Original Pupillometry in Patients with Leber Congenital Amaurosis: Feasibility Study and Residual Retinal Cell Function
Leber Congenital Amaurosis (LCA) (OMIM 204000) is a devastating, hereditary photoreceptor disease. It is estimated to be responsible for 5% of all inherited retinal disease [1,2] and characterized by severe visual impairment at or near birth, wandering nystagmus, abnormal pupillary responses (amaurotic pupils), a severely decreased or absent electroretinogram (ERG), and high refractive errors [3,4]. At least twenty retinal genes have currently been associated with LCA, en-coding retinal proteins that participate in a wide variety of retinal functions. For example, mutant LCA proteins lead to faulty and aberrant retinal phototransduction (GUCY2D), retinoid cycling (RPE65 and LRAT), ciliary function of the photoreceptor connecting cilium function (RPGRIP, TULP1, LCA5 and CEP290), foveal development (NMNAT1), Muller cell and photoreceptor morphogenesis (CRB1), and other pathological changes (e.g. AIPL1). By pre-genome histological studies of cadaver eyes, LCA was documented to represent at least three major retinal disease types, ranging from a dysfunction, to a degeneration and an aplasia . Recently, Jacobson et al ,performed retinal architecture studies by spectral domain optical coherence tomography SD OCT and suggested that at least four types of LCA can be distinguished, ranging from LCA with nor-mal retinal architecture, to severe degeneration, toretinal thickening, to preserved foveal cones.Our hypothesis is that pupillometry may be able to distinguish these differences in underlying LCA gene defects and their resulting retinal architecture and cellular changes. As we are entering a new era of therapies for retinal degenerations with a wide variety of treatment developments and advancing human clinical trials, there is a critical and urgent need for objective, non-invasive clinical endpoints of visual function. In LCA patients, the clinical gold standard objective test, ERG, often reveals non-recordable signals, which may make it difficult to follow up progression and potential responses to treatments objectively [7-9]. Other gold standard clinical endpoints are subjective tests such as visual acuity (VA), and visual fields (static HVF and kinetic GVF). However, these two clinical endpoints do not fully portray the entire and accurate range of macular and retinal function [10, 11].
Pupillary light response testing as an objective test for retinal cell function has raised interest recently, particularly following the ability of relative selective stimulation of different retinal cell populations by extrinsic light stimuli. The pupillometry information conveyed to the midbrain arises indirectly from the outer retina (rod and cone activation) or directly from within the inner retina (melanopsin activation) . Intrinsically photosensitive melanopsin retinal ganglion cells (IpRGCs) are a specialized subset of ocular neurons capable of intrinsic phototransduction using a unique photopigment (melanopsin), and can contribute to the pupillary response. The purpose of this study is to determine feasibility and reproducibility of pupillometry in young children with LCA, nystagmus and a wide variety of underlying disease mechanism.
Five controls, (NC) (10 eyes, ages ranged from 25 to 40 years old) and thirteen genetically de fined LCA children and young adults (26 eyes, ages ranged from 11 to 31 years old) were recruited. The study was approved by the McGill University Health Center (MUHC) and Montreal Children’s Hospital (MCH) review boards, and all subjects had an informed consent in compliance with MUHC standards. Comprehensive ophthalmic examinations were conducted, with retinal optical coherence topography OCT (HRA+OCT Spectralis, Heidelberg Engineering, Franklin, MA), Goldmann visual fields (Goldmann, Haag-Streit Diagnostics), electroretinogaphy ERG (ColorDome Ganzfeld electroretinogram, Diagnosys, Lowell, MA), and pupillometry (View Point EyeTracker PC-60, Arrington Research Inc., Scottsdale, AZ) performed in all subjects.
LCA patients were genotyped by a variety of molecular genetic techniques, including whole exome sequencing and Sanger sequencing for confirmation. In our study we used an established protocol by Kardon et al12 with a further modification of adding a bright light stimulus of 515 nm wavelength, which falls between the previously studied 640±10 nm and 467±17 nm wavelengths, in an effort to use it as a control and compare pupil response to unselective stimulation (peak ocular spectral sensitivity wavelength is 507nm in scotopic environment, and 555nm in photopic environment) .
During the clinic visits, our subjects underwent a period of dark adaptation period of 15 minutes (by wearing a set of completely dark goggles), followed by pupillometry during which they wore the dual channel binocular eye frame pupillometer (ViewPoint EyeTracker PC-60, Arrington Research Inc., Scottsdale, AZ). This pupillometer consists of a miniature video cameras for each eye with infrared light diode illuminators mounted on a lightweight eye frame (Figure 1). The ERG module (ColorDome Ganzfeld electroretinogram, Diagnosys, Lowell, MA) was used to in-troduce the controlled light stimulus to subjects in scotopic conditions. The spectral bands of light for this study were 640 ±10 nm (red spectrum) to test cone function, the 467±17 nm (blue spectrum) to test rod and ganglion cell function, and 515 nm (white light) for unselective cell stimulation. The light stimulus paradigm consisted of the first light stimulus at 1 cd/m2 for 13 seconds, followed by an increased stimulus intensity to 10 cd/m2 for another 13 seconds, then a further increase in stimulus intensity to 100 cd/m2 foranother 13 seconds. A minimum interval of 60 seconds wash out period in complete darkness was used between different spectral bands testing. The paradigm was designed to use the red spectrum stimulus first, followed by the blue spectrum stimulus, and ends by using the white light stimulus. Each eye was tested separately starting with the right eye, then the left eye, then testing both eyes simultaneously.
A connection system to transfer the analogue output of the light stimulus source machine (Diagnosys) into a digital signal for the pupilometer machine was designed by the Biomedical engineers at the Montreal Children’s Hospital. The connection system allows the pupillometr system to detect precisely when the light stimuli were emitted or had a n increment in intensity, which was essential in outlining different cell types’ pupil response (unpublished methods).
Vertical and horizontal measurements of the pupil were recorded,followed by ellipse (2 dimensional shape) area size calculation using the following formula [(Width x Length) x PI/4 = (W x L) x 3.14/4 = (W x L) x 0.8], arbitrary unites were used. Ellipse area formula was used instead of circle area formula given the non-circular shape of the pupils in some patients (figure 1). Percentage of change between pupillary area size measurements was used to ensure accuracy and overcome any possible variability in distance between pupils and recording cameras between patients. Statistical analysis was conducted using “IBM SPSS statistics 20” software at McGill University facilities.
All LCA patients of all genotypes had some rod, cone and ganglion cell function as measured by pupillometry. A summary of all thirteen patient’s pupil responses in this study are documented in the scatter plot (figure 2), while the actual pupillometry waves are captured in (figure 3). We identified bi-allelic LCA mutations in eight different genes; LCA5, NMNAT1, CRB1, TULP1, CRX, RDH12, RPE65 and AIPL1, reflecting a diverse range of underlying disease mechanisms and severity.
Rod, cone and ganglion cell responses were successfully measured in response to different light stimuli (Table 1). IpRGCs selective stimulation was found to generate the most prominent pupil response in controls and LCA cohorts, except in a single patient with NMNAT1 mutations. Controls had a range of pupillary light response to different stimuli (Figure 4). Cone cells stimulation caused pupillary size reduction ranging from 55% to 81%. On rod cells stimulation, the pupillary size reduction ranged between 55% and 79%. IpRGCs stimulation resulted in the most pupillary size reduction in each subject, ranging between 83% and 91%. We were able to measure cone responses in all 13 patients. Pupillary response to cone cells stimulus was detected in all genotypes, despite non detectable ERG measurements (e.g. Subjects 1 and 3), ranging from -30% dilation (p<0.001) (RPE65) to 64.5% constriction (p<0.001) (NMNAT ).
Patients with lower central visual acuity were found to have the least cone cell stimuli response, for example subject 5 (CRX) had a visual acuity of counting fingers, with a maximum pupillary cone response of 7% OD (p<0.001), subject 11 (CRB1) had hand motion visual acuity, with a maximum cone pupillary response of 4% OS (p<0.001).
Mean pupi ls’ constriction percentages compared to baseline.
Table 1. Mean arbitrary percentages of pupils’ constriction during continuous light exposure (13 seconds) compared to baseline measurements before light stimulation. * ipRGCs: Intrinsically photosensitive melanopsin retinal ganglion cells.
Figure 1. The pupillometry device. Pupillometer device computer screenshots of pupils in the upper half, with round pupil measurement (left top side), and near oval measurement (right top side). Lower half showing the mounted cameras and infrared light sources on control subject #2
Figure 2. All LCA patients’ pupil response compared to normal. Scatter chart, showing pupils response in all pupils (controls and participating subjects). OD pupils are demonstrated on the left side of the graph. OS pupils are on demonstrated on the right side of the graphFigure 3. All LCA patients’ pupil response. Graph chart showing pupilometer responses of all 13 patients, illustrating the OD response (upper graph) and the OS response (lower graph). Red ar- rows represent red light stimulus at 1, 10, and 100 cd/m2. Blue arrows represent blue light stimulus at 1, 10, and 100 cd/m2. Black arrows represent bright white stimulus at 1, 10, and 100 cd/m2. Notice the ladder step change in the pupils’ size in response to increasing light stimulus intensities. We can also notice the maximal constriction on ipRGCs stimulation.
Figure 4. Pupilometer results of a normal control NC#2. This figure demonstrates the pupil response to the three different wavelengths at three different intensities. Red arrows represent the 640 ±10 nm (red spectrum) to test cone cell function at 1 cd/m2 for 13 seconds, followed by an increased intensity to 10 cd/m2 for 13 seconds, then a maximum intensity at 100 cd/m2for 13 seconds. An interval of complete darkness followed for 60 seconds. The same process was repeated using 467±17 nm (blue spectrum) to test rod cell function and ipRGCs (blue arrows). Bright white light (515 nm) was in the same manner to act as a control and simulate unselective 260 cell stimulation (black arrows).
Figure 5. Comparing ERG, to OCT and pupilometer in a patient. This is subject #3 OD. ERG of OD on the top section is a dark-adapted rod ERG. The middle image is a spectral domain OCT of the same eye with complete retinal atrophy. The bottom part represents pupilometer recordings of OD.
Rod cell responses were detected in all subjects ranging from -5% OS (p<0.001) (CRB1) to 70.5% OD (p<0.001) in (NMNAT1). Ganglion cell stimulus resulted in the highest pupillary response in the majority of subjects, ranging from -6% dilatation (p<0.001) (CRB1) to 87% (p<0.001) (RPE65).
Some patients had cone responses that were affected more than rod and ganglion cell responses (likely representing a cone-rod degeneration (CRD) pattern), as in LCA5 or CRX patients (p<0.001). Other LCA patients had rod responses that were more affected than cone and ganglion cell responses (likely representing a rod-cone degeneration (RCD) pattern), as in patients with TULP1 or AIPL1 mutations (p<0.001). In certain genotypes (CRB1, and RPE65), we had several pupil response phenotypes for the same gene mutation, where cone, or rod cells responses were the most affected. Knowing that LCA is a photoreceptor disease that affects outer retinal layers, predicting that cone and rod responses would be the most affected. However, in our NMNAT patient, the right eye had the least ganglion cell response 32% despite 64.5% and 70.5% response on cone and rod cells stimuli respectively (p<0.001).pointing to a more severe inner retinal degeneration. We found several examples of patients with nondetectable ERGs, very thin retinas on OCT, but reliable and repeatable pupil responses (figure 5). This documents the existence of viable functioning retinal cells that were not known to exist in these advanced cases of retinal degeneration.
Pupillometry is possible and repeatable in blind LCA children with various genotypes and ages. All children revealed some rod, cone and ganglion cell function. Pupillometry appears to be feasible and reproducible in children with LCA, severe visual loss, nystagmus and a wide range of genotypes, despite non-recordable ERGs and very thin retina on OCT.
Patients had their pupils tested and measured during their clinic visit, however giving that 12 out of the 13 patients travelled internationally to visit our institute and had a short stay, the pupil lometry measurements were multiply repeated during the same clinic visit.
Differences in baseline pupil measurements between controls and between patients were observed, this can be explained by:1- actual difference in baseline pupil size between participants, or 2- the distance between the pupillometry cameras mounted on goggles and pupils may vary between patients and impact pupils’ measurements. For these reasons, the attention was directed toward measuring the percentage of change in pupillary response compared to baseline, rather than actual pupil size in each subject. Few patients showed ne gative mean pupillary size com pared to their baseline dark-adapted pupil size, which we could not explain but found to be statis tically significant. (Table 1)
Human visual perception is in large part generated by three classes of cone photoreceptors (the L, M, and S cones) with peak sensitivities at long, middle, and short wavelengths of visible light, respectively . Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopig ment melanopsin, which has a peak spectral sensitivity between that of S and M cones [16, 17]. Among other “non-image-forming” functions in the eye, melanopsin-containing ipRGCs con tribute to a delayed and sustained constriction of the pupil .
Pupillometry may be an endpoint worthy of use in human clinical trials for drug and gene thera py as we were able to measure rod, cone and ganglion cell function in all patients in our cohort. Meanwhile, it provides evidence of residual, viable cells previously thought to be lost, giving new hope for novel therapeutic modalities.
The authors sincerely thank and acknowledge the patients and their families for their enthusiastic participation. RKK acknowledges financial support form the Foundation Fighting Blindness Canada (FFB-C), Center for Institutes of Health Research (CIHR), NIH, and MCH foundation.
FFB Canada, NIH, CIHR, MCH Foundation
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