Abstract
Purpose.:
A sustained pupilloconstriction is often observed after the cessation of a bright visual stimulus. This post-illumination pupil response (PIPR) is produced by the intrinsically photosensitive retinal ganglion cells (ipRGCs). The present study was designed to examine the characteristics of the PIPR in a normal population without ocular disease.
Methods.:
Thirty-seven subjects (mean age, 48.6 years) were tested by presenting a 60°, 10-second light stimulus (13 log quanta/cm2/s retinal irradiance) and recording pupillary responses for 50 seconds after light cessation. The light stimuli (470 [blue] and 623 [red] nm) were presented by an optical system to one eye after dilation, while the consensual pupil response of the fellow, undilated eye was recorded by infrared pupillometry.
Results.:
A positive PIPR was seen in all subjects tested. The population average of the PIPR for 470-nm light was 1.5 mm (SEM 0.10, P < 0.05) and the net PIPR (blue PIPR minus red PIPR) was 1.4 mm (SEM 0.09, P < 0.0001). The net PIPR correlated positively with baseline pupil diameter (P < 0.05), but not significantly with age, race, or sex (P > 0.05) in the test population.
Conclusions.:
All normal subjects displayed a significant PIPR for a 10-second, 470-nm light stimulus, but not a 623-nm stimulus, which is consistent with the proposed melanopsin-mediated response. In most normal individuals, the amplitude of the PIPR was substantial. This test has the potential to be used as a tool in evaluating subjects with inner retinal dysfunction or melanopsin-related disorders.
The pupillary light reflex (PLR), a reflexive constriction of the pupil in response to an increase in ocular illumination, is an indispensable clinical measure of visual, neurologic, and autonomic function. Until recently, it was thought that the PLR was driven by rods and cones.
1,2 In 2000, a novel photopigment, melanopsin, was discovered in the inner retina of both primates and rodents that was later determined to be expressed by a unique class of retinal ganglion cells (RGCs).
3–8 These melanopsin-containing cells, in addition to receiving rod and cone inputs, are intrinsically photosensitive and have therefore been termed ipRGCs.
9
Intracellular recordings of ipRGCs in both rodents and macaques have shown that these cells exhibit a characteristic transient burst of firing at stimulus onset that decays to a plateau of sustained firing that continues well past stimulus cessation.
9–12 Dacey et al.,
11 identified rod-driven, cone-driven, and intrinsic responses in the ipRGCs and determined the relative contribution of these inputs to the firing pattern of these cells. The melanopsin-driven, intrinsic responses of ipRGCs were present at higher irradiances (>11 log quanta/cm
2/s), and the spectral sensitivity of this response closely matched a single pigment action spectrum with a peak at 483 nm.
11 It has been reported that, in humans lacking rods and cones, these ipRGCs may contribute to rudimentary visual awareness, thus challenging the assumption that rod- and cone-based photoreception mediates all visual responses to light.
13 Preliminary data in humans suggest that under photopic conditions, cones contribute only to the initial light-evoked pupillary constriction, whereas rods and intrinsic activation of the ipRGCs contribute substantially to sustained pupilloconstriction.
14 In behaving macaques, after pharmacologic blockade of the classic photoreceptors, significant melanopsin-driven pupillary responses are present both during continuous (10 seconds) light stimuli (light-evoked response) and after light cessation (henceforth termed the post-illumination pupil response [PIPR]).
4 Indeed, since the PIPR was not significantly diminished after pharmacologic blockade in these studies, it must be produced by the melanopsin-driven, intrinsic photoresponse of the ipRGCs.
4
The present study was conducted to determine the characteristics of the PIPR in a sample of normal individuals without ocular disease. This study represents an initial step in the potential use of the PIPR as a clinical test that allows differentiation between disorders affecting photoreceptors and those affecting the RGCs.
10
Data from all tests were stored and analyzed off-line. Traces showing pupillary diameter were displayed, regions of the data were selected for further analysis, (Excel, Microsoft Corp., Redmond, WA; SAS, SAS, Inc., Cary, NC; and MatLab, The MathWorks, Natick, MA), and data plots were generated (SigmaPlot; SPSS, Chicago, IL).
For the analyses, we defined the following measures. Baseline pupil diameter was the average pupil diameter, over a 7-second period, before light stimulus. Initial pupil response was the average pupil diameter over a 4-second period, starting 1 second after the light stimulus onset. Sustained pupil diameter was the average pupil diameter over a period of 30 seconds, starting 10 seconds after light offset. The variables used for the subsequent analyses were:
Descriptive statistics (mean and SE) were calculated for baseline, sustained, initial response, and the PIPR measures. A test for normalcy was performed with the Shapiro-Wilk test. Statistical analyses were performed with linear regression models, and statistical significance was obtained with a nominal
P ≤ 0.05. The blue- and red-light responses were compared by using Student's
t-test and the slopes for regression were tested with the
F test. Relationships between baseline pupil sizes, subject characteristics, and the PIPR parameters were investigated by means of Pearson's correlation coefficients and analysis of covariance (ANCOVA).
A total of 45 subjects were tested for the study. Eight interviewed subjects were excluded due to detected ocular abnormality (n = 5) or unusable data (n = 3). Hence, a total of 37 subjects were used for the analyses (9 men and 9 women of European ancestry and 8 men and 11 women of African ancestry). The subjects' average age was 48.6 years and ranged from 26 to 80 years.
Average pupil diameters of the subjects were plotted against time for both control and test stimuli (623 and 470 nm), respectively (
Figs. 2A,
2B). As expected, given the spectral sensitivity of melanopsin (
Fig. 2C), the 470-nm stimulus evoked a substantial PIPR after light offset. However, despite producing substantial light-evoked pupillary responses at both 13 and 13.5 log quanta/cm
2/s (
Figs. 2A,
2B), the 623-nm stimulus produced no significant PIPR. The PIPRs to both the blue and red stimuli were plotted against the baseline pupil diameters (
Fig. 2D; blue light,
R 2 =0.354; red light
R 2 =0.157). The slope of the PIPR as a function of baseline pupil diameter, for blue light, was significantly different from 0 (
P < 0.05).
The values for the baseline pupil diameter, sustained pupil diameter, and PIPR measures for red (control) and blue (test) lights are shown in
Table 1. The mean response for the blue stimulus was 1.5 mm (SEM 0.10,
P < 0.05) with a net PIPR of 1.4 mm (SEM 0.09,
P < 0.001).
Table 1. Pupil Measurements in Test Subjects
Table 1. Pupil Measurements in Test Subjects
| Red (R) | Blue (B) | Net (B − R) |
Baseline | 5.0 (0.15) | 5.0 (0.14) | 0.0 (0.05) |
Sustained | 4.8 (0.15) | 3.4 (0.11) | 1.4 (0.11) |
PIPR | 0.2 (0.03) | 1.5 (0.10)* | 1.4 (0.09)† |
To investigate the influence of baseline pupil diameter on the PIPR values, we grouped the study population into tertiles (3.3–4.3 mm [group 1], 4.4–5.4 mm [group 2], and 5.5–6.5 mm [group 3]), and plotted them against the PIPR as box plots with error bars (
Fig. 3). An ANCOVA, autoregressive model, showed that the PIPR was significantly different between the test and control in all the groups (
P < 0.05), although the power of the test substantially increased (90%,
P < 0.001) when the baseline pupil diameters were greater than 4.4 mm (groups 2 and 3).
We calculated the difference between the test and control measures (net PIPR) and used this for further analyses. To control for the influence of baseline pupil diameter on the PIPR amplitude, we calculated the change in the PIPR as a percentage of baseline pupil diameter (PIPR change). The PIPR change for the red and blue stimuli was calculated, with the difference between the two measures being the net PIPR change. The mean of the net PIPR change showed a reduction of 27% (SEM 1.44,
P < 0.001), for the test population.
Figure 4 demonstrates that, whereas the net PIPR (
Fig. 4A,
R 2 = 0.345,
P < 0.05) correlated positively with baseline pupil diameter, the net PIPR change (
Fig. 4B,
R 2 = 0.066,
P > 0.05) did not.
We investigated the relationship between the initial pupil response, baseline pupil diameter, and net PIPR characteristics.
Figure 5A demonstrates the relation between initial pupil response and baseline pupil diameter (
R 2 = 0.612,
P < 0.001). The net PIPR (
Fig. 5B,
R 2 = 0.033,
P > 0.05) and net PIPR change (
Fig. 5C,
R 2 = 0.018,
P > 0.05) plotted against initial pupil response to light stimulus did not correlate significantly.
To examine the reliability of the test, we repeated the experiment in five subjects. We found a very high correlation (R 2 = 0.98) of both the baseline pupil diameters and the PIPR values, measured on different days. In all subjects, we also calculated area measures of the PIPR and proportional area change in the PIPR. The results were similar to those found with linear measures.
To examine the effect of age, we plotted baseline pupil diameter, net PIPR, and net PIPR change against age (in years;
Fig. 6). The baseline pupil diameters decreased significantly with increasing age (
R 2 = 0.166,
P < 0.05;
Fig. 6A), but there was no correlation of the net PIPR (
R 2 = 0.04,
P > 0.05;
Fig. 6B) or net PIPR change (
R 2 = 0.002,
P > 0.05;
Fig. 6C) with age when adjusted for baseline pupillary diameter.
There was no statistically significant difference between the men and women (P > 0.05) or between the individuals of European and African ancestry (P > 0.05) in the net PIPR values (results not shown).
The goal of this study was to investigate the PIPR in a sample of individuals with no ocular disease. Gamlin et al.
4 have reported a melanopsin-driven PIPR in a small sample of macaques and humans. Our results demonstrate a positive melanopsin-driven PIPR in all 37 subjects tested. Overall, we found the average PIPR for a 470-nm stimulus to be 1.5 mm (SEM = 0.10 mm), which is consistent with the results of the original study.
4 More specifically, the stimulus used in the present study was 13 log quanta/cm
2/s and had an area 0.44 log units greater than that used in the original study. Assuming linear summation, this result corresponds to a retinal irradiance of ∼13.5 log quanta/cm
2/s in the original study; this retinal irradiance resulted in an average PIPR to a 470-nm stimulus of 1.7 mm in humans and 1.5 mm in monkeys.
4
We found that the magnitude of the net PIPR varied from 0.5 to 2.3 mm in our sample population. The small PIPRs in some of the subjects were not the result of unresponsive pupils, since these subjects showed substantial light-evoked pupilloconstriction (Initial pupil response). However, our initial analyses showed that the subjects with smaller baseline pupils tended to display smaller PIPRs. To remove this interaction from our subsequent analyses, we found that the data could be normalized by plotting the percentage of PIPR change as a function of the baseline pupil measure. Even with this correction, we observed a significant range in the percentages of PIPR change. In particular, a few subjects displayed minimal PIPRs. It is possible that these subjects represent a small percentage of the general population that possesses a reduced PIPR. Such reduced responses could be seen in individuals with defects in the
OPN4 gene, which codes for melanopsin.
20 They could also be seen in the subjects in whom the intrinsic photoresponse was reduced in magnitude because of a reduced expression of melanopsin. In either of these cases, given such reduced melanopsin-driven pupillary responses, the possibility that these subjects could also display circadian or sleep/wake abnormalities would be interesting to explore.
It is well known that baseline pupil diameters tend to get smaller with age,
17–19,21–24 and this age-related miosis is seen over a wide range of illuminance levels.
21 Furthermore, it has been suggested that the magnitude of light-evoked pupillary responses is reduced with age.
22 Our results indicate that, after adjustment for baseline pupil diameter, age was not a significant contributing factor to the magnitude of the PIPR, independent of variation in pupil size. However, a recent study of light-induced melatonin suppression showed an age-related loss in sensitivity to short-wavelength light.
24 It is thought that such light-induced melatonin suppression is driven by the ipRGCs,
25 which therefore suggests that there is an age-related reduction in ipRGC signaling. Of note, such a decrease in ipRGC signaling may be expected to result in age-related mydriasis rather than the miosis that is actually seen. It is clear that more extensive study is needed to investigate the effect of age on ipRGC mediated pupillary responses. Although there have been some previous reports of racial differences, particularly as they relate to iris color, in pupillary response magnitude,
25,26 we found no effect of race or sex on the net PIPR.
In conclusion, using a newly developed, wide-field optical system, we have demonstrated that all normal subjects display a PIPR with wavelength sensitivities consistent with melanopsin-mediated mechanism. This response is substantial in most normal individuals. Therefore, this system may have potential to be used as a clinical tool in evaluating patients with inner retinal dysfunction or melanopsin-related disorders.
Supported by National Institutes of Health Grants EY09380 (PDG) and P30 EY03039 (PDG); The EyeSight Foundation of Alabama; and a Research to Prevent Blindness Physician Scientist Award (CAG).
Disclosure:
L. Kankipati, None;
C.A. Girkin, None;
P.D. Gamlin, None
The authors thank all the subjects for participating; Bobbie Hill (Glaucoma Service, EFH) for invaluable help in recruitment; and Jerry Millican, David McDougal, Abidin Yildrim, and Mark Bolding for help in the development of the test apparatus.