DOI: 10.5061/dryad.9kd51c5g5
title: Pupil and masking responses to light as functional measures of retinal degeneration in mice Mus Musculus

description: 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.
description: 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. <i>rd1</i>, <i>Rd2</i> and wild-type
 control mice
 (C3Sn.BLiA-<i>Pde6b</i><sup>+</sup>/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<sup>-2</sup>s<sup>-1</sup>,
 then 12-hours dark, except when undergoing experiments. Food and water
 were available <i>ad libitum</i>. Light measurements were made
 using a PM103 power meter (Macam Photometrics Ltd, Livingston,
 UK). <i>Retinal histopathology </i>was
 assessed in Wild-type, <i>rd1</i> and <i>Rd2</i>
 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. <i>The electroretinogram
 </i>was recorded in Wild-type (n = 6), <i>rd1</i> (n =
 8), and <i>Rd2</i> 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). <i>Negative masking responses
 </i>were assessed in Wild-type, <i>rd1</i>, and
 <i>Rd2</i> mice according to previously described protocols
 [6]. Mice<i> </i>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<sup>-2</sup>s<sup>-1</sup>, 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<sup>-2</sup>s<sup>-1</sup> 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. <i>Pupillary light reflexes</i>
 were assessed in wild-type (n = 9), <i>Rd2
 </i>(<i>n</i> = 10), and<i> rd1</i>
 (<i>n</i> = 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<sup>-2</sup>s<sup>-1</sup> 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<i> </i>unpaired 2-tailed parametric<i>
 t</i>-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<i> </i>Welch’s <i>t</i>-test of response
 amplitude at the highest irradiance for red and for blue
 stimuli.