Optomotry

Mice were tested for visual acuity 1 week before, and 3 and 13 weeks after ONC. Visual acuity was tested using the OptomotryTM (Cerebral Mechanics, Inc., Medicine Hat, Alberta, Canada). Mice were placed on an elevated platform in an arena surrounded by four high definition video screens. Black and white 100% contrast square-wave gratings of different spatial frequencies were presented in a semi-shuffled order. In the optokinetic response, mice make head turns in the temporo-nasal direction of the stimulus direction. If the stimulus is clockwise, the head turn is from left to right and driven by the left eye. If the stimulus is counterclockwise, the head turn is from right to left and driven by the right eye. The experimenter was blind to spatial frequency and to the direction of movement presented. If no head turn was signaled within 2 minutes, a pattern of lower spatial frequency was presented. If a head turn was observed, the next higher spatial frequency was presented to establish the acuity threshold. For the final test at 13 weeks, the left, non-affected eye was sutured shut, one day prior acuity testing to avoid any compensation from that eye. The mice were anesthetized with xylazine/ketamine as above, the skin around the eye was swabbed with 70% ethanol, avoiding to contact the eye and the lid sutured closed with two sutures of 6/0 silk.

Dark-adapted ERGs were used to assess inner and outer retinal neuronal function (see Supplementary Materials). ERG measurements were taken at baseline (before the crosslinking procedure), and at 7 and 14 days after microbead injection (Fig. 1C). We analyzed amplitudes of the positive scotopic threshold and negative scotopic threshold signals at –6.0 log cd s/m², the b-wave at –3.0 log cd s/m² (b-wave), and the third oscillatory potential (OP3) at 2.1 log cd s/m², because these amplitudes have been shown to be significantly decreased in rodent models of glaucoma.³⁵ (link)^–39 (link)
Visual function was assessed via quantitative analysis of OMR thresholds of spatial frequency and contrast sensitivity (CS) (see Supplementary Materials, OptoMotry; Cerebral-Mechanics, Lethbridge, AB, Canada). The OMR was measured at baseline (before crosslinking treatment), day 0 (7 days after crosslinking procedure, but before microbead injection that day), day 7, and day 14 (Fig. 1C), following a protocol similar to that of Prusky et al. and Douglas et al.⁴⁰ (link)^,41 (link)

Visual acuity as a function of spatial frequency threshold assessment of mice was measured by a trained observer/operator inducing and recording visual, reflexive, tracking behavior (OKT) of individual mice using a virtual optomotor system (OptoMotry; Cerebral Mechanics, Inc., Lethbridge, Alberta, Canada). Briefly, a mouse was placed on a platform inside a chamber with four walls comprised of computer monitors displaying vertical dark and white lines in motion. The perceived visual environment was that of being inside a revolving cylinder of vertical stripes. The mouse moved its head left or right (depending on the direction the lines were spinning) in order to visually follow the movement of the lines in a reflexive tracking motion.^{16 (link),17 (link)} The spatial frequency in cycles per degree (i.e., the thickness of the dark lines) was progressively narrowed in a staircase pattern by the trained observer/operator until the mouse no longer made detectable head-tracking movements. The highest spatial frequency threshold at which tracking motions were still present was recorded as the visual acuity capability of the mouse. OKT assessments were conducted under photopic conditions and at 100% line contrast 1 week and 4 weeks following toxic light exposure.^{16 (link),18 (link)}