2.3. Pupillary light reflex (PLR)

The PLR was evaluated using a Ganzfeld controlled by an RETI–port pupillometer (Roland Consult, Germany). The stimuli consisted of 1s blue (470 nm) and red (631 nm) light flashes presented alternately at 2-min intervals. To assess the contribution of cone photoreceptors, we used a 631-nm flash at 2.4 log cd.m^–2, with a blue background (470 nm at 0.78 log cd.m^–2). For rods, we used 470-nm flashes at –3 log cd.m^–2 without background light. For the melanopsin response measured by the post-illumination pupil response (PIPR), we used 470-nm flashes at 2.4 log cd.m^–2, also with no background light. A photopically matched stimulus at 631 nm, a region that causes no or minimal melanopsin activation, was also presented for comparison. The difference between the response to the red and blue lights evidences the melanopsin response in the PLR. This protocol was based on Park et al. (2011).

Measurements of the PLR were carried out with an infrared eye-tracking camera system at a 60-Hz sample rate of real-time pupil recording. The subjects were dark-adapted for 10 min prior to the beginning of the measurements.

The PLR data were analyzed offline using a self-written script programed in OriginPro ^® (v. 8.5.1 SR2, OriginLab Corp., Northampton, MA, USA). First, a median filter with a 300-ms time window was applied to remove eye blinks. In the case of long eye blinks or eye closure, the artifact was removed manually.

The parameters obtained are shown in Figure 1. The baseline was obtained by the median pupil size of filtered PLRs of the 3 s prior to each stimulus onset. The PLRs were then normalized by the baseline pupil size. The relative pupil size was defined as the absolute pupil size (mm)/baseline pupil size (mm). The pupil size amplitude was obtained as the baseline divided by the minimum pupil size. Peak latency was the time for the pupil to reach its minimum size. Recovery time was the time for the pupil to reach 75% of its baseline size. The PIPR was expressed as the median pupil size from 6 to 8 s after the stimulus offset. The PIPR parameter was determined only for the melanopsin-activating stimulus (high-intensity blue light) as this parameter reflects the melanopsin function of the ipRGC, while the other parameters were measured for all stimuli (Kelbsch et al., 2019).

Features of the pupillary light reflex. The baseline (B) was obtained as the median of the pupil size of the 3 s before the stimuli. The stimulus (S) was presented as a step of light with 1-s duration. Amplitude (A) was defined as the maximum constriction reached by the pupil after the stimulus. Latency (L) was the time between the beginning of the stimulus and the moment when the pupil reached its maximum constriction. Recovery time was calculated from the pupil maximum constriction until the pupil had returned to 75% of its baseline size (A*0.75). The post-illumination pupil response (PIPR) was calculated as the median pupil size recorded in the period of 6–8 s after the start of the stimulus.

The subjects underwent spectral-domain OCT (SD-OCT) scanning using a commercially available device (Cirrus HD-OCT, Zeiss, Germany). The scanning protocol involved the acquisition of a 6 × 6-mm cube scan of the macula at a scan density of 512 × 128 pixels. The criteria for acceptable OCT images are as follows: the absence of large eye movements, defined as an abrupt shift completely disconnecting a large retinal vessel; consistent signal intensity level across the scan; and absence of the black band.