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Variables selected in the Cabanillas' program were: a Basal pupil diameter mean pupil size from second 0 until immediately before stimulus emission, mm ; b Latency time before constant reaction to light stimulus, ms ; c Amplitude of response difference before light emission and maximum constriction, mm ; and, d Velocity of constriction maximum speed during pupil constriction. In agreement with Bitsios et al. Blue nm , green nm , and red nm monochromatic lights were obtained by the use of narrow frame-mounted interference band-pass filters Edmund Optics, New Jersey, USA.
Intensity of the white light was attenuated by the use of a neutral density frame-mounted filter Edmund Optics, New Jersey, USA , thus ensuring that all wavelength-emitted intensities were the same. Experimental Protocol Measurements were always done between 10 am and 4 pm, in a dimly lit room, with a 0.
Subjects were positioned 1 m from the pupillometer's camera.
Before recording pupillary light reflexes, subjects were adapted to the dark for 10 min in a dark room. The fixation point was established at distance-vision, thus avoiding direct near-vision possible bias source. Light stimulus was emitted after 6 s, in order to obtain stable fixation conditions. The total time for recording was 16 s. As in Daneault et al. The order of the four wavelengths of light emissions was randomized. Statistical Analysis Generalized Linear Model GLM included three categorical factors: wavelength, age group, and participant code nested into the age group.
Participant code was included in order to minimize inter-individual variability, thus improving statistical power.
GLM was carried out for three dependent variables: Latency, Amplitude of response, and Velocity of constriction, as these variables are parameters derived from pupil light-reflex, unlike basal pupil diameter, which was always recorded before light stimulations with the different wavelengths.
For amplitude of response, participant code was substituted by the covariate baseline pupil diameter, to rule out its influence on the amplitude of the response. GLM included interactions between wavelength and age group.
Significance in these effects of interaction for the variables analyzed would mean that the differences showed in the responses according to wavelength also depended on the age group and vice versa.
Consequently, it would allow us to analyze the influence of factors separately for each variable and perform three additional statistical analyses: Randomized Complete Block Design RCBD Student's t-test and ANCOVA the latter was only for amplitude of response, including baseline pupil diameter as covariate. Firstly, regarding wavelength influence, a RCBD was carried out for both age groups to highlight the fact that the responses to different wavelengths originate from the same group of individuals paired data.
In each age-group, wavelength and participant code were used as factors. When statistical significance for wavelength was obtained, Fisher Least Significant Difference LSD test was used for a post-hoc analysis. Lastly, to analyze the effect of age in an independent manner, data for each variable depending on the age group for each wavelength was compared by means of Student's t-tests.
In relation to amplitude of response, however, and given that basal pupil diameter differs between the age groups, this variable was used as a covariate in an ANCOVA analysis.
In addition, Student's t-tests were also used to determine the effect of age on the dark-adapted basal pupil diameter. Results Mean mesopic basal pupil diameter across all subjects was 5. Basal pupil diameter was always recorded before light stimulation with any of the different light wavelengths used in this study; thus, it was a non-wavelength dependant variable. As to the influence of age on this pupillary variable, an unpaired Student's t-test was carried out.
The overall analysis for velocity of constriction did not show any significance for either of the two factors age and wavelength , nor for their interaction. This non-statistically significant interaction could reflect certain lack of statistical power. As the GLM-analysis yielded statistically and near-statistically significant differences in study variables, as shown in Figures 2A,B,C graphic interactions are seen between wavelengths and groups of age , we were encouraged to conduct RCDB-, ANCOVA-, and Student's tests-analyses for all three variables involved in the pupil light reflex.
We did this in order to study the influence of the wavelength on the age groups and the influence of age on wavelength in an independent fashion, as reported above.
Interaction graphics for A amplitude of response; B latency; and, C velocity of constriction. In this age group, all amplitudes of response were of statistically significant difference to each other.
Influence of wavelength on amplitude of response in A younger group; and B older group.
Standard errors are shown with error bars. Additionally, latency in blue nm conditions was significantly longer compared to white light. Effects of the Age in the Different Wavelength Conditions In relation to the amplitude of the pupillary response between age groups, ANCOVA analysis was carried out, with pupil basal diameter as a covariate to rule out this potentially confounding factor.
No statistically significant differences in latency between groups were found for any other wavelength in the study. Velocity of pupillary constriction between groups by Student's t-test did not show any statistically significant differences, except for green nm wavelength. Values for all pupillary parameters obtained with the four study-light divided by age.
Discussion One of the most promising goals of pupillometry is to detect deficits of pupillary light-reflex as an early sign of certain severe diseases. Pupil light reflex can be used as a non-invasive, ocular predictor for C-ANS integrity Bremner, In this study we report the influence of different light-wavelengths and age groups on relevant pupil reflex response parameters in a large sample.
Pupillometric research in large populations groups has often been carried out only with the stimulus of white light Fotiou et al. However, after the discovery of ipRGCs, pupillometric studies have shown the importance of including color stimulation Hattar et al.
Blue light is often used to measure ipRGC function Herbst et al.
Although some studies have evaluated chromatic stimuli to understand the mechanisms involved in the pupil's response to color Barbur et al. Across all subjects, pupillary light response had greatest amplitude and shortest latency in white and green nm light conditions.
In addition, the longest wavelength red, nm always elicited reduced amplitude responses and longer latency values than any other light stimulus, which may reflect lower sensitivity to this particular wavelength in mesopic conditions. Various studies Herbst et al.
This is in line with our results, which have shown that, across both age-groups, in mesopic conditions too, blue wavelength caused greater constriction than red, even after short and decreased intensity stimulation.
Moreover, older subjects 46—78 years showed statistically significant lower velocities of constriction to green nm and longer latency values for white light than younger subjects 18—45 years.
Although amplitude of response to red nm also showed differences, these were only of small significance. These results could reflect a functional defect in the pupil light reflex with age.
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