Pupillometric analysis for assessment of gene therapy in Leber Congenital Amaurosis patients

Background: Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration. Methods: Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images (correcting aberrations due to the inability of these severely visually impaired patients to fixate), and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison. Results: The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye. Conclusions: Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA

A high-resolution binocular video-oculography system: assessment of pupillary light reflex and detection of an early incomplete blink and an upward eye movement

Background: The pupillary light reflex characterizes the direct and consensual response of the eye to the perceived brightness of a stimulus. It has been used as indicator of both neurological and optic nerve pathologies. As with other eye reflexes, this reflex constitutes an almost instantaneous movement and is linked to activation of the same midbrain area. The latency of the pupillary light reflex is around 200 ms, although the literature also indicates that the fastest eye reflexes last 20 ms. Therefore, a system with sufficiently high spatial and temporal resolutions is required for accurate assessment. In this study, we analyzed the pupillary light reflex to determine whether any small discrepancy exists between the direct and consensual responses, and to ascertain whether any other eye reflex occurs before the pupillary light reflex. Methods: We constructed a binocular video-oculography system two high-speed cameras that simultaneously focused on both eyes. This was then employed to assess the direct and consensual responses of each eye using our own algorithm based on Circular Hough Transform to detect and track the pupil. Time parameters describing the pupillary light reflex were obtained from the radius time-variation. Eight healthy subjects (4 women, 4 men, aged 24–45) participated in this experiment. Results: Our system, which has a resolution of 15 microns and 4 ms, obtained time parameters describing the pupillary light reflex that were similar to those reported in previous studies, with no significant differences between direct and consensual reflexes. Moreover, it revealed an incomplete reflex blink and an upward eye movement at around 100 ms that may correspond to Bell’s phenomenon. Conclusions: Direct and consensual pupillary responses do not any significant temporal differences. The system and method described here could prove useful for further assessment of pupillary and blink reflexes. The resolution obtained revealed the existence reported here of an early incomplete blink and an upward eye movement.The authors acknowledge the support of the Spanish Ministerio de Economía y Competitividad through the project BIA2011-22704 and the Generalitat Valenciana through the projects GV/2013/009 and PROMETEO/2011/021. A. B. Roig acknowledges a grant from Cajamurcia

Automating, Analyzing and Improving Pupillometry with Machine Learning Algorithms

The investigation of the pupillary light reflex (PLR) is a well-known method to provide information about the functionality of the autonomic nervous system. Pupillometry, a non-invasive technique, was applied to study the PLR alterations in a new, schizophrenia-like rat substrain, named WISKET. The pupil responses to light impulses were recorded with an infrared camera; the videos were automatically processed and features were extracted from the pupillograms. Besides the classical statistical analysis (ANOVA), feature selection and classification were applied to reveal the significant differences in the PLR parameters between the control and WISKET animals. Based on these results, the disadvantages of this method were analyzed and the measurement setup was redesigned and improved. The pupil segmentation method has also been adapted to the new videos. 2564 images were annotated manually and used to train a fully-convolutional neural network to produce pupil mask images. The method was evaluated on 329 test images and achieved 4% median relative error. With the new setup, the pupil detection became reliable and the new data acquisition offers robustness to the experiments

Psychophysiological indices of recognition memory

It has recently been found that during recognition memory tests participants’ pupils dilate more when they view old items compared to novel items. This thesis sought to replicate this novel ‘‘Pupil Old/New Effect’’ (PONE) and to determine its relationship to implicit and explicit mnemonic processes, the veracity of participants’ responses, and the analogous Event-Related Potential (ERP) old/new effect. Across 9 experiments, pupil-size was measured with a video-based eye-tracker during a variety of recognition tasks, and, in the case of Experiment 8, with concurrent Electroencephalography (EEG). The main findings of this thesis are that: - the PONE occurs in a standard explicit test of recognition memory but not in “implicit” tests of either perceptual fluency or artificial grammar learning; - the PONE is present even when participants are asked to give false behavioural answers in a malingering task, or are asked not to respond at all; - the PONE is present when attention is divided both at learning and during recognition; - the PONE is accompanied by a posterior ERP old/new effect; - the PONE does not occur when participants are asked to read previously encountered words without making a recognition decision; - the PONE does not occur if participants preload an “old/new” response; - the PONE is not enhanced by repetition during learning. These findings are discussed in the context of current models of recognition memory and other psychophysiological indices of mnemonic processes. It is argued that together these findings suggest that the increase in pupil-size which occurs when participants encounter previously studied items is not under conscious control and may reflect primarily recollective processes associated with recognition memory

Wearable multi-color RAPD screening device

In this work, we developed a wearable, head-mounted device that automatically calculates the precise Relative Afferent Pupillary Defect (RAPD) value of a patient. The device consists of two RGB LEDs, two infrared cameras, and one microcontroller. In the RAPD test, the parameters like LED on-off durations, brightness level, and color of the light can be controlled by the user. Upon data acquisition, a computational unit processes the data, calculates the RAPD score and visualizes the test results with a user-friendly interface.Multiprocessing methods used on GUI to optimize the processing pipeline. We have shown that our head-worn instrument is easy to use, fast, and suitable for early-diagnostics and screening purposes for various neurological conditions such as RAPD, glaucoma, asymmetric glaucoma, and anisocoria

{MEYE}: Web-app for translational and real-time pupillometry

Pupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, in- cluding autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is noninvasive and allows for longitudinal experiments sup- porting temporal specificity; however, its measure requires dedicated setups. Here, we introduce a convolu- tional neural network that performs online pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for the nonspecialist and nontechnical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and setup time reduction. The tested model performances indicate that the tool is sensitive enough to detect both locomotor-induced and stimulus-evoked pupillary changes, and its output is compara- ble to state-of-the-art commercial devicesPupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, including autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is noninvasive and allows for longitudinal experiments supporting temporal specificity; however, its measure requires dedicated setups. Here, we introduce a convolutional neural network that performs online pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for the nonspecialist and nontechnical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and setup time reduction. The tested model performances indicate that the tool is sensitive enough to detect both locomotor-induced and stimulus-evoked pupillary changes, and its output is comparable to state-of-the-art commercial devices

Standards in Pupillography

The number of research groups studying the pupil is increasing, as is the number of publications. Consequently, new standards in pupillography are needed to formalize the methodology including recording conditions, stimulus characteristics, as well as suitable parameters of evaluation. Since the description of intrinsically photosensitive retinal ganglion cells (ipRGCs) there has been an increased interest and broader application of pupillography in ophthalmology as well as other fields including psychology and chronobiology. Color pupillography plays an important role not only in research but also in clinical observational and therapy studies like gene therapy of hereditary retinal degenerations and psychopathology. Stimuli can vary in size, brightness, duration, and wavelength. Stimulus paradigms determine whether rhodopsin-driven rod responses, opsin-driven cone responses, or melanopsin-driven ipRGC responses are primarily elicited. Background illumination, adaptation state, and instruction for the participants will furthermore influence the results. This standard recommends a minimum set of variables to be used for pupillography and specified in the publication methodologies. Initiated at the 32nd International Pupil Colloquium 2017 in Morges, Switzerland, the aim of this manuscript is to outline standards in pupillography based on current knowledge and experience of pupil experts in order to achieve greater comparability of pupillographic studies. Such standards will particularly facilitate the proper application of pupillography by researchers new to the field. First we describe general standards, followed by specific suggestions concerning the demands of different targets of pupil research: the afferent and efferent reflex arc, pharmacology, psychology, sleepiness-related research and animal studies