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Pupillary Responses to High-Irradiance Blue Light Correlate with Glaucoma Severity

  • Annadata V. Rukmini
    Affiliations
    Program in Neuroscience and Behavioral Disorders, Duke–National University of Singapore Graduate Medical School, Singapore, Republic of Singapore
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  • Dan Milea
    Affiliations
    Program in Neuroscience and Behavioral Disorders, Duke–National University of Singapore Graduate Medical School, Singapore, Republic of Singapore

    Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Republic of Singapore
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  • Mani Baskaran
    Affiliations
    Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Republic of Singapore

    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore
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  • Alicia C. How
    Affiliations
    Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Republic of Singapore
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  • Shamira A. Perera
    Affiliations
    Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Republic of Singapore
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  • Tin Aung
    Affiliations
    Singapore Eye Research Institute, Singapore National Eye Center, Singapore, Republic of Singapore

    Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore
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  • Joshua J. Gooley
    Correspondence
    Correspondence: Joshua J. Gooley, PhD, Program in Neuroscience and Behavioral Disorders, Duke–National University of Singapore Graduate Medical School Singapore, 8 College Road Singapore 169857, Republic of Singapore.
    Affiliations
    Program in Neuroscience and Behavioral Disorders, Duke–National University of Singapore Graduate Medical School, Singapore, Republic of Singapore

    Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore
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      Purpose

      To evaluate whether a chromatic pupillometry test can be used to detect impaired function of intrinsically photosensitive retinal ganglion cells (ipRGCs) in patients with primary open-angle glaucoma (POAG) and to determine if pupillary responses correlate with optic nerve damage and visual loss.

      Design

      Cross-sectional study.

      Participants

      One hundred sixty-one healthy controls recruited from a community polyclinic (55 men; 151 ethnic Chinese) and 40 POAG patients recruited from a glaucoma clinic (22 men; 35 ethnic Chinese) 50 years of age or older.

      Methods

      Subjects underwent monocular exposure to narrowband blue light (469 nm) or red light (631 nm) using a modified Ganzfeld dome. Each light stimulus was increased gradually over 2 minutes to activate sequentially the rods, cones, and ipRGCs that mediate the pupillary light reflex. Pupil diameter was recorded using an infrared pupillography system.

      Main Outcome Measures

      Pupillary responses to blue light and red light were compared between control subjects and those with POAG by constructing dose-response curves across a wide range of corneal irradiances (7–14 log photons/cm2 per second). In patients with POAG, pupillary responses were evaluated relative to standard automated perimetry testing (Humphrey Visual Field [HVF]; Carl Zeiss Meditec, Dublin, CA) and scanning laser ophthalmoscopy parameters (Heidelberg Retinal Tomography [HRT]; Heidelberg Engineering, Heidelberg, Germany).

      Results

      The pupillary light reflex was reduced in patients with POAG only at higher irradiance levels, corresponding to the range of activation of ipRGCs. Pupillary responses to high-irradiance blue light associated more strongly with disease severity compared with responses to red light, with a significant linear correlation observed between pupil diameter and HVF mean deviation (r = −0.44; P = 0.005) as well as HRT linear cup-to-disc ratio (r = 0.61; P < 0.001) and several other optic nerve head parameters.

      Conclusions

      In glaucomatous eyes, reduced pupillary responses to high-irradiance blue light were associated with greater visual field loss and optic disc cupping. In POAG, a short chromatic pupillometry test that evaluates the function of ipRGCs can be used to estimate the degree of damage to retinal ganglion cells that mediate image-forming vision. This approach could prove useful in detecting glaucoma.

      Abbreviations and Acronyms:

      HRT (Heidelberg Retinal Tomography), HVF (Humphrey Visual Field), ipRGC (intrinsically photosensitive retinal ganglion cell), PIPR (postillumination pupillary response), PLR (pupillary light reflex), POAG (primary open-angle glaucoma), RGC (retinal ganglion cell), RAPD (relative afferent pupillary defect), VF (visual field)
      The pupillary light reflex (PLR) is often used to assess the integrity of the visual system. Until recently, however, the photoreceptor pathways that drive pupillary light responses were not well characterized. The afferent limb of the PLR is thought to be mediated solely by retinal ganglion cells (RGCs) that contain the short wavelength-sensitive photopigment melanopsin.
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      Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.
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      Melanopsin is required for non-image-forming photic responses in blind mice.
      Although melanopsin-containing RGCs are intrinsically photosensitive, they are also activated extrinsically by rod and cone photoreceptors located in the outer retina.
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      Genetic ablation of melanopsin RGCs in mice eliminates pupillary responses to light, indicating that these cells serve as a necessary conduit for light information to reach the olivary pretectal nucleus in the midbrain.
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      Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision.
      Growing evidence indicates that measuring pupillary responses to different wavelengths and irradiances of light (i.e., chromatic pupillometry) can be used to assess inner versus outer retinal degeneration, because intrinsically photosensitive RGCs (ipRGCs) and visual photoreceptors differ in their response properties. In the absence of rod–cone input, melanopsin cells are preferentially sensitive to blue light (λmax, approximately 480 nm), respond sluggishly to light onset and offset, and are less sensitive to light than rods and cones.
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      Melanopsin and rod-cone photoreceptors play different roles in mediating pupillary light responses during exposure to continuous light in humans.
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      By comparison, rods are most sensitive to bluish-green light (λmax, approximately 500 nm), the photopic visual system is most sensitive to green light (λmax, 555 nm), and rod–cone photoreceptors are capable of driving fast pupillary responses. The wavelength and irradiance of light exposure therefore can be manipulated to stimulate preferentially rods, cones, or the intrinsic melanopsin response, thus providing a window onto the status of each photoreceptor cell type. For example, blue-light stimuli can be used to activate rods preferentially at low irradiances and melanopsin at high irradiances, whereas red-light stimuli can be used to target preferentially the activation of middle- and long-wavelength–sensitive cones.
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      As such, chromatic pupillometry using blue-light and red-light stimuli could be used to detect photoreceptor dysfunction associated with different types of retinal diseases.
      Recent studies suggest that melanopsin-containing RGCs are damaged in glaucoma, similar to nonmelanopsin RGCs that mediate image-forming vision.
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      The post-illumination pupil response is reduced in glaucoma patients.
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      Monochromatic pupillometry in unilateral glaucoma discloses no adaptive changes subserved by the ipRGCs.
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      Glaucoma is a major cause of blindness, and hence early detection and treatment are important for slowing the progression of the disease. Patients often seek treatment late in the disease, however, because it is asymptomatic initially. The pupillary light reflex is impaired in severe glaucoma,
      • Feigl B.
      • Mattes D.
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      • Zele A.J.
      Intrinsically photosensitive (melanopsin) retinal ganglion cell function in glaucoma.
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      The post-illumination pupil response is reduced in glaucoma patients.
      but there is limited evidence regarding whether reduced pupillary light responses correlate with glaucoma severity, as measured by visual field (VF) testing and anatomic correlates of optic nerve damage. To address this limitation, we developed and tested a pupillometry-based protocol for evaluating photoreceptor dysfunction in which the light stimulus (blue or red) is increased gradually over time to assess sustained pupillary responses across a wide range of irradiances. The aim of our study was to determine if this chromatic pupillometry test can be used to detect loss of function of ipRGCs in patients with primary open-angle glaucoma (POAG) and if the degree of impairment in the PLR correlates with disease severity.

      Methods

       Subjects

      Three hundred subjects 50 years of age or older were recruited to undergo a chromatic pupillometry test. Subjects were recruited over a 2-month period (August–September 2013) from a larger study involving more than 2000 volunteers who underwent a standardized ophthalmic examination at a community polyclinic. At the time of enrollment, subjects were either visiting the polyclinic for minor health issues (nonocular) or accompanying another patient at the clinic. The aim of the larger study was to evaluate the incidence of ocular abnormalities in older patients seeking outpatient medical care. The eye examination consisted of tests for visual acuity, intraocular pressure measurement by Goldmann applanation tonometry, automated refraction to assess refractive error, a slit-lamp examination, iris and fundus photography, and an examination by a study ophthalmologist. The chromatic pupillometry test was performed before examination by the ophthalmologist, and hence the researcher performing the pupillary recording was not aware of the ophthalmic status of subjects at the time of testing.
      Forty-eight individuals diagnosed with POAG were recruited over a 5-month period (October 2013–February 2014) from glaucoma clinics at the Singapore National Eye Centre. Patients with POAG were defined by the following criteria: the presence of glaucomatous optic neuropathy (defined as a loss of neuroretinal rim with a vertical cup-to-disc ratio of >0.7 or an intereye asymmetry of >0.2, notching attributable to glaucoma, or both) with compatible VF loss (defined below), open angles on gonioscopy, and absence of secondary causes of glaucomatous optic neuropathy. Intraocular pressure before the start of glaucoma treatment was assessed by Goldmann applanation tonometry. In addition to the ophthalmic tests described for the polyclinic study, the glaucoma patients underwent standard automated perimetry (Humphrey Visual Field [HVF] Analyzer II model 750; Carl Zeiss Meditec, Dublin, CA) and Heidelberg Retinal Tomography (HRT 3; Heidelberg Engineering, Heidelberg, Germany), performed either on the day of chromatic pupillometry testing or within the preceding 3 months. Humphrey Visual Field testing was performed with near refractive correction using the 24-2 Swedish interactive thresholding algorithm with stimulus size III. Repeat testing was performed if false-positive or false-negative responses exceeded 33% or if the fixation loss rate was more than 20%. Patients who could not achieve these reliability criteria were ineligible for the study. A glaucomatous VF defect was defined by the presence of a glaucoma hemifield test result outside normal limits and the presence of at least 3 contiguous, nonedge test points within the same hemifield on the pattern deviation probability plot at P < 0.05, with at least 1 point at P < 0.01, excluding points directly above and below the blind spot. Glaucoma severity was graded according to the Hodapp-Parrish-Anderson scale,
      • Hodapp E.
      • Parrish R.K.
      • Anderson D.R.
      Clinical decisions in glaucoma.
      in which subjects with mean deviation less than −6 dB were classified as having early VF loss, mean deviation between −6 dB and −12 dB as moderate VF loss, and mean deviation more than (i.e., more negative than) −12 dB as severe VF loss. For HRT, global and segmental disc and cup areas were analyzed using the standard reference plane. In most cases, the POAG disease severity was known at the time that the pupillometry test was performed.
      Control subjects and POAG patients were excluded from chromatic pupillometry testing if they had undergone previous intraocular surgery. Additional exclusionary criteria for individuals with POAG included significant nuclear sclerosis of more than grade 2 severity on slit-lamp examination; severe retinal or ocular comorbid conditions including, but not limited to, diabetic retinopathy and age-related macular degeneration; and clinically significant pupillary abnormalities (except for relative afferent pupillary defects). Because the ophthalmic health of subjects recruited from the polyclinic was determined after chromatic pupillometry testing, data from participants who failed to meet the above criteria were excluded post hoc from the analyses. Therefore, the number of subjects with normal ocular health was not determined a priori, whereas the sample size of the group with POAG was determined before study recruitment. Demographic information was collected using interviewer-administered questionnaires. The study was approved by the SingHealth Centralized Institutional Review Board, and all participants provided written informed consent. Research procedures adhered to ethical principles outlined in the Declaration of Helsinki.

       Chromatic Pupillometry

      Before each light exposure, participants were seated with their head position fixed by a chinrest for at least 1 minute in a dark environment. To measure the direct PLR, light was administered to one eye using a modified Ganzfeld dome (Labsphere, Inc, North Sutton, NH), with the other eye covered by a patch. Subjects were exposed to a blue-light stimulus (469 nm) or red-light stimulus (631 nm), with the order of exposure randomized and counterbalanced. Narrow-bandwidth light was provided using light-emitting diodes (Nichia Corporation, Tokushima, Japan) that were controlled using a function generator (Keithley Instruments, Inc, Cleveland, OH). Current was applied to the light-emitting diodes over a 2-minute period using a logarithmically increasing function in 14 000 steps. Hence, the light stimulus was perceived as increasing gradually in intensity over time, ranging from 6.8 log photons/cm2 per second to 13.8 log photons/cm2 per second at the level of the cornea. The maximum light level corresponded to 12.27 lux and 28.30 μW/cm2 for the blue-light stimulus and 20.43 lux and 21.67 μW/cm2 for the red-light stimulus. Light levels were calibrated using a portable radiometer (ILT1700 radiometer; International Light Technologies, Peabody, MA), with the sensor placed at the level of the subject's eyes during light exposure. The light exposure was followed by a 1-minute period of darkness, during which the subject remained seated with his or her head in the dome. This was followed immediately by the next light exposure sequence for the other color of light (1 minute of darkness, 2 minutes of light, and 1 minute of darkness). In the event that a quality eye image could not be obtained for one of the trials, participants underwent a third light exposure sequence (7 of 348 subjects). During each 4-minute light exposure sequence, subjects wore a head-mounted eye tracking device that recorded monocular pupil diameter at a rate of 120 samples per second (ETL-100H Pupillometry Lab; ISCAN, Inc, Woburn, MA). The eye tracker was configured to measure pupillary responses of the left eye in all study participants, with the exception of 5 glaucoma patients who had detectable disease only in the right eye.

       Data Analysis and Statistics

      Pupillometry recordings from all participants were coded and processed without knowledge of the patient's ocular health status or POAG disease severity. For each light exposure, pupil diameter measurements were processed to remove blink artifacts in the recording, and then expressed as a percentage of the median pupil diameter during the preceding dark period. Data were reduced by taking the median pupillary constriction response in 0.5 log unit bins from 7 to 14 log photons/cm2 per second, resulting in 14 data points per light exposure sequence. The interaction of irradiance with wavelength (blue vs. red) or with ocular health (controls vs. glaucomatous eyes) on pupillary responses was assessed using a 2-way repeated-measures analysis of variance. For those comparisons in which the omnibus test reached statistical significance, pairwise multiple comparison procedures were performed using the Holm-Sidak method. Differences in age between controls and patients with POAG were assessed using an unpaired Student's t test assuming unequal variance. Within the POAG patient group, the strength of the linear relationship between pupillary constriction and clinical measures (e.g., HVF mean deviation and linear optic cup-to-disc ratio) was assessed using Pearson's correlation analysis. For all statistical tests, the threshold for significance was set at α = 0.05. Statistics were performed using Sigmaplot software version 12.0 (Systat Software, Inc, San Jose, CA) and SPSS software version 22 (IBM Corp, Armonk, NY).

      Results

       Subject Characteristics

      Of the 300 subjects who were studied in the polyclinic, 86 individuals were excluded from the analysis because of diagnosis of an ophthalmic condition (e.g., severe cataracts, primary angle-closure glaucoma, diabetic retinopathy) and were referred to an ophthalmologist for a follow-up assessment. In the remaining 214 subjects with normal-for-age ocular health, we excluded data from 43 participants based on the criterion that the baseline pupil diameter measured in darkness differed between light exposure trials by more than 10%. Because pupillary light responses were measured relative to baseline, the 10% cutoff ensured that the magnitude of pupillary constriction could be compared reliably between blue-light and red-light exposures and across groups with normal ocular health and POAG. Technical problems resulted in loss of data in an additional 10 individuals. Therefore, a total of 161 subjects were included in the control group, among whom 107 individuals had mild cataracts. Pupillary responses in these subjects were similar to those observed in individuals without cataracts (F1,159 < 2.30; P > 0.13, for main effect of group for both colors of light). Of the 48 POAG patients recruited from the glaucoma clinic, baseline pupil diameter was unstable in 7 individuals, and data loss occurred during the pupillometry recording in 1 participant. We therefore included data from 40 patients in our analyses, comprising persons with early (n = 19), moderate (n = 10), and severe (n = 11) POAG. Patients with POAG were 4 years older on average (t = 3.6; P < 0.001) relative to controls and included a greater proportion of men and ethnic Chinese individuals (Table 1).
      Table 1Subject Characteristics
      Subject GroupNo.No. of MenNo. of Chinese IndividualsAge (Mean ± Standard Deviation)
      Controls1615515159.8±6.2
       No cataract54364956.4±5.1
       Mild cataract1071910261.5±6.0
      Glaucoma (stage)40223563.8±6.1
       Early1991662.3±6.3
       Moderate1041064.9±6.1
       Severe119965.5±5.6

       Chromatic Pupillometry in Control Subjects

      In response to the gradually increasing light stimulus (Fig 1A), the onset of pupillary constriction in control subjects occurred much earlier for blue light relative to red light (Fig 1B, C). Pupil diameter closely tracked the change in irradiance over time for both exposures, followed by a similar time course of redilation after light offset. Pupillary light responses measured over time then were converted to dose-response curves by grouping results in 0.5-log unit bins based on photon density (Fig 1D). The threshold for pupillary constriction was between 9.0 and 9.5 log photons/cm2 per second in response to blue light, as compared with 10.5 to 11.0 log photons/cm2 per second for exposure to red light. Pupillary constriction responses were significantly greater in response to blue light across a wide range of irradiances, from 9.0 to 13.0 log photons/cm2 per second (F13,2080 = 62.18; P < 0.001 for interaction; t > 3.35 and P < 0.05 for all pairwise comparisons), with the greatest difference in spectral responses observed in the middle of this range (Fig 1E).
      Figure thumbnail gr1
      Figure 1Graphs showing chromatic pupillometry results in control patients with normal-for-age ocular health. A, Patients were exposed to a 4-minute light exposure sequence consisting of 1 minute of darkness, 2 minutes of monocular exposure to a gradually increasing blue-light (469 nm) or red-light (631 nm) stimulus, and 1 minute of darkness after light offset. B, Representative pupillary constriction responses are shown for a participant who underwent the pupillometry test. C, Average pupillary response, expressed as a percentage of the dark pupil diameter, across 161 control subjects. D, Dose-response curves for pupillary constriction during exposure to blue light versus red light, with data grouped in 0.5-log unit bins. Asterisks indicate significant differences in percentage of pupillary constriction between light conditions. E, Mean difference in the pupillary constriction response for blue light versus red light, demonstrating greater responses to blue light across a 4-log unit range, from 9.0 to 13.0 log photons/cm2 per second. In (C), (D), and (E), the mean ± standard error of the mean is shown.

       Chromatic Pupillometry in Patients with Glaucoma

      Similar to control subjects, the onset of pupillary constriction in patients with POAG occurred earlier for the blue-light stimulus. This was evident across different disease stages of POAG, ranging from early to severe (Fig 2A–C). As with controls, glaucomatous eyes showed stronger pupillary responses to blue light in the irradiance range from 9.0 to 13.0 log photons/cm2 per second (F13,505 = 9.89; P < 0.001 for interaction; t > 2.02 and P < 0.05 for all pairwise comparisons; Fig 2D), and the greatest difference in pupillary constriction relative to red light was in the middle of this irradiance range (Fig 2E). Overall, dose-response curves to blue light and red light were similar in shape between controls and patients with POAG; hence, the wavelength dependency of pupillary responses seemed to be preserved in glaucomatous eyes.
      Figure thumbnail gr2
      Figure 2Graphs showing chromatic pupillometry results in patients with primary open-angle glaucoma (POAG). Representative pupillary constriction responses are shown for patients with (A) early, (B) moderate, or (C) severe glaucoma. The pupillary constriction response was reduced in patients with greater disease severity. As in , patients were exposed to 1 minute of darkness, 2 minutes of monocular exposure to a gradually increasing blue-light (469 nm) or red-light (631 nm) stimulus, and 1 minute of darkness. D, Dose-response curves for pupillary constriction during exposure to blue light versus red light, assessed in 40 patients with POAG. Asterisks indicate significant differences in the response between light conditions. E, Patients exhibited greater responses to blue light in the range from 9.0 to 13.0 log photons/cm2 per second, based on the mean difference in pupillary constriction for blue light versus red light. In (D) and (E), the mean ± standard error of the mean is shown.

       Deficits in Pupillary Responses to Light in Primary Open-Angle Glaucoma at Higher Irradiances

      Next, we evaluated whether the magnitude of pupillary light responses differed in controls and patients with POAG. At lower irradiances, there was no difference between groups in pupillary constriction for blue-light and red-light stimuli (Fig 3A, B). As the corneal irradiance was increased beyond 11.5 log photons/cm2 per second, however, the relative response in patients with POAG became increasingly impaired. For both colors of light, there was a significant interaction between group (POAG vs. controls) and irradiance such that the difference in pupillary constriction between healthy and glaucomatous eyes was greatest at the highest irradiances tested (F13,2587 > 11.38; P < 0.001 for blue- and red-light stimuli; t > 2.73 and P < 0.05 for all pairwise comparisons of more than 11.5 log photons/cm2 per second). To address the possibility that the difference in pupillary responses between groups may be driven by small differences in age or other patient characteristics, a secondary analysis was performed in which 40 individuals were selected randomly from the control group who were matched for age (±2 years), sex, ethnicity, and order of light exposure with patients who had POAG. Consistent with our previous analysis, pupillary constriction was impaired at higher irradiances in glaucomatous eyes when these patients were matched with similar control participants (F13,1014 > 5.42; P < 0.001; t > 2.28 and P < 0.05 for all pairwise comparisons of more than 11.5 log photons/cm2 per second for 469-nm light and for comparisons of more than 12.0 log photons/cm2 per second for 631-nm light).
      Figure thumbnail gr3
      Figure 3Graphs showing impaired pupillary constriction responses in patients with primary open-angle glaucoma. Dose-response curves for pupillary constriction for controls (n = 161, black traces) and patients with glaucoma (n = 40) who were exposed to (A) blue 469-nm light (blue trace), and (B) red 631-nm light (red trace). For both colors of light, the magnitude of the pupillary light reflex was reduced in glaucomatous eyes as the irradiance of light was increased (>11.5 log photons/cm2 per second). Pupil diameter is expressed as a percentage of the dark pupil measured before each light exposure. Asterisks show significant differences in pupillary responses between controls and patients with glaucoma. The mean ± standard error of the mean is shown.
      To explore whether pupillary responses differed for controls versus POAG patients with early, moderate, or severe disease, an analysis of variance was performed using the maximum pupillary constriction values for blue-light and red-light stimuli. The PLR differed significantly across groups during exposure to blue light (F3,197 = 13.72; P < 0.001), such that pupillary constriction was greater for control subjects versus POAG patients with moderate or severe VF loss (P < 0.025 for both comparisons, Fisher's least significant difference [LSD]), whereas the difference between control subjects and individuals with early glaucoma failed to reach statistical significance by the smallest of possible margins (P = 0.050). Pupillary constriction also differed across groups during exposure to red light (F3,197 = 10.02; P < 0.001), such that responses in control subjects were greater than those observed for POAG patients with early, moderate, or advanced VF loss (P < 0.045 for each paired comparison, Fisher LSD). It should be noted, however, that the sample size was small across different disease severity groups, and the experiment was not designed to have a balanced number of participants across groups.

       Correlation between Pupillary Responses in Glaucoma and Clinical Measures

      Based on deficits in the PLR observed in patients with POAG, we tested whether the pupillary constriction response to high-irradiance blue light (>13.5 log photons/cm2 per second) correlated with clinical measures used for assessing severity of glaucoma.
      • Zangwill L.M.
      • Weinreb R.N.
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      • et al.
      Baseline topographic optic disc measurements are associated with the development of primary open-angle glaucoma: the Confocal Scanning Laser Ophthalmoscopy Ancillary Study to the Ocular Hypertension Treatment Study.
      Pupil diameter (expressed as a percentage of the dark pupil) exhibited a significant correlation with HVF mean deviation (Fig 4A; r = −0.44; P = 0.005) and linear cup-to-disc ratio assessed by HRT (Fig 4B; r = 0.61; P ≤ 0.001). That is, impaired pupillary responses were associated with greater VF loss and cupping of the optic disc. Notably, the correlation between the PLR and the linear cup-to-disc ratio was just as strong as the correlation between HVF mean deviation and the linear cup-to-disc ratio (r = −0.59; P ≤ 0.001). By comparison, intraocular pressure before treatment for glaucoma did not correlate significantly with pupillary constriction, HVF mean deviation, or the linear cup-to-disc ratio (|r| < 0.14 and P > 0.40 for all comparisons). Also, the pattern standard deviation obtained via HVF testing did not correlate with pupillary responses to blue light or red light (|r| < 0.31 and P > 0.49 for both comparisons). Next, we systematically evaluated the relationship between the magnitude of pupillary constriction at different irradiances with HVF mean deviation and optic nerve head parameters assessed by HRT. As the irradiance of light was increased, the strength of the correlation between pupillary constriction and HVF and HRT measures also increased. For blue-light and red-light stimuli, a moderate correlation was observed for the PLR and HVF mean deviation by the end of the light exposure (Fig 5). By comparison, the relationship between pupillary responses and HRT parameters was much stronger for blue light relative to red light, with correlation coefficients reaching their highest levels near the end of the blue-light stimulus for several measures including cup area (r = 0.60; P < 0.001), cup volume (r = 0.56; P < 0.001), cup-to-disc area ratio (r = 0.69; P < 0.001), and mean retinal nerve fiber layer thickness (r = −0.47; P = 0.022).
      Figure thumbnail gr4
      Figure 4Scatterplots showing the correlation between pupillary responses and glaucoma severity. In patients with primary open-angle glaucoma, the pupillary constriction response to high-irradiance blue light (>13.5 log photons/cm2 per second) correlated significantly with visual field loss assessed by (A) Humphrey Visual Field (HVF) testing, as well as (B) the linear cup-to-disc ratio determined by Heidelberg Retinal Tomography. The linear regression line is shown with 95% confidence intervals. dB = decibels.
      Figure thumbnail gr5
      Figure 5Pupillary constriction responses correlated with clinical measures used to diagnose glaucoma. The heat maps show Pearson's correlation coefficient (absolute values) for visual field testing and optic nerve head parameters versus pupillary constriction in a group of 40 patients with primary open-angle glaucoma. Correlations with pupillary responses to blue light (469 nm) and red light (631 nm) are shown in 0.5-log unit bins from 7 to 14 log photons/cm2 per second. Warmer colors (i.e., more red) indicate higher correlation coefficient values. Results for Humphrey Visual Field (HVF) analysis and Heidelberg Retinal Tomography correlated most strongly with the magnitude of pupillary constriction during exposure to high-irradiance blue light. dB = decibels; RNFL = retinal nerve fiber layer.

      Discussion

      Our results suggest that the functional integrity of ipRGCs and their efferent projections can be evaluated by measuring pupillary responses to a light stimulus that is increased gradually over time. Using this approach, it was possible to construct dose-response curves to light over a short time interval. Because melanopsin-dependent pupillary responses have a relatively high threshold of activation and are short-wavelength sensitive, the ramp-up stimulus that we used allows for examination of rod–cone and melanopsin-dependent RGC activation using a single light-exposure sequence. In glaucomatous eyes, deficits in the PLR were observed primarily at higher irradiances, and responses to blue light correlated most strongly with VF loss severity and cupping of the optic disc. These findings suggest that in POAG, the magnitude of impairment of melanopsin-dependent pupillary responses potentially can be used to estimate the degree of damage to RGCs that mediate image-forming vision.
      Although it has been suggested that ipRGCs may be spared preferentially in animal models with experimentally induced ocular hypertension,
      • Li R.S.
      • Chen B.Y.
      • Tay D.K.
      • et al.
      Melanopsin-expressing retinal ganglion cells are more injury-resistant in a chronic ocular hypertension model.
      we and other investigators have found that pupillary light responses are impaired in patients with glaucoma.
      • Feigl B.
      • Mattes D.
      • Thomas R.
      • Zele A.J.
      Intrinsically photosensitive (melanopsin) retinal ganglion cell function in glaucoma.
      • Kankipati L.
      • Girkin C.A.
      • Gamlin P.D.
      The post-illumination pupil response is reduced in glaucoma patients.
      • Nissen C.
      • Sander B.
      • Milea D.
      • et al.
      Monochromatic pupillometry in unilateral glaucoma discloses no adaptive changes subserved by the ipRGCs.
      Here, deficits in pupillary responses to blue light were specific to the irradiance range associated with activation of melanopsin-containing RGCs.
      • Gamlin P.D.
      • McDougal D.H.
      • Pokorny J.
      • et al.
      Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells.
      • Gooley J.J.
      • Ho Mien I.
      • St Hilaire M.A.
      • et al.
      Melanopsin and rod-cone photoreceptors play different roles in mediating pupillary light responses during exposure to continuous light in humans.
      • Lucas R.J.
      • Hattar S.
      • Takao M.
      • et al.
      Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice.
      Because rod-driven pupillary light responses are thought to be mediated solely through ipRGCs,
      • Guler A.D.
      • Ecker J.L.
      • Lall G.S.
      • et al.
      Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision.
      • Altimus C.M.
      • Guler A.D.
      • Alam N.M.
      • et al.
      Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities.
      it may be expected that loss of ipRGC function also would result in reduced responses to low-irradiance light. Under the lighting conditions used in the present study, however, the amount of rod input to ipRGCs in POAG patients seemed to be sufficient to drive a normal PLR in the scotopic visual range. Therefore, deficits in ipRGC function measured by pupillometry may not relate directly to loss of peripheral vision that occurs in glaucoma. Our findings could be explained in part by regional differences in cell density, synaptic contacts, and receptive fields of melanopsin cells versus RGCs that mediate vision.
      • Dacey D.M.
      • Liao H.W.
      • Peterson B.B.
      • et al.
      Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN.
      Pupillary responses to red light in the photopic visual range suggest involvement of cone photoreceptors,
      • Gooley J.J.
      • Ho Mien I.
      • St Hilaire M.A.
      • et al.
      Melanopsin and rod-cone photoreceptors play different roles in mediating pupillary light responses during exposure to continuous light in humans.
      but also could be mediated by rods.
      • Altimus C.M.
      • Guler A.D.
      • Alam N.M.
      • et al.
      Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities.
      Notably, patients with POAG showed normal spectral responses to light, i.e., the difference in pupillary constriction for blue light versus red light was similar to that of controls, suggesting that the relative contribution of melanopsin and rod–cone photoreceptors to the PLR was not substantially affected by the disease.
      Growing evidence indicates that chromatic pupillometry-based approaches can be used to detect abnormalities that associate with damage either to the photoreceptor layer or to RGCs in different types of retinal diseases.
      • Feigl B.
      • Zele A.J.
      Melanopsin-expressing intrinsically photosensitive retinal ganglion cells in retinal disease.
      In this study, pupillary responses were examined across much finer steps in irradiance compared with prior work,
      • Kardon R.
      • Anderson S.C.
      • Damarjian T.G.
      • et al.
      Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.
      • Kardon R.
      • Anderson S.C.
      • Damarjian T.G.
      • et al.
      Chromatic pupillometry in patients with retinitis pigmentosa.
      • Leon L.
      • Crippa S.V.
      • Borruat F.X.
      • Kawasaki A.
      Differential effect of long versus short wavelength light exposure on pupillary re-dilation in patients with outer retinal disease.
      which made it possible to determine the dose response across a wide range of irradiance levels. In principle, this approach should perform better at detecting subtle deficits in the PLR, hence providing more detailed information regarding the origin and magnitude of the underlying neuroanatomic abnormality. Light stimuli also were equated by photon density, which is better for making inferences regarding the contribution of different photoreceptor types to the light response.
      • Lucas R.J.
      • Peirson S.N.
      • Berson D.M.
      • et al.
      Measuring and using light in the melanopsin age.
      Several studies have used the postillumination pupillary response (PIPR) to evaluate melanopsin-dependent RGC responses.
      • Feigl B.
      • Mattes D.
      • Thomas R.
      • Zele A.J.
      Intrinsically photosensitive (melanopsin) retinal ganglion cell function in glaucoma.
      • Kankipati L.
      • Girkin C.A.
      • Gamlin P.D.
      The post-illumination pupil response is reduced in glaucoma patients.
      • Park J.C.
      • Moura A.L.
      • Raza A.S.
      • et al.
      Toward a clinical protocol for assessing rod, cone, and melanopsin contributions to the human pupil response.
      Because the ipRGCs respond sluggishly to changes in lighting, the PIPR is thought to reflect sustained activation of the melanopsin pathway after light offset.
      • Gamlin P.D.
      • McDougal D.H.
      • Pokorny J.
      • et al.
      Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells.
      • Kankipati L.
      • Girkin C.A.
      • Gamlin P.D.
      Post-illumination pupil response in subjects without ocular disease.
      In patients with advanced glaucoma, the PIPR to high-irradiance blue light is blunted relative to the response in normal subjects, and the difference in the PIPR between blue-light and red-light stimuli correlates inversely with HVF mean deviation in patients with glaucomatous eyes.
      • Feigl B.
      • Mattes D.
      • Thomas R.
      • Zele A.J.
      Intrinsically photosensitive (melanopsin) retinal ganglion cell function in glaucoma.
      • Kankipati L.
      • Girkin C.A.
      • Gamlin P.D.
      The post-illumination pupil response is reduced in glaucoma patients.
      Glaucoma also can be detected based on a relative afferent pupillary defect (RAPD), in which asymmetric pupillary responses are observed because of a difference in disease progression between eyes.
      • Chang D.S.
      • Boland M.V.
      • Arora K.S.
      • et al.
      Symmetry of the pupillary light reflex and its relationship to retinal nerve fiber layer thickness and visual field defect.
      • Chang D.S.
      • Xu L.
      • Boland M.V.
      • Friedman D.S.
      Accuracy of pupil assessment for the detection of glaucoma: a systematic review and meta-analysis.
      Automated RAPD pupillometry systems have been used to screen for patients with glaucoma who exhibit asymmetric disease
      • Chang D.S.
      • Arora K.S.
      • Boland M.V.
      • et al.
      Development and validation of an associative model for the detection of glaucoma using pupillography.
      • Ozeki N.
      • Yuki K.
      • Shiba D.
      • Tsubota K.
      Pupillographic evaluation of relative afferent pupillary defect in glaucoma patients.
      • Tatham A.J.
      • Meira-Freitas D.
      • Weinreb R.N.
      • et al.
      Detecting glaucoma using automated pupillography.
      ; however, complementary approaches are still needed because many patients show bilateral damage to the optic nerve without an apparent RAPD. Although the aforementioned pupillometry-based methods show promise for detecting impaired photic transmission by ipRGCs, further improvements are needed if they are to be used to screen reliably for glaucoma in population studies. Similarly, our light exposure protocol likely requires further optimization if it is to be used for distinguishing early stages of glaucoma from normal ocular health. It is possible that combining our approach with other methods (e.g., measuring the PIPR and RAPD) could be used to improve detection of glaucoma, which remains to be tested.
      A limitation of our study is that light stimuli were calibrated at the level of the cornea, rather than attempting to correct for prereceptoral filtering by the cornea and lens. In the age group studied here (50 years and older), lens yellowing and mild cataracts are common, which would be expected to reduce the amount of short-wavelength light that reaches the retina.
      • Kessel L.
      • Lundeman J.H.
      • Herbst K.
      • et al.
      Age-related changes in the transmission properties of the human lens and their relevance to circadian entrainment.
      In addition, because pupillary constriction was short-wavelength sensitive and measurements were performed for the same eye that was being exposed to light, the smaller pupil in the blue-light condition further limited the number of blue photons reaching the retina. Therefore, our method likely underestimated the relative sensitivity of pupillary responses to blue light versus red light at the level of retinal photoreceptors. Given that the primary deficit we observed in POAG patients was a reduction in the pupillary response at high irradiances, a single blue-light exposure sequence may be sufficient if our approach is to be adapted for use as a screening test. The red-light stimulus nonetheless may be useful for detecting diseases that affect cone function.
      • Kardon R.
      • Anderson S.C.
      • Damarjian T.G.
      • et al.
      Chromatic pupillometry in patients with retinitis pigmentosa.
      Although previous studies suggest that pupillary responses to blue-light and red-light stimuli are reproducible across different times of day in healthy subjects (intraclass correlation coefficient, ≥0.7 for maximum pupillary constriction),
      • Herbst K.
      • Sander B.
      • Milea D.
      • et al.
      Test-retest repeatability of the pupil light response to blue and red light stimuli in normal human eyes using a novel pupillometer.
      test–retest reliability was not assessed in the present study. Also, subjects in the control group did not undergo VF testing or HRT; hence, these participants were not included in analyses on the relationship between pupillary light responses with visual loss and cupping of the optic disc. It also should be highlighted that deficits in the PLR observed in POAG patients may be similar for other conditions affecting ipRGC transmission. For example, it was shown recently that nonarteritic anterior ischemic optic neuropathy is associated with an abnormal PIPR to blue light, which is consistent with dysfunction of ipRGCs.
      • Herbst K.
      • Sander B.
      • Lund-Andersen H.
      • et al.
      Unilateral anterior ischemic optic neuropathy: chromatic pupillometry in affected, fellow non-affected and healthy control eyes.
      Hence, a reduction in pupillary responses to high-irradiance blue light does not necessarily imply that a patient has glaucoma, but rather suggests reduced ipRGC activation or optic nerve transmission. In future work, it will be important to characterize pupillary responses further using our testing protocol in other patient populations with damage to the optic nerve, including other types of glaucoma. It will also be important to determine whether our results are generalizable to patient groups that differ in ethnicity and age, because our subject pool included predominantly ethnic Chinese individuals over a limited age range.
      Our study provides proof of concept that a short-duration chromatic pupillometry test can be used to assess loss of ipRGC function associated with POAG. Because gradual loss of peripheral vision in glaucomatous eyes can go unnoticed for many years, patients often seek treatment after substantial and irreversible damage to the optic nerve has occurred. We and others therefore have explored chromatic pupillometry as an approach for detecting early retinal dysfunction. A major advantage of pupillometry-based methods is that they can be adapted for population screening in the form of portable devices, for example, as a desktop system or incorporated into specialized goggles,
      • Ozeki N.
      • Yuki K.
      • Shiba D.
      • Tsubota K.
      Pupillographic evaluation of relative afferent pupillary defect in glaucoma patients.
      • Herbst K.
      • Sander B.
      • Milea D.
      • et al.
      Test-retest repeatability of the pupil light response to blue and red light stimuli in normal human eyes using a novel pupillometer.
      • Lorenz B.
      • Strohmayr E.
      • Zahn S.
      • et al.
      Chromatic pupillometry dissects function of the three different light-sensitive retinal cell populations in RPE65 deficiency.
      that can provide objective data on the integrity of the pathway from the retina to the midbrain. If pupillometry systems are shown to provide reliable detection of ocular diseases using short testing protocols, such low-cost and patient-friendly technologies could be used to screen for damage to the visual system in population studies and community samples. This would allow for identification of at-risk individuals who should undergo a comprehensive ophthalmic examination to treat or halt the progression of POAG or other ocular diseases.

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