FIGURE 6 | Protocol for assessing photoreceptor health using 1-s light flashes. Pupillary responses were assessed in patients with Leber congenital amaurosis (LCA), using light stimuli designed to stimulate preferentially rods, cones, or melanopsin. (A) Pupillary responses to the rod-weighted light stimulus (465 nm, −3 log cd/m2 after 10 min of dark adaptation) were non-recordable in LCA patients. (B) Responses to the cone-weighted light stimulus (642 nm, 1 log cd/m2 on a blue background of 0.78 log cd/m2 ) were reduced in LCA patients compared with healthy controls. (C) The post-illumination pupillary response to the melanopsin-weighted stimulus (2.6 log cd/m2 ) was intact in LCA patients but the amplitude was reduced. Gray traces show the range of pupillary responses in the control group. The figure is redrawn and modified with permission from (67) .

responses between groups. The PIPR after exposure to bright blue light was also only modestly reduced in patients with LHON, suggesting that ipRGC function was preferentially spared relative to conventional retinal ganglion cells involved in image-forming vision. Similarly, in another study that measured the PIPR after a 1-s flash of bright blue light, the post-stimulus pupil size (at 6 s after light offset) was not different between HON patients (n = 11) and healthy controls (76).

Several studies have suggested that ipRGCs are resistant to neurodegeneration in mitochondrial optic neuropathies (i.e., LHON and autosomal dominant optic atrophy) (77–79), but they are vulnerable in other, more common types of optic neuropathies (i.e., patients with ischemic, inflammatory, or glaucomatous optic neuropathies). In a series of patients with anterior ischemic optic neuropathy (AION; n = 18) the sensitivity of pupillary responses to blue light (464 nm, −4.0 to −1.0 cd/m2 in 0.5 log unit steps with intervening dark periods) and red light (635 nm, 1.0 to 2.5 log cd/m2 in 0.5 log unit steps under light adaptation to 90 cd/m2 ) was in the normal range, but the PIPR after exposure to bright blue light was impaired in eyes affected by AION, compared with contralaterally unaffected eyes and healthy control eyes (80). These findings indicate that ipRGCs are damaged following ischemic injury to the optic nerve. Several studies have also shown that the PIPR to a 1 s flash of short-wavelength light is impaired in patients with glaucomatous optic neuropathy compared with healthy controls (76, 81–84). Moreover, in glaucoma patients (n = 38), the magnitude of the PIPR (normalized pupil size measured 6 s after light offset) after exposure to a bright blue light stimulus (470 nm, 2.4 log cd/m2 ) correlated with the magnitude of visual field loss assessed by standard automated perimetry (SAP), and RNFL thickness assessed by OCT (81). Similar results were obtained in another study in patients with glaucoma (n = 46) in which the consensual pupillary light reflex was assessed after monocular exposure to either a full-field or superonasal-quadrant light stimulus (464 nm, 2.9 log cd/m2 ) (84). The PIPR amplitude (6 s after light offset) in glaucomatous eyes was associated with visual field deficits and RNFL thinning, suggesting that reduced melanopsin-dependent pupillary responses might be used as a proxy to estimate the loss of conventional retinal ganglion cells involved in image-forming vision.

### Assessing ipRGC Function Using the PIPR After a 10-Second or 20-Second Light Stimulus

The melanopsin-dependent PIPR was first demonstrated in humans and macaques using a 10-s light stimulus protocol (47). Prior work had shown that macaque ipRGCs studied in vitro continued to fire long after the offset of a 10-s light exposure (23). Hence, subsequent clinical studies have also investigated the PIPR using a 10-s light stimulus, with average pupil size measured from 10 to 40 s after light offset. Using this approach, the consensual PIPR was characterized in healthy individuals (n = 45) after exposing the other eye to a bright blue light or red light stimulus, centered on the pupil in Maxwellian view (470 nm vs. 623 nm; retinal irradiance of 13 log quanta/cm2 /s with a fully-dilated pupil using a mydriatic agent) (85). Although there were substantial individual differences in the magnitude of pupillary constriction, all participants exhibited a sustained PIPR based on the change in pupil size after exposure to blue light relative to red light, adjusted for the percentage change in pupil size relative to the baseline pupil diameter (i.e., the net PIPR change, determined by subtracting the blue PIPR percentage value from the red PIPR percentage value). Similar methods have been used to examine ipRGC function in patients with glaucoma (86, 87). After exposure to 10 s of bright light (488 vs. 610 nm; corneal irradiance, 14.2 log quanta/cm2 /s), the net PIPR to blue light vs. red light was reduced in patients with advanced glaucoma (n = 11) compared with healthy controls (**Figures 7A,B**), but there was no difference in the PIPR between patients with early glaucoma (n = 14) and healthy controls (87). In another study, the net PIPR change in glaucoma patients (n = 16) after exposure to bright light (470 vs. 623 nm; retinal irradiance of 13 log quanta/cm2 /s) was linearly correlated with the magnitude of visual field loss (**Figure 7C**), demonstrating that ipRGC dysfunction in glaucoma is associated with disease severity (86). In patients with type 2 diabetes without diabetic retinopathy (n = 7), the PIPR to bright light (488 vs. 610 nm; corneal irradiance, 14.2 log quanta/cm2 /s) was also reduced and associated with the duration of diabetes (88), suggesting that ipRGC dysfunction may occur in diabetes prior to onset of visual loss. Similarly, in patients with age-related macular degeneration (AMD; n = 2), the PIPR to bright light (464 vs. 635 nm; corneal irradiance, 15 log photons/cm2 /s) was reduced in both early and advanced, neovascular AMD compared with controls (89).

In other studies, the PIPR to a bright 20-s light stimulus has been used to examine ipRGC function. In a patient with LHON with unilateral visual loss, sustained pupillary constriction after light offset (460 vs. 660 nm; 2.0 or 2.5 log cd/m2 ) did not differ between the affected eye and the healthy eye, suggesting that ipRGC function was resistant to effects of the disease (90). In contrast, in patients with unilateral AION (n = 10), pupillary responses of the affected eye were reduced compared with the contralateral, non-affected eye, both during and after exposure to 20-s of bright light (470 vs. 660 nm; 2.5 log cd/m2 ) (91). In patients with choroideremia (n = 18), which is characterized by progressive degeneration of the outer retina, the peak pupillary constriction response was also reduced during exposure to bright blue light or red light (463 vs. 643 nm; 100 lux), but the PIPR was intact and lasted longer compared with healthy individuals (92). Together, these results are consistent with studies that used 1-s light flashes (see previous section), showing that the PIPR is reduced in diseases that result in damage to ipRGCs, but remains largely intact in diseases that primarily affect the outer retina.

## Chromatic Pupillometry Methods That use Continuously Presented Light

### Assessing Photoreceptor Function Using Stepwise Increases in Light Intensity

The pupillary light reflex shows a phasic (i.e., transient) constriction response at the beginning of a continuous light stimulus, followed by a tonic (i.e., sustained) response that is lower in amplitude. Hence, deficits in outer retinal function can be assessed by the phasic response to a step of blue or red light, and deficits in inner retinal function can be assessed by the tonic pupillary response and the PIPR to high-intensity blue light. These response characteristics can be investigated using protocols in which the light intensity is increased stepwise over time. Using a Ganzfeld system to administer light to the eye continuously under mesopic conditions (with the non-stimulated eye covered), the direct pupillary response to a sequence of increasing blue or red light stimuli was measured over a 2 log unit range, with each light step presented for 13 s and matched for photopic luminance (467 and 640 nm; 0, 1, and 2 log cd/m2 ; **Figure 7**) (93). The transient pupillary response was defined as the maximum constriction response measured within 180– 500 ms after light onset, and the sustained pupillary response was defined as pupillary constriction at the 13th second of stimulation for each light step (with each measure adjusted for pre-stimulus pupil size). After characterizing pupillary responses in healthy individuals, this protocol was tested in a patient with retinitis pigmentosa with loss of rod function and reduced cone function based on electroretinography, a patient with achromatopsia in whom cone function was disrupted due to a mutation in the CNGA3 gene, and a patient with unilateral AION with severe visual loss in the affected eye (93). The patient with retinitis pigmentosa showed deficits in phasic and tonic pupillary responses to the rod-weighted dim blue light stimulus, whereas the melanopsin-weighted response to bright blue light was in the normal range. Responses to moderate-to-bright red light were also attenuated, which is consistent with reduced cone function. In the achromat, pupillary responses to blue light were comparable to healthy controls, suggesting normal function of rods and melanopsin, whereas the cone-weighted response to red light was on the lower end of the normal range, perhaps due to activation of rods (see below). In the AION patient, pupillary responses in the affected eye were markedly reduced for all light stimuli, indicating reduced function of ipRGCs and their input from the outer retina.

This protocol was subsequently evaluated in a group of patients with retinitis pigmentosa (n = 32) (62), in whom the transient pupillary response to dim blue light (467 nm; 0 log cd/m2 ) was defined as the rod-weighted light response, and the transient pupillary response to bright red light (640 nm; 2 log cd/m2 ) was defined as the cone-weighted response. The melanopsin-dependent pupillary response was assessed by sustained pupillary constriction at the end of the bright blue light stimulus (467 nm; 2 log cd/m2 ). Under these testing conditions, patients with a non-recordable or abnormal scotopic/photopic electroretinogram showed reduced transient pupillary responses to rod- and cone-weighted light stimuli, as well as a reduced tonic response to the melanopsin-weighted light stimulus. However, patients with retinitis pigmentosa showed a slower and more sustained PIPR after exposure to the melanopsinweighted stimulus, as compared with patients with normal vision. Consistent with these findings, another study that used the same protocol found that patients with mutations in the RPE65 gene (n = 11) exhibited reduced transient pupillary responses to rod- and cone-weighted light stimuli, a small reduction in sustained pupillary constriction during exposure to the melanopsin-weighted stimulus, and a prolonged PIPR after light offset compared with healthy controls (68). In a pair of achromats, pupillary responses were in the normal range for the rod-weighted stimulus, but were attenuated for the coneweighted stimulus. The finding that achromats could still respond to red light is likely explained by activation of rods by the cone-weighted stimulus because total loss of cone function was confirmed in other experiments in the same individuals (68).

The stepwise pupillometry protocol has also been compared between patients with degeneration of the outer retina (n = 23; retinitis pigmentosa, LCA, corneoretinal dystrophy of Bietti, cone-rod dystrophy, or Stargardt disease) and optic nerve disease (n = 13; ischemic optic neuropathy or compression lesion of the optic nerve) (63). Pupillary constriction responses to rod-, cone-, and melanopsin-weighted light stimuli were reduced in patients with either outer or inner retinal disease compared with healthy controls (**Figures 8A,B**). However, the pupillary re-dilation response after blue-light offset was much slower in

FIGURE 7 | Protocol for assessing melanopsin-dependent pupillary responses using the post-illumination pupillary light response (PIPR). The PIPR was assessed after exposure to 10 s of bright blue light or red light (488 or 610 nm; 14.2 log quanta/cm2 /s). (A) In individuals with normal vision, the PIPR to blue light was greater than the PIPR to red light for at least 30 s after light offset. (B) In patients with glaucoma, the PIPR to blue light was reduced relative to red light, indicating reduced light transmission from melanopsin-containing retinal ganglion cells. (C) In glaucomatous eyes, the net PIPR change (the difference in the PIPR to blue light vs. red light, adjusted for baseline pupil size) correlated with visual field loss assessed by Humphrey visual field mean deviation. Panels (A,B) are redrawn with permission from (87). Panel (C) is redrawn with permission from (86).

patients with outer retinal disease compared with patients with optic nerve disease or healthy controls, which is consistent with other studies in which the stepwise light protocol was used (62, 68). In contrast, most studies that have used a 1-s flash of blue light to elicit the PIPR have found a relatively normal response after light offset in patients with outer retinal disease (60, 67, 72, 73), with only one study reporting a greater PIPR relative to controls (68). The difference in the PIPR across light exposure protocols may be related to the pre-exposure lighting conditions. In the stepwise light protocol, patients are exposed to continuously presented light and hence the PIPR is measured after light adaptation. Similar to results using the stepwise light protocol, a prolonged time course of pupillary re-dilation has been observed in patients with outer retinal disease after the offset of a bright blue light stimulus lasting 20 s or longer (i.e., after light adaptation) (38, 92). Even for a 1-s flash of bright blue light, the PIPR has been shown to be extended in patients with retinitis pigmentosa or LCA if the stimulus is presented after light adaption, rather than dark adaption (60). Together, these studies suggest that the stepwise pupillometry protocol can be used to differentiate loss of outer retinal function vs. melanopsin-dependent ipRGC function, by measuring transient pupillary responses at the start of each light step and the PIPR after light adaptation.

### Assessing Photoreceptor Function Using Ramp-Up Light Protocols

Given that rods and cones are more sensitive to light than the melanopsin-dependent ipRGC response, photoreceptors in the outer retina and inner retina can be activated sequentially by using a gradually increasing light stimulus (i.e., a rampup light protocol). Based on irradiance-response curves for the pupillary light reflex, damage to the outer retina can be assessed by reduced pupillary responses to light stimuli at lower irradiances, i.e., below the threshold of activation for the melanopsin-dependent pupillary response (low-to-moderate intensity blue light or moderate-to-high intensity red light). By comparison, damage to the inner retina can be detected by measuring the pupillary response during exposure to continuous, high-intensity blue light. This approach has been tested using a modified Ganzfeld system to administer a gradually-increasing blue or red light stimulus (469 or 631 nm; corneal irradiance from 7 to 14 log photons/cm2 /s) to one eye over a 2-min period (with the other eye covered), in order to construct irradianceresponse curves for the direct pupillary light reflex (58). In patients with glaucoma (n = 40) with different stages of disease severity (Early, n = 19; Moderate, n = 10; Severe, n = 11), pupillary responses were impaired for the blue light stimulus at irradiances corresponding with the range of activation of the melanopsin-dependent response (>11.5 log photons/cm2 /s), and the difference between patients and healthy controls was greatest at the highest irradiances tested (**Figure 9A**). Pupillary responses in glaucoma patients were also reduced for the red light stimulus at moderate-to-high intensities of red light (>11.5 log photons/cm2 /s), suggesting reduced transmission from rod/cone photoreceptors to ipRGCs (**Figure 9B**). In contrast, the pupillary response in a patient with retinitis pigmentosa with no light perception was markedly reduced for the blue light stimulus at low-to-moderate light intensities (<13 log photons/cm2 /s), and there was no detectable response to the red light stimulus (**Figures 9A,B**), which is consistent with loss of rod and cone function (94). However, the amplitude of his pupillary constriction response was in the normal range for high intensity blue light, suggesting preserved melanopsin-dependent ipRGC function. In the glaucoma patients, the magnitude of pupillary constriction to high-irradiance blue light (>13.5 log photons/cm2 /s) was inversely correlated with Humphrey visual field mean deviation and optic disc cupping assessed using Heidelberg Retinal Tomography (**Figures 9C,D**) (58). These results suggest that ipRGC responses in glaucoma can be used to estimate damage to retinal ganglion cells that mediate image-forming vision.

In a later study, the ramp-up light exposure protocol was tested in a group of patients with early-stage glaucoma (n = 46; permission from (63).

visual field mean deviation of −6 dB or better on automated perimetry) (95). Pupillary light responses were reduced in patients compared with healthy controls at moderate-to-high intensities of blue and red light (>11.0 log photons/cm2 /s). In glaucomatous eyes, the maximum pupillary constriction amplitude correlated with RNFL thickness, but unsurprisingly, not with the amount of visual field loss. Hence, ipRGC dysfunction or cell loss can be detected in early stages of glaucoma and is associated with structural correlates of disease progression. In another study that used the ramp-up light protocol, patients with autosomal-dominant optic atrophy (n = 5) showed pupillary responses that were comparable to healthy controls (96), which is consistent with other studies that have found preserved ipRGC function in mitochondrial disease (77).

## Summary of Chromatic Pupillometry Methods Used to Assess Photoreceptor Health

Chromatic pupillometry methods can detect dysfunction of photoreceptors in diseases affecting either the outer retina or the inner retina. Clinical protocols that measure pupillary responses to 1-s light flashes allow for testing of rod function using a dim blue light stimulus after dark adaption, while cone function can be tested using a moderate-to-bright red light stimulus under light adaptation. This approach has been used to demonstrate loss of rod and/or cone function in outer retinal disease (e.g., retinitis pigmentosa and LCA). Melanopsin-dependent ipRGC function can be assessed by measuring the PIPR after exposure to a bright blue light stimulus. The PIPR after dark adaptation is reduced in patients with optic nerve disease (e.g., glaucoma and AION), with the notable exception of mitochondrial disease (e.g., LHON and autosomal dominant optic atrophy) in which ipRGCs appear to be preferentially spared. In stepwise light protocols, the phasic pupillary response to light stimulus onset, which is dominated by rods/cones, is impaired in outer retinal disease. In contrast, the sustained pupillary response to bright blue light, which is dominated by melanopsin, is impaired in diseases affecting ipRGC function, whereas the subsequent PIPR is prolonged in patients with outer retinal disease. In protocols in which light intensity is increased gradually over time (ramp-up light protocol), the tonic pupillary light reflex is impaired during exposure to dim-to-moderate intensity blue light or red light in outer retinal disease, whereas sustained pupillary constriction to high-irradiance blue light is impaired in inner retinal disease. In glaucoma, melanopsin-dependent pupillary responses correlate with visual field loss and anatomic correlates of optic nerve damage, suggesting that pupillometry methods can be used to estimate the degree of damage to ipRGCs and conventional retinal ganglion cells.

## LIMITATIONS AND CONSIDERATIONS OF CHROMATIC PUPILLOMETRY

Chromatic pupillometry is currently the only approach that can be used to assess rapidly the health of rods, cones, and melanopsin using a single light protocol. However, the contribution of outer retinal photoreceptors to phasic and tonic components of the pupillary light reflex is still not fully understood. Based on studies using the silent substitution method to selectively modulate the activity of different photoreceptor types, S-cones and M-cones may provide inhibitory input to ipRGCs that mediate the pupillary light reflex, whereas stimulation of L-cones and melanopsin induces pupillary constriction (55, 97, 98). A subset of melanopsin cells in the macaque retina has also been shown to exhibit an S-Off, (L+M)-On type of color-opponent receptive field (23). These findings have potential implications for interpreting the effects of blue light and red light stimuli on pupillary responses in chromatic pupillometry protocols, in which multiple photoreceptor types (i.e., rods, S-cones, M-cones, L-cones,

to a continuously presented light stimulus that was increased gradually over a 2-min period (469 or 631 nm; from 7 to 14 log photons/cm2 /s). (A) Pupillary responses to blue light were reduced in patients with glaucoma at higher irradiances compared with controls. In contrast, pupillary responses were reduced at dim-to-moderate light intensities in a patient with retinitis pigmentosa (RP) without rod/cone function, but were normal at the highest irradiances tested. (B) Pupillary responses to red light were also reduced in patients with glaucoma at higher irradiances, whereas there was no detectable response in the RP patient. In glaucomatous eyes, the magnitude of pupillary constriction during exposure to high-irradiance blue light (>13.5 log photons/cm2 /s) correlated with (C) visual field loss determined by Humphrey Visual Field (HVF) mean deviation, and (D) optic disc cupping determined by Heidelberg Retinal Tomography. In (C,D), the linear regression line is shown with 95% CIs. Data for glaucoma patients are replotted and modified with permission from (58). Data for the RP patient are replotted and modified with permission from (94).

and melanopsin) may be activated simultaneously and interact in complex ways at the level of ipRGCs. Outer retinal photoreceptors and melanopsin also differ in their time-course of dark adaptation and light adaptation (39, 99). Therefore, pre-exposure lighting conditions (e.g., the duration of prior darkness or exposure to light) can influence results of chromatic pupillometry testing (100, 101). Differences in the distribution of rods, cones, and melanopsin-containing retinal ganglion cells in the retina also gives rise to regional differences in spatial summation and ipRGC responses to light (102, 103). This may have implications for chromatic pupillometry protocols that administer light to different parts of the visual field to assess retinal health, e.g., central-field, hemi-field, quadrant-field, or annular light stimulation (84, 104–106).

Although chromatic pupillometry methods can be used to localize damage to the outer retina or the inner retina, they do not provide information on the specific disease type. For example, similar deficits in pupillary light responses are observed between patients with retinitis pigmentosa and LCA, and between patients with glaucoma or AION. Therefore, additional ophthalmic tests are required to establish an accurate diagnosis and its underlying pathophysiology. Pupillary light responses are also impaired in non-ocular diseases that result in demyelination or degeneration of the optic nerve, or altered autonomic nervous system function. For example, phasic pupillary light responses and the PIPR are reduced in patients with multiple sclerosis or Parkinson's disease (107–109). Efferent pathway defects from the midbrain to the pupils can also give rise to impaired pupillary constriction responses despite intact photoreceptor responses (110, 111). Moreover, the PIPR is reduced in patients with Seasonal Affective Disorder (SAD) or non-seasonal depression (112, 113), in whom there are no pathological changes in optic nerve function. These observations may be related to effects of the number of daylight hours on the amplitude of the PIPR (113, 114), perhaps through modulation of melanopsin protein levels and/or the sensitivity of ipRGCs to light. Relatedly, genetic variation in the melanopsin gene modulates pupillary responses to bright blue light and risk of SAD (112, 115–117). The PIPR and other pupillary light responses are also influenced by circadian phase (118, 119), sleep deprivation (119), and some types of medication (120). Additionally, cortical visual pathways are thought to be involved in modulating pupillary light responses (121, 122). Collectively, these studies show that results of chromatic pupillometry testing are influenced not only by the stimulation of retinal photoreceptors, but also by other biological pathways and disease processes.

An important concern regarding the use of chromatic pupillometry is whether it is necessary to adjust for effects of aging on pupillary responses. In aging, decreased sympathetic activity and smaller pupil size (i.e., age-related miosis) are thought to contribute to reduced pupillary constriction responses to light (123). When pupillary responses are measured relative to the dark-adapted pupil size, however, there is little effect of age on the amplitude of pupillary constriction (85, 124, 125). Similarly, age-dependent yellowing of the lens, which reduces transmission of short-wavelength light, does not appear to affect melanopsin-dependent pupillary responses. Rather, baselineadjusted pupillary light responses are similar between young and older adults (124, 125), and the magnitude of the PIPR to blue light vs. red light is preserved across adulthood (85, 126, 127). Moreover, there is no effect of mild cataract (125) or narrow irido-corneal angles (128) on spectral responses of the pupil, and no effect of refractive error on the PIPR (127). Together, these results indicate that melanopsin-dependent pupillary responses are relatively stable in healthy aging. The pupillary light reflex also shows good test-retest reliability when assessed in the same individuals over short time intervals (68, 129). However, pupillary light responses in healthy individuals show substantial between-subject differences (85, 93), and usually overlap with responses in patients with mild-to-moderate retinal or optic nerve disease. Therefore, further optimization of chromatic pupillometry protocols may be necessary to differentiate reliably individuals with normal health vs. those with retinal or optic nerve disease.

A challenge in interpreting and comparing chromatic pupillometry studies is that different methods have been applied for measuring and delivering light to the eyes. In this review, we report light stimuli as they were described in the original research articles in which different units of light measurement were used (lux, cd/m2 , and log photons/cm2 /s). Units based on the photopic luminosity function (e.g., lux or cd/m2 ) are familiar to engineers and vision researchers, but should not be used for nonvisual light responses including the pupillary light reflex (130). This is because it is well established that rods and melanopsin (not only cones) contribute to pupillary light responses in the photopic visual range, including sustained pupillary constriction and the PIPR. Instead, researchers should be encouraged to report either the power distribution (µW/cm2 ) or photon density (log photons/cm2 /s) of their light stimulus, and/or calculate the α-opic illuminance values (α-opic lux) to provide an estimate of the effective illuminance for each of the 5 human photopigments. Doing so will make it easier to replicate experimental conditions across studies and to interpret the relative contribution of different photopigments to pupillary responses. Different types of light delivery systems have also been used in chromatic pupillometry studies. Most studies have administered light using a Ganzfeld system, or directed light through the pupil in Maxwellian view. While Ganzfeld systems are relatively easy to build and implement in pupillometry studies, the retinal illuminance is limited and cannot be specified by the experimenter for different stimulus conditions. In Maxwellian view, a high retinal illuminance field can be readily obtained and the size of the entry pupil can be controlled, but these systems require precise alignment of the eye, which is often achieved by using a bite bar to stabilize the participant's eye position (131). Hence, there are trade-offs that must be taken into consideration when choosing the type of light delivery system used for chromatic pupillometry.

Another challenge in comparing pupillometry studies is that different methods have been used for measuring pupillary responses. Some studies have measured the direct pupillary light reflex while covering the other eye (i.e., the pupil of the stimulated eye is recorded), whereas other studies have measured the consensual pupillary light reflex (i.e., the stimulated eye is dilated with a mydriatic agent and the pupil of the non-stimulated eye is recorded). Because the consensual light response is greater when the pupil of the stimulated eye is dilated, rather than constricted (132), the size of the pupil exposed to light must be taken into consideration when interpreting results of chromatic pupillometry. Additionally, many different types of pupillary response metrics have been used in chromatic pupillometry studies. For example, there are several metrics that have been used to quantify the PIPR, including the 6-s PIPR, area under the curve, re-dilation velocity, and the plateau of the PIPR based on the best-fit exponential model of the data. Some of these measures may be better than others at capturing the melanopsin-dependent component of the PIPR vs. the mixed contribution of rods/cones and melanopsin during the early phase of pupillary re-dilation. The reliability of these PIPR metrics has been shown to differ in healthy participants, with lower coefficients of variation for the 6-s PIPR and the plateau of the PIPR (50). Hence, some PIPR metrics are likely better than others, and this may also depend on the type of disease being examined. The PIPR amplitude also varies by stimulus duration, with shorter light stimuli (1 s) producing larger responses than longer light stimuli (10 or 30 s) (50). As such, a PIPR testing paradigm that utilizes a 1-s light stimulus and either a 6-s or plateau PIPR metric might prove most useful in clinical applications, as remains to be tested. Moving forward, researchers using chromatic pupillometry should strive to develop a set of consensus standards for light stimuli and pupillary response metrics that can be used to readily compare results across different studies and types of disease.

## FUTURE APPLICATIONS OF CHROMATIC PUPILLOMETRY METHODS

Early detection of retinal and optic nerve diseases is important for treating and preventing loss of vision. However, gradual loss of peripheral vision can go unnoticed for years. For example, patients with glaucoma often seek treatment after substantial and irreversible damage to the optic nerve has occurred. Based on results of chromatic pupillometry testing, patients with early-stage glaucoma show deficits in pupillary responses to bright blue light compared with healthy controls (84, 95). Such findings raise the possibility that pupillometry testing could be used to screen for early optic nerve dysfunction. An advantage of chromatic pupillometry methods is that they can be readily incorporated into portable testing systems for population screening. For example, chromatic pupillometry devices could be used in a polyclinic setting or in geriatric clinics to identify patients with suspected retinal or optic nerve disease. Such patients could then be directed to undergo a comprehensive ophthalmic examination to determine the origin of their impaired pupillary light response. Additionally, chromatic pupillometry methods can potentially be used in patients who have difficulty communicating or who are unable to follow procedures for visual field testing. Following diagnosis of the underlying condition, chromatic pupillometry testing could be used periodically to track progression of the disease and effects of treatment.

The pupillary light reflex can be used to test for intact melanopsin-dependent ipRGC responses in patients who are blind. Notably, a standard penlight examination is inadequate for this purpose, with results that are often unreliable (133). For example, pupillary light responses were studied extensively in a pair of blind patients with no light perception who were previously described as having no detectable pupillary light response based on penlight examination by an ophthalmologist (37, 38). Clinical testing of optic nerve function should therefore include conditions that are appropriate for assessing melanopsindependent responses, i.e., exposure to high-irradiance blue light on a background of darkness. In the future, this may be especially important for identifying blind patients with intact optic nerve function who should be considered as candidates for gene therapy trials to restore vision (see below). Additionally, melanopsin-dependent pupillary light responses could be used as a surrogate measure for other responses mediated by ipRGCs (119, 134, 135), in order to screen for blind patients with intact circadian photoreception who should expose themselves to light-dark cues to entrain to the 24-h solar day. Chromatic pupillometry methods can also be used to assess photoreceptor health in veterinary medicine, as demonstrated in dogs with sudden acquired retinal degeneration syndrome and optic nerve disease (136–138).

With the development of technologies for restoring vision in blind individuals, there is a need for standard clinical tests that can be used to help select suitable candidates and to estimate the degree of recovery of non-visual photoreception after treatment. Chromatic pupillometry methods may be useful for testing photoreceptor health in degenerative diseases (e.g., retinitis pigmentosa and LCA), in which mutations in photoreceptorspecific or non-photoreceptor-specific cells in the retina result in rod cell death, followed by loss of cones. Given that retinal ganglion cells and other retinal neuronal cell types can survive for long periods after blindness, vision can be partially restored by rendering the remaining cells photosensitive. This can potentially be achieved by surgically-implanted subretinal prostheses that can electrically stimulate retinal ganglion cells, injection of smallmolecule photoswitches to bestow light sensitivity to retinal ganglion cells, and gene therapy to express light-regulated ion channels, transporters, or receptors (e.g., melanopsin or microbial opsins) in retinal neurons. These approaches have been tested in blind mice, demonstrating restoration of some behavioral light responses, and improved pupillary responses to light at low-to-moderate intensities (139–143). Similarly, gene therapy has been used to treat LCA in blind mice with impaired ability to regenerate visual photopigments (by restoring function of lecithin:retinol acyl transferase), which resulted in increased sensitivity of the pupillary light reflex by about 2.5 log units (144). Parallel findings have been reported for RPE65-associated LCA in humans, in whom viral delivery of the normal RPE65 gene to the retina resulted in sustained improvement of subjective and objective measures of vision (145, 146), as well as an increase in sensitivity of pupillary responses to light that lasted for at least 3 years after follow-up. These studies demonstrate that gene therapy for restoring vision also results in improvement in the pupillary light reflex. In future studies, chromatic pupillometry protocols can potentially be used to quantify the degree of recovery of non-visual photoreceptor pathways in blind patients who undergo gene therapy or other treatments to restore vision.

In summary, chromatic pupillometry methods have the potential to improve detection and management of diseases affecting the retina or optic nerve. Previous studies have characterized the differential role of outer retinal photoreceptors and melanopsin in mediating the pupillary light reflex. This has led to development and testing of short-duration protocols for assessing pupillary responses in patients with retinal or optic nerve disease. Clinical studies have provided proof-of-concept that pupillometry can be used to localize loss of function to photoreceptors in the outer retina or inner retina in patients whose disease status was already known. We are now in the position to exploit these research findings to test prospectively the ability of chromatic pupillometry to detect abnormalities in ipRGC function. Future large-scale studies should therefore focus on optimizing, standardizing, and adapting chromatic pupillometry protocols for early detection of retinal and optic nerve diseases, and for monitoring disease progression or recovery after treatment.

## DATA AVAILABILITY STATEMENT

This review article summarizes published data. Requests for datasets should be directed to the authors of the original research articles.

## AUTHOR CONTRIBUTIONS

AR, DM, and JG made substantial contributions to the conception and/or design of the work, acquisition, analysis, and interpretation of data, drafting and/or revising the manuscript critically for important intellectual content. AR, DM, and JG provided final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

## REFERENCES

## FUNDING

This work was supported by the Duke-NUS Signature Research Program funded by the Agency for Science, Technology and Research, Singapore, and the Ministry of Health, Singapore; the National Medical Research Council, Singapore (NMRC/NIG/1000/2009 and NMRC/CIRG/1401/2014); the Singapore National Eye Centre Health Research Endowment Fund, Singapore (R1005/20/2013); and the Biomedical Research Council, Singapore (01-TCRP-2010 and TCR0101674).

## ACKNOWLEDGMENTS

We thank research coordinators at the Singapore Eye Research Institute for their assistance in carrying out some of the studies described in this review article.

form two morphologically distinct populations. J Compar Neurol. (2016) 524:2845–72. doi: 10.1002/cne.23995

the age-related optic properties of the lens. BMC Ophthalmol. (2012) 12:4. doi: 10.1186/1471-2415-12-4

**Conflict of Interest Statement:** DM and JG filed a patent application for some of the chromatic pupillometry test procedures described in this manuscript.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Rukmini, Milea and Gooley. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Melanopsin Retinal Ganglion Cells and Pupil: Clinical Implications for Neuro-Ophthalmology

#### Chiara La Morgia1,2 \*, Valerio Carelli 1,2 and Michele Carbonelli 1

1 Unità Operativa Complessa Clinica Neurologica, IRCCS Istituto delle Scienze Neurologiche di Bologna, Ospedale Bellaria, Bologna, Italy, 2 Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, Italy

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs that mediate many relevant non-image forming functions of the eye, including the pupillary light reflex, through the projections to the olivary pretectal nucleus. In particular, the post-illumination pupil response (PIPR), as evaluated by chromatic pupillometry, can be used as a reliable marker of mRGC function in vivo. In the last years, pupillometry has become a promising tool to assess mRGC dysfunction in various neurological and neuro-ophthalmological conditions. In this review we will present the most relevant findings of pupillometric studies in glaucoma, hereditary optic neuropathies, ischemic optic neuropathies, idiopathic intracranial hypertension, multiple sclerosis, Parkinson's disease, and mood disorders. The use of PIPR as a marker for mRGC function is also proposed for other neurodegenerative disorders in which circadian dysfunction is documented.

#### Edited by:

Andrew J. Zele, Queensland University of Technology, Australia

#### Reviewed by:

Prakash Adhikari, Queensland University of Technology, Australia Jason C. Park, University of Illinois at Chicago, United States Birgit Sander, University of Copenhagen, Denmark

#### \*Correspondence:

Chiara La Morgia<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuro-Ophthalmology, a section of the journal Frontiers in Neurology

Received: 02 September 2018 Accepted: 19 November 2018 Published: 07 December 2018

#### Citation:

La Morgia C, Carelli V and Carbonelli M (2018) Melanopsin Retinal Ganglion Cells and Pupil: Clinical Implications for Neuro-Ophthalmology. Front. Neurol. 9:1047. doi: 10.3389/fneur.2018.01047 Keywords: melanopsin retinal ganglion cells, light, pupil, neurodegeneration, optic nerve, optic neuropathies, Alzheimer, Parkinson

## INTRODUCTION

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs expressing the photopigment melanopsin (1, 2). They constitute about 0.2–1% of total RGCs and contribute to the photoentrainment of circadian rhythms, through their projections to the suprachiasmatic nucleus (SCN), but also to other anatomical structures devoted to non-image forming functions of the eye. These include pupil regulation through their projections to the olivary pretectal nucleus (OPN) in the midbrain (3–5) and brain structures relevant for emotional processing (6). Recent data support the notion that distinct subpopulations of mRGCs mediate different functions in the central nervous system, including circadian rhythm regulation and pupil light reflex (PLR) through their projections to the OPN (5, 7).

The mRGC contribution to the pupil function has been extensively investigated over the recent years and it is now clear that rods mediate mainly the transient pupil contraction, whereas mRGCs contribute to the steady-state pupil constriction (8–11). In fact, mRGCs are characterized by a unique property, which is the capability of firing without fatigue in response to continuous stimulation, consistent with the intrinsic activation of these cells (12). In particular, post-illumination pupil response (PIPR), measured after 1.7 s from onset of the light stimulus, and its magnitude can be considered as specific measures of mRGC function (13). Different protocols, using different light paradigms and experimental setting of stimulation, have been tested and now established to assess in vivo PLR mediated by mRGCs (13, 14). Specifically, the contribution of mRGCs to pupil response has been evaluated using blue (470 nm) and red (640 nm) light, being the blue light able to maximally stimulating mRGCs (13–15).

The PLR mediated by mRGCs has been investigated in different ophthalmological conditions including glaucoma (16–18), retinitis pigmentosa (19), diabetes (20), Leber's congenital amaurosis (14), age-related macular degeneration (21), and ischemic optic neuropathies (22, 23). Moreover, various neurological and psychiatric disorders have been evaluated, including hereditary optic neuropathies (24–29), seasonal affective disorder (SAD) (30), idiopathic intracranial hypertension (IIH) (31, 32), multiple sclerosis (MS) (33), and Parkinson's disease (PD) (34).

In this review we will focus on pupillometry findings in neuro-ophthalmological disorders in which pupil and circadian functions have been investigated. In particular, we include disorders affecting the optic nerve such as glaucoma and hereditary optic neuropathies, neurodegenerative disorders with optic nerve involvement and circadian dysfunction and affective disorders for which a relevant role of mRGCs has been postulated. We will highlight the potential role of mRGCmediated pupil function as an in vivo objective tool and possible biomarker for evaluating mRGC function in different neurodegenerative disorders.

## MELANOPSIN RGCs AND PUPIL IN GLAUCOMA AND ANTERIOR ISCHEMIC OPTIC NEUROPATHY

Glaucoma is a chronic optic neuropathy characterized by loss of peripheral visual field secondary to a progressive and extensive loss of RGCs and their optic nerve fibers (35). The pathophysiology of glaucoma is not yet completely understood, even though two common and pivotal events are the increase in intraocular pressure and impaired microcirculation (vascular deregulation), both preceding the RGC death (36). Previous studies in monkey models of glaucoma reported that all classes of RGCs are susceptible to injury or damage since the early stages of the disease including the sub-population of mRGCs (37). Concordantly, recent clinical studies have shown high prevalence of sleep and circadian disorders, as well as depression in glaucoma patients, implying that the mRGCdriven photoentrainment of circadian rhythms may be affected in patients with glaucoma (38–41).

In the last years, several studies were published aimed at measuring in vivo the integrity of mRGC system in glaucoma by assessing the PLR (16–18). Overall, the results and the conclusions of these studies have been frequently inconsistent because of the different protocols and methodology adopted for chromatic pupillography. In fact, many variables may affect the results, such as time of dark adaptation, light stimulus (duration, intensity, and wavelength), time to measure the intrinsic melanopsin-mediated PIPR, direct or consensual pupil stimulation, and so on (see **Table 1**).

Nonetheless, it is now clearly proven that the PLR, and particularly the PIPR, is altered in moderate and advanced stages of glaucoma, despite the use of different chromatic illumination paradigms (16–18, 27, 42, 43). These findings are also correlated with functional and structural features of the glaucomatous pathology, as demonstrated by the fact that PIPR is inversely correlated with the mean deviation in the visual fields (17, 42, 43). Moreover, an inverse correlation between PLR to high-irradiance blue light and optic disc cupping measured by Heidelberg Retinal Tomography was found (42), and the reduction of PLR to blue and red light correlates with retinal nerve fiber layer (RNFL) thinning (44, 45). These results are in line with a study demonstrating that there is a correlation between the severity of the glaucoma and the reduction of the PIPR (16). This is concordant with the knowledge that in glaucoma the central 10 degrees of the retina, where the mRGCs are more concentrated, are affected only in the last stages of the disease. However, in the last 2 years, a new method of light delivery (quadrant field pupillometry) (44), and a new light stimulation protocol (increasing light regimens) (45) were used to better investigate the pre-perimetric and early-stage glaucoma. By stimulating only the portion of the retina most precociously affected in glaucoma it was shown that the superonasal quadrant PIPR differentiates patients suspected of having glaucoma and with early glaucoma from healthy controls, and this finding correlated with RNFL thinning measured by OCT (44). Furthermore, by increasing logarithmically the light stimuli intensity, PLR is reduced in patients with early-stage glaucoma compared with controls at moderate to high irradiances with both blue and red light, and the maximal pupillary constriction amplitude is correlated to the RNFL thickness (45). To highlight the possible correlation of different measurements of mRGC functions, it is also worth mentioning that in advanced glaucoma, individuals with greater light-induced melatonin suppression (a measure of the retinohypothalamic tract function) have also a smaller PIPR (27).

Finally, a functional damage of the mRGC-mediated PLR has been reported in the affected eyes of patients with unilateral or bilateral anterior ischemic optic neuropathy (AION), specifically 10 patients with unilateral non-arteritic ischemic optic neuropathy (NAION), 1 bilateral NAION, and 7 patients with bilateral AION associated with optic disc drusen, compared to the unaffected and control eyes (22). Differently, previous studies failed to demonstrate differences in the PLR between NAION and control eyes (23). Furthermore, if the bright blue stimuli were presented bilaterally and simultaneously to both eyes, bilateral AION patients showed, through binocular summation, the same post-stimulus pupil size of patients with unilateral AION and controls (22).

## MELANOPSIN RGCs AND PUPIL IN HEREDITARY MITOCHONDRIAL OPTIC NEUROPATHIES

Mitochondrial optic neuropathies are inherited disorders of the optic nerve due to mitochondrial DNA (mtDNA) mutations affecting the mitochondrial-encoded subunits of complex I of the respiratory chain complex, pathogenic for Leber's hereditary Optic Neuropathy (LHON) or to mutations of the nuclear gene OPA1 causing Dominant Optic Atrophy (DOA) (46, 47). These inherited mitochondrial disorders are characterized by the selective loss of RGCs, in particular those originating the TABLE 1 | Pupillometry findings in glaucoma and in anterior ischemic optic neuropathy.

papillo-macular bundle, thus leading to optic atrophy secondary to mitochondrial dysfunctions with the invariable outcome of severe visual loss (46). In both disorders, previous data suggested the maintenance of the PLR even in the chronic stage of the disease, pointing to a pupil-visual dissociation (48, 49). In fact, in these disorders recent histological studies demonstrated a La Morgia et al. MRGCs and Pupil in Neurology

relative preservation of mRGCs compared to the massive loss of regular RGCs in both LHON and DOA, which supports the maintenance of the PLR in these patients (50). At this regard, interestingly, a previous post mortem study demonstrated the relative sparing of the retinofugal fibers to the pretectum in a LHON case, supporting the maintenance of the mRGC projections to the pretectum, which constitute the afferent pathway of the PLR (51). The reasons for the robustness of mRGCs in mitochondrial optic neuropathies are still unknown and under investigation, even though the possible role of peculiar metabolic properties, including the size of the soma, has been proposed (50, 52, 53). More recently, pupillometric studies showed a relative maintenance of the mRGC-mediated pupil response in LHON and DOA patients (24–29) (**Table 2**). Similarly to the maintenance of the PLR a preserved lightinduced melatonin suppression has been demonstrated in LHON and DOA patients supporting a relative preservation of these cells in hereditary mitochondrial optic neuropathies (50). Interestingly, the preservation of mRGCs and PLR has also been demonstrated in an OPA1-mouse model (54).

## MELANOPSIN RGCs AND PUPIL IN OTHER NEUROLOGICAL DISORDERS

In the last years the mRGC-mediated pupil light response has been investigated in various neurological disorders, including IIH, MS, and PD (31–34).

In a cohort of 13 IIH patients compared to 13 controls it was reported a significant reduction of PLR under melanopsin and rod paradigms in IIH subjects, suggesting the potential use of these parameters as an objective measure of RGC dysfunction in IIH (31). However, the abnormal mRGC-driven PLR has not been reported in a cohort of drug naïve IIH patients (32).

A significant reduction of the sustained pupil response to blue light in the eyes with thinner ganglion cell layer (GCL) + inner plexiform layer (IPL) was demonstrated in a group of 24 MS patients, in particular in those with a previous history of optic neuritis, compared to 15 controls (33). The authors proposed the use of the sustained pupil response to light mediated by mRGCs as a surrogate biomarker for neurodegeneration, including the retinohypothalamic tract, in MS patients (33). In consideration that mRGCs are a fundamental conduit for circadian photoentrainment, the sustained PLR to light may be used as a surrogate marker for RHT integrity and consequently for circadian measurements including melatonin rhythm. This may be relevant for potential light therapeutic interventions in these patients (33). Congruently, previous studies demonstrated an abnormal melatonin rhythm in MS patients (55).

An attenuated PIPR for short wavelength and reduced pupil constriction amplitude for long wavelength stimulation was described in a group of 17 early PD patients compared to a control group (34). Pupil metrics in this group were not influenced by disease severity, sleep quality, medications, or OCT measurements and were controlled for unrest pupil conditions. The authors proposed the pupil response mediated by mRGCs as potential biomarker for non-motor symptoms in PD, such as sleep and circadian dysfunction (34). In fact, there is a large body of evidence supporting the occurrence of circadian dysfunction in PD (56).

Finally, a recent study reported the occurrence of PLR dysfunction in R6/2 and Q175 Huntington's disease (HD) mouse models, with a prevalent contribution of cone dysfunction in young-middle-aged mice and of mRGCs in old mice (57). HD is a neurodegenerative disorder in which circadian dysfunction is a prominent and early disease trait pointing again to a possible mRGC dysfunction (56, 58).

Based on these recent findings, it seems reasonable that other neurological disorders, for which there is evidence of circadian dysfunction and mRGC pathology, such as Alzheimer's disease (AD) (59, 60), HD (56–58), and possibly others, may present an abnormal mRGC-driven PLR.

## MELANOPSIN RGCs AND PUPIL IN AFFECTIVE DISORDERS

SAD is a psychiatric condition characterized by the recurrence of depression in winter, in relation to low levels of ambient light in this season (61). Even if the etiology of this disorder is still elusive, the possible role of individual seasonal variation in retinal sensitivity, and in particular of retinal subsensitivity in SAD has been proposed (62–64). Moreover, a polymorphism in the melanopsin (OPN4) gene (P10L) has been associated with SAD, suggesting that mRGCs and sensitivity to light may play a relevant role in the pathogenesis of SAD (65). Based on these premises, Roecklein and coauthors investigated the PIPR in 15 individuals with SAD compared to 15 controls. They found a reduced PIPR and a lower PIPR percent change in response to blue light in SAD subjects compared to controls, implying an abnormal mRGC-mediated response to light, as measured by PLR in SAD (30). Moreover, the PIPR response after blue light varied in relation to the OPN4 I394T genotype, another polymorphic variant, suggesting again a possible influence of genetic predisposition in modulating the sensitivity to light in SAD (30). Interestingly, this polymorphic variant has also been found to influence the steady-state pupil diameter in controls (66).

Differently, the melanopsin-mediated PIPR measurements were not significantly different between eight patients with non-seasonal depression and 13 age-matched healthy controls matched for day-light exposure (67). This finding possibly implies a different pathophysiological mechanism in SAD and non-seasonal depression. However, another study using a different light stimulation protocol, showed an abnormal PIPR in both seasonal-depressed and non-seasonal depressed patients (68).

## DISCUSSION

Intrinsically photosensitive retinal ganglion cells, the mRGCs, are unique photoreceptors located in the inner retina, which TABLE 2 | Pupillometry findings in neurological disorders.

express the photopigment melanopsin (1, 2, 7). The presence of melanopsin makes these cells maximally sensitive to blue light at 470–480 nm and able to spontaneously spike for a long period, even when isolated from the surrounding retinal structures (7, 12). The mRGCs are crucial for nonimage forming functions of the eye including circadian photoentrainment, sleep and melatonin synthesis, and PLR. Of particular importance, in this context, is the possibility of using some pupil metrics, such as the PIPR, as a specific signature of mRGC function in vivo (8, 13, 56) for ophthalmological and neurodegenerative disorders, which may present circadian dysfunction. In fact, mRGCs contribute mainly to the sustained component of the PLR and, using blue wavelength light, it is possible to isolate the melanopsin contribution to the PLR.

The availability of the mRGC-mediated PLR as a tool to indirectly test the circadian system status, as recently proposed (69), opens new avenues in the analysis of circadian, sleep, and non-motor features in many neurodegenerative disorders. Interestingly, it has been demonstrated in 15 healthy subjects, using combined evaluations including pupillometry, actigraphy, light sensors and body temperature, a close inverse relationship between pupil light response metrics and circadian status (70). In particular, for the pupil recordings it was used a protocol in which the right eye was dilated and different light stimuli including different light wavelength were tested (5 min stimuli) with 40 min of darkness between the light stimulations. Pupil parameters were analyzed using ad-hoc software. For the actigraphic recordings the subjects wore an actigraph with light sensor and non-parametric circadian measures, such as intradaily variability, interdaily stability, relative amplitude, L5 and M5, were obtained (70). The authors proposed the Circadian Status Index as an integrative measure to unify three aspects (robustness, timing, and level) of the three circadian rhythm measures (temperature, activity, and light), as well as a global parameter for pupil metrics (circadian photoreception PLR). However, the authors found an inverse relationship between the pupil and circadian metrics. These contradictory findings between circadian status robustness and the PLR might be referred to individual differences in the M1 cell population of mRGCs. Larger studies, more uniform light stimulation protocols and the inclusion of more circadian and pupil metrics are warranted to analyze the possible correlations between pupil metrics and circadian status. In fact, the current available pupillometric studies all suffer the limitation of great heterogeneity of stimulation protocols and consequent lack of reproducibility of their results. Similarly, all these studies are generally underpowered by the limited number of subjects analyzed.

Finally, since mRGCs are contributing to other non-visual functions of the eye, and different class of mRGCs have different projections to the CNS contributing to different functions (5), it must be emphasized that the finding of an abnormal mRGC-mediated PLR does not mean necessarily a global dysfunction of these cells. Overall, the availability of an easily accessible metric for mRGC function, in conjunction with other tests, such as melatonin suppression test, actigraphic recordings, and functional MRI, may represent a comprehensive strategy to further exploring the function of these cells in patients with different neuro-ophthalmological conditions.

## REFERENCES

## CONCLUSIONS AND FUTURE DIRECTIONS

In conclusion, the use of PLR mediated by mRGCs, as a measure of mRGC function, is of particular relevance for neurodegenerative disorders for which there is already evidence of circadian and sleep dysfunction, such as PD, AD, and HD. Similarly, it might be also relevant for other neurological disorders with evidence of circadian dysfunction such as frontotemporal dementia (71), Lewy-Body dementia (72), Progressive Supranuclear Palsy (73, 74), and possibly prion diseases, in particular fatal familial insomnia (75). Moreover, the study of PLR mediated by mRGCs might be particularly intriguing for conditions, in which light sensitivity is a predominant feature, such as photophobia (76, 77) and photosensitivity in epilepsy (78). At this regard, an abnormal PLR has been recently documented in migraineous photophobic subjects, even though it was not specifically assessed the mRGC contribution to PLR (79, 80).

Overall, after adequate standardization of light protocols, the availability of an easy accessible tool to assess mRGC function, as a surrogate marker for more general non-image forming functions of the eye, including circadian rhythms and sleep, is a particularly promising biomarker for neurodegenerative disorders.

## AUTHOR CONTRIBUTIONS

CLM and MC were responsible for conception, design, drafting, and revision of the manuscript. VC was responsible for conception and revision of the manuscript.

## FUNDING

This work was supported by the Ministry of Health Young Researcher project (GR-2013-02358026) (to CLM) and by the Italian Ministry of Health and of Research and the Gino Galletti Foundation (to VC).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 La Morgia, Carelli and Carbonelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Light-Induced Pupillary Responses in Alzheimer's Disease

Pratik S. Chougule1 , Raymond P. Najjar 1,2, Maxwell T. Finkelstein1 , Nagaendran Kandiah3,4 and Dan Milea1,2,5 \*

*1 Department of Visual Neurosciences, Singapore Eye Research Institute, Singapore, Singapore, 2 The Ophthalmology & Visual Sciences ACP, Duke-National University of Singapore (NUS) Medical School, Singapore, Singapore, 3 Department of Neurology, National Neuroscience Institute, Singapore, Singapore, 4 Duke-National University of Singapore (NUS), Singapore, Singapore, 5 Singapore National Eye Centre, Singapore, Singapore*

The impact of Alzheimer's disease (AD) on the pupillary light response (PLR) is controversial, being dependent on the stage of the disease and on the experimental pupillometric protocols. The main hypothesis driving pupillometry research in AD is based on the concept that the AD-related neurodegeneration affects both the parasympathetic and the sympathetic arms of the PLR (cholinergic and noradrenergic theory), combined with additional alterations of the afferent limb, involving the melanopsin expressing retinal ganglion cells (mRGCs), subserving the PLR. Only a few studies have evaluated the value of pupillometry as a potential biomarker in AD, providing various results compatible with parasympathetic dysfunction, displaying increased latency of pupillary constriction to light, decreased constriction amplitude, faster redilation after light offset, decreased maximum velocity of constriction (MCV) and maximum constriction acceleration (MCA) compared to controls. Decreased MCV and MCA appeared to be the most accurate of all PLR parameters allowing differentiation between AD and healthy controls while increased post-illumination pupillary response was the most consistent feature, however, these results could not be replicated by more recent studies, focusing on early and pre-clinical stages of the disease. Whether static or dynamic pupillometry yields useful biomarkers for AD screening or diagnosis remains unclear. In this review, we synopsize the current knowledge on pupillometric features in AD and other neurodegenerative diseases, and discuss potential roles of pupillometry in AD detection, diagnosis and monitoring, alone or in combination with additional biomarkers.

Keywords: Alzheimer's disease, dementia, pupillary light response, chromatic pupillometry, melanopsin expressing intrinsically photosensitive retinal ganglion cells, Parkinson's disease, post -llumination pupil response, cholinergic deficit

## INTRODUCTION

Dementia is a global epidemic and has become a public health priority. Alzheimer's disease (AD) is the most common cause for dementia worldwide (1), accounting for 50–70% of dementia cases. The two major neuropathological landmarks of AD are deposition of insoluble amyloid-β (Aβ) plaques and formation of neurofibrillary tangles, composed of hyperphosphorylated tau proteins. These pathologic abnormalities are found in the central nervous system, as well as in the retina (2–4). The pathophysiology of AD is poorly understood, but a common hypothesis postulates that aggregation of Aβ is a pre-requisite for tau accumulation, neurodegeneration, and ultimately, to clinical manifestations. Clinical features of AD include progressive cognitive decline, affecting

#### Edited by:

*Andrew J. Zele, Queensland University of Technology, Australia*

#### Reviewed by:

*Shlomo Dotan, Hadassah Medical Center, Israel Chiara La Morgia, IRCCS Istituto delle Scienze Neurologiche di Bologna (ISNB), Italy Kathryn Ariel Roecklein, University of Pittsburgh, United States*

> \*Correspondence: *Dan Milea<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuro-Ophthalmology, a section of the journal Frontiers in Neurology*

Received: *12 November 2018* Accepted: *25 March 2019* Published: *12 April 2019*

#### Citation:

*Chougule PS, Najjar RP, Finkelstein MT, Kandiah N and Milea D (2019) Light-Induced Pupillary Responses in Alzheimer's Disease. Front. Neurol. 10:360. doi: 10.3389/fneur.2019.00360*

**239**

memory, learning, language, visuospatial abilities, and executive functions, but also deterioration of sleep and normal circadian rhythms (5). Most often extensive and largely irreversible neuronal histopathological changes precede clinical features of AD (6), which may explain the current failure of all disease modifying agents in this condition. For these reasons, it is believed that early diagnosis of AD is crucial for early and effective therapeutic interventions, improving AD outcomes.

Several in vivo biomarkers have been proposed for early identification of AD pathology, including brain imaging biomarkers (positron emission tomography after Aβ labeling) (7), as well as fluid biomarkers (within the cerebrospinal fluid and, possibly, in the blood) (8). These, and other novel genetic, biological, deep-learning based, or behavioral biomarkers aim to surpass the current performance of classical clinical evaluations in AD, which are subjective, time-consuming and deliver variable results. The eye, which is embryologically, anatomically, and physiologically an extension of the brain, has been an early explored target for identification of neurodegeneration biomarkers in AD (9, 10). Functionally, various ocular biomarkers have been tested for detection and evaluation of AD, such as eye movement recordings and pupillary responses to light or to cognitive load (9–11). Several studies have suggested that AD may be associated with altered pupillary light responses (PLR), as a consequence of abnormalities in the retina and/or the efferent pupillary system. Several arguments support the possible pupillary involvement in AD, including pathological changes in the retina, as well as existence of parasympathetic (cholinergic) and sympathetic (adrenergic) dysfunctions in the disease. Indeed, neurodegeneration commonly affects the locus coeruleus (LC), located in pons and involved in the sympathetic control of pupil size and PLR (2, 12), as well as the Edinger Westphal nucleus (EWN) (4, 13), involved in the parasympathetic control of the pupil. Pupillometry is an easy, non-invasive and affordable tool, allowing the evaluation of the PLR in AD and other ocular and neurological diseases. Whether static or dynamic pupillometry yields useful biomarkers for AD screening or diagnosis remains unclear. In this review, we synopsize the current knowledge on pupillometric features in AD and other neurodegenerative diseases, and discuss potential roles of pupillometry in AD detection, diagnosis and monitoring, alone or in combination with additional biomarkers.

## THE NEUROPHYSIOLOGY OF THE PUPILLARY LIGHT RESPONSE

## Afferent and Efferent Pathways Governing the Pupillary Light Response

The pupil size is under the control of a closed autonomic loop. The pupil constrictor and dilator muscles receive antagonistic impulses from the parasympathetic (cholinergic) and sympathetic (adrenergic) autonomic nervous systems, respectively (**Figure 1**). The PLR is also dependent on the integrity of the retina, and in particular on the integrity of

the intrinsically photosensitive melanopsin expressing retinal ganglion cells (mRGCs) (15). Although the mRGCs are activated by rods and cones, they are also intrinsically photosensitive through the melanopsin photopigment, subserving the PLR via central projections to the olivary pretectal nucleus (OPN) (16), which projects to the EWN, as demonstrated in non-human primates. The parasympathetic EWN is a cholinergic nucleus in the oculomotor complex, at the origin of preganglionic efferent neurons which synapse in the ciliary ganglion, subsequently innervating the constrictor pupillae (14, 17). The mRGCs also project to the central circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN), which governs various bodily circadian rhythms and projects to the sympathetic locus coeruleus (LC), located in pons (**Figure 1**) (18, 19) [for review see (20)]. The sympathetic efferent system, including LC and SCN, regulates the resting pupil size at different levels of background illumination (17), by controlling the tone of the dilator muscle. Beyond the intervention of these motor structures, the pupillary size and function can be modulated by supranuclear neuronal influences. Other possible confounding factors, affecting the size of the pupils include the respiration rate (21), emotional status (22), vigilance (23), and age (24).

## Retinal Photoreception and Chromatic Pupillometry

Recorded using an infra-red pupilometer, the PLR is governed by rods, cones and mRGCs. The intrinsically photosensitive mRGCs, located in the inner retina, produce a sustained constriction of the pupil in response to bright blue light which persists even after light offset; in addition to integrating signals from rods and cones (25). Different light wavelengths, at different intensities stimulate specifically different retinal photoreceptors (26–29). Thus, chromatic pupillometry, using different wavelengths and light intensities has been used for the evaluation of inner and outer retina integrity, in various conditions (26–29).

## PATHOPHYSIOLOGY OF LIGHT-INDUCED PUPILLARY RESPONSES IN AD

## Cholinergic Deficit in AD

Loss of cholinergic neurons is a common event in AD, possibly leading to alteration of cognitive processes. Specific loss of cholinergic neurons, mainly located in the medial septum and in the para-hippocampal area, is associated with memory impairment (30), but also with other cognitive deficits seen in AD (31, 32). The hypothesis of cholinergic deficit fails however, to explain other impairments in AD, i.e., disruption of circadian rhythms, sleep, and executive functions.

AD affects the cholinergic EWN, which is the central brainstem sub-nucleus of the oculomotor complex, involved in the control of the pupil constriction. Pathologic studies have shown that the EWN is affected even at early stages of AD, displaying deposition of Aβ amyloid plaques and neurofibrillary tangles (4, 13, 33). AD is associated with increased glutaminyl cyclase activity, resulting in formation of highly neurotoxic Aβ amyloid precursors (pyroglutamate Aβ peptide), identified in the EWN and in the preganglionic cholinergic PLR-governing neurons, as well as in other cholinergic neurons in the nucleus basalis of Meynert (34). In AD, the EWN neurons, display a decrease in their total dendritic length per neuron as well as a severe loss of distal dendritic branches and dendritic spines, leading to severe decrease in synaptic contacts (35). It has been suggested that these pathological changes of the EWN may be an early and specific feature of AD and they may result in decreased cholinergic control of pupillary responses (35). Unfortunately, little is known about the involvement of other parasympathetic structures involved in the PLR, i.e., the olivary pretectal nucleus and ciliary ganglia.

## Adrenergic Deficit in AD

The LC modulates pupil size in 2 possible ways; by direct stimulation of preganglionic sympathetic neurons, as well as by inhibitory regulation of the EWN (**Figure 1**) (17, 36). Various factors stimulating the LC may modulate the PLR. For example, anxiety, associated with excitation of the LC (37, 38) or drugs increasing noradrenergic output to the EWN like noradrenaline re-uptake inhibitors (14, 39), lead to an increased sympathetic response on PLR like prolonged latency, reduced amplitude of constriction and faster redilation. Conversely, drugs inhibiting LC activity like clonidine (α2-adrenoceptor agonist) cause pupillary miosis and reduce the sympathetic effect on PLR (40–42). In monkeys, an electrophysiologically detectable activity in LC has been associated with mydriasis at rest (43).

Patients with AD and mild cognitive impairment (MCI), considered as the pre-clinical stage for AD, undergo significant loss of noradrenergic neurons in the LC (55 and 30%, respectively), compared to healthy controls, a finding which may impact the PLR (44). Neuronal loss in the LC of patients with AD may lead to decreased sympathetic supply to the iris and reduce the baseline pupil size (45).

## Retinal Changes in AD

Aging is associated with optical (46, 47) photoreceptoral and retinal neuronal changes (48–50). Optically, in spite of decreased lens transmittance for short wavelength blue light in aging and cataract, the mRGCs induced pupillary response by blue light are well-preserved (51–53), and the pupillary responses are reduced irrespective of the wavelength of light. Although, aging has been associated with axonal and retinal ganglion cell loss (54), AD has been associated with greater thinning of retinal nerve fiber layer compared to age matched healthy controls (55–57), suggesting an accelerated loss of RGCs in AD patients. Pathological studies have shown presence of Aβ amyloid plaques in the retina of AD patients (58, 59) including in the inner layers of retina (3). These changes were often associated with blood vessel abnormalities and areas of cellular degeneration, similar to what is seen histologically in the brain of patients with AD (60, 61).

Moreover, retinas of patients with advanced AD display not only histological evidence of mRGC loss, but also selective deposition of Aβ amyloid plaques within these cells, which subserve the PLR (3). Less is known about the early selective loss of mRGC and possible mRGC Aβ deposition, occurring in AD. Occurrence of such a phenomenon should allow discrimination between normal aging patients and AD, using chromatic pupillometry. Chromatic pupillometry has been used in other conditions as a marker of mRGC integrity (62). In primary open angle glaucoma, a condition associated with histological mRGC loss (63), abnormal melatonin secretion profile (64) and sleep and circadian rhythm dysfunction (65), various pupillometric studies have shown abnormal PLR responses (28, 66–70). Conversely, in mitochondrial hereditary optic neuropathies, mRGCs are resistant to neurodegeneration, explaining the relatively preserved chromatic pupillometry parameters (71–73) and melatonin profiles (74). It is possible that mRGC loss, alone or combined with neuronal loss occurring in the suprachiasmatic nuclei, may be associated with circadian rhythm dysfunctions which can occur even at early stages of AD (3, 5).

## FEATURES OF THE PUPILLARY LIGHT RESPONSES IN AD

## Baseline Pupil Diameter

The consequence of AD on the pupillary diameter at rest has been controversial in various studies, probably due to methodological differences, i.e., measurement conditions and sample sizes. A few

#### TABLE 1 | Summary of studies on PLR in AD.

*BPD, baseline pupillary diameter in mm; LoC, latency of constriction in seconds; AC, amplitude of constriction; MCV, Maximum constriction velocity; MCA, maximum constriction acceleration; AD, Alzheimer's disease; PSD, Parasympathetic deficiency; SD, Sympathetic deficiency;* ↓*, decreased;* ↑*, increased;* ↔*, Not significant; NA, Not applicable/available; PLR, pupillary light response; MMSE, Mini-Mental State Examination;* \**n, Sample size of patients included in the study (excluding controls).*

studies with small sample size (range 9 to 23 AD patients) have reported reduced baseline pupillary diameters in AD compared to those of healthy subjects (45, 75–77). Other studies did not find any difference in baseline pupil diameters between AD, MCI and controls (78, 79), however, the groups were not age-matched in the study with largest sample size (n = 66 AD, 42 MCI) (78), while AD patients in the other study were using cholinergic and anti-depressant medications which may alter the baseline pupil size (n = 15 AD) (79). An increased baseline pupillary diameter has also been reported in AD (n = 20 AD), but no details of age of the two groups were mentioned (80) (**Table 1**).

## Constriction Phase

Most pupillometric studies in patients with AD have reported results compatible with parasympathetic deficiency, translating to increased latency of pupillary constriction to light, decreased constriction amplitude, reduced mean constriction velocity and faster redilation after light offset (45, 75, 76, 83, 85) (**Table 1**). Pupil constriction velocity is obtained as the first derivative of change in pupil size with respect to time and acceleration as the second derivative (change in constriction velocity with respect to time) (**Figure 2**). Patients with AD typically display decreased maximum velocity of constriction (MCV) and maximum constriction acceleration (MCA) compared to controls, suggesting a parasympathetic deficiency. Amongst all pupillometric features, MCA and MCV have been reported as the most accurate parameters to differentiate AD patients from healthy controls (75, 76, 83). Nonetheless, other studies have failed to find such differences between AD patients and healthy individuals (78, 80). These differences may be the result of the different illumination protocols used, since studies using white light typically are associated with larger constriction amplitudes and shorter latencies (24), compared to studies using red light at 660 nm (80) or 585 nm (78). Considering that different studies have used different intensity and wavelength stimuli, this effect cannot be completely attributed to the wavelength alone.

However, other studies have demonstrated that when photon density is kept constant, shorter wavelength light produced greater constriction amplitude than longer wavelength (86, 87).

In a recent study in cognitively normal pre-clinical AD subjects, diagnosed on the basis of high cortical binding potential of Aβ amyloid on PET imaging and/or low CSF Aβ amyloid levels, there was no significant difference in any of the pupillary parameters in response to light emitting diodes (LEDs) producing a white flash of 180 W for 525 ms, compared to healthy controls (84). Only a marginal difference was observed in PLR in pre-clinical stages, suggesting that the effect of AD on PLR in pre-clinical stages is very small and may not be detectable. This suggests that PLR using achromatic light stimulus may be a valid biomarker for established Alzheimer's disease but it may have limited clinical utility in screening for AD in pre-clinical stages, perhaps due to the very small disease effect on PLR (84).

A pupillometric study, using non-Maxwellian retinal stimulation with repetitive, brief, long wavelength light (585 nm), aimed to compare patients with MCI, AD and normal controls, but failed to find inter-group differences (evaluating baseline diameters, constriction amplitudes, and constriction latencies), after adjustment for age (78). However, the use of specific repetitive light stimuli induced stronger pupillary responses in controls in terms of relative constriction amplitude (difference in the PLR measurements of the first and last stimuli), compared to AD and MCI patients, suggesting that repetitive pupillary stimulation might be a more appropriate stimulus to discriminate patients with AD and MCI from controls (78). It was proposed that repetitive stimuli caused a fatigue of the sympathetic inhibitory system to the parasympathetic pathway revealing the true effect of parasympathetic system alone on PLR. Smaller relative miosis and amplitude of constriction in AD patients suggests a decreased parasympathetic innervation to the pupillary system which may not be detected in the presence of inhibitory sympathetic system. Although relative amplitude was independent of age, the difference between MCI and control groups was not statistically significant while between AD and controls may fail to reach statistical significance if Bonferroni correction is applied. Moreover, its definition and calculation is not clearly explained in the study. Constriction phase due to different wavelength light stimuli in AD patients has not been studied yet.

## Pupillary Redilation Phase

The pupillary redilation phase has been explored in several studies, but these results are sometimes difficult to compare, given the variable definitions of this parameter such as (i) percentage of pupillary redilation after 3.5 s of white flash light offset (75–77), (ii) 75% redilation time (45, 77), and (iii) average dilation velocity (77, 84). The majority of these studies have reported a faster pupillary redilation phase [analogous to decreased Post-illumination pupil response (PIPR)] in AD patients compared to controls, making it the most consistent PLR feature in AD patients (45, 75–77). In a study which extensively studied the redilation phase, the percentage recovery at 3.5 s and the 75% redilation time was significantly greater in AD patients compared to aging controls. Although the mean dilation velocity (mm/sec) was slower in AD patients, it can be ignored since the pupil size was not calculated as a percentage of baseline pupil diameter which was significantly smaller in AD (77). A recent study in preclinical AD alone did not find a significant increase in dilation velocity compared to controls following an achromatic light stimulus, however, it is not clear whether the measurements were adjusted to baseline pupil diameter (84). The faster pupillary redilation after light offset has been attributed to the diminished parasympathetic tonus associated with the cholinergic deficit, translating into failure to maintain a tonic pupillary constriction. An alternative explanation, which was not yet considered in previous studies, is that the accelerated pupillary redilation may be the result of selective mRGC loss in AD, causing an abnormal, faster PIPR as seen in other conditions affecting the mRGC, such as glaucoma (66). This hypothesis is in line with the findings of another study, which did not find any difference of the redilation phase between a group of AD patients and controls, after exposure to red light (660 nm) (80). Indeed, red light at medium high intensities is less prone to stimulate the mRGC and might have failed to explore their dysfunction. Although, redilation was not found to be different in preclinical AD patients in a recent study using a 180 W achromatic stimulus for 525 ms (84), chromatic pupillometry studying different parameters like constriction amplitude or PIPR to blue light, can still be a viable option in such cases due to selective mRGCs loss as reported in AD patients (3). No study till date has investigated pupillometric signs of mRGC dysfunction or impaired PIPR to blue light in AD patients and needs to be explored in the future. However, these interpretations are speculative, it is well-possible that the faster redilation in AD patients may be the result of an interplay between the two factors, i.e., the mRGC loss and the cholinergic deficit. It is noticeable that mRGC loss in AD should result in selective faster redilation after offset of blue light (460 nm), which specifically stimulates the mRGCs. On the opposite, faster redilation due to cholinergic deficiency should be independent of wavelength of stimulus used, occurring after offset of both blue and red light. Future studies taking into account these factors, should be able to disentangle the respective role of the afferent vs. efferent system in the pupillary redilation phase.

## A POTENTIAL ROLE FOR PUPILLOMETRY IN A MULTI-MODAL APPROACH FOR DETECTING AD?

## Generic Screening and Diagnostic Tests for AD

The gold standard for AD diagnosis is still based on pathology findings. However, in clinical practice, screening for AD or cognitive impairment uses various questionnaires and interactive tests, including the Mini Mental Scoring Examination (MMSE) or the Montreal cognitive assessment test (MoCA). These tests have numerous limitations, including language barriers, geographical adaptability, subjectivity, long implementation time, and the necessity for constant supervision by trained and skilled personnel. Several objective tests have been developed for early AD detection and diagnosis including cerebro-spinal fluid (CSF) analysis to measure beta amyloid (Aβ), total tau proteins and phosphorylated tau peptides quantification (8, 88), and magnetic resonance imaging (MRI) imaging of the brain and positron emission tomography (PET) scan measuring the brain Aβ plaque burden (6, 7, 89). These modalities are expensive, invasive and potentially dangerous (90, 91). In addition, they detect only structural and not functional changes in AD.

From the published studies, pupillometric evaluations may not suffice for early AD detection. However, they may constitute an adjunctive method in a, multimodal approach, combining (i) novel, cognitive, visuospatial, and memory tests involving portable virtual reality devices, (ii) retinal imaging for detection of neuronal loss and/or amyloid deposition, and (iii) objective functional outcomes provided by targeted color pupillometry.

## PLR and Genetic Mutations

Apolipoprotein E is a fat-binding protein involved in the metabolism of fat, produced by APOE gene found on chromosome 19, being the only genetic factor associated with the common late onset AD. APOE mutation is not a causative mutation, but is rather considered as a risk factor for AD (92, 93). Although the PLR is not directly influenced by the APOE ε4 carrier status (78), their combination may increase the area under the curve for the combined test performance (83).

A pupillometric study has evaluated participants from a single family harboring an Amyloid-Beta Precursor Protein genetic mutation (APPGlu693Gln) (6 carriers with no cognitive impairment and 6 non-carriers) (94). This mutation results in a rare form of autosomal dominant Alzheimer's disease with phenotypical penetration approaching 100% and which is responsible for an early onset of AD. The pupillometric assessment yielded a slower pupil 75% recovery time in mutation carriers compared to non-mutation carriers. Globally, pupillometric changes were detected in pre-symptomatic carriers of the mutations, but were not statistically significant.

## PLR and Cognitive Assessment Tools

MMSE is routinely used to screen elderly subjects for dementia and has a AUROC of 0.89 (95). PLR in patients with AD having higher MMSE and Wechsler Memory Scale (better cognition) scores correlated moderately with MCV, MCA and latency of constriction (82). On repetitive pupillary stimulation, higher MMSE correlated with larger increase in amplitude and relative amplitude and greater decrease in the latency (p < 0.05) of constriction (78). These outcomes suggest that the pupillary light response may depend on the severity of the disease and can be used for monitoring the disease progression. However, combined efficacy of MMSE and PLR has not been explored as both tools are practical, easy, non-invasive, and affordable and may yield better accuracy if combined together, compared to individual outcomes.

## PLR and CSF Abnormalities

Decrease in pupillary constriction amplitude with repetitive stimulation in AD patients correlated with lower Aβ42 protein levels (p = 0.01) and a trend with higher tau levels in CSF (p = 0.08) (78). This suggests a possible association between cholinergic deficit, decreased Aβ42 protein levels and a trend with higher tau levels in CSF which supports a causative role of Aβ amyloid plaques in central cholinergic deficit (92). To date, the efficacy of a combined PLR-CSF screening method remains unknown.

## PLR and Topical Weak Anticholinergic Eye Drops

In a highly controversial study, Scinto et al. reported that patients with AD exhibit larger pupil dilation compared to age-matched controls after instillation of diluted anticholinergic eyedrops (Tropicamide 0.01%) (96) Several studies have contradicted this finding (97–99) which could be due to ethnicity, age, ocular penetration of drug, properties of the solution and background luminance (79). A combination of topical weak anticholinesterase and PLR showed significant reduction in constriction amplitude for AD and Parkinson's patients compared to controls, but no significant difference between Alzheimer's and Parkinson's patients was found, while latency of constriction was similar within the 3 groups (79). However, others did not find any such significant difference in PLR pre or post weak anticholinergic eyedrops use, between AD patients and controls (80). Hence, the use of weak anticholinergic eyedrops may not improve the efficacy of PLR in detecting AD, since it may not give consistent results and decrease in amplitude of constriction is noted in AD even without using topical anticholinesterase.

## LIMITATIONS OF PREVIOUS PUPILLOMETRIC STUDIES IN AD

Most of the previously published pupillometric studies in AD have various methodologic limitations. The intensity, light wavelength and duration of light exposure were variable in all the above mentioned studies. Yet, these parameters can affect, independently, or in combination, the PLR outcomes (24, 100). Therefore, there is a high need for standardization of experimental conditions in AD studies, similar to what has been described in studies using light therapy (101) and in other animal studies (102). Most current pupillometric studies agree of the need for standardized analysis of baseline pupil diameters (103, 104). Interestingly, most of the previous PLR studies in AD have not normalized the baseline diameter in their subjects, making any comparison very difficult. In a few studies evaluating pupillometric results in AD, there was no agematching between the groups of patients and controls (78, 79, 83). Indeed, the decreased pupillary diameter with age (105, 106) may constitute a confounding factor. Last, but not least, the severity of AD was rarely taken into account in the evaluation of the PLR.

## Effect of Cholinergic Medications

Only a few, small sample studies have reported the effect on the PLR of cholinergic drugs, commonly used in AD (79, 81). Thus, AD patients without cholinergic medications displayed larger baseline pupillary diameter, reduced pupillary miosis and higher number of oscillations at rest, compared to AD patients on cholinergic treatments and to healthy controls. Patients on medications had a greater latency of onset of constriction compared to both the controls and the medication free AD patients (81). However, other study in AD patients, have not found an effect of cholinergic medications on pupillary miosis but supported the increase in latency of constriction (79). PLR in AD patients on cholinergic medications behaved more like controls with no significant difference in constriction amplitude and baseline pupil diameter than their medication free counterparts (81). Taken together, these findings suggest that cholinergic medications might improve the pupillary responses in AD patients. Due to the very small sample size of these studies, it is difficult to conclude regarding a possible effect of cholinergic medications on the PLR in these patients. Additional studies are needed to understand the effect of cholinergic medications on PLR.

## Effect of Ocular Co-morbidities

The most common ocular condition associated with aging is cataract which can attenuate the PLR response to both red and blue light. But the senescence of the lens does not selectively reduce the mRGCs responses to intense blue light and is wellpreserved, in spite of its decreased lens transmittance in aging and cataract (51). Different retinal and optic nerve conditions can affect PLR and using chromatic pupillometry it is possible to localize the loss of photoreceptoral function i.e., inner or outer retina (107). Primary open angle glaucoma is associated with decreased PLR in response to exposure to both red and blue light with decreased PIPR for blue light (28, 70), while retinal dystrophies affecting rods and cones lead to decreased PLR responses to red and low intensity blue light with an increased PIPR to bright blue light stimulus (108, 109). Diabetic retinopathy and age-related macular degeneration can also affect the PLR (110, 111), but there is little indication to what extent these PLR alterations might be disease-specific, or whether they may confound co-existence of AD in the aging population.

## PLR IN OTHER NEURODEGENERATIVE DISORDERS

Autonomic nervous system dysfunction has been described in Parkinson's disease (PD) (112), including cholinergic deficit (113), Various PLR abnormalities have been described in PD, including reduced amplitude of constriction, increased latency and decreased velocity and acceleration of constriction, while the baseline pupil diameter may be increased or not significantly different compared to healthy controls (**Table 2**) (76, 114). Pupillary unrest has also been increased in PD patients which were not on medications compared to healthy individuals or in treated patients (115), Pupillary redilation has not been

*BPD, baseline pupillary diameter in mm; LoC, latency of constriction in seconds; AC, amplitude of constriction; MCV, Maximum constriction velocity; MCA, maximum constriction acceleration; PD, Parkinson's disease; PSD, Parasympathetic deficiency; SD, Sympathetic deficiency;* ↓*, decreased;* ↑*, increased;* ↔*, Not significant; NA, Not applicable/available; mRGC, Melanopsin expressing retinal ganglion cells; PLR, pupillary light response;* \**n, Sample size of patients included in the study (excluding controls).*

significantly different in PD studies using white flash light stimuli (114, 115). However, a recent chromatic pupillometry study in PD patients has suggested that the PIPR following a short wavelength blue light elicits a faster redilation compared to healthy controls. This finding is consistent with loss of mRGCs in PD (116), possibly related to deposition of α-synuclein in the retinal ganglion cells in the inner plexiform layer of the retina (117, 118). An alternative explanation might be related to reduction in the dopamine expression in the amacrine cells which relay information from rods and cones to mRGCS.

Autonomic nervous system dysfunction has also been described in dementia with Lewy bodies and to a lesser extent in fronto-temporal dementia, which can be associated with retinal abnormalities (119, 120). However, the specific effects of autonomic dysfunctions and retinal changes on pupillary light reflexes have not yet been studied in these disorders.

## SUMMARY

In summary, MCV and MCA appear to be the most accurate PLR features, but also the least studied, while redilation velocity/rate (corresponding to PIPR) appears to be the most consistently altered PLR feature in AD. In conjunction with other features (baseline pupillary diameter, amplitude and latency of constriction), these parameters predominantly suggest parasympathetic deficiency, associated with mRGCs dysfunction. Longitudinal and adequately designed studies are necessary to validate the use of pupillometry in the early detection and followup of AD. Further studies are needed to establish the respective contribution of retinal (afferent) vs. efferent pupillary pathways in the alteration of the pupillary responses, for which chromatic pupillometry can potentially be used and translated into clinical application. Studies may also be designed to investigate the effect of cholinergic medication on PLR in AD patients and the potential use of artificial intelligence on pupillometric traces and video recordings. Using low-cost hardware, pupillometry can now easily be implemented in both remote tele-ophthalmology settings (121), as well as in continuous home monitoring (122). Combined with cognitive game-based investigations and wearables (123, 124), pupillometry may allow a more accurate screening, follow-up, and management of patients with AD.

## AUTHOR CONTRIBUTIONS

PC conducted the review of literature. PC, DM, and RN wrote the manuscript. All authors (PC, RN, DM, MF, and NK) reviewed and approved the manuscript.

## FUNDING

This work was supported by the National Medical Research Council, Singapore (NMRC/CIRG/1401/2014) to DM.

## REFERENCES

Alzheimer's and Parkinson's disease. Int J Psychophysiol. (2003) 47:95–115. doi: 10.1016/s0167-8760(02)00122-8

124. Katz S, Marshall BL. Tracked and fit: FitBits, brain games, and the quantified aging body. J Aging Stud. (2018) 45:63–8. doi: 10.1016/j.jaging.2018.01.009

**Conflict of Interest Statement:** DM has a patent application based on a pupillometry protocol (PCT/SG2015/050494): A method and system for monitoring and/or assessing pupillary responses. DM and RN have a patent application based on a hand held device for ophthalmic and neurological screening (PCT/SG2018/050204): Hand held ophthalmic and neurological screening device.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chougule, Najjar, Finkelstein, Kandiah and Milea. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Buzzing Sympathetic Nerves: A New Test to Enhance Anisocoria in Horner's Syndrome

Rawan Omary 1 , Christopher J. Bockisch1,2,3, Klara Landau1 , Randy H. Kardon4 and Konrad P. Weber 1,2 \*

*1 Department of Ophthalmology, University Hospital Zurich, University of Zurich, Zurich, Switzerland, 2 Department of Neurology, University Hospital Zurich, University of Zurich, Zurich, Switzerland, 3 Department of ENT, University Hospital Zurich, University of Zurich, Zurich, Switzerland, 4 Department of Ophthalmology, University of Iowa and Veterans Medical Center, Iowa City, IA, United States*

#### Edited by:

*Andrew J. Zele, Queensland University of Technology, Australia*

#### Reviewed by:

*Jorge Kattah, University of Illinois at Chicago, United States Mark Paine, Royal Brisbane and Women's Hospital, Australia Elemer Szabadi, University of Nottingham, United Kingdom*

> \*Correspondence: *Konrad P. Weber<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuro-Ophthalmology, a section of the journal Frontiers in Neurology*

Received: *26 October 2018* Accepted: *28 January 2019* Published: *21 February 2019*

#### Citation:

*Omary R, Bockisch CJ, Landau K, Kardon RH and Weber KP (2019) Buzzing Sympathetic Nerves: A New Test to Enhance Anisocoria in Horner's Syndrome. Front. Neurol. 10:107. doi: 10.3389/fneur.2019.00107* Introduction: Patients with suspected Horner's syndrome having equivocal pupil dilation lag and pharmacologic testing may undergo unnecessary MR imaging and work up in the case of false positive pupil test results. Our goal was to increase the diagnostic accuracy of pupillometry by accentuating the inter-ocular asymmetry of sympathetic innervation to the iris dilator with surface electrical stimulation of the median nerve using a standard electromyography machine. We hypothesized that an accentuated difference in sympathetic response between the two eyes would facilitate the diagnosis of Horner's syndrome.

Methods: Eighteen patients with pharmacologically proven Horner's syndrome were compared to ten healthy volunteers tested before and after monocular instillation of 0.2% brimonidine tartrate ophthalmic solution to induce pharmacological Horner's syndrome. Pupillary responses were measured with binocular pupillometry in response to sympathetic activation by electrical stimulation of the median nerve in darkness and at various times after extinction of a light stimulus. Sudomotor sympathetic responses from the palm of the stimulated arm were recorded simultaneously.

Results: In subjects with Horner's syndrome and pharmacologically induced unilateral sympathetic deficit, electrical stimulation in combination with the extinction of light greatly enhanced the anisocoria during the evoked pupil dilation, while there was no significant increase in anisocoria in healthy subjects. The asymmetry of the sympathetic response was greatest when the electrical stimulus was given 2 s after termination of the light or under constant low light conditions. When given 2 s after termination of light, the electrical stimulation increased the mean anisocoria from 1.0 to 1.2 mm in Horner's syndrome (*p* = 0.01) compared to 0.22–0.26 mm in healthy subjects (*p* = 0.1). In all subjects,

the maximal anisocoria induced by the electrical stimulation appeared within a 2 s interval after the stimulus. Correspondingly, the largest change in anisocoria between light and dark without electrical stimulation was seen between 3 and 4 s after light-off. While stronger triple stimulation further enhanced the anisocoria, it was less well tolerated.

Conclusions: Electrical stimulation 2 s after light-off greatly enhances the sensitivity of pupillometry for diagnosing Horner's syndrome. This new method may help to rule in or rule out a questionable Horner's syndrome, especially if the results of topical pharmacological testing are inconclusive.

Keywords: Horner's syndrome, pupillometry, sympathetic activation, electrical stimulation, anisocoria, brimonidine

## INTRODUCTION

The clinical diagnosis of Horner's syndrome (HS) relies on the classical triad of ipsilateral pupillary miosis, blepharoptosis, and facial anhidrosis, which result from an interruption of the sympathetic innervation to the eye and ocular adnexa. Pupillary dilation lag, which is considered to be the most specific feature of Horner's syndrome, is not routinely used for diagnosis due to the difficulty in detecting it with clinical certainty, leaving pharmacologic testing with cocaine or apraclonidine as the gold standard despite their own limitations of availability, added time to a clinic visit, and correct interpretation. The resulting diagnostic uncertainty in borderline cases often leads to patient anxiety, unnecessary neuroimaging and workup.

Previous attempts to use automated pupillometry for the diagnosis of HS through the detection of pupillary dilation lag have shown very high specificity yet the sensitivity was low (1).

Knowing that unilateral Horner's syndrome occurs due to a sympathetic innervation defect in one eye, we suggest that by delivering a generalized sympathetic stimulation to both eyes, we can cause enhancement of the anisocoria in patients with HS but not in healthy subjects.

General sympathetic activation can be achieved through a painful stimulus (2, 3), such as that caused by an electrical surface stimulation to the median nerve at the wrist using a standard electromyography (EMG) machine. Using automated pupillometry, we look for an increase in the anisocoria and difference in pupil dilation velocity in reaction to the enhanced sympathetic activation, to more sensitively detect a unilateral sympathetic innervation deficit.

Patients with HS can have different degrees of ocular sympathetic deficit, depending on the underlying site of nerve damage or the duration of HS. In order to control for those variables, we treated one eye of healthy subjects with 0.2% brimonidine tartrate ophthalmic solution, which is known to induce a sympathetic block in the eye owing to the alpha-2 adrenergic agonist effect (4, 5), blocking the release of norepinephrine, resulting in a complete pharmacological HS.

With this new method, our goal is to enhance the diagnostic accuracy of pupillometry for the diagnosis of Horner's syndrome with electrically induced sympathetic activation.

## MATERIALS AND METHODS

## Protocol Approval, Registrations, and Patient Consents

Written informed consent was obtained from all participants, and the protocol was approved by the Zurich cantonal ethics committee, Switzerland (BASEC-Nr. 2016-02151), in accordance with the Helsinki Declaration.

## Participants

Participants were tested at the University Hospital of Zurich between October 2017 and August 2018. Eighteen patients (8 female) with proven Horner's syndrome (HS) (age mean 59 years, range 38–83 years) and 10 healthy volunteers (4 female, age mean 39 years, range 25–52 years) took part in the study. Etiologies for HS included: long-standing and new onset HS of unknown etiology, surgically induced HS, cervical lesions, and internal carotid artery dissection. Inclusion criteria for the patients' group were: older than 18 years, unilateral HS previously confirmed using cocaine eye drops test (6), no past ocular surgery or trauma with residual iris sphincter damage, and no topical or systemic medications that could affect pupillary responses. Healthy subjects' inclusion criteria: older than 18 years, no past pupillary disorders, no past ocular surgery or trauma, no chronic topical or systemic medications use. Exclusion criteria for both groups: the presence of a cardiac pacemaker or defibrillator.

## Study Design

Case-control study.

## Experimental Procedure

A binocular pupillometer (DP-2000, Neuroptics, Irvine, CA, USA) was used to produce light stimuli and record the pupils of all subjects. Electrical stimulation was provided using a standard electromyography (EMG) machine (Nicolet Viking Quest, Natus Medical Incorporated, Pleasanton, CA, USA).

With the participant sitting and looking into the eyepieces of the pupilometer, binocular pupillary recording (frame rate 30 Hz) was done for each test paradigm (**Figure 1**). In a dark and quiet examination room, the participants were asked to look straight ahead into the video cameras for about 1–1.5 min depending on the test paradigm, during which they were asked to close

their eyes for 4 s at the end of each repetition to prevent ocular irritation and blinking at critical recording times. An electrical stimulus was delivered using the stimulator of the EMG machine to the median nerve at the wrist (**Figure 1A**), similar to that used in the sympathetic skin response test (SSR) (7). Conducting gel [Ten20, Weaver and Company, Aurora, USA] for the tip of the stimulator, as well as a grounding sticker electrode [Neuroline Ground, Ambu, Copenhagen, Denmark] at the back of the hand, were used, as in the standard SSR test. The electric current of a single electrical stimulus was chosen to be 55 mA during 0.2 ms, and a triple stimulus was defined as three consecutive single stimuli (12 Hz). The devices were synchronized so that the pupillometer triggered the EMG machine to deliver an electrical stimulus at specified times as programmed for each test paradigm.

The SSR was recorded from electrodes at both sides of the hand (**Figure 1A**) in 2 patients and 2 healthy volunteers as a reference. SSR represents the potential generated in skin sweat glands in response to sympathetic stimulation. SSR in response to median nerve stimulation was recorded simultaneously to the pupil reaction to compare the timing of the pupillary reflex dilation to the SSR.

Patients with HS received the stimulation to the ipsilateral median nerve (same side as the HS), and healthy volunteers were assigned randomly to receive the electrical stimulation to the right or left median nerve. The purpose of the electric stimulus was to induce a general sympathetic activation through the associated pain, rather than a direct activation of the median nerve (2, 3).

After completing the initial tests, healthy volunteers were treated with 0.2% brimonidine tartrate ophthalmic solution to one eye only, assigned randomly, and the tests were all repeated 45 min after the drop instillation. Brimonidine is an alpha-2 adrenergic agonist, which causes inhibition of norepinephrine secretion, resulting in a sympathetic block to the iris dilator (4, 5), resembling the pupil's state in a complete HS.

## Test Paradigms

Pupil responses were recorded in response to cycles of light and dark, to electrical stimulation during constant low (0.1 log-lux) and high (2.5 log-lux) illumination, and to electrical stimulation at different time points during the cycles of light and dark. Examples of test paradigms are shown in **Supplementary Videos 1**–**5**.

Each subject first underwent baseline binocular pupil recordings, without electrical stimulation. The main paradigm consisted of cycles with 4 s of white light-on (3 log-lux) followed by 20 s of darkness, and was repeated at least four times. Pupil responses were also recorded for 17 s of constant light stimulation with levels of 0.1 and 2.5 log lux. Next, the paradigms were repeated with the addition of electrical stimulation. For the cyclic paradigm, the stimulation occurred at different time points (at 0.5 s before, simultaneously with, and 2 s after the termination of light). Only one stimulation was presented per light/dark cycle, and four repetitions of each stimulation time point were included. In the constant light paradigms, the electrical stimulation occurred after 4 s of constant light stimulation.

To avoid an "order bias" which can be caused by response habituation after repeated nerve stimulation (8), the test paradigms with electrical stimulation were performed in a randomized order for each subject, and a 5–10 min break with turning the room light on and engaging the subject in a conversation were taken half way through the experiment.

In order to assess the pupils' reaction to different levels of electrical stimulation, three of the aforementioned test paradigms, namely the electrical stimulus given at 0.5 s before and 2 s after light-off, and the electrical stimulation alone paradigm with 0.1 log-lux light intensity, were repeated with a triple stimulus, performed last in each testing session.

## Data Analysis and Statistics

Videos recorded by the pupillometer were analyzed with custom programs written in MATLAB and the Image Processing and Statistics toolboxes 2016b (The MathWorks Inc., Natick, Massachusetts, United States). Pupils were found by thresholding the image, the pupil edge identified with the MATLAB function "bwboundaries.m," and an ellipse was fitted to the edge (9). The vertical diameter of the fitted ellipse was used as a measure of pupil size, since this will not change with horizontal eye movements, which seemed more common than vertical

FIGURE 2 | The effect of electrical stimulation ("buzzing") in a patient with Horner's syndrome. The left column shows the pupil response to a cycle of light and dark alone, and the right column shows the response with the addition of electrical stimulation delivered 2 s after light-off (A) Pupils size over time in a patient with HS, as seen on pupillometry with light/dark alone. The dark lines are the means of 5 trials, and the shaded region represents ±1 standard deviation. Time zero indicates extinction of the light, after which the difference between the pupils starts to increase. (B) The difference in mean pupil size, showing a maximal difference of about 0.8 mm, 4 s after the light is turned off (C) Pupil dilation velocity over time for the same test: a difference is noted in the early dilation phase between the healthy and affected pupils. (D–F) When an electrical stimulation is introduced at 2 s after light-off, a clear increase in anisocoria (D,E) and second dilation velocity peak (arrow) of the healthy eye (F) appear.

eye movements in our paradigms (a fixation target was not present during recording, and subjects were reminded to try and maintain straight-ahead gaze as necessary). Recordings of artificial pupils were used to calibrate the pupilometer and to convert pupil diameter from pixels to millimeters. The measured values were removed (usually owing to full or partial blinks or large eye movements) if the fitted ellipse deviated too far from a circle (ratio of major to minor axes >1.3), if the pupil diameter was <1.25 or >8 mm, if pupil constriction/dilation velocity exceeded 10 mm/s, or the duration of the eyes being open was <0.5 s. Entire trials were rejected if more than one third of the data of either eye was lost.

Pupillary dilation lag was defined as the change in anisocoria (difference in pupil diameter) between 5 and 15 s after the light stimulus was removed (1). For patients, we always measured anisocoria as the healthy pupil size minus the affected pupil size; for healthy subjects we took the absolute value of the difference in pupil size. Pilot experiments showed that the effect of electrical stimulation was limited to the 2 s period following stimulation, so we defined the electrically-induced anisocoria as the maximum anisocoria during this period. To determine the effect of electrical stimulation during trials where there was a changing light stimulus, compared to a baseline measure of ansiocoria, we also calculated the maximum anisocoria during the 2 s interval on

the equivalent trials without electrical stimulation. For constant light-on trials, we used the anisocoria just prior to electrical stimulation as the baseline.

To determine the optimal time point for measuring anisocoria that differentiates patients from healthy subjects without electrical stimulation, we calculated the relative change in anisocoria between light and dark over 1 s for each second after light-off. The relative change in anisocoria was defined as the median anisocoria during that time interval minus the anisocoria at the end of the light-on period. The area under the receiver operating characteristic curve (AUC) was then calculated at each time interval.

To evaluate the effect of electrical stimulation, we performed paired t-tests with Holm's correction for multiple comparisons within each subject group. In order to evaluate which electrical stimulation condition produced the most consistent differences from non-electrical stimulation conditions, we calculated the mean differences to z-scores (mean difference/standard deviation of the pair differences). Larger z-scores thus indicated that electrical stimulation produced a larger consistent effect, and could be produced either by a larger mean difference, or by smaller variability.

## RESULTS

We measured 18 patients with Horner's syndrome (HS) and 10 healthy volunteers before and after monocular instillation of brimonidine drops. Using automated pupillometry, we compared the anisocoria and difference in pupillary dilation velocity with and without electrical stimulation.

**Figure 2** shows a representative example of the pupil light responses of a patient with left HS without (left column) and with (right column) electrical stimulation delivered 2 s after the extinction of light. The healthy right pupil (red) of this patient dilated normally in the dark, expanding from a little more than 2 mm in diameter in the light to about 5 mm after 15 s (**Figure 2A**). The affected left pupil (blue), however, dilated similarly in the first second, but then dilation slowed, with a maximum anisocoria of about 0.8 mm appearing about 4 s after light off (**Figure 2B**). In **Figure 2C** the dilation velocity graph for each pupil shows that the right healthy pupil (red) has a larger dilation velocity in the first 3 s after light off. Electrical stimulation 2 s after light off increased the dilation in the healthy right pupil, but had no discernable effect in the affected left pupil (**Figure 2D**), resulting in an increase in maximum anisocoria to about 1.4 mm (**Figure 2E**) as well as an increase in the difference in pupillary dilation velocity between the healthy and affected pupil (**Figure 2F**, arrow).

We also tested the effect of electrical stimulation at different times relative to light-off. **Figure 3** shows the average responses of one patient during different test paradigms. A single electrical stimulus in low light (0.1 log-lux) (**Figure 3B**) produced a small change in anisocoria, whereas the triple stimulus during similar light conditions produced a noticeable dilation response in the healthy eye (**Figure 3C**) causing a larger increase in anisocoria. When electrical stimulation occurred 0.5 s before (**Figure 3D**) or with (**Figure 3E**) the light extinction, the effect of the stimulus was smaller and no discernable increase in anisocoria was noticed. Electrical stimulation at 2 s after light off (**Figure 3F**) produced the largest increase in anisocoria as compared to the other test paradigms.

FIGURE 5 | stimulus 2 s after light-off in a light and dark cycle results in a second dilation peak (arrow) yet does not provoke a difference between the pupils as compared to light and dark alone. In a patient with Horner's syndrome (C,D) as well as in a healthy subject treated with brimonidine (E,F), electrical stimulation results in an increase in the anisocoria as compared to a similar test paradigm of light and dark alone (arrows).

To test the effect of stimulus intensity, we applied three electrical stimuli in quick succession at 12 Hz (triple stimulus). **Figure 4** shows an example of mean pupil responses in a patient with HS to the triple stimulation compared to the single stimulation during a cycle of light and dark. The increase in both the anisocoria (**Figure 4A**) and difference in pupil dilation velocity (**Figure 4B**) produced by the single stimulus were further increased in the same patient when a triple stimulus was given (**Figures 4C,D**).

A representative example of mean pupil responses to electrical stimulation (triple stimulus condition) compared to the condition without electrical stimulation is shown in **Figure 5** in a healthy subject (**Figures 5A,B**), a patient with HS (**Figures 5C,D**), and a healthy subject treated with brimonidine (**Figures 5E,F**). The triple-stimulus produced a prominent increase in anisocoria in the HS patient and the subject with brimonidine for a couple of seconds, whereas no increase or induction of anisocoria was seen in the healthy subject as a result of the stimulation.

Average anisocoria traces for each of the groups are shown in **Figure 6** (**Figures 6A–C** during light and dark cycles, **Figures 6D–F** in constant light-on conditions). As expected, there was little measured anisocoria in healthy subjects, whereas substantial increases in anisocoria were noted in HS patients and healthy subjects with brimonidine in response to electrical stimulation as compared to without. Note that in our patients during cycles of light and dark without electrical stimulation, the average anisocoria was largely constant in the period from 5 s after light off (1.0 mm) to the end of the trial (0.9 mm) (**Figure 6A**).

We observed that the effect of electrical stimulation was generally confined to 2 s after the stimulus. Therefore, we assessed the effect of the electrical stimulation by measuring the maximum anisocoria within 2 s after electrical stimulation, and compared it to the anisocoria during the same time interval in the conditions without electrical stimulation. **Figure 7** shows the measured anisocoria in each condition after electrical stimulation (color bars), along with the associated condition without electrical stimulation for each test paradigm (yellow bars) for comparison. **Figure 7** also shows a measurement of dilation lag as it was previously defined in literature (1, 10) as the change in anisocoria from 5 to 15 s after light off. Of note is that none of our HS patients or brimonidine subjects had an average dilation lag of 0.4 mm or larger with these parameters. In subjects treated with brimonidine the anisocoria slightly increased over time after light off, giving a negative result when calculating the dilation lag using this method.

For HS patients, electrical stimulation produced a significant increase in anisocoria in all test paradigms except for the

patient (A) and brimonidine (B) groups. Stimulation during constant light on of 2.5 log-lux intensity did not increase the anisocoria in any of the 3 groups.

single electrical stimulus given 0.5 s before light off during a cycle of light and dark (**Figure 7A**). The largest increase in anisocoria was found when electrical stimulation occurred 2 s after light off (mean = 1.3 mm, standard deviation (SD) = 0.4, p < 0.01 for difference from baseline t-test) for triple stimulus, and 1.2 mm for single stimulus (SD = 0.4, p < 0.0001) as compared to 1 mm (SD = 0.41, p < 0.0001) without stimulus. Electrical stimulation also significantly increased the anisocoria in the constant light condition, particularly with the lower light intensity of 0.1 log-lux. Subjects treated with brimonidine showed the same pattern of results as the HS, though the amount of anisocoria was higher. Healthy subjects did not, in general, show an increase in anisocoria with electrical stimulation (because we took the absolute difference in pupil size for healthy subjects, any change in anisocoria was likely just an increase in variability). Overall, the increase in anisocoria for the healthy group in response to a single and triple electrical stimuli given 2 s after light of was similar and equal to 0.04 (p > 0.1). Within the healthy group, three subjects had some physiological anisocoria (mean 0.3 mm). The mean increase in anisocoria for those subjects in response to a single and triple electrical stimuli 2 s after light-off was 0.07 and 0.09 mm, respectively, and for subjects without physiological

anisocoria was 0.03 and 0.015 mm, respectively (p = 0.5 and 0.42).

We also determined the best time point after light off at which the change in anisocoria, relative to light on, best differentiated HS from healthy subjects based on pupillometry without electrical stimulation. The time interval after light termination which gave the greatest area under the receiver operating characteristic curve (AUC) and the best discrimination between patients with HS and healthy subjects, was 3–4 s after light-off, with an AUC of 0.98 (**Figure 8**). For this time interval, the best discriminating criterion (cut-off value for relative change in anisocoria) was 0.4 mm. Note, however, that AUC was >0.97 for all time intervals between 3 and 8 s, and was above 0.9 for all time intervals except the first second after light off. The AUC for pupillary dilation lag (change in anisocoria from 5 to 15 s after light-off) was only 0.5, and the AUC for all test paradigms with electrical stimulation was more than 0.97.

To determine which of the electrical stimulation conditions gave the largest consistent change in anisocoria, thus taking within-subject variability into account, we converted the differences (electrical stimulation minus no stimulation) into standardized z-scores. The largest z-score was given by the single electrical stimulus under 0.1 log-lux constant light condition (z

FIGURE 7 | Summary of buzzing effect. Each bar shows the measured anisocoria in response to electrical stimulation compared to the baseline anisocoria (yellow bars) for each matched condition with no electrical stimulation. (A) In Horner's syndrome patients, electrical stimulation produced a significant increase in aniscoria in all conditions except for the single stimulus given 0.5 s before light off. (B) Except for the constant bright light condition (2.5 log-lux), healthy subjects did not show any significant increase in anisocoria in the test paradigms as compared to parallel no electrical stimulation paradigms. (C) Subjects treated with brimonidine showed similar reaction patterns to electrical stimulation as seen in HS group, yet with larger increases in anisocoria.

= 2.6). For the light and dark cycles, the triple stimulus given 2 s after light off condition (z = 1.1) was best, though just slightly larger than the single stimulus 2 s after light off condition.

## DISCUSSION

## Summary of Results