damage to the brain and retina is often attributed to the redox ability of iron to generate dangerous free radicals, which exacerbates local oxidative stress and neuronal damage. Iron chelators are compounds designed to scavenge labile iron, aiding to regulate iron bioavailability. Recently there has been growing interest in the application of chelating agents for treatment of diseases including neurodegenerative conditions, characterized by increased oxidative stress. This article reviews both clinical and preclinical evidence relating to the effectiveness of iron chelation therapy in conditions of iron dyshomeostasis linked to neurodegeneration in the brain and retina. The limitations as well as future opportunities iron chelation therapy are discussed.

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.

Alzheimer's disease is characterized by the aberrant deposition of protein in the brain and is the leading cause of dementia worldwide. Increasingly, there have been reports of the presence of these protein hallmarks in the retina. In this study, we assayed the retina of 5xFAD mice, a transgenic model of amyloid deposition known to exhibit dementia-like symptoms with age. Using OCT, we found that the retinal nerve fiber layer was thinner in 5xFAD at 6, 12, and 17 months of age compared with wild-type littermates, but the inner plexiform layer was thicker at 6 months old. Retinal function showed reduced ganglion cell responses to light in 5xFAD at 6, 12, and 17 months of age. This functional loss was observed in the outer retina at 17 months of age but not in younger mice. We showed using immunohistochemistry and ELISA that soluble and insoluble amyloid was present in the retina and brain at all ages. In conclusion, we report that amyloid is present in brain and retina of 5xFAD mice and that the pattern of neuronal dysfunction occurs in the inner retina at the early ages and progresses to encompass the outer retina with age. This implies that the inner retina is more sensitive to amyloid changes in early disease and that the outer retina is also affected with disease progression.

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.

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.

Purpose : The retina, an accessible outpouching of the central nervous system, may manifest cortical changes that occur with Parkinson’s disease (PD), lending itself as a potential biomarker. PD is characterised by reduced dopamine levels, a neurotransmitter found in amacrine cells. Human PD patients have also shown structural changes in the outer retina. This work aims to determine if retinal function and structure are altered in a murine model of PD and whether deficits can be ameliorated with L-DOPA treatment. Methods : A PD model was induced in adult C57BL6/J mice using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 4x i.p. injections, 20mg/kg) and vehicle control and examined at day 21 and 45. Another MPTP group was administered L-DOPA (L-3,4-dihydroxyphenylalanine 0.2 mg/ml) or control in their drinking water and assessed at day 45 (n=12–15/group). In ketamine:xylazine anaesthetised (80:10mg/kg) mice full-field dark- and light-adapted electroretinography (ERG) was assessed to target dopamine-related responses. Optical coherence tomography (OCT) was used to quantify thickness of retinal layers. Retinal and cortical tissue were collected for immunohistochemical assessment of changes in tyrosine hydroxylase (TH)and imaged using confocal microscopy. Data (mean±SEM) were compared using unpaired ANOVA and t-tests as appropriate. Results : At day 21 no retinal changes were found. At day 45 dark and light adapted ERGs showed slower amacrine cell responses (oscillatory potential, p<0.05), a finding which reversed with L-DOPA treatment (p<0.05). Other components of the ERG were unchanged. TH staining showed a trend towards decreased retinal levels in MPTP mice but this did not reach significance (p=0.10). Reduced levels of TH were found in the ventral hippocampus of MPTP mice compared with control (p<0.05). OCT revealed thinning of the outer plexiform layer at day 45, and the L-DOPA group exhibited a thinning of the outer nuclear layer (p<0.05). Conclusions : This

Purpose : We exploit the improved spatial resolution and signal-to-noise gain of ultra high field (7T) magnetic resonance imaging (MRI) with a dedicated eye coil for more accurate morphometric measurements of the optic nerve ~2.5mm behind the globe. Methods : Coronal T2-weighted oblique images (TR=2000ms, TE=64ms, FOV=155mm, matrix=384 x 384, slice thickness=0.7mm, scan time=2’34”) through the optic nerve were obtained in 21 healthy adults (20-41 years, 11 emmetropes: +0.75 to -0.50D, 10 myopes: -4.5 to -12D) using a 7T Siemens Magnetom scanner (Erlangen, Germany) and 6-channel eye coil (MRI.TOOLS GmbH, Berlin, Germany). Horizontal and vertical outer diameter of the optic nerve, subarachnoid space (fluid gap) and optic sheath were measured by hand using biomedical imaging software (OsiriX, Pixmeo, Switzerland) (Figure). Significant motion artefacts were avoided with customised fixation and preparation techniques. Results : Horizontal and vertical measurements were similar so were averaged. Right and left eye diameters did not differ and were highly correlated (optic nerve: Pearson r=0.9, p<0.001; fluid gap: r=0.8, p<0.001; optic sheath: r=0.7, p<0.001); hence we report left eye data only. Optic nerve diameter (average of horizontal and vertical diameters) ranged from 2.8-4.1mm in emmetropes and 1.5-4.2mm in myopes and correlated with refractive error (Spearman r=0.46, p=0.04). Similarly, fluid gap diameter (emmetropes: 3.6-5.5mm, myopes: 2.5-5.6mm), but not optic sheath diameter (emmetropes: 4.5-6.8mm, myopes: 4.2-6.8mm), correlated with refractive error (r=0.47, p=0.03). Conclusions : Ultra high field MRI with thinner slices enables more accurate demarcation of the optic nerve, surrounding fluid/subarachnoid space and optic sheath without overlapping of neighbouring anatomy (minimal partial volume artefact). Our 7T MRI-derived normative measurements of optic nerve, fluid gap and sheath diameter are comparable with published reports in healthy observers obtained at

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].

Purpose : Progress in the development of therapies to enhance remyelination in demyelinating diseases has been hampered by a lack of appropriate preclinical models - functional measures are often lacking or variable. We sought to develop a tractable and reproducible model of optic nerve demyelination with precise structural and functional measures. Methods : Oligodendrocytes of MBP-DTR 100a transgenic mice express diphtheria toxin receptor (DTR) and systemic diphtheria toxin (DT) administration induces diffuse demyelination of the central nervous system. In the present study we used retrobulbar DT injection to induce focal demyelination of the optic nerves of 3-month-old MBP-DTR 100a mice. Dose optimisation: anaesthetised mice underwent unilateral retrobulbar DT injection with 5, 10 or 15ng/kg DT (n=7 per dose, 1 µL per injection). Tissues were harvested three weeks after injection. Time-course study: Following baseline visual evoked potential (VEP) recording, electroretinogram (ERG) and optical coherence tomography (OCT), mice underwent retrobulbar DT injection with 15ng/kg DT or 1µL PBS. Follow-up measurements were taken at 2 (n=5 DT, 5 PBS), 4 (n=6 DT, 6 PBS), 8 (n=9 DT, 9 PBS) or 12-weeks (n=7 DT, 7 PBS). Animals were culled at each timepoint for tissue analysis. Tissue analysis: Optic nerves were resin embedded, sectioned (1µm) and stained with toluidine blue for myelin analysis, or cryosectioned for immunofluorescence, and retinas were flat-mounted for ganglion cell counts. Results : 3 weeks after injection with 15ng/kg DT, optic nerves showed colocalisation of activated caspase 3 & olig2, consistent with the apoptosis of oligodendroglia. Gliosis and axonal degeneration were evident.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy. MicroRNAs (miRNAs) are master regulators of gene expression that play important roles in retinal neovascularization. Here, we investigated the retinal miRNA expression profile in a rat model of oxygen-induced retinopathy (OIR) through miRNA-Seq. We found that miR-143-3p, miR-126-3p, miR-150-5p and miR-145-5p were significantly down-regulated in the retina of OIR rats, and directly involved in the development of retinal neovascularization. Of these identified miRNAs, miR-143 is enriched in retina and was first reported being associated with pathological retinal angiogenesis. Our RNA-Seq data further suggested that miR-143 alleviates retinal neovascularization by mediating the inflammation/stress pathways via Fos. Moreover, the computational analysis indicated that Transforming Growth Factor-beta Activated Kinase 1 (TAK1) is involved in several key pathways associated with the dysregulated miRNAs. The pharmacological inhibition of TAK1 suppressed angiogenesis in vitro and retinal neovascularization in vivo. Our data highlight the utility of next-generation sequencing in the development of therapeutics for ocular neovascularization and further suggest that therapeutic targeting the dysregulated miRNAs or TAK1 may be a feasible adjunct therapeutic approach in patients with retinal neovascularization.

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.

Executive function deficits in Alzheimer’s disease (AD) occur early in disease progression and may be predictive of cognitive decline. However, no preclinical studies have identified deficits in rewarded executive function in the commonly used APP/PS1 mouse model. To address this, we assessed 12-26 month old APP/PS1 mice on rewarded reversal and/or extinction tasks. 16-month-old, but not 13- or 26-month-old, APP/PS1 mice showed an attenuated rate of extinction. Reversal deficits were seen in 22-month-old, but not 13-month-old APP/PS1 animals. We then confirmed that impairments in reversal were unrelated to previously reported visual impairments in both AD mouse models and humans. Age, but not genotype, had a significant effect on markers of retinal health, indicating the deficits seen in APP/PS1 mice were directly related to cognition. This is the first characterisation of rewarded executive function in APP/PS1 mice, and has great potential to facilitate translation from preclinical models to the clinic.

The zebrafish (Danio rerio) is a popular vertebrate model for studying visual development, especially at the larval stage. For many vertebrates, post-natal visual experience is essential to fine-tune visual development, but it is unknown how experience shapes larval zebrafish vision. Zebrafish swim with a moving texture; in the wild, this innate optomotor response (OMR) stabilises larvae in moving water, but it can be exploited in the laboratory to assess zebrafish visual function. Here, we compared spatial-frequency tuning inferred from OMR between visually naïve and experienced larvae from 5 to 7 days post-fertilisation. We also examined development of synaptic connections between neurons by quantifying post-synaptic density 95 (PSD-95) in larval retinae. PSD-95 is closely associated with N-methyl-D-aspartate (NMDA) receptors, the neurotransmitter-receptor proteins underlying experience-dependent visual development. We found that rather than following an experience-independent genetic programme, developmental changes in visual spatial-frequency tuning at the larval stage required visual experience. Exposure to motion evoking OMR yielded no greater improvement than exposure to static form, suggesting that increased sensitivity as indexed by OMR was driven not by motor practice but by visual experience itself. PSD-95 density varied with visual sensitivity, suggesting that experience may have up-regulated clustering of PSD-95 for synaptic maturation in visual development.

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.

Purpose : Retinal vascular resistance is constantly regulated by both myogenic and metabolic mechanisms. While most studies have investigated these mechanisms separately, how they interact to impact the vascular resistance is unclear. We considered whether hypercapnia (HC) modified the effect of ocular perfusion pressure (OPP) lowering, induced by lowering blood pressure (BP) or increasing intraocular pressure (IOP) on retinal vessel diameter (Ø). Methods : In pentobarbital anesthetized Brown Norway rats, breathing ~30% O2 air, normocapnia (NC) and hypercapnia (HC) were achieved by controlled ventilation. A gradual decrease in OPP, at the same rate for BP lowering or IOP elevation, was induced by drawing blood (1ml/min) from a femoral artery or by increasing the IOP manometrically from 10 to 70 mmHg (9.78 mmHg/min) in two subgroups for each NC (BP=7, IOP=9) and HC (BP=9, IOP=5). Arterial CO2 partial pressure (pCO2) was measured. In all groups, image sequences centered on the optic nerve were acquired with a confocal scanning laser ophthalmoscope (cSLO) every 1.5 minutes until OPP was < 20 mmHg. Arteries and veins at 1-disc diameter from optic nerve were analyzed. Change in Ø (%) was normalized to its own baseline (before pressure manipulation) and differences between NC and HC groups were compared. Results : Average pCO2levelwas 35 ±5 mmHg (n=12) in NC and 77 ±18 mmHg (n=10) in HC (p<0.001), and pO2 was >80 mmHg for all animals. In the NC group, lowering BP induced progressive arterial and venous dilation (P<0.0001 and 0.005, respectively. Fig 1); increasing IOP caused vasodilatation in arteries (P<0.0001), but not in veins (P>0.05, Fig 2). Vasodilation was evident when OPP dropped to 50-60 mmHg for both BP and IOP modification. During HC, IOP and BP induced arterial vasodilation was significantly attenuated and venous diameter showed greater compression (P<0.0001 both, 2-way ANOVA) compared to the NC groups. Conclusions : Carbon dioxide levels significantly modifie

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.

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 : The

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) is used by some bacteria and most archaea to protect against viral phage intrusion and has recently been adapted to allow for efficient editing of the mammalian genome. Whilst CRISPR/Cas-based technology has been used to modify genes in mammalian cells in vitro, delivery of CRISPR/Cas system into mammalian tissue and/or organs is more difficult and often requires additional vectors. With the use of adeno-associated virus (AAV) gene delivery system, active CRISPR/Cas enzyme can be maintained for an extended period of time and enable efficient editing of genome in the retina in vivo. Herein we outline the method to edit the genome in mouse retina using a dual AAV vector -mediated CRISPR/Cas9 system.

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 c

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.

PURPOSE: To predict the lead time (difference in time taken for a visual field [VF] defect to be detected) obtained when using stimulus sizes within or near the size of the critical area of spatial summation (Ac), and to test these predictions using sensitivity measurements from a cohort of glaucoma patients. METHODS: Thirty-seven patients with early open-angle glaucoma and 60 healthy observers underwent VF testing on the Humphrey Field Analyzer in full threshold mode using Goldmann stimulus sizes I to V (GI-V) across the 30-2 test grid. We used the sensitivities measured using GI to V in healthy patients to predict the lead time gained by using stimulus sizes within the size of Ac at all locations within the 30-2 grid. Then, we used sensitivities measured in the glaucoma patients to test this predictive model. RESULTS: Median lead time to VF defect detection when using stimulus sizes within Ac compared with stimulus sizes larger than Ac was 4.1 years across the 30-2 test grid (interquartile range, 3.1 and 5.1 years). Sensitivities of the glaucoma patients showed good agreement with the predictive model of lead time gained (77.5%-84.3% were within ±3 dB). CONCLUSIONS: Our model predicted substantial lead time differences when using stimulus sizes within or near Ac. Such stimulus sizes could potentially detect VF defects, on average, 4 years earlier than current paradigms. TRANSLATIONAL RELEVANCE: Stimulus sizes within or near Ac may be more suitable for early detection of glaucomatous VF defects. Larger stimulus sizes may be more suitable for later monitoring of established disease.

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.

PURPOSE: The number of subjects needed to establish the normative limits for visual field (VF) testing is not known. Using bootstrap resampling, we determined whether the ground truth mean, distribution limits, and standard deviation (SD) could be approximated using different set size (x) levels, in order to provide guidance for the number of healthy subjects required to obtain robust VF normative data. METHODS: We analyzed the 500 Humphrey Field Analyzer (HFA) SITA-Standard results of 116 healthy subjects and 100 HFA full threshold results of 100 psychophysically experienced healthy subjects. These VFs were resampled (bootstrapped) to determine mean sensitivity, distribution limits (5th and 95th percentiles), and SD for different 'x' and numbers of resamples. We also used the VF results of 122 glaucoma patients to determine the performance of ground truth and bootstrapped results in identifying and quantifying VF defects. RESULTS: An x of 150 (for SITA-Standard) and 60 (for full threshold) produced bootstrapped descriptive statistics that were no longer different to the original distribution limits and SD. Removing outliers produced similar results. Differences between original and bootstrapped limits in detecting glaucomatous defects were minimized at x = 250. CONCLUSIONS: Ground truth statistics of VF sensitivities could be approximated using set sizes that are significantly smaller than the original cohort. Outlier removal facilitates the use of Gaussian statistics and does not significantly affect the distribution limits. TRANSLATIONAL RELEVANCE: We provide guidance for choosing the cohort size for different levels of error when performing normative comparisons with glaucoma patients.

High intraocular pressure is the most well documented glaucoma risk factor; however many patients develop and/or show progression of glaucoma in its absence. It is now thought that in some instances, ocular perfusion pressure (blood pressure - intraocular pressure) may be as important as intraocular pressure alone. Thus, systemic hypertension would be protective against glaucoma. Epidemiological studies, however, are inconclusive. One theory of why hypertension may not protect against elevated intraocular pressure in spite of increasing ocular perfusion pressure is that with time, morphological changes to the vasculature and autoregulatory failure outweigh the benefits of improved perfusion pressure, ultimately leading to poor retinal and optic nerve head blood supply. In this study we showed the presence of increased wall:lumen ratio and wall area of the ophthalmic artery in rats with chronic hypertension in addition to failure of retinal autoregulation in response to acute modification of ocular perfusion pressure. Subsequently we found that in spite of dramatically increasing ocular perfusion pressure, chronic systemic hypertension failed to protect retinal structure and function from a rodent model of glaucoma.

PURPOSE: This pilot study considered whether intraocular pressure (IOP) lowering could reverse ganglion cell dysfunction in a rat model of chronic ocular hypertension. METHODS: A circumlimbal suture was applied in one eye to induce ocular hypertension (n = 7) in Long-Evans rats. The contralateral eye served as an untreated control. After 8 weeks of IOP elevation the suture was removed to lower IOP for the remaining 7 weeks. Electroretinogram (ERG) and optical coherence tomography (OCT) were measured at baseline, 2, 4, 8, 12 and 15 weeks. Retinae were collected for histology at week 15. RESULTS: In sutured eyes, IOP was elevated by 7-11 mmHg above control eyes (12 ± 0.2 mmHg [standard error of the mean]). Eight weeks of chronic IOP elevation resulted in a reduction of the ganglion cell mediated positive Scotopic Threshold Response (pSTR, -25 ± 7% of baseline), as well as smaller photoreceptor (-7 ± 4%) and bipolar cell mediated responses (-6 ± 5%). After suture removal, IOP recovered to normal. By 15 weeks the a-wave (0 ± 6%), b-wave (-2 ± 6%) and pSTR had recovered back to baseline (from -25 ± 7% to -4 ± 6%). The retinal nerve fiber layer was thinned by -9 ± 3% at week 8 and showed no further decline at week 15 (-10 ± 2%). Cell numbers in the ganglion cell layer were similar between suture removal and control eyes at week 15 (3543 ± 478 vs 4057 ± 476 cells mm-2 ). CONCLUSIONS: The circumlimbal suture model might be a useful platform to study the reversibility of neuronal dysfunction from chronic IOP challenge.

Although changes in vessel diameter following gas perturbation have been documented in retinal arterioles and venules, these responses have yet to be quantified in the smallest vessels of the human retina. Here, using in vivo adaptive optics, we imaged 3-25 µm diameter vessels of the human inner retinal circulation and monitored the effects of altered gas-breathing conditions. During isocapnic hyperoxia, definite constrictions were seen in 51% of vessel segments (mean ± SD for pre-capillary arterioles -9.5 ± 3.0%; capillaries -11.8 ± 3.3%; post-capillary venules -6.3 ± 2.8%); these are comparable with responses previously reported in larger vessels. During isoxic hypercapnia, definite dilations were seen in 47% of vessel segments (mean ± SD for pre-capillary arterioles +9.8 ± 1.5%; capillaries +13.7 ± 3.8%; post-capillary venules +7.5 ± 4.2%); these are proportionally greater than responses previously reported in larger vessels. The magnitude of these proportional changes implies that the capillary beds themselves play an important role in the retinal response to changes in carbon dioxide levels. Interestingly, the distribution of microvascular responses shown here differs from our previously reported responses to flicker stimulation, suggesting differences in the way blood supply is coordinated following gas perturbation and altered neural activity.

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 an

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.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

Infrared (IR) spectroscopy has been used to quantify chemical and structural characteristics of a wide range of materials including biological tissues. In this study, we examined spatial changes in the chemical characteristics of rat retina in response to intraocular pressure (IOP) elevation using synchrotron infrared microscopy (SIRM), a non-destructive imaging approach. IOP elevation was induced by placing a suture around the eye of anaesthetised rats. Retinal sections were collected onto transparent CaF2 slides 10 days following IOP elevation. Using combined SIRM spectra and chemical mapping approaches it was possible to quantify IOP induced changes in protein conformation and chemical distribution in various layers of the rat retina. We showed that 10 days following IOP elevation there was an increase in lipid and protein levels in the inner nuclear layer (INL) and ganglion cell layer (GCL). IOP elevation also resulted in an increase in nucleic acids in the INL. Analysis of SIRM spectra revealed a shift in amide peaks to lower vibrational frequencies with a more prominent second shoulder, which is consistent with the presence of cell death in specific layers of the retina. These changes were more substantial in the INL and GCL layers compared with those occurring in the outer nuclear layer. These outcomes demonstrate the utility of SIRM to quantify the effect of IOP elevation on specific layers of the retina. Thus SIRM may be a useful tool for the study of localised tissue changes in glaucoma and other eye diseases.

Objective. To examine whether retinal electrophysiology is a useful surrogate marker of drug penetrance into the central nervous system (CNS). Materials and Methods. Brain and retinal electrophysiology were assessed with full-field visually evoked potentials and electroretinograms in conscious and anaesthetised rats following systemic or local administrations of centrally penetrant (muscimol) or nonpenetrant (isoguvacine) compounds. Results. Local injections into the eye/brain bypassed the blood neural barriers and produced changes in retinal/brain responses for both drugs. In conscious animals, systemic administration of muscimol resulted in retinal and brain biopotential changes, whereas systemic delivery of isoguvacine did not. General anaesthesia confounded these outcomes. Conclusions. Retinal electrophysiology, when recorded in conscious animals, shows promise as a viable biomarker of drug penetration into the CNS. In contrast, when conducted under anaesthetised conditions confounds can be induced in both cortical and retinal electrophysiological recordings.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder resulting in dementia and eventual death. It is the leading cause of dementia and the number of cases are projected to rise in the next few decades. Pathological hallmarks of AD include the presence of hyperphosphorylated tau and amyloid protein deposition. Currently, these pathological biomarkers are detected either through cerebrospinal fluid analysis, brain imaging or post-mortem. Though effective, these methods are not widely available due to issues such as the difficulty in acquiring samples, lack of infrastructure or high cost. Given that the eye possesses clear optics and shares many neural and vascular similarities to the brain, it offers a direct window to cerebral pathology. These unique characteristics lend itself to being a relatively inexpensive biomarker for AD which carries the potential for wide implementation. The development of ocular biomarkers can have far implications in the discovery of treatments which can improve the quality of lives of patients. In this review, we consider the current evidence for ocular biomarkers in AD and explore potential future avenues of research in this area.

An increasing number of studies indicate that the optic nerve head of the eye is sensitive not only to changes in intraocular pressure (IOP), but also to intracranial pressure (ICP). This study examines changes to optic nerve and retinal structure in a rat model in response to a range of IOP and ICP levels using optical coherence tomography. Furthermore, we examine the functional sequelae of these structural changes by quantifying the effect of pressure changes on the electroretinogram. IOP elevation (10-90 mmHg) induces progressive deformation of the optic nerve head and retinal surface (P < 0.05), compression of the retina (P < 0.05) and bipolar cell (b-wave), and retinal ganglion cell (scotopic threshold response) dysfunction (P < 0.05). Simultaneously altering ICP (-5 to 30 mmHg) modifies these IOP-induced responses, with lower ICP (-5 mmHg) exacerbating and higher ICP (15-30 mmHg) ameliorating structural and functional deficits. Thus, the balance between IOP and ICP (optic nerve pressure gradient, ONPG = IOP - ICP) plays an important role in optic nerve integrity. Structural and functional parameters exhibit a two-phase relationship to ONPG, with structural changes being more sensitive to ONPG modification (threshold = -0.6 to 11.3 mmHg) compared with functional changes (threshold = 49.7-54.6 mmHg). These findings have implications for diseases including glaucoma, intracranial hypertension, and long-term exposure to microgravity.