Validation of an automated method for studying retinal capillary blood flow.

Two major approaches for tracking cellular motion across a range of biological tissues are the manual labelling of cells, and automated analysis of spatiotemporal information represented in a kymograph. Here we compare these two approaches for the measurement of retinal capillary flow, a particularly noisy application due to the low intrinsic contrast of single red blood cells (erythrocytes). Image data were obtained using a flood-illuminated adaptive optics ophthalmoscope at 750 nm, allowing the acquisition of flow information over several cardiac cycles which provided key information in evaluating tracking accuracy. Our results show that in addition to being much faster, the automated method is more accurate in the face of rapid flow and reduced image contrast. This study represents the first validation of commonly used kymograph approaches to capillary flow analysis.

Adaptive optics imaging in inherited retinal diseases: A scoping review of the clinical literature.

Adaptive optics (AO) imaging enables direct, objective assessments of retinal cells. Applications of AO show great promise in advancing our understanding of the etiology of inherited retinal disease (IRDs) and discovering new imaging biomarkers. This scoping review systematically identifies and summarizes clinical studies evaluating AO imaging in IRDs. Ovid MEDLINE and EMBASE were searched on February 6, 2023. Studies describing AO imaging in monogenic IRDs were included. Study screening and data extraction were performed by 2 reviewers independently. This review presents (1) a broad overview of the dominant areas of research; (2) a summary of IRD characteristics revealed by AO imaging; and (3) a discussion of methodological considerations relating to AO imaging in IRDs. From 140 studies with AO outcomes, including 2 following subretinal gene therapy treatments, 75% included fewer than 10 participants with AO imaging data. Of 100 studies that included participants' genetic diagnoses, the most common IRD genes with AO outcomes are CNGA3, CNGB3, CHM, USH2A, and ABCA4. Confocal reflectance AO scanning laser ophthalmoscopy was the most reported imaging modality, followed by flood-illuminated AO and split-detector AO. The most common outcome was cone density, reported quantitatively in 56% of studies. Future research areas include guidelines to reduce variability in the reporting of AO methodology and a focus on functional AO techniques to guide the development of therapeutic interventions.

Does vision therapy for visual information processing improve academic performance? A randomised clinical trial.

PURPOSE: To determine whether a typical vision therapy (VT) programme designed to improve visual information processing (VIP) skills is effective in improving these skills and/or academic performance. METHODS: We used a double-blind, randomised clinical trial to compare VIP VT to placebo training. Participating schools referred a sample of 579 early primary school children identified as being within the lower third of their class for literacy. From the referred sample, we identified 247 children eligible to participate (passed visions and auditory processing screening, and VIP performance <34th percentile), 94 of whom participated. Matching IQ, school grade and sex was achieved by sorting hierarchically on these values and then alternately allocating to VT or placebo groups. Both programmes ran for 10 weeks and consisted of 33 h working at home and 4 h working in office. The VT programme was indicative of that employed in Australian paediatric optometry practices, with the placebo programme containing similar activities, except targeting skills within a child's competencies and with specific VIP development activities removed. The main outcome measures were score change on three standardised educational tests (reading comprehension, spelling and mathematics) and six VIP tests, both immediately post-intervention (PI) and 6 months later. RESULTS: Sixty-nine children completed the programmes. The VT programme produced no significant improvement in the three educational tests or in five of the six VIP tests compared to the control. The VT programme improved visual sequential memory (VSM) by a moderate amount compared to the control (Cohen's d = 0.57 and 0.52, immediately PI and at 6 months, respectively: p < 0.03 and p < 0.02). CONCLUSIONS: The VIP and academic performance benefits from a VT programme were largely identical to those from a control programme, both immediately and 6-month PI. Placebo effects and general effects such as improvements in executive function and/or regression-to-the-mean could be mistaken for specific programme effectiveness.

Flow Heterogeneity and Factors Contributing to the Variability in Retinal Capillary Blood Flow.

Purpose: Capillary flow plays an important role in the nourishment and maintenance of healthy neural tissue and can be observed directly and non-invasively in the living human retina. Despite their importance, patterns of normal capillary flow are not well understood due to limitations in spatial and temporal resolution of imaging data. Methods: Capillary flow characteristics were studied in the retina of three healthy young individuals using a high-resolution adaptive optics ophthalmoscope. Imaging with frame rates of 200 to 300 frames per second was sufficient to capture details of the single-file flow of red blood cells in capillaries over the course of about 3 seconds. Results: Erythrocyte velocities were measured from 72 neighboring vessels of the parafoveal capillary network for each subject. We observed strong variability among vessels within a given subject, and even within a given imaged field, across a range of capillary flow parameters including maximum and minimum velocities, pulsatility, abruptness of the systolic peak, and phase of the cardiac cycle. The observed variability was not well explained by "local" factors such as the vessel diameter, tortuosity, length, linear cell density, or hematocrit of the vessel. Within a vessel, a moderate relation between the velocities and hematocrit was noted, suggesting a redistribution of plasma between cells with changes in flow. Conclusions: These observations advance our fundamental understanding of normal capillary physiology and raise questions regarding the potential role of network-level effects in explaining the observed flow heterogeneity.

Effect of hydroxychloroquine or chloroquine and short wavelength light on in vivo retinal function and structure in mouse eyes.

CLINICAL RELEVANCE: The use of chloroquine or hydroxychloroquine can lead to both acute and chronic changes to both retinal structure and function. BACKGROUND: Chloroquine (CQ) and hydroxychloroquine (HCQ) have the potential for retina toxicity. The acute impact of short-term drug exposure (2-4 weeks) on in vivo retinal structure and function and assess whether short wavelength light exposure further exacerbates any structural and functional changes was assessed in a murine model. METHODS: Adult C57BL/6 J mice received intraperitoneal injection of vehicle or hydroxychloroquine (10 mg/kg) 3 times per week for 2 or 4 weeks, or chloroquine for 4 weeks (10 mg/kg). Over this period, animals were exposed to room light (8 hours) or short-wavelength light 4 hours per day (4 hours of normal room light) for 5 days each week. Retinal changes were assessed using electroretinography (ERG), in vivo optical coherence tomography (OCT) imaging. RESULTS: Short-term low-dose HCQ and CQ treatment led to RPE thickening and elongation of photoreceptors. These structural changes were associated with a no dysfunction in the case of HCQ treatments and widespread functional changes (photoreceptor sensitivity, bipolar cell amplitude and oscillatory potential amplitude) in the case of CQ treatment. Exposure to low intensity short-wavelength light does not appear to alter the effect of HCQ or CQ. CONCLUSIONS: HCQ and CQ treatment has acute effects on both retinal structure and function, effects that were not exacerbated by short wavelength light exposure. Whether chronic short wavelength light exposure exacerbates these changes require further study.

Optimizing Retinal Thermofusion in Retinal Detachment Repair: Achieving Instant Adhesion without Air Tamponade.

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 retina over the 2-week follow-up period. Conclusions: We showed that a short preliminary dehydrating laser treatment of a retinal tear margin before traditional laser photocoagulation creates an immediate intraoperative waterproof retinopexy adhesion independent of tamponade and a wound-healing response. This approach potentially will allow rapid postoperative recovery regardless of the tear location and improved vision.

The influence of retinal image motion on the perceptual grouping of temporally asynchronous stimuli.

Briefly presented stimuli can reveal the lower limit of retinal-based perceptual stabilization mechanisms. This is demonstrated in perceptual grouping of temporally asynchronous stimuli, in which alternate row or column elements of a regular grid are presented over two successive display frames with an imperceptible temporal offset. The grouping phenomenon results from a subtle shift between alternate grid elements due to incomplete compensation of small, fixational eye movements occurring between the two presentation frames. This suggests that larger retinal shifts should amplify the introduced shifts between alternate grid elements and improve grouping performance. However, large shifts are necessarily absent in small eye movements. Furthermore, shifts follow a random walk, making the relationship between shift magnitude and performance difficult to explore systematically. Here, we established a systematic relationship between retinal image motion and perceptual grouping by presenting alternate grid elements (untracked) during smooth pursuit of known velocities. Our results show grouping performance to improve in direct proportion to pursuit velocity. Any potential compensation by extraretinal signals (e.g., efference copy) does not seem to occur.

The influence of perceptual stabilisation on perceptual grouping of temporally asynchronous stimuli.

Even during fixation, our eyes constantly make small, involuntary eye movements that cause the retinal image to be swept across our retinae. Despite this, our world appears completely stable, due to powerful perceptual stabilisation mechanisms. Whether these mechanisms are of functional consequence for visual performance remains largely unexplored, however. We directly tested this by using a perceptual grouping task, where physically aligned alternate grid elements were presented with an imperceptible temporal offset. Observers' abilities to reliably group the grid into rows (or columns) is posited to arise from the failure in compensation of retinal slip arising from the small eye movements that occur during the temporal offset, effectively introducing a spatial shift in the arrangement of grid elements. We incorporated this perceptual grouping task within the on-line jitter illusion, which temporarily disables perceptual stabilisation mechanisms through a 10 Hz flickering annulus of random noise (Vision Res 43 (2003) 957-969). Observers' abilities to correctly group the grid stimulus were measured with and without perceptual stabilisation mechanisms engaged (i.e. non-flickering vs. flickering annulus). Grouping performance was better when eye movements were perceived, suggesting that the influence of retinal slip is increased when perceptual stabilisation mechanisms are disabled. We therefore find that perceptual stabilisation can measurably influence visual function, in addition to its perceptual effects.

Masking of random-walk motion by flicker, and its role in the allocation of motion in the on-line jitter illusion.

Typically, perceptual stabilization mechanisms make us unaware of the retinal image motion produced by the small, involuntary eye movements our eyes constantly make during fixation. The breakdown of perceptual stability is demonstrated by the on-line jitter illusion, in which a circular static pattern appears to jitter coherently when surrounded by a flickering annular pattern. Although both regions of the stimulus are subject to retinal motion from eye movements, the visual system attributes this motion to the central static region in the form of visual jitter, while the surrounding flickering region remains perceptually stable. We investigated factors influencing this allocation of motion and reference frame in the on-line jitter illusion. The flickering of the surround was found to impair the detection of simultaneous random-walk motion in this area, giving a detection reliability of around 80% for motion approximating that from fixational eye movements. Changes to spatial texture and location of flicker (centre vs. surrounding annulus) had little effect on the final percept. However, use of a nonconcentric stimulus resulted in a marked reduction in apparent jitter in all subjects. Our results suggest for the on-line jitter illusion, allocation of motion and reference frame is influenced by the general principle that, if one region surrounds another, the surrounding region tends to be allocated as the frame of reference. When this factor is controlled for, spatial textures, location of flicker, and the masking of motion by flicker have a smaller but measurable influence on the final percept.

Reactivity in the human retinal microvasculature measured during acute gas breathing provocations.

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.

Evidence of Flicker-Induced Functional Hyperaemia in the Smallest Vessels of the Human Retinal Blood Supply.

Regional changes in blood flow are initiated within neural tissue to help fuel local differences in neural activity. Classically, this response was thought to arise only in larger arterioles and venules. However, recently, it has been proposed that a) the smallest vessels of the circulation make a comparable contribution, and b) the response should be localised intermittently along such vessels, due to the known distribution of contractile mural cells. To assess these hypotheses in human neural tissue in vivo, we imaged the retinal microvasculature (diameters 3-28 µm) non-invasively, using adaptive optics, before and after delivery of focal (360 µm) patches of flickering visible light. Our results demonstrated a definite average response in 35% of all vessel segments analysed. In these responding vessels, the magnitude of proportional dilation (mean ± SEM for pre-capillary arterioles 13 ± 5%, capillaries 31 ± 8%, and post-capillary venules 10 ± 3%) is generally far greater than the magnitudes we and others have measured in the larger retinal vessels, supporting proposition a) above. The dilations observed in venules were unexpected based on previous animal work, and may be attributed either to differences in stimulus or species. Response heterogeneity across the network was high; responses were also heterogeneous along individual vessels (45% of vessel segments showed demonstrable locality in their response). These observations support proposition b) above. We also observed a definite average constriction across 7% of vessel segments (mean ± SEM constriction for capillaries -16 ± 3.2%, and post-capillary venules -18 ± 12%), which paints a picture of dynamic redistribution of flow throughout the smallest vessel networks in the retina in response to local, stimulus-driven metabolic demand.

Frequency-doubling illusion under scotopic illumination and in peripheral vision.

PURPOSE: The authors sought to determine whether frequency-doubling illusion (FDI) could be perceived under scotopic illumination at central and peripheral retinal locations. For comparison, perception of the FDI at the central and peripheral retina under photopic illumination was also evaluated. METHODS: Five subjects matched the apparent spatial frequency of counterphase flickering sinusoidal gratings with stationary sinusoidal gratings presented foveally and out to 20 degrees eccentricity under photopic and scotopic illumination conditions. Two spatial frequencies (0.25 and 0.50 cpd) were used at four temporal frequencies (2, 8, 15, and 25 Hz). Subsequent experiments explored the range of spatial and temporal frequency stimulus conditions under which the scotopic FDI might be observed. RESULTS: Under scotopic illumination conditions, the apparent spatial frequency of eccentrically presented 0.25- and 0.50-cpd flickering gratings gradually increased as a function of flicker frequency and approaches "doubling" at 15 Hz. Under photopic conditions, the apparent spatial frequency of 0.25-cpd flickering at 25 Hz was approximately doubled in all four primary meridians at central and peripheral eccentricities. The final experiment showed that the spatiotemporal range under which the scotopic FDI could be seen was similar to the photopic illumination condition reported earlier. CONCLUSIONS: Scotopic FDI is similar to photopic FDI at the central and the peripheral retina. This suggests that similar mechanisms are responsible for generating the illusion under both photopic and scotopic illumination conditions.

Spatial structure of the frequency doubling illusion.

We sought to determine (1) the perceived spatial frequency of flickering sinusoidal gratings as a function of temporal frequency at contrasts down to detection threshold levels, and (2) the influence of two different matching criteria on reported size matches to identify changes in the apparent spatial structure introduced by counterphase flicker. To determine if spatial structure was observable at detection threshold contrasts, we simultaneously measured contrast detection and orientation-identification thresholds of flickering gratings. We then measured the apparent size of flickering grating bars at sub-threshold to supra-threshold contrast levels using two matching criteria: (1) by adjusting the spatial frequency of a stationary comparison grating and (2) by adjusting the distance between two parallel thin lines. For all subjects, contrast detection and orientation-identification thresholds were similar, indicating that sufficient spatial structure is observable at detection threshold to make judgements of local spatially defined features. With increasing flicker rate, the apparent periodicity of low (0.25 and 0.50cpd) spatial frequency test gratings increased. At detection threshold contrast levels and below, all observers reported difficulty in performing the match and results were variable. Size matches using stationary gratings were consistently smaller than those made using the line targets, which is suggestive of distortion in perceived spatial structure at the nodal region of flickering gratings.

Two expressions of "surround suppression" in V1 that arise independent of cortical mechanisms of suppression.

The preferred stimulus size of a V1 neuron decreases with increases in stimulus contrast. It has been supposed that stimulus contrast is the primary determinant of such spatial summation in V1 cells, though the extent to which it depends on other stimulus attributes such as orientation and spatial frequency remains untested. We investigated this by recording from single cells in V1 of anaesthetized cats and monkeys, measuring size-tuning curves for high-contrast drifting gratings of optimal spatial configuration, and comparing these curves with those obtained at lower contrast or at sub-optimal orientations or spatial frequencies. For drifting gratings of optimal spatial configuration, lower contrasts produced less surround suppression resulting in increases in preferred size. High contrast gratings of sub-optimal spatial configuration produced more surround suppression than optimal low-contrast gratings, and as much or more surround suppression than optimal high-contrast gratings. For sub-optimal spatial frequencies, preferred size was similar to that for the optimal high-contrast stimulus, whereas for sub-optimal orientations, preferred size was smaller than that for the optimal high-contrast stimulus. These results indicate that, while contrast is an important determinant of spatial summation in V1, it is not the only determinant. Simulation of these experiments on a cortical receptive field modeled as a Gabor revealed that the small preferred sizes observed for non-preferred stimuli could result simply from linear filtering by the classical receptive field. Further simulations show that surround suppression in retinal ganglion cells and LGN cells can be propagated to neurons in V1, though certain properties of the surround seen in cortex indicate that it is not solely inherited from earlier stages of processing.

The relationship between temporal phase discrimination ability and the frequency doubling illusion.

The frequency doubling illusion (FDI) occurs when a low spatial frequency sinusoidal grating is modulated at high temporal frequencies--its apparent spatial frequency increases. A recent study suggests that this illusion is perceived due to a frequency-dependent loss of temporal phase encoding ability. We sought to elucidate the relationship between temporal phase encoding and the FDI by exploring the spatiotemporal characteristics of temporal phase discrimination (TPD) thresholds using a novel stimulus comprising three grating patches presented simultaneously in a triangular pattern. A reference grating was presented superiorly, and six degrees below two gratings (one a copy of the reference) were each randomly presented in one of two fixed positions. The odd grating had abutting regions of spatial half-cycles with alternate half-cycles locked in temporal phase. The temporal phase difference between adjoining half-cycles was varied between 0 degrees and 180 degrees via QUEST staircase--subjects had to identify which lower stimulus appeared different from the reference grating. TPD thresholds were measured for 0.25, 0.50, and 2.20 cpd stimulus at six temporal frequencies (1 to 28 Hz) at 2x, 4x, and 8x orientation identification contrast thresholds. For all subjects, thresholds were variable at low contrasts. At higher contrasts, TPD thresholds increase for 0.25 and 0.50 cpd gratings with increasing flicker rate. These data support the idea that frequency-dependent loss of temporal phase encoding ability could possibly underlie the FDI.

Activity-dependent maintenance and growth of dendrites in adult cortex.

Whereas it is widely accepted that the adult cortex is capable of a remarkable degree of functional plasticity, demonstrations of accompanying structural changes have been limited. We examined the basal dendritic field morphology of dye-filled neurons in layers III and IV of the mature barrel cortex after vibrissal-deafferentation in adult rats. Eight weeks later, the tendency for these neurons to orient their dendritic arbors toward the center of their home barrel was found to be disrupted by the resultant reduced activity of thalamocortical innervation. Measures of spine density and total dendritic length were normal, indicating that the loss of dendritic bias was accompanied by growth of dendrites directed away from the barrel center. This finding suggests that in the mature cortex, the apparently static structural attributes of the normal adult cortex depend on maintenance of patterns of afferent activity; with the corollary that changes in these patterns can induce structural plasticity.

Topographic plasticity in primary visual cortex is mediated by local corticocortical connections.

The placement of monocular laser lesions in the adult cat retina produces a lesion projection zone (LPZ) in primary visual cortex (V1) in which the majority of neurons have a normally located receptive field (RF) for stimulation of the intact eye and an ectopically located RF (displaced to intact retina at the edge of the lesion) for stimulation of the lesioned eye. Animals that had such lesions for 14-85 d were studied under halothane and nitrous oxide anesthesia with conventional neurophysiological recording techniques and stimulation of moving light bars. Previous work suggested that a candidate source of input, which could account for the development of the ectopic RFs, was long-range horizontal connections within V1. The critical contribution of such input was examined by placing a pipette containing the neurotoxin kainic acid at a site in the normal V1 visual representation that overlapped with the ectopic RF recorded at a site within the LPZ. Continuation of well defined responses to stimulation of the intact eye served as a control against direct effects of the kainic acid at the LPZ recording site. In six of seven cases examined, kainic acid deactivation of neurons at the injection site blocked responsiveness to lesioned-eye stimulation at the ectopic RF for the LPZ recording site. We therefore conclude that long-range horizontal projections contribute to the dominant input underlying the capacity for retinal lesion-induced plasticity in V1.

Local signals from beyond the receptive fields of striate cortical neurons.

We examined in anesthetized macaque how the responses of a striate cortical neuron to patterns inside the receptive field were altered by surrounding patterns outside it. The changes in a neuron's response brought about by a surround are immediate and transient: they arise with the same latency as the response to a stimulus within the receptive field (this argues for a source locally in striate cortex) and become less effective as soon as 27 ms later. Surround signals appeared to exert their influence through divisive interaction (normalization) with those arising in the receptive field. Surrounding patterns presented at orientations slightly oblique to the preferred orientation consistently deformed orientation tuning curves of complex (but not simple) cells, repelling the preferred orientation but without decreasing the discriminability of the preferred grating and ones at slightly oblique orientations. By reducing responsivity and changing the tuning of complex cells locally in stimulus space, surrounding patterns reduce the correlations among responses of neurons to a particular stimulus, thus reducing the redundancy of image representation.