Functional Assessment of the Melanopsin-Containing Retinal Ganglion Cells in Progressive Supranuclear Palsy Using Chromatic Pupillometry
Conditions: PSP - Progressive Supranuclear Palsy, PD - Parkinson's Disease, AD - Alzheimer's Disease, ALS (Amyotrophic Lateral Sclerosis)
Sponsor: Massachusetts General Hospital

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v. 09/18/2017 
 
PARTNERS HUMAN RESEARCH COMMITTEE 
DETAILED PROTOCOL 
 
PRINCIPAL / OVERALL INVESTIGATOR  
Shirley H. Wray, MD, PhD, FRCP 
 
PROTOCOL TITLE  
Functional Assessment of the Melanopsin-Containing Retinal Ganglion Cells in 
Progressive Supranuclear Palsy Using Chromatic Pupillometry 
 
FUNDING 
None 
 
VERSION DATE 
09/18/2017 
 
I. 
BACKGROUND AND SIGNIFICANCE  
a. Historical background  
In 1963, Richardson, Steele and Olszewaki published a landmark clinical 
report on 8 cases of supranuclear ophthalmoplegia, pseudobulbar palsy, 
nuchal dystonia and dementia and established the syndrome of heterogeneous 
system degeneration as a clinicopathological entity now known as progressive 
supranuclear palsy (PSP) (1). 
By 1972, when Steele revisited the subject (2), 73 cases (22 with 
post-mortem examinations) had been described in the medical literature with 
examples discovered across the U.S. I encountered approximately 3 to 4 new 
cases yearly among patients referred for eye movement examinations. 
Rare familial clusters have been described in which the heredity pattern is 
compatible with autosomal dominant transmission with incomplete penetrance 
(3). Rojo and co-workers have described 12 pathologically-confirmed 

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pedigrees (4) and noted the variable phenotypical expression of the disease 
within a single pedigree. 
The disease has a characteristic onset in the sixth decade (range 45 to 75 
years) with some combination of impaired balance, abrupt falls, visual 
disturbances, slurred speech, dysphasia and vague changes in personality. 
Akinesia is responsible for most of these parkinsonian symptoms described 
by patients, all of which relate to slowness and difficulty in initiating, 
implementing, and maintaining motor movements. Akinesia is readily evident 
during simple observation. When the patient is asked to carry out a specific 
motor task and it may lead to a misdiagnosis of Parkinson’s disease (PD) in an 
estimated 5% of patients who actually have PSP (5-7).  
PSP is caused by a novel silent mutation in exon 10 of the tau gene, 
and like frontotemporal dementia and Alzheimer’s disease, it is a tauopathy (8).  
Two distinct phenotypes have been recognized in pathologically proven 
cases (9). One is called Richardson’s syndrome and is characterized by early 
falls, cognitive decline and vertical gaze abnormalities (10) and a second which 
more closely resembles PD with rigidity, tremor and some response to 
treatment with levodopa (9). 
 
Spatial covariance analysis has been used with F-flurodeoxyglucose (FDG) 
positron emission tomography (PET) to identify the disease-related metabolic 
patterns that can serve as biomarkers in assessing PD, atypical PD, and PSP 
(11). 
 
Slowing of voluntary vertical saccades, either down, up or both (10) are a 
diagnostic marker of PSP (10) and later impairment of voluntary horizontal 
saccades are characteristic in more than half of the cases (12). A related sign 
has been the finding of hypometric saccades in response to an optokinetic 
drum moving vertically in one direction (usually best seen with stripes moving 
downward) and eventually all voluntary eye movements are lost (12). However, 
a proportion of PSP patients do not demonstrate these eye signs for a year or 
more after the onset of the disease. Furthermore, Adams and Victor followed 
several patients who had no involvement of their eye movements during life but 

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in whom the typical pathological changes of PSP were found unexpectedly (13) 
and this has to be kept in mind in follow-up. 
 
Other diagnostic features of PSP are slow hypometric horizontal saccades, 
selective defects of visual tracking (14), loss of convergence, and disruption of 
steady gaze by square-wave jerks. Eyelid disorders include reduced blink rate, 
blepharospasm, repetitive blinking in response to flashlight stimulus (failure to 
habituate), and impaired initiation of eyelid opening (apraxia) (15). Delayed 
initiation of eyelid opening in PSP results from metabolic impairment of the 
motor network and cortical degeneration of the frontal lobes (16). One can, in 
advanced cases of PSP, visualize, on MRI of the brain, atrophy of the rostral 
midbrain tegmentum. When detected by mid-sagittal MRI, it looks like the bill of 
a hummingbird and is referred to as the “hummingbird” sign (17). In PSP, the 
sign is due to atrophy of the rostral and caudal midbrain tegmentum and to a 
relative increase in the length of the interpeduncular fossa over that of the 
anteroposterior diameter of the midbrain tegmentum. Its presence on MRI 
helps establish the diagnosis (18) (see Figure 1) (17).   
 
    
 
 
Figure 1. Brain MRI mid-sagittal plain. (A) Normal control. (B) PSP (Progressive supranuclear 
palsy), PD (Parkinson’s Disease). The region, including the most rostral midbrain, the midbrain 

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tegmentum, the pontine base and the cerebellum, appears to correspond to the bill, crown, 
body and wing, respectively, of a hummingbird (e.g., the “hummingbird sign”). (17) 
 
Measurement of the midbrain tegmentum on midsaggital MRI section is 
also considered a reliable way of differentiating PSP from other Parkinsonian 
diseases (15, 16). 
 
b. Previous pre-clinical or clinical studies leading up to, and 
supporting, the proposed research 
    One of the most absorbing features of the eye is the pupil’s reaction to light 
(19, 20). Changes in pupil size in response to a light stimulus, the pupil light 
reflex (PLR), are based on a functional balance between the parasympathetic 
nervous system and the sympathetic nervous system, and balance between 
two neurotransmitters - acetylcholine and noradrenaline (19, 20). The method 
used to optimize pupil measurements, characterize their velocity and determine 
the sensitivity of pupil latency as a function of the light stimulus is a critically 
important element in determining the diagnostic potential of the PLR (21).   
The PLR is driven by intrinsically photosensitive retinal ganglion cells 
(ipRGCs) containing melanopsin (22-24). Melanopsin is a novel photo-pigment 
in the inner retina of both primates and rodents which was discovered only in 
the last ten years (25-26). Melanopsin has since been found to provide a 
unique class of ipRGCs with the capability to act as a third set of photoreceptors 
(27-29), allowing these cells to fire action-potentials on their own when 
stimulated by light (30-32). Melanopsin-mediated ipRGC photoactivity provides 
sustained coding of ambient light irradiance for circadian rhythm (33), regulates 
tonic pupil size and mediates the pupil light reflex hitherto thought to be 
primarily driven by rods and cones. 
 
Melanopsin-containing 
ipRGCs 
show 
prolonged 
depolarization 
after-potential in the dark in response to melanopsin-activating blue light 
leading to a sustained pupil constriction beyond the offset of light stimulus (34).  
Lei et al. (35-37) have now demonstrated that melanopsin containing ipRGCs 

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are selectively sensitive to short wavelength light, with a maximum absorption 
at around 480nm. 
 
Non-visual light detection and photo transduction occur primarily in a small 
group of ipRGCs (27, 29-31) and at least three of several subtypes of ipRGC 
demonstrate distinct anatomic projections with clear functional differences  
(32-34). One major site of ipRGC projection is the suprachiasmatic nucleus, the 
central pacemaker for circadian rhythm in the hypothalamus (39-41) (see 
Figure 2) (41). 
 
 
 
 
 
 
 
 
 
Figure 2. The melanopsin mediated ipRGC pathways projecting to the suprachiasmatic nucleus 
in the hypothalamus and to the olivary pretectal nucleus in the midbrain. (41)  
 
 
Another site of ipRGC projection is the olivary pretectal nucleus (OPN), the 
central integrator of the PLR (42).  
The ipRGCs receive or project modulatory signals from and to neighboring 
cells, including rods and cones which themselves act as local circadian clock 
cells in the retina (43, 44). The relative contribution of rods, cones, and intrinsic 
melanopsin to the ipRGC activity at any moment varies with light 
characteristics, such as wavelength, intensity, and exposure duration (25, 35, 
40, 41). 
 
Three main parameters of the PLR are distinguishable on a 
pupillogram: (1) the immediate pupillary constriction of the dark- or dim light- 
adapted pupil to the abrupt onset of a light stimulus (transient constriction) 

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which is largely due to signalling from activated rod-cones; (2) a more 
sustained state of the pupillary constriction during continuous light stimulation 
to which all photoreceptive elements may contribute; and (3) under certain 
conditions an extended phase of pupillary constriction that persists for some 
time after stimulus light termination (45, 46). This post-stimulus sustained pupil 
constriction derives primarily from intrinsic melanopsin activation of the 
ipRGCSs and is specific to the melanopsin signaling pathway. It has been 
called the ‘post-illumination pupil response’ (PIPR) (47). When it follows 
exposure to bright blue light, it is almost entirely attributable to melanopsin 
ipRGC activity and can therefore be used to estimate functioning of the 
melanopsin signaling pathway itself (48). 
Bergamin and Kardon used a binocular infrared video pupillometer to 
record the dynamic pupil response and time the onset (latency) of pupil 
constriction to a light stimulus in normal subjects (mean age 34). Latency to the 
onset of constriction was defined as the time of maximum constriction 
acceleration (49). The improved technique developed in their study showed 
that pupil latency becomes shorter as a function of light intensity in a 
predictable, non-linear function, and shows less change at higher intensities. 
This has been confirmed by others (46). 
The latency period is composed of two separate mechanisms:  an 
irreducible minimizing latency built into the motor system of the iris, and a 
variable additional delay due to retinal discharges (from ipRGCs) and their 
temporal summation in the midbrain (pretectal olivary nucleus) (49). The 
weaker the stimulus, the longer the latency period (49). 
Latency as a function of light intensity is reproducible in a given eye and a 
similar response is present in the normal right and left eyes. Stergious et al. (47) 
based their pupillometric study of patients with PD, with and without cognitive 
impairment, on Bergamin and Kardon’s study. The characteristic V-shaped 
PLR response to three pupillometric parameters is shown in Figure 3:  A) 
Amplitude, B) Maximum constriction velocity, and C) Maximum constriction 
acceleration (47). 

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Figure 3. This picture shows the characteristic V-shared PLR response in three pupillometric 
parameters: A) Amplitude, B) Maximum Constriction Velocity, C) Maximum Constriction 
Acceleration in three different subjects: 1) a healthy 70-year-old control, 2) a 73-year-old PD 
patient without cognitive disorders, and 3) a 69-year-old PD patient with coexisting cognitive 
impairment. All the aforementioned parameters are impaired in both PD patients, especially in 
the patient with cognitive impairment. (47)  
 
The three parameters were studied in three different subjects: 1) a health 
70-year-old control, 2) a 73-year-old PD patient without cognitive impairment, 
and 3) a 69-year-old PD patient with co-existing cognitive impairment. The 
parameters were impaired in both PD patients, especially in patient 3 with 
cognitive impairment (47). 
 
These pupillometric findings were not, however, attributed to the functional 
impairment or degeneration of amacroinic, dopaminergic cells in the retina. The 
investigators hypothesized that they were attributable to an additional central 
cholinergic deficit (47). At the time, the evidence did not exist to attribute these 
findings to a degenerative process affecting the ipRGCs (44). 
 
c. Rationale behind the proposed research and potential benefits to 
patients and/or society 
The rationale behind using chromatic pupillometry, a novel methodology, as 
an objective in vivo identifier of PSP is based in part on:  

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1) 
Clinicopathological correlation between the key clinical signs of a 
supranuclear gaze palsy with pathological verification that the 
degenerative process affects the pretectum and rostral midbrain, 
2) 
The melanopsin-signaling pathway from ipRGCs in the eye projects to 
the OPN in the midbrain, 
3) 
Chromatic pupillometry is a non-invasive technique suitable for 
elderly subjects with or without dementia. 
 
Because spectral sensitivity or intrinsic melanopsin-mediated 
phototransduction is narrow with a peak at around 482 nm (short wavelength 
light) (35-37), there is a higher threshold to light compared with rod-mediated 
and cone-mediated phototransduction. The activation of ipRGCs has a longer 
latency, a characteristic pattern of discharge of action potentials and a 
discharge rate which builds relatively slowly a maximal sustained firing rate that 
is linearly proportional to light intensity. When the bright light is left on, the firing 
rate of ipRGCs is remarkably steady and sustained without fatigue or 
adaptation to the continuous light stimulation.  When the light is turned off, 
ipRGCs do not immediately stop firing but gradually decrease their firing rate 
until they cease. This long observed sustained pupil constriction, sustained 
after the cessation of a short wavelength high intensity light stimulus, is now 
considered a characteristic electrophysiological signature of melanopsin-driven 
ipRGC intrinsic photoactivity (35-37). Table 1 illustrates the differences in 
pupil-to-light responses of three types of photoreceptors. 
 
 
CONE 
ROD 
ipRGC 
Functions 
photopic, central 
vision 
scotopic, peripheral 
vision 
pupillary response, 
circadian rhythm 
Number 
5 million 
120 million 
4000-12000 
Location 
photoreceptor layer photoreceptor layer 
ganglion cell/inner 

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(outer retina) 
(outer retina) 
plexiform layers 
(inner retina) 
Photopigment 
lodopsin 
rhodopsin 
melanopsin 
Photosensitivity 
low 
extremely high 
low 
Receptive field 
very small 
small 
very large 
Peak absorption 
wavelength (nm) 
420 (S), 534(M), 
564 (L) 
498 
482 
Patterns of light 
pupillary response 
rapid onset, early 
adaptation 
rapid onset, early 
adaptation 
delayed onset and 
prolonged firing 
 
Table 1. Properties of Three Types of Photoreceptors: Cones, Rods and melanopsin retinal 
gangion cells (35-37)  
 
These developments have led to a novel chromatic pupillometry technique 
to measure the PIPR as an index of intrinsic ipRGC activity. This technique 
utilizes color stimuli and infrared pupil tracking systems to measure differential 
pupil responses under continuous light stimulation at selected wavelengths 
over a range of intensities.  
Kankipati et al. (45) showed that the PIPR can be induced in vivo in normal 
human subjects with a chromatic pupillometry technique. Kardon et al. (43) 
provided evidence that a clinical chromatic pupillometry protocol can assess 
differentially the contributions of the rod, cone and IPRGC pathways to the 
pupillary light response. Their test protocol was refined by Park et al. (24) to 
isolate rod, cone and ipRGC contributions to the pupillary light reflex. In an 
experimental series, Shaobo Lei and his co-authors (35-37) further developed 
a comprehensive full-field chromatic pupillometry system to investigate the 
contributions of rod/cone-driven extrinsic ipRGC activity and melanopsin-driven 
intrinsic ipRGC activity to the pupillary light reflex. Using this protocol, a robust 
melanopsin-driven PIPR can be induced with a short flash of blue light of only 
400 ms duration, making the test clinically friendly with good test-retest 
reliability (46). The potential benefit to the patients center on the use of 

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chromatic pupil responses, especially the blue light induced melanopsin-driven 
PIPR, to investigate the role of ipRGCs in a wide range of disease populations, 
including retinitis pigmentosa, achromatopsia, glaucoma and ischemic optic 
neuropathy. The evidence also suggests that the PIPR correlates with other 
anatomical and functional parameters in diseases that primarily affect retinal 
ganglion cells such as glaucoma (50).  
 
II. 
SPECIFIC AIMS  
    The specific aim of this study is to investigate rod, cone and melanopsin 
driven pupillary light response in individuals with PSP and in age-matched 
healthy control individuals and individuals with other neurogenerative diseases, 
using 
chromatic 
pupillometry, 
with 
special 
interest 
in 
assessing 
melanopsin-driven PIPR as an identifier for PSP.  
 
    This study addresses the following hypotheses:  
1) Chromatic pupil responses, including rod/cone-driven rapid phase 
constriction and melanopsin-driven PIPR, are reduced in subjects with 
PSP compared to age-matched normal healthy control subjects, 
2) Pupil parameters of the melanopsin-driven PIPR are abnormal in PSP 
subjects without supranuclear palsy, which is indicative of a subclinical 
physiological deficit of the OPN in the early stages of PSP.  
 
If these hypotheses are upheld, chromatic pupillometry to measure the 
PIPR promises to be a reliable in vivo, non-invasive, convenient and 
inexpensive technique to detect asymptomatic pupillomotor impairment in 
advance of diagnostic oculomotor signs and deterioration of cognitive function. 
 
III. 
SUBJECT SELECTION  
 
Male and female subjects diagnosed with PSP (age 55 years and over) and 
followed at the MGH’s Gerontology Research Unit (GRU) and/or the MGH’s 

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Frontotemporal Disorders Unit directed by Dr. Brad Dickerson (Department of 
Neurology) will be recruited for the study. Age-matched healthy control subjects 
will be recruited from participants enrolled in the Alzheimer’s Disease Research 
Center’s (1999p003693) longitudinal research cohort studies that are 
conducted at the GRU. Age-matched subjects with Parkinson’s disease or 
Alzheimer’s disease will also be recruited from those who are enrolled in 
related research studies conducted in the MGH Neurology Department as a 
comparison group. For data analyses and cross-validation purposes, subjects 
with neuroimaging research data will be targeted for enrollment. 
No individuals will be excluded from study participation on the basis of sex, 
race, ethnicity or any other characteristics (e.g., sexual orientation) that bear no 
effect on the integrity of data for research. 
 
a) 
Inclusion Criteria 
 
1) Individuals that meet the clinical criteria for PSP. Core features include: 
• Recurrent falls and unsteady gait 
• Axial and nuchal rigidity 
• Pseudobulbar palsy  
• Bilateral lid retraction 
• Supranuclear vertical gaze palsy 
• Atrophy of the midbrain tegmentum (the hummingbird sign on  
brain MRI, 
 
2) Individuals that fit the criteria for the second PSP phenotype (which 
resembles PD) that has asymmetric findings, tremors and poor 
responses to treatment with Levodopa, 
 
3) Individuals that meet the clinical criteria for PD with: 
• Progressive bradykinesia 
• Postural instability and frequent falls 
• Festinating gait with loss of associated movements  
• Cogwheel rigidity and mask-like face 
• Rest tremor, 
 

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4) Individuals who carry a diagnosis of Alzheimer’ disease who present with 
progressive impairment of memory and cognitive domains such as 
language and visuospatial perception.  
 
 
 
 
 
Diagnoses will be confirmed by the review of health/medical records of 
patients recruited from the Frontotemporal Disorders Unit clinic. In the case of 
participants recruited from research studies, diagnoses will be confirmed by the 
review of the research diagnoses indicated on the individuals’ research 
records. 
 
b)  Exclusion Criteria 
 
1) Individuals who are frail or in questionable health,  
2) Individuals with cataracts or with posterior pole ocular pathology such as 
age-related macular degeneration and optic neuropathies, including open 
angle high intraocular pressure glaucoma,  
   3) Individuals with photophobia (i.e., painful light sensitivity) when exposed 
to bright light, including those with ophthalmological conditions such as 
keratitis (herpes simplex), uveitis or Achrometopsia, 
4) Individuals with advanced dementia with inability to sit erect, hold the 
eyes open, incontinence,  
5) Individuals with epilepsy, 
6) Individuals diagnosed with major depression or other severe psychiatric 
disorders 
 
Study investigators will use their best clinical judgment when selecting 
potential participants for the study. 
 
IV. 
SUBJECT ENROLLMENT  
a) 
Methods of enrollment 

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Potential study participants will first learn about the study from a clinician of 
the Frontotemporal Disorders Unit or from study staff of related research 
studies in which these individuals are already enrolled. The health/medical 
records of patients treated at the Frontotemporal Disorders Unit clinic will be 
reviewed in order to identify potential participants who meet the 
inclusion/exclusion criteria of the study. For age-matched control subjects 
enrolled in protocol 1999p003693, the Center’s study staff will ask these 
subjects if they would be interested in an ‘eye-test for detecting brain diseases’ 
study that Dr. Shirley Wray is conducting. If so, subjects will be requested to 
leave their contact information so that Dr. Wray or one of her study staff may be 
able to reach them.  Individuals from the Frontotemporal Disorders Unit clinic 
who express interests in learning more about the study and/or to enroll in the 
study will be referred to the study PI (Dr. Shirley Wray) or research staff for 
initial screening and the informed consent process. 
    All potential participants will undergo a brief neuro-opthalmological 
examination (20-30 minutes) by the study PI prior to the start of the study. 
 
 b) Procedure for informed consent 
    Written informed consent for all subjects will be obtained either by a 
board-certified clinician or by a trained research assistant/study coordinator 
under the close supervision of the study PI. Informed consent from 
surrogates for adults who do not have decision-making capacity will only be 
obtained by a licensed physician investigator. Subjects who are consented 
by a non-physician investigator will always be given the opportunity to 
discuss any questions they may have with the study PI.  Potential subjects 
will always be given as much as time as necessary to consider study 
enrollment. Enrolled subjects will be assigned a randomized study ID and 
names or other identifiers (except dates of testing) will never be released to 
anyone who is not an approved study staff on the protocol. All study staff will 
be trained in research ethics on an ongoing basis. 

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    For individuals who may have impaired decision-making capacities 
for research participation, their capacity for initial or ongoing consent/assent will 
be evaluated by our licensed physician-investigators at study enrollment and 
throughout the study. In line with published guidelines on assessing 
competency, we generally regard individuals with a Clinical Dementia Rating 
(CDR) score of 0 or 0.5 as competent to consent for research participation.  
Individuals with global CDR scores of 1.0 or 2.0 are regarded as having 
impaired 
decision-making 
capacities. 
For 
impaired 
subjects, 
the 
PHRC-preferred order of surrogates from whom we will obtain informed 
consent - based on the ‘substituted judgment’ of surrogates - will be followed.  
Study staff will document the relationship of the surrogate to the subject in the 
latter’s research records.  
 
Subjects will not be approached for brain donation/autopsy as part of the 
study. 
 
V. 
STUDY PROCEDURES  
a) 
Study visits 
 
Study visits will be at baseline and approximately at the one-year mark. 
Subjects will primarily be tested at the MGH Gerontology Research Unit located 
in Charlestown. Each study test session, including time for dark adaptation, will 
take no more than 30 minutes. Subjects will not be paid to participate in the 
study. 
 
b) Drugs to be used  
 
Not applicable.  
 
c) Device to be used  
The NeurOptics DP-2000 Multi-Chromatic Desktop Binocular 
Pupillometer (see Figure 3) will be used to test the subjects in this 
non-invasive human study. Pupillometry is a well-established clinical 

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technique in departments of ophthalmic electroretinography to evaluate 
patients with night blindness and for other diseases of the retina. 
 
    DP-2000 is a Class I device and complies with part 15 of the Federal 
Communications Commission (FCC) Rules.  
 
 
Dr. Pat Fortune (VP, Market Sectors) of the Partners Innovation Office is 
processing the necessary agreements with device manufacturer NeurOptics, 
Inc (Dr. Claudio Privitera, PhD) and external collaborator Dr. Shaobo Lei MD, 
MSc, PhD, of the University of Toronto regarding Intellectual Property (IP) 
and related compliance issues in conjunction with the study PI. 
 
 
FIGURE 3. The DP-2000 desktop multi-chromatic desktop binocular pupillometer showing the 
subject’s pupils well in focus in the center of the GUI. 
 
The DP-2000 Pupillometer has the following features: 
 Binocular dual-camera system measures both eyes at once 
 Multi-chromatic (4 ultra-bright color light stimulus:  White, green, blue 
and red) 
 Video stream captured at 30 Hz, 0.05mm pixel resolution 
 Stimulates direct, consensual, or both eyes simultaneously 

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 Pupil variables:  Latency, diameter at onset and peak of constriction, 
constriction and dilation velocity, 75% recovery time 
 Light intensities defined in Lux or pure radiometric units (W/m2); can be 
adjusted over ~ 5 log-unit range by increments of 0.1 log-units 
 Automatic tracking and pupil detection 
 Waveform of light stimulation can be customized using a simple 
graphical interface or defined in a file using Microsoft Excel™ 
 Saves pupil profile and light stimulus data in a file (Excel™ compatible) for 
external/independent analysis 
     Source:  https://neuroptics.com/dp-2000-binocular-pupillometer/   
 
Appendix A contains the pupillary measurement variables.  
 
d) Pupillometry Testing Procedures  
All subjects and age-matched normal controls will be tested with a 
Simplified Chromatic Pupillometry Protocol where rod, cone and melanopsin 
driven pupil responses will be isolated with the following 3 stimulation 
conditions: 
1. 
4 lux red light for 1 s (dark-adapted cone response) 
2. 
4 lux blue light for 1 s (dark-adapted rod response) 
3.   200 lux red light for 1 s (cone response) 
4.   200 lux blue light for 1 s  (melanopsin response) 
 
The test will be done monocularly (if both eyes are eligible, only one eye will 
be randomly selected to be tested); the non-tested eye will be covered by an 
eye-patch. After 20 minutes of dark adaptation, condition 1 and 2 will be 
presented in the dark 3 times at 15 s intervals, then conditions 2-3 will be 
presented in alternating fashion at 1 minute intervals, for 3 times each. The 
protocol, including time for dark adaptation, will be approximately 30 minutes or 
less. 
 

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e) Data to be collected and when the data is to be collected  
Pupil diameter data will be recorded in real time at 30 Hz sample rate. 
Pupillometry data will be stored on an approved password-protected, malware 
protection-enabled computer/laptop that is stored in a secured, limited-access 
testing room located in the GRU. Data will be backed-up on secured drives on a 
regular basis. 
 
VI. 
BIOSTATICIAL ANALYSIS  
 
a) Specific data variables and pupil measurement variables  
Please see below. 
 
b) Study endpoints 
 
Primary endpoints 
1. Maximal Pupil Constriction: the smallest pupil size following light 
stimulation. This parameter primarily represents rapid phase extrinsic ipRGC 
activity driven by rods and cones through synaptic input.  
2. Post-illumination pupil response (PIPR): measured pupil diameter over 
a period of 20 seconds, from 10 to 30 seconds after the offset of light 
stimulation. Previous work found that the cone-driven pupil responses 
subsided within 10 seconds after the offset of light stimuli, so this 
measurement is expected to represent “pure” melanopsin-driven ipRGC 
activity. 
 
 Secondary endpoints: 
Secondary pupil variables are summarized in Table 2. 
 
Broadcast Function 
Range 
Delta= % Change 
% of change (Size-MIN)/Size as a% 
LAT= Latency of constriction 
Time of onset of constriction 

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following initiation of the light 
stimulus 
 
ACV= Constriction Velocity 
Average velocity of how the pupil 
diameters is constricting measured 
in millimeters per second 
MCV= Maximum Constriction 
Velocity 
Maximum velocity of how the pupil 
diameter is constricting measured 
millimeters per second 
ADV= Dilation Velocity 
The average pupillary velocity 
when, after having reached the 
peak of constriction, the pupil tends 
to recover and to dilate back to the 
initial resting size, measured in 
millimeters per second 
T75= Time to reach 75% recovery The time to reach 75% of the 
original baseline pupil diameter 
after the peak of the constriction 
       Table 2. Secondary pupillary measurement variables 
 
All of the data from the DP-2000 laptop will be de-identified/coded and 
transferred by secured-file-transfer means to co-investigators Dr. Shaobo Lei, 
MD, MSc, PhD, Department of Ophthalmology and Vision Sciences, University 
of Toronto, Toronto, ON, Canada and by Dr. Claudio Privitera, PhD, of 
NeurOptics, Inc for analyzes and measurement of pupil variables.  
Dr Lei and Dr. Privitera are experienced clinical/research pupillometric 
experts and their collaboration is essential to ensure accurate measurements 
of the pupil variables and to provide advice to the study PI if any adjustment is 
needed to the research protocol. 
  No Protected Health Information (PHI) or identifiers will be shared with Dr. 
Lei and/or Dr. Privitera except for dates of tests. 

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c) Statistical Methods 
The results collected with the help of Drs. Lei and Privitera will be 
analyzed by our own biostatisticians at MGH. Categorical variables (sex) will 
be compared with Cochra’s Q test. Primary and secondary outcomes will be 
compared using repeated measure ANOVA.  Each ANOVA will have Group 
as a between-subjects factor (2 levels: AD patients, controls), and Testing 
Condition (4 levels: conditions 1-4 ) as a repeated within-subject factor. Main 
effects and interactions will be analyzed further using Tukey’s HSD post-hoc 
tests.  
 
d) Power analysis (e.g., sample size, evaluable subjects, etc.) 
It is expected that a sample size of approximately 20 will be adequate to 
detect differences between subjects with PSP and normal healthy age- and 
sex- matched control subjects. A smaller sample size of approximately 12 
subjects with other neurodegenerative diseases will be used as a pilot study 
group for comparison purposes. 
 
VII. 
RISKS AND DISCOMFORTS  
a) Complications of a non-surgical procedure 
     
Not applicable 
 
b) Drug side effects  
Not applicable 
 
c)  Device complications/malfunctions  
    Care and attention to cleaning and maintenance of equipment will be 
carried out by the operator. The Pupillometer does not require any regularly 
schedule maintenance. If the device is not working properly or is damaged, the 
PI will contact NeurOptics Customer Service. 

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The Pupillometer will only be used by trained study personnel. If a problem 
is recognized while operating the device, the device will be removed from use 
and referred to qualified personnel for servicing. During the testing session, a 
caregiver may remain with the subject at all times together with the tester.  
 
d) Complications 
The pupillometry test described is non-contact and non-invasive. Aside 
from the fact that the bright light may be bothersome for some photophobic 
subjects, there are no foreseeable physical harms to participating in the study. 
The test does not induce headache, dizziness, syncope, epilepsy or panic 
attacks. 
 
e) Psychological Risks 
The subject’s caregiver will be invited to sit in the room while the subject is 
dark-adapted and throughout the test. If the subject experiences any feelings 
of stress, anxiety, discomfort, and an/or fatigue, the patient or the caregiver 
can stop the test at any time.   
 
f) 
Radiation Risks 
None. 
 
VIII. 
POTENTIAL BENEFITS  
a) Potential benefits to participating individuals 
There are no direct benefits to subjects from participating in this 
research.  
 
b) Potential benefits to society 
The ultimate benefit of this research is the development of a non-invasive 
test to contribute additional diagnostic data in the evaluation of individuals 
suspected of having a progressive neurodegenerative disease. 

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The researchers’ observations and experiences will aid in the development 
of an in vivo noninvasive, cost-effective diagnostic chromatic pupillometer to 
monitor disease-modifying therapies in tau-associated neurodengerative 
dementia.  
 
IX. 
MONITORING AND QUALITY ASSURANCE  
The research team will meet regularly to monitor and assure the validity and 
integrity of the data and adherence to the IRB-approved procedures. Quality 
assurance, the safety and monitoring of outcomes are ultimately the 
responsibility of the study PI - Shirley H. Wray, MD, PhD, FRCP.  Should an 
adverse event occur, it will be reported to the IRB in accordance with the IRB’s 
reporting procedures and timelines. 
 
X.  APPENDIX  
 
NeurOptics DP-2000 Multi-Chromatic Desktop Binocular Pupillometer 
The video stream infrared device is captured at 30Hz. The video frame is 
digitized into 640X480 pixels with 8-bit gray level resolution. The profile of light 
stimulated can be customized using a graphic user interface (GUI). Pupillary 
variables are automatically analyzed, reported graphically in a special window 
and saved into a results file. Light stimulation has four different chromatic 
options: Red (622 nm), Green (528 nm), Blue (463 nm) and White. Light is 
emitted through a diffusing screen (approx. 50° x 35° of visual angle).  
The system includes a laptop that has specific technical characteristics and 
cannot be replaced with a different model. Pupillary distance can be adjusted to 
fit the subject’s eyes by turning the knob located below the camera. Camera 
height can be adjusted be pressing the button on the arm and lifting the camera 
to the desired position.  
All possible options to operate the program are listed on the left extreme 
side of the GUI. The live video from the two cameras is displayed in the same 
GUI. Pupil tracking is represented by a circle fitting the contour of the pupil – the 

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subject’s left eye is displayed on the right and the subject’s right eye is 
displayed on the left. This is to maintain the natural perspective of the examiner 
facing the subject during the examination. The left pupil (OS) is tracked with a 
yellow circle and the right pupil (OD) with a green circle. This association 
(left-yellow and right-green) is also used for display purposes. Light stimulus 
waveform and the corresponding pupil response dynamics are shown together 
in the graph located at the bottom of the GUI. Note: the software version can be 
checked by pressing the Version button.  
 
Subject ID 
The first two windows on the top of the list serve to record a subject’s 
randomized ID number and information regarding the current measurement. 
This information is attached to the master data result file produced at the end of 
the measurement and will be used for defining the name of the result file. The 
ID number of each subject will allow the operator to re-check previous studies 
as needed.  
 
Edit Stimulus Profile 
The Edit Stimulus Profile button serves to define and to save a new light 
stimulus profile or to load a predefined profile. The basic unit of a profile is a 
light step during which a light pulse is flashed to the eye and then turned off. 
Light pulses can be repeated a number of times, thereby forming a section. 
Pulses and/or sections can be concatenated in order to define the final light 
stimulus profile. The Edit Stimulus Profile GUI is used to define all the elements 
of the light stimulus profile.  
 
Preparing the subject for the scan and checking the tracking  
The subject will be dark-adapted, sitting in a dark room for 3 minutes with 
the tester, and with their caregiver if requested. They will then be positioned 
close to the camera, with the eye in front of the lens, visible and well in focus in 
the center of the video display in the main GUI. It is advisable to slightly rotate 

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the two cameras (as opposed to keeping the cameras perfectly straight and 
parallel) for a better fit to the subject’s eyes. A green circle should automatically 
track the right eye (displayed on the left) and a yellow circle tracks the left 
(displayed on the right). It is very important to carefully check the accuracy of 
the tracking before initiating the test. If the circles are not precisely around the 
pupil or there is no circle or there is only a red circle moving erratically around 
the video frames and tacking other portions of the image or the green (or 
Yellow) circle turns occasionally into red – all of these are signs of bad tracking 
and a few corrective steps are necessary before initiating the measurement.  
 
 Right IR – Left IR 
Each camera has two pairs of IR LEDs (0 and 1) that can be toggled using 
the IR buttons for the left and right channel. This can be useful for small pupils, 
if one of the IR corneal reflections is overlapping on the pupil, disrupting the 
tracking. Switching to the other pair of IRs can, often, resolves the problem and 
improves the visibility of small pupils. 
 
Results obtained from the Pupillometer scan will be used for information or 
scientific research investigation only, not for clinical diagnostic purposes. The 
subject’s ID will be recorded on the main screen to enable recall of subject data. 
 
XI.      REFERENCES  
1. Richardson JC, Stelle J, Olszewski J. Supranuclear ophthalmoplegia, 
pseudobulbar palsy, nuchal dystonia and dementia. Trans Am Neurol Assoc. 
1963:88:25. 
2. Steele JC. Famillial progressive supranuclear palsy. Brain. 1972;95:693. 
3. Brown J, Lantos P, Stratton M, Roques P, Rossor M. Famillial progressive 
supranuclear palsy. J Neurol Neurosurg Psychiatry. 1993;56:473. 
4. Rojo A, Pernaute RS, Fontan A, Ruiz PG, Honnorat J, Lynch T, Chin S, 
Gonzalo I, Rabano A, Martinez A, Daniel S, Pramsteller P, Morris H, Wood N, 
Lees A, Tagbernero C, Nyggard T, Jackson AC, Hanson A, de Yebenes JG. 
Clinical genetics of familial progressive supranuclear palsy. Brain. 1999; 
122:1233. 

24 
 
 
v. 09/18/2017 
5. Agid Y, Javoy-Agid F, Ruberg M. Pillon B, Dubois B, Duyckaerts C, Hauw JJ, 
Baron JC, Scatton B. Progressive supranuclear palsy: anatomoclinical and 
biochemical considerations. pp. 191-206. in Yahr MD, Bergmann KJ (eds): 
Advances in Neurology. Vol. 45. Raven, New York, 1986. 
6. Albers DS, Augood SJ. New insights into progressive supranuclear palsy. 
Trends Neurosci. 2001;24:347-352. 
7. Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of 
idiopathic Parkinson's disease: a clinico-pathological study of 100 cases. J 
Neurol Neurosurg Psychiatry. 1992;55;181-184 
8. Stanford PM, Halliday GM, Brooks WS, Kwok JB, Storey CE, Creasey H, 
Morris JG, Fulham MJ, Schofield PR. Progressive supranuclear palsy 
pathology caused by a novel silent mutation in exon 10 of the tau gene: 
Expansion of the disease phenotype caused by tau gene mutations. Brain. 
2000;123(Pt 5):880-893. 
9. Wiliams DR, deSilva R, Paviour DC, Pittman A, Watt HC, Kilford L, Holton JL, 
Revesz T, Lees AJ. Characteristics of two distinct clinical phenotypes in 
pathologically proven progressive supranuclear palsy: Richardson's syndrome 
and PSP-parkinsonism. Brain. 2005;128(Pt6):1247-1258. 
10. Eckert T, Tang C, Ma Y, Brown N, Lin T, Frucht S, Feigin A, Eidelberg D. 
Abnormal metabolic networks in atypical parkinsonism. Mov Disord. 
2008;323(5): 727-733. 
11. Friedman DI, Jankovic J, McCrary JA. Neuro-ophthalmic Findings in 
Progressive Supranuclear Palsy. J Clin Neuro-ophthalmol. 1992;12(2): 
104-109. 
12. Daniel SE, De Bruin VM, Lees AJ. The clinical and pathological spectrum of 
Steele-Richardson-Olszewski syndrome (progressive supranuclear palsy): a 
reappraisal. Brain. 1995;118:759-770. 
13. Joshi AC, Riley DE, Mustari MJ, Cohen ML, Leigh RJ. Selective defects of 
visual tracking in progressive supranuclear palsy (PSP): Implications for 
mechanisms of motion vision. Vis Res. 2010; 50(8):761-771. 
14. Adams and Victor's Principles of Neurology. Ropper AH, Brown RH. 8th 
Edition. Chapter 39 p.927. McGraw-Hill 2005. 
 
15. Smith D, Ishikawa T, Dhawan V, Winterkorn JS, Eidelberg D. Lid opening 
apraxia is associated with medial frontal hypometabolism. Mov Disord. 1995; 
10:341-344. 
 
16. Adair JC, Williamson DJG, Heilman KM. Eyelid opening apraxia in focal 

25 
 
 
v. 09/18/2017 
cortical degeneration [letter]. J Neurol Neurosurg Psychiat. 1995; 58:508-509. 
 
17. Iwata M. Humming-bird appearance of mid-brain in MRI of progressive 
supranuclear palsy. Annual Report of the Resarch Committee of CNS 
Degenerative Diseases. The Minister of Health and Welfare of Japan; Osaka 
University. 1994: 38-40. 
18. Oba H, Yagishita A, Terada H, Barkovich AJ, Kutomi K, Yamauchi T, Furui 
S, Shimizu T, Uchigata M, Matsumura K, Sonoo M, Sakai M, Takada K, 
Harasawa A, Takeshita K, Kohtake H, Tanaka H, Suzuki S.. New and reliable 
MRI diagnosis for progressive supranuclear palsy. Neurology, 2005;64(12): 
2050-2055. 
19. Loewenfeld, LE 1999.  The pupil: anatomy, physiology and clinical 
applications. Vol 1. Butterworth-Heinemann, Boston, pp. 83-273. 
 
20. Bitsios P, Prettyman R, Szabadi E. Changes in Autonomic Function with 
Age: A Study of Pupillary Kinetics in Healthy Young and Old People. Age Aging. 
1996;25:432-38. 
 
21. Wilhelm, H Wilhelm B. Clinical applications of pupillography. J 
Neurophthalmol. 2003; 23(1): 42-49. 
 
22. Gamlin PD, McDougal DH, Pokorny J, Smith VC, Yau KW, Dacey DM. 
Human and macaque pupil responses driven by melanopsin-containing retinal 
ganglion cells. Vision Res. 2007;47:946-54. (note: same as 26) 
  
23. Lei S, Goltz HC, Chandrakumar M, Wong AM. Full-field chromatic 
pupillometry for the assessment of the postillumination pupil response driven 
by melanopsin-containing retinal ganglion cellsfull-field chromatic pupillometry. 
Investigative Ophthalmology & Visual Science 2014;55:4496-4503. (note: 
same as 35) 
 
24. Park JC, Moura AL, Raza AS, Rhee DW, Kardon RH, Hood DC. Toward a 
clinical protocol for assessing rod, cone, and melanopsin contributions to the 
human pupil response. Invest Ophthalmol Vis Sci. 2011;52:6624-35. 
 
25. Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD. A 
novel human opsin in the inner retina. The journal of Neuroscience 
2000;20:600-605. 
 
26. Gamlin PD, McDougal DH, Pokorny J, Smith VC, Yau KW, Dacey DM. 
Human and macaque pupil responses driven by melanopsin-containing retinal 
ganglion cells. Vision Res. 2007;47:946-54. (note: same as 22) 
 
27. Lucas RJ, Douglas RH, Foster RG. Characterization of an ocular 

26 
 
 
v. 09/18/2017 
photopigment capable of driving pupillary constriction in mice. Nat Neurosci. 
2001;4:621-26. 
 
28. Kawasaki A, Munier FL, Leon L, Kardon RH. Pupillometric quantification of 
residual rod and cone activity in Leber’s congenital amaurosis. Arch 
Ophthalmol. 2012;130:798-800. 
 
29. Dacey DM, Liao HW, Peterson BB, Robinson FR, Smith VC, Pokorny J, Yau 
KW, Gamlin PD. Melanopsin-expressing ganglion cells in primate retina signal 
color and irradiance and project to the LGN. Nature. 2005;433:749-54. 
 
30. Do MTH, Yau KW. Intrinsically photosensitive retinal ganglion cells. Physiol 
Rev. 2010;90:1547-81. 
 
31. Hattar S, Liao HW, Takao M, Berson DM, Yau KW. Melanopsin-containing 
retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. 
Science. 2002;295:1065-70. (note: same as 40) 
 
32. Hattar S, Lucas RJ, Mrosovsky N, Thompson, S, Douglas, RH, Hankins, 
MW, Lem, J, Giel, M, Hofmann, F, Foster, RG, Yau, KW. Melanopsin and 
rod-cone photoreceptor systems account for all major accessory visual 
functions in mice. Nature. 2003; 424:75-81. (note: same as 38) 
 
33. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells 
that set the circadian clock. Sci Ophthalmol. 2002;106:98-99. 
 
34. Lucas RJ. Mammalian inner retinal photoreception. Curr Biol. 
2013;23:R125-R33. 
 
35. Lei S, Goltz HC, Chandrakumar M, Wong AM. Full-field chromatic 
pupillometry for the assessment of the postillumination pupil response driven 
by melanopsin-containing retinal ganglion cellsfull-field chromatic pupillometry. 
Investigative Ophthalmology & Visual Science 2014;55:4496-4503. (note: 
same as 23) 
 
36. Lei S, Goltz HC, Chandrakumar M, Wong AM. Test–Retest Reliability of 
Hemifield, Central-Field, and Full-Field Chromatic Pupillometry for Assessing 
the Function of Melanopsin-Containing Retinal Ganglion CellsTest–Retest 
Reliability of Chromatic Pupillometry. Investigative Ophthalmology & Visual 
Science 2015;56:1267-1273.  
 
37. Lei S, Goltz HC, Sklar JC, Wong AM. The absence of attenuating effect of 
red light exposure on pre-existing melanopsin-driven post-illumination pupil 
response. Vis Res. 2016;124:59-65. 
 

27 
 
 
v. 09/18/2017 
38 . Hattar S, Lucas RJ, Mrosovsky N, Thompson, S, Douglas, RH, Hankins, 
MW, Lem, J, Giel, M, Hofmann, F, Foster, RG, Yau, KW. Melanopsin and 
rod-cone photoreceptor systems account for all major accessory visual 
functions in mice. Nature. 2003; 424:75-81. (note: same as 32) 
 
40. Hattar S, Liao HW, Takao M, Berson DM, Yau KW. Melanopsin-containing 
retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. 
Science. 2002;295:1065-70. (note: same as 31) 
 
41. Benson DM. Strange vision:  ganglion cells are circadian photoreceptors. 
Trens Neurosci. 2003; 26:314-20.  
 
42. Baver SB, Pickard GE, Sollars PJ, Pickard GE. Two types of melanopsin 
retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic 
nucleus and the olivary pretectal nucleus. Eur J Neurosci. 2008;27:1763–1770. 
 
43. Kardon R, Anderson SC, Damarjian TG, Grace EM, Stone E, Kawasaki A. 
Chromatic pupil responses: preferential activation of the melanopsin-mediated 
versus outer photoreceptor-mediated pupil light reflex. Ophthalmology. 
2009;116:1564-73 
 
44. Markwell EL, Feigl B, Zele AJ. Intrinsically photosensitive melanopsin 
retinal ganglion cell contributions to the pupillary light reflex. Clin Exp Optom. 
2010;93:137-49. 
45. Kankipati L, Girkin CA, Gamlin PD. Post-illumination pupil response in 
subjects without ocular disease. Investigative Ophthalmology & Visual Science 
2010;51:2764-2769. 
 
46. Herbst K, Sander B, Lund-Anderson, Broendsted AE, Kessel L, Hansen MS, 
Kawasaki A. Intrinsically photosensitive retinal ganglion cell function in relation 
to age: a pupillometric study in humans with special reference to the 
age-related optic properties of the lens. BMC Ophthalmol. 2012;12:4. 
 
47. Stergiou V, Fotiou D, Tsiptsios D, Haidich B, Nakou M, Giantselidis C, 
Karlovasitou A. Pupillometric findings in patients with Parkinson's disease and 
cognitive disorder. Int J Psychophysiol. 2009;72:97-101. 
 
48. Van der Meijden et al. Post-illumination pupil response after blue light: 
Reliability of optimized melanopsin-based phototransduction assessment. 
Experimental Eye Research. 2015;139:73-80.  
 
49. Bergamin O, Kardon RH. Latency of the Pupil Light Reflex: Sample Rate, 
Stimulus Intensity, and Variation in Normal Subjects. Invest Ophthalmol Vis 
Sci. 2003; 44:1546-54.  
 

28 
 
 
v. 09/18/2017 
50. Kankipati L, Girkin CA, Gamlin PD. The post-illumination pupil response is 
reduced in glaucoma patients. Invest Ophthalmol Vis Sci. 2011;52:2287-92.