Contrast Sensitivity of ON and OFF Human Retinal Pathways in Myopia.

The human visual cortex processes light and dark stimuli with ON and OFF pathways that are differently modulated by luminance contrast. We have previously demonstrated that ON cortical pathways have higher contrast sensitivity than OFF cortical pathways and the difference increases with luminance range (defined as the maximum minus minimum luminance in the scene). Here, we demonstrate that these ON-OFF cortical differences are already present in the human retina and that retinal responses measured with electroretinography are more affected by reductions in luminance range than cortical responses measured with electroencephalography. Moreover, we show that ON-OFF pathway differences measured with electroretinography become more pronounced in myopia, a visual disorder that elongates the eye and blurs vision at far distance. We find that, as the eye axial length increases across subjects, ON retinal pathways become less responsive, slower in response latency, less sensitive, and less effective and slower at driving pupil constriction. Based on these results, we conclude that myopia is associated with a deficit in ON pathway function that decreases the ability of the retina to process low contrast and regulate retinal illuminance in bright environments.

A clinically viable approach to restoring visual function using optogenetic gene therapy.

Optogenetic gene therapies offer a promising strategy for restoring vision to patients with retinal degenerative diseases, such as retinitis pigmentosa (RP). Several clinical trials have begun in this area using different vectors and optogenetic proteins (Clinical Identifiers: NCT02556736, NCT03326336, NCT04945772, and NCT04278131). Here we present preclinical efficacy and safety data for the NCT04278131 trial, which uses an AAV2 vector and Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using electroretinograms (ERGs). Safety was assessed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), electroretinograms (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed.

A Robust Microbead Occlusion Model of Glaucoma for the Common Marmoset.

Purpose: To establish a robust experimental model of glaucoma in the common marmoset (Callithrix jacchus), a New World primate, using an intracameral microbead injection technique. Methods: Elevated intraocular pressure (IOP) was induced by an injection of polystyrene microbeads. Morphologic changes in the retina and optic nerve of glaucomatous eyes were assessed and electroretinogram (ERG) recordings were performed to evaluate functional changes. Results: Microbead injections induced a sustained IOP elevation for at least 10 weeks in a reproducible manner. At the end of the 10-week experimental period, there was significant loss of retinal ganglion cells (RGCs) in all quadrants and eccentricities, although it was more prominent in the mid-peripheral and peripheral regions. This was consistent with a thinning of the Retinal nerve fiber layer (RNFL) seen in spectral domain optical coherence tomography scans. Surviving RGCs showed marked changes in morphology, including somatic shrinkage and dendritic atrophy. Retinas also showed significant gliosis. The amplitude of the ERG photopic negative response, with subsequent a- and b-wave changes, was reduced in glaucomatous eyes. The optic nerve of glaucomatous eyes showed expanded cupping, disorganization of the astrocytic matrix, axonal loss, and gliosis. Conclusions: We developed a robust and reproducible model of glaucoma in the marmoset. The model exhibits both structural and functional alterations of retina and optic nerve characteristic of glaucoma in humans and animal models. Translational Relevance: The glaucoma model in the marmoset described here forms a robust method to study the disease etiology, progression, and potential therapies in a nonhuman primate, allowing for more effective translation of animal data to humans.

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma.

Purpose: We examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes. Methods: We used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function. Results: We found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage. Conclusions: Together, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

Clinical electrophysiology of the optic nerve and retinal ganglion cells.

Clinical electrophysiological assessment of optic nerve and retinal ganglion cell function can be performed using the Pattern Electroretinogram (PERG), Visual Evoked Potential (VEP) and the Photopic Negative Response (PhNR) amongst other more specialised techniques. In this review, we describe these electrophysiological techniques and their application in diseases affecting the optic nerve and retinal ganglion cells with the exception of glaucoma. The disease groups discussed include hereditary, compressive, toxic/nutritional, traumatic, vascular, inflammatory and intracranial causes for optic nerve or retinal ganglion cell dysfunction. The benefits of objective, electrophysiological measurement of the retinal ganglion cells and optic nerve are discussed, as are their applications in clinical diagnosis of disease, determining prognosis, monitoring progression and response to novel therapies.

ISCEV standard for clinical multifocal electroretinography (mfERG) (2021 update).

The multifocal electroretinogram (mfERG) is an electrophysiological test that allows the function of multiple discrete areas of the retina to be tested simultaneously. This document, from the International Society for Clinical Electrophysiology of Vision (ISCEV), presents an updated and revised ISCEV standard for clinical mfERG and defines minimum protocols for basic clinical mfERG recording and reporting so that responses can be recognized and compared from different laboratories worldwide. The major changes compared with the previous mfERG standard relate to the minimum length of m-sequences used for recording, reporting of results and a change in document format, to be more consistent with other ISCEV standards.

Stable isotopes of H, C and N in mice bone collagen as a reflection of isotopically controlled food and water intake.

2H/1H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of 2H, 13C, 15N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the 2H, 13C, 15N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ∼81 % of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ∼17 %. Air humidity contributed an unspecified amount. Additionally, we monitored 13C and 15N-enrichment in bone collagen and found strong linear correlations with the 2H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers' collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.

Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc.

ISCEV extended protocol for the photopic negative response (PhNR) of the full-field electroretinogram.

The International Society for Clinical Electrophysiology of Vision (ISCEV) Standard for full-field electroretinography (ERG) describes a minimum procedure, but encourages more extensive testing. This ISCEV extended protocol describes an extension to the ERG Standard, namely the photopic negative response (PhNR) of the light-adapted flash ERG, as a well-established technique that is broadly accepted by experts in the field. The PhNR is a slow negative-going wave after the b-wave that provides information about the function of retinal ganglion cells and their axons. The PhNR can be reduced in disorders that affect the innermost retina, including glaucoma and other forms of optic neuropathy. This document, based on existing literature, provides a protocol for recording and analyzing the PhNR in response to a brief flash. The protocol includes full-field stimulation, a frequency bandwidth of the recording in which the lower limit does not exceed 0.3 Hz, and a spectrally narrowband stimulus, specifically, a red flash on a rod saturating blue background. Suggested flash strengths cover a range up to and including the minimum required to elicit a maximum amplitude PhNR. This extended protocol for recording the PhNR provides a simple test of generalized retinal ganglion cell function that could be added to standard ERG testing.

Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy.

Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma.

The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma.

Intensity response function of the photopic negative response (PhNR): effect of age and test-retest reliability.

PURPOSE: To assess the effect of age and test-retest reliability of the intensity response function of the full-field photopic negative response (PhNR) in normal healthy human subjects. METHODS: Full-field electroretinograms (ERGs) were recorded from one eye of 45 subjects, and 39 of these subjects were tested on two separate days with a Diagnosys Espion System (Lowell, MA, USA). The visual stimuli consisted of brief (<5 ms) red flashes ranging from 0.00625 to 6.4 phot cd.s/m2, delivered on a constant 7 cd/m2 blue background. PhNR amplitudes were measured at its trough from baseline (BT) and from the preceding b-wave peak (PT), and b-wave amplitude was measured at its peak from the preceding a-wave trough or baseline if the a-wave was not present. The intensity response data of all three ERG measures were fitted with a generalized Naka-Rushton function to derive the saturated amplitude (V max), semisaturation constant (K) and slope (n) parameters. Effect of age on the fit parameters was assessed with linear regression, and test-retest reliability was assessed with the Wilcoxon signed-rank test and Bland-Altman analysis. Holm's correction was applied to account for multiple comparisons. RESULTS: V max of BT was significantly smaller than that of PT and b-wave, and the V max of PT and b-wave was not significantly different from each other. The slope parameter n was smallest for BT and the largest for b-wave and the difference between the slopes of all three measures were statistically significant. Small differences observed in the mean values of K for the different measures did not reach statistical significance. The Wilcoxon signed-rank test indicated no significant differences between the two test visits for any of the Naka-Rushton parameters for the three ERG measures, and the Bland-Altman plots indicated that the mean difference between test and retest measurements of the different fit parameters was close to zero and within 6% of the average of the test and retest values of the respective parameters for all three ERG measurements, indicating minimal bias. While the coefficient of reliability (COR, defined as 1.96 times the standard deviation of the test and retest difference) of each fit parameter was more or less comparable across the three ERG measurements, the %COR (COR normalized to the mean test and retest measures) was generally larger for BT compared to both PT and b-wave for each fit parameter. The Naka-Rushton fit parameters did not show statistically significant changes with age for any of the ERG measures when corrections were applied for multiple comparisons. However, the V max of BT demonstrated a weak correlation with age prior to correction for multiple comparisons, and the effect of age on this parameter showed greater significance when the measure was expressed as a ratio of the V max of b-wave from the same subject. CONCLUSION: V max of the BT amplitude measure of PhNR at the best was weakly correlated with age. None of the other parameters of the Naka-Rushton fit to the intensity response data of either the PhNR or the b-wave showed any systematic changes with age. The test-retest reliability of the fit parameters for PhNR BT amplitude measurements appears to be lower than those of the PhNR PT and b-wave amplitude measurements.

Radionuclide bone scan SPECT-CT: lowering the dose of CT significantly reduces radiation dose without impacting CT image quality.

The CT component of SPECT-CT is required for attenuation correction and anatomical localization of the uptake on SPECT but there is no guideline about the optimal CT acquisition parameters. In our department, a standard CT acquisition protocol was changed in 2013 to give lower radiation dose to the patient. In this study, we retrospectively compared the effects on patient dose as well as the CT image quality with current versus older CT protocols. Ninety nine consecutive patients [n=51 Standard dose 'old' protocol (SDP); n=48 lower dose 'new' protocol (LDP)] with lumbar spine SPECT-CT for bone scan were examined. The main differences between the two protocols were that SDP used 130 kVp tube voltage and reference current-time product of 70 mAs whereas the LDP used 110 kVp and 40 mAs respectively. Various quantitative parameters from the CT images were obtained and the images were also rated blindly by two experienced nuclear medicine physicians for bony definition and noise. The mean calculated dose length product of the LDP group (121.5±39.6 mGy.cm) was significantly lower compared to the SDP group patients (266.9±96.9 mGy.cm; P<0.0001). This translated into a significant reduction in the mean effective dose to 1.8 mSv from 4.0 mSv. The physicians reported better CT image quality for the bony structures in LDP group although for soft tissue structures, the SDP group had better image quality. The optimized new CT acquisition protocol significantly reduced the radiation dose to the patient and in-fact improved CT image quality for the assessment of bony structures.

Test-retest reliability of the multifocal photopic negative response.

PURPOSE: To assess the test-retest reliability of the multifocal photopic negative response (mfPhNR) of normal human subjects. METHODS: Multifocal electroretinograms were recorded from one eye of 61 healthy adult subjects on two separate days using a Visual Evoked Response Imaging System software version 4.3 (EDI, San Mateo, California). The visual stimulus delivered on a 75-Hz monitor consisted of seven equal-sized hexagons each subtending 12° of visual angle. The m-step exponent was 9, and the m-sequence was slowed to include at least 30 blank frames after each flash. Only the first slice of the first-order kernel was analyzed. The mfPhNR amplitude was measured at a fixed time in the trough from baseline (BT) as well as at the same fixed time in the trough from the preceding b-wave peak (PT). Additionally, we also analyzed BT normalized either to PT (BT/PT) or to the b-wave amplitude (BT/b-wave). The relative reliability of test-retest differences for each test location was estimated by the Wilcoxon matched-pair signed-rank test and intraclass correlation coefficients (ICC). Absolute test-retest reliability was estimated by Bland-Altman analysis. RESULTS: The test-retest amplitude differences for neither of the two measurement techniques were statistically significant as determined by Wilcoxon matched-pair signed-rank test. PT measurements showed greater ICC values than BT amplitude measurements for all test locations. For each measurement technique, the ICC value of the macular response was greater than that of the surrounding locations. The mean test-retest difference was close to zero for both techniques at each of the test locations, and while the coefficient of reliability (COR-1.96 times the standard deviation of the test-retest difference) was comparable for the two techniques at each test location when expressed in nanovolts, the %COR (COR normalized to the mean test and retest amplitudes) was superior for PT than BT measurements. The ICC and COR were comparable for the BT/PT and BT/b-wave ratios and were better than the ICC and COR for BT but worse than PT. CONCLUSION: mfPhNR amplitude measured at a fixed time in the trough from the preceding b-wave peak (PT) shows greater test-retest reliability when compared to amplitude measurement from baseline (BT) or BT amplitude normalized to either the PT or b-wave amplitudes.

Quantitative (99m)Tc DTPA renal transplant scintigraphic parameters: assessment of interobserver agreement and correlation with graft pathologies.

Various (99m)Tc DTPA scintigraphic quantitative parameters for renal graft function assessment have been recommended, but none is universally accepted. In this study, 439 dynamic renal transplant scintigraphies (DRTS) were retrospectively analysed. In the first set of studies, four observers analysed the 47 random DRTS and interobserver agreement of eleven derived parameters was assessed. In the other set of studies, 181 instances of DRTS, performed on 127 recipients with renal biopsies within five days of each other were selected for correlation with pathology. Hilson's Perfusion index (HI), ΔP, P:Pl, P:U & T10 were selected for this analysis. The pathologies were categorized into renal vascular compromise (RVC; n = 20), acute tubular necrosis (ATN; n = 40), vascular rejection (VR; n = 34), interstitial rejection (IR; n = 33), normal (NOR; n = 36) and unclassified pathologies (n = 18). A majority of the parameters showed good Intraclass correlation (ICC). HI differentiated well between grafts with RVC and the remainder of the study cohort, (p < 0.0001; AUC = 0.84); at a cut-off > 278, it had 84% sensitivity and 78% specificity (Likelihood ratio = 3.8). At < 278, it had 98% 'negative' predictive value for RVC. HI also showed reasonable association with VR (p = 0.02; AUC = 0.62) and IR (p = 0.009; AUC = 0.65). However, significant overlap of HI values between various subgroups was noted. Other parameters had good ICC but were not effective in differentiating graft pathologies. Of the measured parameters, only HI proved to be useful for the pathological assessment, particularly in the identification of vascular compromise. This parameter, however, has lower specificity in differentiating the other pathologies.

A GPR18-based signalling system regulates IOP in murine eye.

BACKGROUND AND PURPOSE: GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N-arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn-CBD). The presence and/or function of any GPR18-based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55. EXPERIMENTAL APPROACH: IOP was measured in mice following topical application of Abn-CBD, NAGly or the GPR55/GPR18 agonist O-1602, alone or with injection of the GPR18 antagonist, O-1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS-MS. KEY RESULTS: GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn-CBD and NAGly, but not O-1602, significantly reduced IOP in all mice tested. The antagonist, O-1918, blocked the effects of Abn-CBD and NAGly. CONCLUSIONS AND IMPLICATIONS: We present evidence for a functional GPR18-based signalling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn-CBD and NAGly, reduce IOP independently of CB1, CB2 or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications.

ISCEV standard for clinical pattern electroretinography (PERG): 2012 update.

The pattern electroretinogram (PERG) is a retinal response evoked by a contrast-reversing pattern, usually a black and white checkerboard, which provides information about macular and retinal ganglion cell function. This document from the International Society for Clinical Electrophysiology of Vision (www.iscev.org) is a scheduled revision of the ISCEV PERG Standard, which updates and replaces the 2007 update and all earlier versions. The standard defines a single minimum stimulus and recording protocol for clinical PERG testing to assist practitioners in obtaining good quality responses and to facilitate inter-laboratory comparison. The present revision tightens stimulus specifications, expands on steady-state PERG recording, addresses visual stimulus display distinctions (CRT vs. LCD), and provides a more explicit definition of response components.

Retinal intrinsic optical signals in a cat model of primary congenital glaucoma.

PURPOSE: To examine the impact of reduced inner retinal function and breed on intrinsic optical signals in cats. METHODS: Retinal intrinsic optical signals were recorded from anesthetized cats with a modified fundus camera. Near infrared light (NIR, 700-900 nm) was used to illuminate the retina while a charge-coupled device (CCD) camera captured the NIR reflectance of the retina. Visible stimuli (540 nm) evoked patterned changes in NIR retinal reflectance. NIR intrinsic signals were compared across three subject groups: two Siamese cats with primary congenital glaucoma (PCG), a control Siamese cat without glaucoma, and a control group of seven normally pigmented cats. Intraocular pressure (IOP), pattern electroretinogram, and optical coherence tomography measurements were evaluated to confirm the inner retinal deficit in PCG cats. RESULTS: Stimulus-evoked, NIR retinal reflectance signals were observed in PCG cats despite severe degeneration of the nerve fiber layer and inner retinal function. The time course, spectral dependence, and spatial profile of signals imaged in PCG cats were similar to signals measured from normal and Siamese control cats. CONCLUSIONS: Despite increased IOP, reduced nerve fiber layer thickness and ganglion cell function, intrinsic optical signals persist in cats affected with PCG. The mechanisms giving rise to intrinsic signals remain despite inner retinal damage. Signal strength was reduced in all Siamese cats compared to controls, suggesting that reduced intrinsic signals in PCG cats represent a difference between breeds rather than loss of ganglion cells. These results corroborated previous findings that retinal ganglion cells are not the dominant source of intrinsic optical signals of the retina.