Background: Pre-clinical testing of retinal pathology and treatment efficacy depends on reliable and valid measures of retinal function. The electroretinogram (ERG) and tests of visual acuity are the ideal standard, but can be unmeasurable while useful vision remains. Non-image-forming responses to light such as the pupillary light reflex (PLR) are attractive surrogates. However, it is not clear how accurately such responses reflect changes in visual capability in specific disease models. The purpose of this study was to test whether measures of non-visual responses to light correlate with previously determined visual function in two photoreceptor degenerations.

Methods: The sensitivity of masking behavior (light induced changes in running wheel activity) and the PLR were measured in 3-month-old wild-type mice (WT) with intact inner retinal circuitry, Pde6b-rd1/rd1 mice (rd1) with early and rapid loss of rods and cones, and Prph2-Rd2/Rd2 mice (Rd2) with slowly progressing loss of rods and cones.

Results: In rd1 mice, negative masking had increased sensitivity, positive masking was absent, and the sensitivity of the PLR was severely reduced. In Rd2 mice, positive masking identified useful vision at higher light levels, but there was a limited decrease in the irradiance sensitivity of negative masking and the PLR, and the amplitude of change for both underestimated the reduction in irradiance sensitivity of image-forming vision.

Conclusions: Together these data show that in a given disease, two responses to light can be affected in opposite ways, and that for a given response to light, the change in the response does not accurately represent the degree of pathology. However, the extent of the deficit in the PLR means that even a limited rescue of rod/cone function might be measured by increased PLR amplitude. In addition, positive masking has the potential to measure effective treatment in both models by restoring responses or shifting thresholds to lower irradiances.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the New Mexico Tech Institutional Animal Care and Use Committee. All procedures were non-invasive and mice were lightly sedated when restraint stress would affect measurements.

To constrain variables, mice of the same age and C3H genetic background were studied under the same conditions. rd1, Rd2 and wild-type control mice (C3Sn.BLiA-Pde6b⁺/DnJ) were sourced from the Jackson Laboratories (Bar Harbor, ME) and then bred on site. Mice were maintained in a repeating cycle of 12-hours fluorescent white light at ~20μWcm^-2s^-1, then 12-hours dark, except when undergoing experiments. Food and water were available ad libitum. Light measurements were made using a PM103 power meter (Macam Photometrics Ltd, Livingston, UK).

Retinal histopathology was assessed in Wild-type, rd1 and Rd2 mice according to previously described protocols [15]. Mice were humanely sacrificed (n = 3 each), eyes were collected and fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO) for 4 hours, then transferred to 1% phosphate buffered saline (PBS) and stored at 4 °C. After removal of the lens, eyes were infiltrated and embedded in acrylamide solution, then embedded in Tissue Freezing Medium (General Data, Cincinnati, OH) and sectioned at 7μm along the anterior-posterior axis on a Shandon FE Cryostat (Thermo Fisher scientific, Waltham, MA). Sections were stained with hematoxylin-eosin and images recorded on a Leica ICC50 HD.

The electroretinogram was recorded in Wild-type (n = 6), rd1 (n = 8), and Rd2 mice (n = 8) according to previously described protocols [7]. Mice were dark-adapted then sedated with ketamine:xylazine (100:20 mg/kg). Pupils were dilated using Tropicamide 1% (Falcon Pharmaceuticals, Fort Worth, TX) and corneas moistened using GenTeal (Novartis, East Hannover, NJ). Animals were positioned on a temperature-regulated platform and electrodes were placed for corneal contact, midline subdermal reference, and ground. Five flashes of 4-milliseconds at 25cd.s.m were applied with an inter-stimulus interval of 60 seconds and responses were recorded using a Espion E2 system (Diagnosys LLC, Lowell, MA).

Negative masking responses were assessed in Wild-type, rd1, and Rd2 mice according to previously described protocols [6]. Mice were individually housed in wheel cages (Harvard Apparatus, Holliston, MA) mounted in custom built environmental control cabinets (n = 12 for each genotype, and first test at post-natal day 90 for all mice). Wheel running was continuously recorded using a customized ClockLab data acquisition system (Actimetrics, Inc. Evanston, IL). Between tests, mice were maintained under a cycle of 12-hours fluorescent white light at ~20μWcm^-2s^-1, then 12-hours dark. Animals were allowed to acclimatize to wheel cages for 14 days prior to testing. The testing schedule was a 3-day repeating experimental cycle of pre-pulse baseline day, pulse day, and maintenance day. On pulse days, starting one hour after daily dark onset, light at a defined irradiance was applied to mice in their home cage for 1-hour. Under this protocol, circadian activity remains entrained throughout testing, but acute changes in activity during the dark phase are induced. Cinegel Neutral density film (Rosco, Stamford, CT) was used to regulate the irradiance of the applied light. Six light levels between 0.0003 and 6.0 μWcm^-2s^-1 were applied in a sequence that distributed bright and dim pulses over the course of testing. Changes in activity over the 1-hour light treatment were calculated as % of baseline activity at the corresponding time on the preceding day for each animal.

Variable slope sigmoid dose response curves were fitted to data in Prism (GraphPad, San Diego, CA) with a fixed constant for the minimum set at 0%. The irradiance producing a half maximal response (EC50) and hill-slope were calculated in Prism from fitted curves. Features of fitted curves were then compared by an F-test of a two-fit comparison in Prism. Curves were fitted to the data sets for two genotypes independently and then to the combined data set for both genotypes; the effect of combining data sets on the quality of fit to a given parameter was then used to calculate if there was a difference.

Pupillary light reflexes were assessed in wild-type (n = 9), Rd2 (n = 10), and rd1 (n = 9) mice according to previously described protocols [26]. Briefly, mice were dark adapted for a minimum of 2-hours then lightly sedated using 46mg/kg ketamine with 4.6mg/kg of xylazine. Sedation avoids the stress-induced pupil dilation of awake restraint, and therefore allows measurement of pupil responses at low light levels. Using infra-red cameras, animals were positioned on a test platform and pupil responses recorded from both eyes using A2000 pupillometer (Neuroptics, Laguna Hills CA). Change in pupil size was recorded on a dark-adapted background to a 1-second red stimulus (622 ± 3nm) then a 1 second blue stimulus (480 ± 3nm), separated by a 59 second inter-stimulus interval: 1-second stimuli test rod/cone-generated pupil responses. Recordings at 0.01, 0.1, 1.0 and 10μWcm^-2s^-1 were separated by a minimum of 3 non-test days.

Recordings with poor pupil acquisition were discarded because pupil ellipse fitting prevents reliable measurement of response peak. Dark-adapted pupil size was compared by unpaired 2-tailed parametric t-test with Welch’s correction for difference Standard deviations between genotypes.

The ‘Initial response’ primarily generated by rod/cone activation, was defined as the maximal constriction within 2 seconds of stimulus onset. Pupil constriction was converted to percent constriction against baseline for each animal and test, then plotted as an irradiance-response function, and fitted with a sigmoidal dose response in Prism (GraphPad, San Diego, CA), using 0% pupil constriction as constrained minimum, and 95% pupil constriction as a constrained maximum, reflecting the range of pupil constriction in mice. Full dose-response curves were not established so comparison was by Welch’s t-test of response amplitude at the highest irradiance for red and for blue stimuli.