Optometry and Vision Sciences - Research Publications
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ItemTime-Frequency Analysis of ERG With Discrete Wavelet Transform and Matching Pursuits for Glaucoma.(Association for Research in Vision and Ophthalmology (ARVO), 2022-10-03)PURPOSE: To examine the performance of two time-frequency feature extraction techniques applied to electroretinograms (ERGs) for the prediction of glaucoma severity. METHODS: ERGs targeting the photopic negative response were obtained in 103 eyes of 55 patients with glaucoma. Features from the ERG recordings were extracted using two time-frequency extraction techniques based on the discrete wavelet transform (DWT) and the matching pursuit (MP) decomposition. Amplitude markers of the time-domain signal were also extracted. Linear and multivariate adaptive regression spline (MARS) models were fitted using combinations of these features to predict estimated retinal ganglion cell counts, a measure of glaucoma disease severity derived from standard automated perimetry and optical coherence tomography imaging. RESULTS: Predictive models using features from the time-frequency analyses-using both DWT and MP-combined with amplitude markers outperformed predictive models using the markers alone with linear (P = 0.001) and MARS (P ≤ 0.011) models. For example, the proportions of variance (R2) explained by the MARS model using the DWT and MP features with amplitude markers were 0.53 and 0.63, respectively, compared to 0.34 for the model using the markers alone (P = 0.011 and P = 0.001, respectively). CONCLUSIONS: Novel time-frequency features extracted from the photopic ERG substantially added to the prediction of glaucoma severity compared to using the time-domain amplitude markers alone. TRANSLATIONAL RELEVANCE: Substantial information about retinal ganglion cell dysfunction exists in the time-frequency domain of ERGs that could be useful in the management of glaucoma.
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ItemAAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)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.
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ItemThe Effect of Aging on Retinal Function and Retinal Ganglion Cell Morphology Following Intraocular Pressure Elevation(FRONTIERS MEDIA SA, 2022-05-12)Aging and elevated intraocular pressure (IOP) are two major risk factors for glaucomatous optic neuropathy; a condition characterized by the selective, progressive injury, and subsequent loss of retinal ganglion cells (RGCs). We examined how age modified the capacity for RGCs to functionally recover following a reproducible IOP elevation (50 mmHg for 30 min). We found that RGC functional recovery (measured using electroretinography) was complete by 7 days in 3-month-old mice but was delayed in 12-month-old mice until 14 days. At the 7-day recovery endpoint when RGC function had recovered in young but not older eyes, we examined RGC structural responses to IOP-related stress by analyzing RGC dendritic morphology. ON-RGC cell volume was attenuated following IOP elevation in both young and older mice. We also found that following IOP elevation OFF-RGC dendritic morphology became less complex per cell volume in young mice, an effect that was not observed in older eyes. Our data suggest that adaptations in OFF-RGCs in young eyes were associated with better functional recovery 7 days after IOP elevation. Loss of RGC cellular adaptations may account for delayed functional recovery in older eyes.
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ItemUtility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina(MARY ANN LIEBERT, INC, 2019-11-01)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.
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ItemPrediction of glaucoma severity using parameters from the electroretinogram(NATURE PORTFOLIO, 2021-12-13)Glaucoma is an optic neuropathy that results in the progressive loss of retinal ganglion cells (RGCs), which are known to exhibit functional changes prior to cell loss. The electroretinogram (ERG) is a method that enables an objective assessment of retinal function, and the photopic negative response (PhNR) has conventionally been used to provide a measure of RGC function. This study sought to examine if additional parameters from the ERG (amplitudes of the a-, b-, i-wave, as well the trough between the b- and i-wave), a multivariate adaptive regression splines (MARS; a non-linear) model and achromatic stimuli could better predict glaucoma severity in 103 eyes of 55 individuals with glaucoma. Glaucoma severity was determined using standard automated perimetry and optical coherence tomography imaging. ERGs targeting the PhNR were recorded with a chromatic (red-on-blue) and achromatic (white-on-white) stimulus with the same luminance. Linear and MARS models were fitted to predict glaucoma severity using the PhNR only or all ERG markers, derived from chromatic and achromatic stimuli. Use of all ERG markers predicted glaucoma severity significantly better than the PhNR alone (P ≤ 0.02), and the MARS performed better than linear models when using all markers (P = 0.01), but there was no significant difference between the achromatic and chromatic stimulus models. This study shows that there is more information present in the photopic ERG beyond the conventional PhNR measure in characterizing RGC function.
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ItemTargeting Diet and Exercise for Neuroprotection and Neurorecovery in Glaucoma(MDPI, 2021-02)Glaucoma is a leading cause of blindness worldwide. In glaucoma, a progressive dysfunction and death of retinal ganglion cells occurs, eliminating transfer of visual information to the brain. Currently, the only available therapies target the lowering of intraocular pressure, but many patients continue to lose vision. Emerging pre-clinical and clinical evidence suggests that metabolic deficiencies and defects may play an important role in glaucoma pathophysiology. While pre-clinical studies in animal models have begun to mechanistically uncover these metabolic changes, some existing clinical evidence already points to potential benefits in maintaining metabolic fitness. Modifying diet and exercise can be implemented by patients as an adjunct to intraocular pressure lowering, which may be of therapeutic benefit to retinal ganglion cells in glaucoma.
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ItemMeasuring the Photopic Negative Response: Viability of Skin Electrodes and Variability Across Disease Severities in Glaucoma(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-03)PURPOSE: The purpose of this study was to determine the feasibility of measuring the photopic negative response (PhNR) of the full-field electroretinogram (ERG) using skin electrodes compared to conjunctival electrodes and its test-retest variability over a range of disease severities in open-angle glaucoma. METHODS: Recordings were performed twice (100 sweeps each) within the same session in 43 eyes of 23 participants with glaucoma to determine its intrinsic variability. The ratio between the PhNR and B-wave amplitude (PhNR/B ratio) was determined for each trace and computed across 5 to 100 sweeps of each recording. Spectral-domain optical coherence tomography was used to measure the average peripapillary retinal nerve fiber layer (RNFL) thickness. RESULTS: The PhNR/B ratio and its magnitude of variability were not significantly different between skin and conjunctival electrodes (P ≤ 0.197), and the degree of variability decreased substantially with increasing number of sweeps. For skin electrodes, the intraclass correlation coefficient was 0.89 and 0.91 for right and left eyes, respectively. The variability of the PhNR/B ratio decreased with lower RNFL thickness values and larger B-wave amplitudes (P ≤ 0.002). CONCLUSIONS: Skin electrodes are a viable alternative to conjunctival electrodes when measuring the PhNR in open angle glaucoma, and increasing the number of sweeps substantially reduced its intrinsic variability; the extent of variability was also lower with worsening disease severity. TRANSLATIONAL RELEVANCE: The feasibility of performing ERG recordings widely across a range of disease severities in glaucoma can be achieved through using skin electrodes and increasing the number of sweeps performed to improve measurement repeatability.
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ItemShort-Term Changes in the Photopic Negative Response Following Intraocular Pressure Lowering in Glaucoma(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2020-08)PURPOSE: To evaluate the short-term changes in inner retinal function using the photopic negative response (PhNR) after intraocular pressure (IOP) reduction in glaucoma. METHODS: Forty-seven participants with glaucoma who were commencing a new or additional IOP-lowering therapy (treatment group) and 39 participants with stable glaucoma (control group) were recruited. IOP, visual field, retinal nerve fiber layer thickness, and electroretinograms (ERGs) were recorded at baseline and at a follow-up visit (3 ± 2 months). An optimized protocol developed for a portable ERG device was used to record the PhNR. The PhNR saturated amplitude (Vmax), Vmax ratio, semi-saturation constant (K), and slope of the Naka-Rushton function were analyzed. RESULTS: A significant percentage reduction in IOP was observed in the treatment group (28 ± 3%) compared to the control group (2 ± 3%; P < 0.0001). For PhNR Vmax, there was no significant interaction (F1,83 = 2.099, P = 0.15), but there was a significant difference between the two time points (F1,83 = 5.689, P = 0.019). Post hoc analysis showed a significant difference between baseline and 3 months in the treatment group (mean difference, 1.23 µV; 95% confidence interval [CI], 0.24-2.22) but not in the control group (0.30 µV; 95% CI, 0.78-1.38). K and slope were not significantly different in either group. Improvement beyond test-retest variability was seen in 17% of participants in the treatment group compared to 3% in the control group (P = 0.007, χ2 test). CONCLUSIONS: The optimized protocol for measuring the PhNR detected short-term improvements in a proportion of participants following IOP reduction, although the majority showed no change.
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ItemImprovement in inner retinal function in glaucoma with nicotinamide (vitaminB3) supplementation: A crossover randomized clinical trial(WILEY, 2020-09)IMPORTANCE: Retinal ganglion cells endure significant metabolic stress in glaucoma but maintain capacity to recover function. Nicotinamide, a precursor of NAD+ , is low in serum of glaucoma patients and its supplementation provides robust protection of retinal ganglion cells in preclinical models. However, the potential of nicotinamide in human glaucoma is unknown. BACKGROUND: To examine the effects of nicotinamide on inner retinal function in glaucoma, in participants receiving concurrent glaucoma therapy. DESIGN: Crossover, double-masked, randomized clinical trial. Participants recruited from two tertiary care centres. PARTICIPANTS: Fifty-seven participants, diagnosed and treated for glaucoma. METHODS: Participants received oral placebo or nicotinamide and reviewed six-weekly. Participants commenced 6 weeks of 1.5 g/day then 6 weeks of 3.0 g/day followed by crossover without washout. Visual function measured using electroretinography and perimetry. MAIN OUTCOME MEASURES: Change in inner retinal function, determined by photopic negative response (PhNR) parameters: saturated PhNR amplitude (Vmax), ratio of PhNR/b-wave amplitude (Vmax ratio). RESULTS: PhNR Vmax improved beyond 95% coefficient of repeatability in 23% of participants following nicotinamide vs 9% on placebo. Overall, Vmax improved by 14.8% [95% CI: 2.8%, 26.9%], (P = .02) on nicotinamide and 5.2% [-4.2%, 14.6%], (P = .27) on placebo. Vmax ratio improved by 12.6% [5.0%, 20.2%], (P = .002) following nicotinamide, 3.6% [-3.4%, 10.5%], (P = .30) on placebo. A trend for improved visual field mean deviation was observed with 27% improving ≥1 dB on nicotinamide and fewer deteriorating (4%) compared to placebo (P = .02). CONCLUSIONS: Nicotinamide supplementation can improve inner retinal function in glaucoma. Further studies underway to elucidate the effects of long-term nicotinamide supplementation.
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ItemBaseline Detrending for the Photopic Negative Response(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2018-09)PURPOSE: The photopic negative response (PhNR) of the light-adapted electroretinogram (ERG) holds promise as an objective marker of retinal ganglion cell function. We compared baseline detrending methods to improve PhNR repeatability without compromising its diagnostic ability in glaucoma. METHODS: Photopic ERGs were recorded in 20 glaucoma and 18 age-matched control participants. A total of 50 brief, red-flashes (1.6 cd.s/m2) on a blue background (10 photopic cd/m2) were delivered using the RETeval device. Detrending methods compared were: (1) increasing the high-pass filter from 1 to 10 Hz and (2) estimating and removing the trend with an increasing polynomial (order from 1-10) applied to the prestimulus interval, prestimulus and postsignal interval, or the whole ERG signal. Coefficient of repeatability (COR%), unpaired Student's t-test, and area under the receiver operating characteristic curve (AUC) were used to compare the detrending methods. RESULTS: Most detrending methods improved PhNR test-retest repeatability compared to the International Society for Clinical Electrophysiology of Vision (ISCEV) recommended 0.3 to 300 Hz band-pass filter (COR% ± 200%). In particular, detrending with a polynomial (order 3) applied to the whole signal performed the best (COR% ± 44%) while achieving similar diagnostic ability as ISCEV band-pass (AUC 0.74 vs. 0.75, respectively). However, over-correcting with higher orders of processing can cause waveform distortion and reduce diagnostic ability. CONCLUSIONS: Baseline detrending can improve the PhNR repeatability without compromising its clinical use in glaucoma. Further studies exploring more complex processing methods are encouraged. TRANSLATIONAL RELEVANCE: Baseline detrending can significantly improve the quality of the PhNR.