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Chromatic Pupillometry in Patients with Retinitis Pigmentosa
Chromatic Pupillometry in Patients with Retinitis Pigmentosa Randy Kardon, MD, PhD,1,2 Susan C. Anderson, BA,1,2 Tina G. Damarjian, BS,1 Elizabeth M. Grace, BS,1 Edwin Stone, MD, PhD,1,3 Aki Kawasaki, MD4 Objective: To evaluate the chromatic pupillary response as a means of assessing outer and inner retinal function in patients with retinitis pigmentosa (RP). Design: Evaluation of diagnostic technology. Participants: Thirty-two patients with RP and visual loss and 43 normal subjects. Methods: Patients were tested with a chromatic pupillometer using red and blue lights (1, 10, and 100 cd/m2), and their pupil responses were compared with those from 43 normal subjects (reported previously). Visual field and electroretinography (ERG) results were examined and compared with the pupil responses. Main Outcome Measures: The percent pupil contraction of the transient response to a low-intensity (1 cd/m2) blue light and high-intensity (100 cd/m2) red light and the sustained response to a high-intensity blue light was calculated for 1 eye of each subject. Results: The pupil responses to red and blue light at all intensities were recordable in all patients except 1, whose pupil responded only to bright blue light. There was a significant difference of the pupil response between patients with RP and normal subjects in testing conditions that emphasized rod (1 cd/m2 blue light) or cone (100 cd/m2 red light) contribution (P⬍0.001). Patients with a non-recordable scotopic ERG showed significantly reduced pupil responses (P⬍0.001) to low-intensity blue light (1 cd/m2). Patients with a non-recordable or abnormal photopic ERG showed significantly reduced pupil responses (P⬍0.05) to high-intensity red light (100 cd/m2). Patients with a nonrecordable ERG had the most visual field loss and reduced pupil responses. Unexpectedly, patients with RP showed a slower re-dilation of the pupil after termination of bright blue light compared with red light, a pattern not observed in normal subjects. Conclusions: Pupil responses to red and blue light stimuli weighted to favor cone or rod input are significantly reduced in patients with RP but are still recordable in patients having a non-recordable ERG. In addition, outer photoreceptor disease appears to unmask a post-illumination pupillary constriction to bright blue light, most likely mediated by intrinsic activation of melanopsin ganglion cells. Chromatic pupillometry provides a novel, noninvasive method for following retinal functional status, particularly in patients with severe RP and non-recordable ERG. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. Ophthalmology 2011;118:376 –381 © 2011 by the American Academy of Ophthalmology.
The neural origin of the afferent pupillomotor signal derives from a subset of retinal ganglion cells identifiable by their expression of a photopigment called “melanopsin.”1,2 Because melanopsin can directly absorb photic energy and initiate the process of phototransduction, the melanopsinexpressing retinal ganglion cells (MGCs) are capable of generating and discharging an electrical (action) potential in response to light exposure without synaptic input from outer retinal photoreceptors.3 In addition to their capacity for intrinsic activation, the MGCs also are activated by extrinsic signaling originating from the outer retinal photoreceptors.4 – 6 The pupil light reflex evoked with a bright white light stimulus in a non– dark-adapted eye is driven, for the most part, by cone “ON” signals to the MGCs. In a previous article, we described a novel technique for obtaining differential pupil responses using continuous light at selected wavelengths over a range of intensities.7 Such pre-selection of the light intensity, wavelength, and duration was based
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on current knowledge of the differing functional properties of the rods and cones and melanopsin-mediated intrinsic activation and was intended to evoke pupil responses that emphasized activity primarily from 1 input source, that is, rods versus cones versus melanopsin.8 –10 The differential pupil responses to the testing conditions of our previous study suggested that the pupil response obtained in response to a low-intensity blue light stimulus may be a reasonable marker of rod activity. Likewise, the pupil response to a high-intensity red light stimulus was thought to represent a predominantly cone-driven response. The pupil response, particularly the sustained contraction, to continuous highintensity blue light was believed to derive primarily from direct, intrinsic activation of melanopsin ganglion cells. This current article describes the application of the methodology of differential pupil light reflex testing in a nonselected group of patients with RP. We desired to understand how the pupil responses to these selected stimulus condiISSN 0161-6420/11/$–see front matter doi:10.1016/j.ophtha.2010.06.033
Kardon et al 䡠 Pupil and Photoreceptor tions differ between patients with rod and cone dysfunction and subjects with healthy eyes and how the pupil responses in the patients compare with other tests of visual function, such as the electroretinogram (ERG) and kinetic (Goldmann) perimetry.
weighted” pupil response for purposes of this study. Melanopsin activity was estimated from the sustained pupil response to 100 cd/m2 blue light because of its distinctive property of non-fatigability to a steady state light of short (blue) wavelength.
Analysis
Materials and Methods Patients The study was conducted according to the tenets of the Declaration of Helsinki and received approval from the University of Iowa Institutional Review Board committee. All patients gave informed consent for study participation. Thirty-two consecutive patients with a diagnosis of retinitis pigmentosa (RP) underwent pupil testing at the time of their clinic appointment in the Retina Clinic of the University of Iowa (author ES) between 2007 and 2008. Inclusion criteria included abnormal fundus appearance (pigmentary disturbance or atrophy or diffuse arteriolar narrowing observed both clinically and on fundus photographs) and a previously recorded ERG that was abnormal in either scotopic or photopic conditions or both (International Society for Clinical Electrophysiology of Vision protocol; absent or diminution of b-wave amplitude below the 5th percentile or prolongation of implicit times compared with age-matched normal subjects). None of the patients had a second ocular diagnosis involving the retina or optic nerve, or any condition that affected the efferent pupil response. Gender, diagnosis, or genetic defect if known, kinetic visual field assessment using Goldmann perimetry, and ERG results were recorded. The eye with the greater visual field loss was selected for pupil testing.
Chromatic Pupillometry The dynamics of the pupil movement to stepwise increases in intensity of red and blue light were recorded by a computerized infrared pupillometer using a protocol previously described.7 In brief, a ColorDome Ganzfeld ERG apparatus (Diagnosys, Lowell, MA) was used to present a continuous light stimulus (duration specified by software) at a number of predetermined spectral bandwidths. The spectral bands chosen for this study were 640⫾10nm (red light) and 467⫾17 nm (blue light) under mesopic conditions of adaptation. Three stimulus intensities (1, 10, and 100 cd/m2 photopically matched for intensity for blue and red light stimuli) and stimulus duration of 13 seconds were used. For each wavelength, the light stimulus was presented as a continuous stepwise increase in stimulus intensity. In this study, the untested eye was occluded with a patch, and the tested (stimulated) eye was also the monitored eye. A dual-channel binocular eye frame pupillometer worn by the subject (Arrington Research, Scottsdale, AZ) was used to record the pupil diameter of the stimulated eye at 30 times per second. The details of this instrument and the analysis of recordings have been described in a previous publication.7 The main measurement parameter for the pupil response was percent change in pupil size from baseline size. The transient response was operationally defined as the maximal percent change from the baseline pupil size during a time window 180 to 500 ms after the onset of stimulus intensity. The sustained response was the amount that the pupil had remained contracted at the last (13th) second of light stimulation. Figure 1 shows an example of a pupil tracing of a normal subject (available at http://aaojournal.org). We selected the transient pupil responses to 1 cd/m2 blue light and 100 cd/m2 red light to differentiate between rod and cone input to the pupil light reflex and termed these the “rod-weighted” and “cone-
The distribution of pupil responses to each of the aforementioned stimulus conditions was analyzed for all 32 patients and compared with 43 normal subjects using parametic (t test and analysis of variance [ANOVA] for normally distributed data) and nonparametric (Mann–Whitney rank sum test) paired statistical comparisons. SigmaPlot version 11.0 (Systat Software Inc., San Jose, CA) was used for all statistical analyses. The patients with RP were then categorized into 3 groups according to their electrophysiologic status. Group 1 included patients with a non-recordable ERG, Group 2 included patients with an abnormal but recordable ERG (⬍5th percentile of normal), and Group 3 included patients with a normal ERG. This categorization was performed for the scotopic ERG and photopic ERG results separately. The distribution of the “rod-weighted” and “cone-weighted” pupil responses for the 3 groups of patients was examined and compared with 43 normal subjects using 1-way ANOVA (paired contrasts were significant at the Pⱕ0.05 using the Holm–Sidak method for normally distributed data and Dunn’s method for nonparametric data analyzed by rank). The intrinsic, melanopsin-driven pupil response (that is, the sustained response to 100 cd/m2 blue light) also was examined for each of these patient groups. Finally, the distribution of the quantified visual field score for the 3 groups of patients was analyzed using 1-way ANOVA between groups (see details below). The Goldmann visual field (GVF) for each patient was objectively quantified using a manual grid scoring system originally described by Esterman11 and applied to GVF by Kwon et al.12 The grid measures the area contained within the I4e isopter using a template composed of 100 dots. The grid template on a transparency is overlaid on the visual field, and dots that fall completely within the I4e isopter are manually counted. A perfect score using this scoring system is 100. Each GVF was also subjectively assessed for total volume of visual field loss and ranked from greatest to least by 2 of the authors who were each masked to patient and diagnosis (RK and AK). These visual field analysis techniques were correlated with each other and with pupil responses using Spearman and Pearson correlation analyses. The distribution of visual field loss was also compared in each of the 3 ERG groups. Spearman and Pearson correlation analyses were performed. To study the effect of severe photoreceptor disease on the bright blue stimulus offset pupil response, a subset of 13 patients with a non-recordable ERG to the combined response (scotopic response to the maximal International Society for Clinical Electrophysiology of Vision standard white light) was selected for further pupil analysis. The pupil tracings were qualitatively reviewed by 2 of the authors (RK and AK) for delay in re-dilation to baseline size after termination of the 100 cd/m2 bright blue light compared with the equivalent photopically matched red light stimulus. This was best observed as an asymmetry of the pupil redilation slope during the light “OFF” period between the red and blue light stimulus conditions. The superimposed digital pupil tracings from the photopically matched red and blue light stimuli were analyzed by tracing a straight line over the first 20 seconds of the pupil re-dilation phase after light offset. The difference in the slope of the 2 lines was evaluated, and the pupil tracings were ranked in order of slope asymmetry.
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Results Thirty-two patients were included in this study (17 women and 15 men). Diagnoses were RP in 30 patients and Stargardt’s disease in 2 patients. Two patients with RP demonstrated genetic transmission consistent with an autosomal dominant form of RP, 5 patients had an autosomal recessive form, and 4 patients had an X-linked form. A recordable pupil response was obtained in all patients except for 1 patient with X-linked RP who had no light perception, had a flat ERG, showed no pupil response to 100 cd/m2 red light, but had pupil responses to blue light. Overall, there was a significant reduction of the rod-weighted (1 cd/m2 blue) and coneweighted (100 cd/m2 red) pupil responses of the 32 patients (median percent pupil contraction ⫽ 29.6% rod-weighted and 44.5% cone-weighted) compared with 43 normal subjects (median percent pupil contraction ⫽ 39.3% rod-weighted and 53.7% coneweighted). The stimulus condition weighted to favor the intrinsic melanopsin activity (100 cd/m2 blue) also showed reduced pupil response in patients with RP compared with normal subjects (median value ⫽ 50.9% and 56.8%, respectively). The details of the descriptive statistics for patients and normal subjects are shown in Table 1 (available at http://aaojournal.org) and Figure 2. Twenty of 32 patients showed loss of the rod-weighted pupil response below the 25th percentile of normal, whereas 22 patients showed loss of the cone-weighted pupil response below the 25th percentile. Twenty-five of 32 patients had an abnormal b-wave amplitude on ERG under both scoptic and photopic conditions. Thirteen patients had a non-recordable combined response. Grouped by their scotopic ERG results, 17 patients had a non-recordable ERG (Group 1), 8 patients had an abnormal but
Figure 2. Scatterplot distribution of the pupil responses to a chromatic light stimulus obtained from 32 patients with RP superimposed on normative box plots. Left graph shows the transient pupil responses to a blue light presented at 1 cd/m2 (rod-weighted responses), the middle graph shows the transient pupil response to a red light presented at 100 cd/m2 (cone-weighted responses), and the right graph shows the sustained pupil responses to a blue light at 100 cd/m2 (melanopsin-mediated pupil responses). Each box plot shows the median, 75th, 25th, 95th, and 5th percentile levels for the group of 43 normal eyes.
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recordable ERG (Group 2), and 7 patients had a normal ERG (Group 3). The dot-plot distribution of the rod-weighted pupil responses for these 3 groups of patients is shown in Figure 3 (available at http://aaojournal.org).). Grouped by their photopic ERGs, 17 patients had a non-recordable ERG (Group 1), 11 patients had an abnormal but recordable ERG (Group 2), and 4 patients had a normal ERG (Group 3). A dot plot of the coneweighted pupil response for these 3 groups of patients is shown in Figure 4 (available at http://aaojournal.org). The pupil responses were significantly reduced in Group 1, who had the most severe electrophysiologic abnormality (non-recordable scotopic ERG or non-recordable photopic ERG) compared with normal subjects (P⬍0.001 and P⬍0.05, respectively). Group 1 also showed significant reduction in their melanopsin-mediated pupil response, as seen in Figure 5 (available at http://aaojournal.org). There was a significant difference between patients with an abnormal, but not flat, photopic ERG (Group 2) and normal subjects (P⬍0.05). There was no difference in the rod-weighted, cone-weighted, or melanopsinmediated pupil responses of patients with RP with normal ERG compared with those of normal subjects. The patterns of visual field loss in the 32 patients with RP included ring scotoma, generalized constriction, and large central scotoma. The GVF score ranged from 0 to 89 (maximal score possible ⫽ 100). The distribution of the GVF score showed a similar pattern to the pupil responses of patients in Groups 1, 2, and 3 for both scotopic and photopic ERG conditions, shown in Figure 6 (available at http://aaojournal.org). The patients with the non-recordable ERGs tended to have the lowest GVF scores, and the patients with normal ERGs generally had the highest scores. The visual field score of Group 1 was significantly lower (P⬍0.05) than in Groups 2 and 3 for scotopic and photopic conditions. Group 2 patients with abnormal but recordable ERG showed the widest range of GVF scores. There were 13 patients who had a non-recordable combined response on their ERG. The rod-weighted pupil response ranged from contraction amplitudes of 9% to 43%, and their coneweighted pupil response ranged from 0% to 61%. Although, as a group, the melanopsin-mediated pupil response was lower than in the normal eyes, this difference was not significant. Patients with RP commonly showed an asymmetry in the rate of re-dilation after light termination (light OFF period), in which the pupil tended to remain small and constricted to a bright blue light stimulus (100 cd/m2) compared with a photopically matched red light (100 cd/m2). Normal eyes did not show this feature (Fig 1, available at http://aaojournal.org). The re-dilation dynamics were symmetric between the blue and red light stimulus conditions in normal eyes. There was no correlation between the rank score associated with the amount of delayed dilation and the magnitude of the light onset pupil response (rod-weighted or cone-weighted) or the amount of visual field loss (GVF score). There was a good correlation between the GVF rank by subjective assessment (authors RK and AK) and the objective GVF score (P⬍0.05, r2⫽0.75) in this subset of 13 patients, indicating that the dot overly quantification method did well to reflect the estimated overall volume of visual field loss. To better illustrate the patterns of pupil responses observed among the patients with RP with a non-recordable combined ERG response, 3 patient examples are shown. Figures 7 and 8 show 2 patients with a similar amount of severe kinetic visual field loss but very different pupil responses to red and blue light. One patient (Fig 7) shows abnormally reduced rod and cone-weighted pupil contractions but preservation of the melanopsin-mediated pupil response, whereas the other patient (Fig 8) shows pupil responses that are all within the normal range, despite the severe visual field loss and non-recordable combined ERG. Notably, both of these patients show an asymmetry of the pupil re-dilation dynamics during the light OFF period between the red and blue light con-
Kardon et al 䡠 Pupil and Photoreceptor
Figure 7. Patient with X-linked RP and a non-recordable combined response on ERG. The upper left image shows arteriolar narrowing, pigment clumping, and pigment epithelial atrophy in the right eye. Goldmann perimetry (upper right) of the same eye shows a severely constricted visual field. The pupil diameter change in response to stepwise increases to photopically matched red and blue light stimuli (bottom left). The patient’s rod-weighted, cone-weighted, and melanopsin-mediated pupil response superimposed on the normative box plot (bottom right). Note that the rod and cone-weighted responses are severely reduced, to near or below the 5th percentile of normal subjects. The melanopsin-mediated pupil response, however, is preserved. Note the asymmetry in the pupillary re-dilation during the light OFF period on the pupil response curves (bottom left). The pupil is slower to re-dilate back to baseline (shallower slope) after offset of the bright blue light compared with offset of a photopically matched equiluminant red light.
Figure 8. Patient with X-linked RP and a non-recordable ERG. The fundus photograph demonstrates the severe pigment epithelial atrophy with clumping and arteriolar narrowing in the right eye (upper left). Goldmann perimetry of the same eye (upper right) shows severe constriction with a quantified visual field score similar to the patient shown in Figure 7. Yet this patient’s rod-weighted, cone-weighted, and melanopsinmediated pupil responses lie surprisingly within the 25th to 75th percentile range of normal subjects (bottom right). Examination of the pupil response curves (bottom left), however, reveals an asymmetry in the pupil re-dilation dynamics as seen in the light OFF period of the tracing. Note that after exposure to the bright blue light, the pupil tends to stay partially contracted for several seconds and the rate of re-dilation to baseline size is much slower compared with red light exposure.
ditions. After bright blue light exposure, the pupils remained partially contracted and were slower to return to baseline size. Figure 9 is a third patient with RP whose visual field loss is primarily within the central location. The rod-weighted and coneweighted pupil responses are severely reduced, and there is some reduction of the pupil response weighted to melanopsin activation (percent contraction is less than the 25th percentile). Although a delay in return to baseline size after the offset of bright blue light (100 cd/m2) is noted, the asymmetry in pupillary re-dilation dynamics between red and blue light conditions is not as striking as in the 2 patients shown in Figures 7 and 8.
Discussion This study examined the pupil light reflex using a novel protocol with red and blue light stimuli in patients with rod and cone dysfunction. We had previously developed the stimulus conditions based on light wavelength, intensity, and duration intended to bias the input in the afferent pupillomotor signal to favor rods, cones, or melanopsin activation.7 After testing a group of normal subjects, we applied the protocol to a nonselected group of patients with RP and evaluated their pupil responses compared with the normal group. Our assumption was that if our stimulus conditions did indeed represent preferential activation of the outer photoreceptors, our so-called rod-weighted and cone-
Figure 9. Patient with rod-cone dystrophy and a non-recordable combined response on ERG. An abnormal fundus with pigment epithelial atrophy particularly notable in the macula (upper left). Goldmann perimetry (upper right) shows central visual field loss. The patient’s rod-weighted, coneweighted, and melanopsin-mediated pupil response superimposed on the normative box plot (bottom right). Note that the rod and cone-weighted responses are severely reduced and well below the 5th percentile of normal subjects. There is also reduction of the pupil response weighted to melanopsin activation to less than the 25th percentile. There is a mild delay in re-dilation after the bright blue light (100 cd/m2) is turned off, but it is not as pronounced as in the 2 patients shown in Figures 7 and 8.
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Ophthalmology Volume 118, Number 2, February 2011 weighted pupil responses would be reduced in patients with RP, whereas the melanopsin-mediated pupil response would remain preserved, because it does not require signaling from the outer photoreceptors to be activated. Our findings largely support this premise. We found that the median pupil responses of the patients, including the melanopsin-mediated response, were significantly reduced compared with the normal subjects, but their range of responses was large. To look for a relationship between the pupil response and the functional photoreceptor deficit, we reexamined the pupil responses after dividing the patients by the degree of their ERG abnormality (nonrecordable, abnormal, or normal in the scotopic and photopic conditions). In other words, the “rod-weighted” pupil response (transient response to a 1 cd/m2 blue light) was compared with the scotopic ERG and the “cone-weighted” pupil response (transient response to a 100 cd/m2 red light) was compared with the photopic ERG. Not surprisingly, patients with a non-recordable ERG tended to have the greatest loss of pupil response, and the median pupil response (rod-weighted and cone-weighted) from this group was significantly lower than in the other 2 groups (patients with an abnormal ERG and patients with a normal ERG). Likewise, the rod and cone-weighted pupil responses tended to be preserved in those patients with normal ERGs, and the difference from normal subjects was not significant. Although these results are highly promising for use of the chromatic pupil light reflex as an alternative, objective parameter of outer and inner retinal function in disease states, the current test protocol still has not been optimized for isolating rod, cone, and melanopsin-mediated pupil responses. Two findings from this study suggest that the stimulus conditions used in this study are not selective enough in activating a single population of photoreceptors. Among the patients with RP who had an abnormal but recordable scotopic ERG, their rod-weighted pupil response was not significantly different (reduced) compared with normal subjects. Yet these patients had clear evidence of field loss and electrophysiologic dysfunction, and we would have expected their pupil response to be similarly abnormal. Admittedly, there might simply not have been enough patients in this group (n⫽8) to reflect any difference in median values. In addition, the spatial summation of pupil response across the retina may be uniquely different than what is measured with a Ganzfeld ERG and what is measured with kinetic perimetry. Yet it may also be that our measure of rod function, defined in this study as the pupil response to a 1 cd/m2 blue light stimulus, is not a selective enough measure of pure rod activity. That cones are simultaneously activated during this light stimulus condition is suggested by the subjective reporting of blue color perception during testing. We currently are developing more selective stimuli using a much lower-intensity blue stimulus under conditions of dark adaptation for the rod-mediated response and bright red stimulus on a rod-suppressing background stimulus for a cone-mediated pupil response. The other unexpected finding of this study was that the intrinsic, melanopsin-mediated pupil response was significantly reduced in the group of patients with non-recordable scotopic or photopic ERG. From a pathophysiologic stand-
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point, this might suggest that RP in its later stages can cause ganglion cell loss or that outer photoreceptors have an enhancing influence on intrinsic phototransduction in ganglion cells that is lost in the advanced stages of disease.13 But it is also possible that our measure of melanopsin function, the pupil response to continuous bright blue light, does not isolate intrinsic melanopsin activity completely but also includes rod and cone input to the pupil light reflex. Even with sustained, steady bright light stimulation, rods may be contributing a constant level of input to the melanopsin ganglion cell signal, as well as cones. In such a situation, a severe loss of rod or cone function might be the reason for the reduced pupil response to 100 cd/m2 blue light seen in our patients with RP with a non-recordable ERG. Further refinement of the stimulus condition and pupil analysis to light onset and offset is being developed for better isolation of the intrinsic melanopsin-mediated pupil response. Our results provide evidence that the sustained pupil contraction after blue light offset compared with red light offset may provide additional diagnostic information correlating to the status of outer photoreceptor disease. In conclusion, this study demonstrated that patients with RP have reduced pupil responses to red and blue light and that the loss of pupil function mediated by outer retinal photoreceptors appears most substantial in those who had a non-recordable ERG. Yet despite the absence of any recordable electrophysiologic signal of rod or cone function, all these patients except 1 had a recordable pupil response. This suggests a potentially important role for chromatic pupillometry, namely, that it may be a clinical test that extends the dynamic range for monitoring outer photoreceptor function. This is a particularly relevant situation in the treatment of patients who have severely constricted visual fields and a flat ERG. Following the pupil responses to red and blue light may be one way to assess functional recovery of the outer photoreceptors to assess new treatments.14 In addition to the static measure of pupil response to a light stimulus, we observed a delay in pupil re-dilation in the eyes of patients with RP after termination of the bright blue light compared with red light offset in the eyes of patients with RP. In a recent article by Kankipati et al,15 the authors described a post-illumination pupil constriction after offset of a bright blue light in normal subjects and ascribed the phenomenon to melanopsin activity. Because our blue stimulus condition did not evoke a post-illumination constriction in any of our normal subjects, we were surprised to see its occurrence in the patients with RP. This suggests that outer photoreceptors have a modifying influence on the intrinsic MGC signal and that assessment of the light OFF period may add diagnostic information for monitoring outer retinal dysfunction. Although the present test protocol does not yet seem to be maximally optimized for selectively activating rods, cones, and melanopsin-mediated pupil responses independently, the results of our study support the potential usefulness of chromatic pupillometry as a noninvasive method for monitoring outer and inner retinal function and also may be a unique means for following patient status when the ERG has become non-recordable.
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References 1. Hattar S, Lucas RJ, Mrosovsky N, et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 2003;424:76 – 81. 2. Güler AD, Ecker JL, Lall GS, et al. Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision. Nature 2008;453:102–5. 3. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 2002;295: 1070 –3. 4. Jusuf PR, Lee SCS, Hannibal J, Grünert U. Characterization and synaptic connectivity of melanopsin-containing ganglion cells in the primate retina. Eur J Neurosci 2007;26:2906 –21. 5. Wong KY, Dunn FA, Graham DM, Berson DM. Synaptic influences on rat ganglion-cell photoreceptors. J Physiol 2007; 582;279 –96. 6. Schmidt TM, Taniguchi K, Kofuji P. Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development. J Neurophysiol 2008;100:371– 84. 7. Kardon R, Anderson SC, Damarjian TG, et al. Chromatic pupil responses: preferential activation of the melanopsinmediated versus outer photoreceptor-mediated pupil light reflex. Ophthalmology 2009;116:1564 –73.
8. Dacey DM, Liao HW, Peterson BB, et al. Melanopsinexpressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature 2005;433:749 –54. 9. Gamlin PDR, McDougal DH, Pokorny J, et al. Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Res 2007;47:946 –54. 10. McDougal DH, Gamlin PD. The influence of intrinsicallyphotosensitive retinal ganglion cells on the spectral sensitivity and response dynamics of the human pupillary light reflex. Vision Res 2010;50:72– 87. 11. Esterman B. Grid for scoring visual fields. II. Perimeter. Arch Ophthalmol 1968;79:400 – 6. 12. Kwon YH, Kim CS, Zimmerman MB, et al. Rate of visual field loss and long-term visual outcome in primary open-angle glaucoma. Am J Ophthalmol 2001;132:47–56. 13. Wan J, Zheng H, Hu BY, et al. Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina. Neurosci Lett 2006;400:48 –52. 14. Maguire AM, High KA, Auricchio A, et al. Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 2009;374: 1597– 605. 15. Kankipati L, Girkin CA, Gamlin PD. Post-illumination pupil response in subjects without ocular disease. Invest Ophthalmol Vis Sci 2010;51:2764 –9.
Footnotes and Financial Disclosures
Financial Disclosure(s): The author(s) have made the following disclosure(s):
Supported by a Merit Review and Rehabilitation Award from the Veterans Administration, Washington, DC (Dr. Kardon, VA Center of Excellence for the Prevention and Treatment of Vision Loss). Dr. Kardon is the recipient of a Lew Wasserman Scholar Award (Research to Prevent Blindness) and the Pomerantz Chair in Ophthalmology. Dr. Kawasaki was supported in part by a grant from the Foundation for Research in Ophthalmology and Loterie Romande Swiss. Unrestricted support was also received from Research to Prevent Blindness (New York, NY). Dr. Stone receives support as an Investigator of the Howard Hughes Medical Institute and from the Carver Family Center and the Center for Macular Degeneration. This article contains additional online-only material. The following figures should appear online-only: Figures 1 and 3 to 6, and Table 1.
Dr. Kawasaki is a paid consultant by Bayer SpA, Milano Italy. None of the authors have a proprietary interest in any of the instrumentation, technology, or products mentioned in this manuscript.
Correspondence: Aki Kawasaki, MD, Hôpital Ophtalmique Jules Gonin, Avenue de France 15, Lausanne-1004, Switzerland. E-mail: [email protected].
Originally received: February 5, 2010. Final revision: June 21, 2010. Accepted: June 25, 2010. Available online: September 23, 2010.
Manuscript no. 2010-194.
1
Department of Ophthalmology and Visual Science, University of Iowa, Iowa City, Iowa.
2
Veterans Administration Hospitals, Iowa City, Iowa.
3
Howard Hughes Medical Institute, Chevy Chase, Maryland.
4
Hôpital Ophtalmique Jules Gonin, University of Lausanne, Switzerland.
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The neural origin of the afferent pupillomotor signal derives from a subset of retinal ganglion cells identifiable by their expression of a photopigment called “melanopsin.”1,2 Because melanopsin can directly absorb photic energy and initiate the process of phototransduction, the melanopsinexpressing retinal ganglion cells (MGCs) are capable of generating and discharging an electrical (action) potential in response to light exposure without synaptic input from outer retinal photoreceptors.3 In addition to their capacity for intrinsic activation, the MGCs also are activated by extrinsic signaling originating from the outer retinal photoreceptors.4 – 6 The pupil light reflex evoked with a bright white light stimulus in a non– dark-adapted eye is driven, for the most part, by cone “ON” signals to the MGCs. In a previous article, we described a novel technique for obtaining differential pupil responses using continuous light at selected wavelengths over a range of intensities.7 Such pre-selection of the light intensity, wavelength, and duration was based
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© 2011 by the American Academy of Ophthalmology Published by Elsevier Inc.
on current knowledge of the differing functional properties of the rods and cones and melanopsin-mediated intrinsic activation and was intended to evoke pupil responses that emphasized activity primarily from 1 input source, that is, rods versus cones versus melanopsin.8 –10 The differential pupil responses to the testing conditions of our previous study suggested that the pupil response obtained in response to a low-intensity blue light stimulus may be a reasonable marker of rod activity. Likewise, the pupil response to a high-intensity red light stimulus was thought to represent a predominantly cone-driven response. The pupil response, particularly the sustained contraction, to continuous highintensity blue light was believed to derive primarily from direct, intrinsic activation of melanopsin ganglion cells. This current article describes the application of the methodology of differential pupil light reflex testing in a nonselected group of patients with RP. We desired to understand how the pupil responses to these selected stimulus condiISSN 0161-6420/11/$–see front matter doi:10.1016/j.ophtha.2010.06.033
Kardon et al 䡠 Pupil and Photoreceptor tions differ between patients with rod and cone dysfunction and subjects with healthy eyes and how the pupil responses in the patients compare with other tests of visual function, such as the electroretinogram (ERG) and kinetic (Goldmann) perimetry.
weighted” pupil response for purposes of this study. Melanopsin activity was estimated from the sustained pupil response to 100 cd/m2 blue light because of its distinctive property of non-fatigability to a steady state light of short (blue) wavelength.
Analysis
Materials and Methods Patients The study was conducted according to the tenets of the Declaration of Helsinki and received approval from the University of Iowa Institutional Review Board committee. All patients gave informed consent for study participation. Thirty-two consecutive patients with a diagnosis of retinitis pigmentosa (RP) underwent pupil testing at the time of their clinic appointment in the Retina Clinic of the University of Iowa (author ES) between 2007 and 2008. Inclusion criteria included abnormal fundus appearance (pigmentary disturbance or atrophy or diffuse arteriolar narrowing observed both clinically and on fundus photographs) and a previously recorded ERG that was abnormal in either scotopic or photopic conditions or both (International Society for Clinical Electrophysiology of Vision protocol; absent or diminution of b-wave amplitude below the 5th percentile or prolongation of implicit times compared with age-matched normal subjects). None of the patients had a second ocular diagnosis involving the retina or optic nerve, or any condition that affected the efferent pupil response. Gender, diagnosis, or genetic defect if known, kinetic visual field assessment using Goldmann perimetry, and ERG results were recorded. The eye with the greater visual field loss was selected for pupil testing.
Chromatic Pupillometry The dynamics of the pupil movement to stepwise increases in intensity of red and blue light were recorded by a computerized infrared pupillometer using a protocol previously described.7 In brief, a ColorDome Ganzfeld ERG apparatus (Diagnosys, Lowell, MA) was used to present a continuous light stimulus (duration specified by software) at a number of predetermined spectral bandwidths. The spectral bands chosen for this study were 640⫾10nm (red light) and 467⫾17 nm (blue light) under mesopic conditions of adaptation. Three stimulus intensities (1, 10, and 100 cd/m2 photopically matched for intensity for blue and red light stimuli) and stimulus duration of 13 seconds were used. For each wavelength, the light stimulus was presented as a continuous stepwise increase in stimulus intensity. In this study, the untested eye was occluded with a patch, and the tested (stimulated) eye was also the monitored eye. A dual-channel binocular eye frame pupillometer worn by the subject (Arrington Research, Scottsdale, AZ) was used to record the pupil diameter of the stimulated eye at 30 times per second. The details of this instrument and the analysis of recordings have been described in a previous publication.7 The main measurement parameter for the pupil response was percent change in pupil size from baseline size. The transient response was operationally defined as the maximal percent change from the baseline pupil size during a time window 180 to 500 ms after the onset of stimulus intensity. The sustained response was the amount that the pupil had remained contracted at the last (13th) second of light stimulation. Figure 1 shows an example of a pupil tracing of a normal subject (available at http://aaojournal.org). We selected the transient pupil responses to 1 cd/m2 blue light and 100 cd/m2 red light to differentiate between rod and cone input to the pupil light reflex and termed these the “rod-weighted” and “cone-
The distribution of pupil responses to each of the aforementioned stimulus conditions was analyzed for all 32 patients and compared with 43 normal subjects using parametic (t test and analysis of variance [ANOVA] for normally distributed data) and nonparametric (Mann–Whitney rank sum test) paired statistical comparisons. SigmaPlot version 11.0 (Systat Software Inc., San Jose, CA) was used for all statistical analyses. The patients with RP were then categorized into 3 groups according to their electrophysiologic status. Group 1 included patients with a non-recordable ERG, Group 2 included patients with an abnormal but recordable ERG (⬍5th percentile of normal), and Group 3 included patients with a normal ERG. This categorization was performed for the scotopic ERG and photopic ERG results separately. The distribution of the “rod-weighted” and “cone-weighted” pupil responses for the 3 groups of patients was examined and compared with 43 normal subjects using 1-way ANOVA (paired contrasts were significant at the Pⱕ0.05 using the Holm–Sidak method for normally distributed data and Dunn’s method for nonparametric data analyzed by rank). The intrinsic, melanopsin-driven pupil response (that is, the sustained response to 100 cd/m2 blue light) also was examined for each of these patient groups. Finally, the distribution of the quantified visual field score for the 3 groups of patients was analyzed using 1-way ANOVA between groups (see details below). The Goldmann visual field (GVF) for each patient was objectively quantified using a manual grid scoring system originally described by Esterman11 and applied to GVF by Kwon et al.12 The grid measures the area contained within the I4e isopter using a template composed of 100 dots. The grid template on a transparency is overlaid on the visual field, and dots that fall completely within the I4e isopter are manually counted. A perfect score using this scoring system is 100. Each GVF was also subjectively assessed for total volume of visual field loss and ranked from greatest to least by 2 of the authors who were each masked to patient and diagnosis (RK and AK). These visual field analysis techniques were correlated with each other and with pupil responses using Spearman and Pearson correlation analyses. The distribution of visual field loss was also compared in each of the 3 ERG groups. Spearman and Pearson correlation analyses were performed. To study the effect of severe photoreceptor disease on the bright blue stimulus offset pupil response, a subset of 13 patients with a non-recordable ERG to the combined response (scotopic response to the maximal International Society for Clinical Electrophysiology of Vision standard white light) was selected for further pupil analysis. The pupil tracings were qualitatively reviewed by 2 of the authors (RK and AK) for delay in re-dilation to baseline size after termination of the 100 cd/m2 bright blue light compared with the equivalent photopically matched red light stimulus. This was best observed as an asymmetry of the pupil redilation slope during the light “OFF” period between the red and blue light stimulus conditions. The superimposed digital pupil tracings from the photopically matched red and blue light stimuli were analyzed by tracing a straight line over the first 20 seconds of the pupil re-dilation phase after light offset. The difference in the slope of the 2 lines was evaluated, and the pupil tracings were ranked in order of slope asymmetry.
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Results Thirty-two patients were included in this study (17 women and 15 men). Diagnoses were RP in 30 patients and Stargardt’s disease in 2 patients. Two patients with RP demonstrated genetic transmission consistent with an autosomal dominant form of RP, 5 patients had an autosomal recessive form, and 4 patients had an X-linked form. A recordable pupil response was obtained in all patients except for 1 patient with X-linked RP who had no light perception, had a flat ERG, showed no pupil response to 100 cd/m2 red light, but had pupil responses to blue light. Overall, there was a significant reduction of the rod-weighted (1 cd/m2 blue) and coneweighted (100 cd/m2 red) pupil responses of the 32 patients (median percent pupil contraction ⫽ 29.6% rod-weighted and 44.5% cone-weighted) compared with 43 normal subjects (median percent pupil contraction ⫽ 39.3% rod-weighted and 53.7% coneweighted). The stimulus condition weighted to favor the intrinsic melanopsin activity (100 cd/m2 blue) also showed reduced pupil response in patients with RP compared with normal subjects (median value ⫽ 50.9% and 56.8%, respectively). The details of the descriptive statistics for patients and normal subjects are shown in Table 1 (available at http://aaojournal.org) and Figure 2. Twenty of 32 patients showed loss of the rod-weighted pupil response below the 25th percentile of normal, whereas 22 patients showed loss of the cone-weighted pupil response below the 25th percentile. Twenty-five of 32 patients had an abnormal b-wave amplitude on ERG under both scoptic and photopic conditions. Thirteen patients had a non-recordable combined response. Grouped by their scotopic ERG results, 17 patients had a non-recordable ERG (Group 1), 8 patients had an abnormal but
Figure 2. Scatterplot distribution of the pupil responses to a chromatic light stimulus obtained from 32 patients with RP superimposed on normative box plots. Left graph shows the transient pupil responses to a blue light presented at 1 cd/m2 (rod-weighted responses), the middle graph shows the transient pupil response to a red light presented at 100 cd/m2 (cone-weighted responses), and the right graph shows the sustained pupil responses to a blue light at 100 cd/m2 (melanopsin-mediated pupil responses). Each box plot shows the median, 75th, 25th, 95th, and 5th percentile levels for the group of 43 normal eyes.
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recordable ERG (Group 2), and 7 patients had a normal ERG (Group 3). The dot-plot distribution of the rod-weighted pupil responses for these 3 groups of patients is shown in Figure 3 (available at http://aaojournal.org).). Grouped by their photopic ERGs, 17 patients had a non-recordable ERG (Group 1), 11 patients had an abnormal but recordable ERG (Group 2), and 4 patients had a normal ERG (Group 3). A dot plot of the coneweighted pupil response for these 3 groups of patients is shown in Figure 4 (available at http://aaojournal.org). The pupil responses were significantly reduced in Group 1, who had the most severe electrophysiologic abnormality (non-recordable scotopic ERG or non-recordable photopic ERG) compared with normal subjects (P⬍0.001 and P⬍0.05, respectively). Group 1 also showed significant reduction in their melanopsin-mediated pupil response, as seen in Figure 5 (available at http://aaojournal.org). There was a significant difference between patients with an abnormal, but not flat, photopic ERG (Group 2) and normal subjects (P⬍0.05). There was no difference in the rod-weighted, cone-weighted, or melanopsinmediated pupil responses of patients with RP with normal ERG compared with those of normal subjects. The patterns of visual field loss in the 32 patients with RP included ring scotoma, generalized constriction, and large central scotoma. The GVF score ranged from 0 to 89 (maximal score possible ⫽ 100). The distribution of the GVF score showed a similar pattern to the pupil responses of patients in Groups 1, 2, and 3 for both scotopic and photopic ERG conditions, shown in Figure 6 (available at http://aaojournal.org). The patients with the non-recordable ERGs tended to have the lowest GVF scores, and the patients with normal ERGs generally had the highest scores. The visual field score of Group 1 was significantly lower (P⬍0.05) than in Groups 2 and 3 for scotopic and photopic conditions. Group 2 patients with abnormal but recordable ERG showed the widest range of GVF scores. There were 13 patients who had a non-recordable combined response on their ERG. The rod-weighted pupil response ranged from contraction amplitudes of 9% to 43%, and their coneweighted pupil response ranged from 0% to 61%. Although, as a group, the melanopsin-mediated pupil response was lower than in the normal eyes, this difference was not significant. Patients with RP commonly showed an asymmetry in the rate of re-dilation after light termination (light OFF period), in which the pupil tended to remain small and constricted to a bright blue light stimulus (100 cd/m2) compared with a photopically matched red light (100 cd/m2). Normal eyes did not show this feature (Fig 1, available at http://aaojournal.org). The re-dilation dynamics were symmetric between the blue and red light stimulus conditions in normal eyes. There was no correlation between the rank score associated with the amount of delayed dilation and the magnitude of the light onset pupil response (rod-weighted or cone-weighted) or the amount of visual field loss (GVF score). There was a good correlation between the GVF rank by subjective assessment (authors RK and AK) and the objective GVF score (P⬍0.05, r2⫽0.75) in this subset of 13 patients, indicating that the dot overly quantification method did well to reflect the estimated overall volume of visual field loss. To better illustrate the patterns of pupil responses observed among the patients with RP with a non-recordable combined ERG response, 3 patient examples are shown. Figures 7 and 8 show 2 patients with a similar amount of severe kinetic visual field loss but very different pupil responses to red and blue light. One patient (Fig 7) shows abnormally reduced rod and cone-weighted pupil contractions but preservation of the melanopsin-mediated pupil response, whereas the other patient (Fig 8) shows pupil responses that are all within the normal range, despite the severe visual field loss and non-recordable combined ERG. Notably, both of these patients show an asymmetry of the pupil re-dilation dynamics during the light OFF period between the red and blue light con-
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Figure 7. Patient with X-linked RP and a non-recordable combined response on ERG. The upper left image shows arteriolar narrowing, pigment clumping, and pigment epithelial atrophy in the right eye. Goldmann perimetry (upper right) of the same eye shows a severely constricted visual field. The pupil diameter change in response to stepwise increases to photopically matched red and blue light stimuli (bottom left). The patient’s rod-weighted, cone-weighted, and melanopsin-mediated pupil response superimposed on the normative box plot (bottom right). Note that the rod and cone-weighted responses are severely reduced, to near or below the 5th percentile of normal subjects. The melanopsin-mediated pupil response, however, is preserved. Note the asymmetry in the pupillary re-dilation during the light OFF period on the pupil response curves (bottom left). The pupil is slower to re-dilate back to baseline (shallower slope) after offset of the bright blue light compared with offset of a photopically matched equiluminant red light.
Figure 8. Patient with X-linked RP and a non-recordable ERG. The fundus photograph demonstrates the severe pigment epithelial atrophy with clumping and arteriolar narrowing in the right eye (upper left). Goldmann perimetry of the same eye (upper right) shows severe constriction with a quantified visual field score similar to the patient shown in Figure 7. Yet this patient’s rod-weighted, cone-weighted, and melanopsinmediated pupil responses lie surprisingly within the 25th to 75th percentile range of normal subjects (bottom right). Examination of the pupil response curves (bottom left), however, reveals an asymmetry in the pupil re-dilation dynamics as seen in the light OFF period of the tracing. Note that after exposure to the bright blue light, the pupil tends to stay partially contracted for several seconds and the rate of re-dilation to baseline size is much slower compared with red light exposure.
ditions. After bright blue light exposure, the pupils remained partially contracted and were slower to return to baseline size. Figure 9 is a third patient with RP whose visual field loss is primarily within the central location. The rod-weighted and coneweighted pupil responses are severely reduced, and there is some reduction of the pupil response weighted to melanopsin activation (percent contraction is less than the 25th percentile). Although a delay in return to baseline size after the offset of bright blue light (100 cd/m2) is noted, the asymmetry in pupillary re-dilation dynamics between red and blue light conditions is not as striking as in the 2 patients shown in Figures 7 and 8.
Discussion This study examined the pupil light reflex using a novel protocol with red and blue light stimuli in patients with rod and cone dysfunction. We had previously developed the stimulus conditions based on light wavelength, intensity, and duration intended to bias the input in the afferent pupillomotor signal to favor rods, cones, or melanopsin activation.7 After testing a group of normal subjects, we applied the protocol to a nonselected group of patients with RP and evaluated their pupil responses compared with the normal group. Our assumption was that if our stimulus conditions did indeed represent preferential activation of the outer photoreceptors, our so-called rod-weighted and cone-
Figure 9. Patient with rod-cone dystrophy and a non-recordable combined response on ERG. An abnormal fundus with pigment epithelial atrophy particularly notable in the macula (upper left). Goldmann perimetry (upper right) shows central visual field loss. The patient’s rod-weighted, coneweighted, and melanopsin-mediated pupil response superimposed on the normative box plot (bottom right). Note that the rod and cone-weighted responses are severely reduced and well below the 5th percentile of normal subjects. There is also reduction of the pupil response weighted to melanopsin activation to less than the 25th percentile. There is a mild delay in re-dilation after the bright blue light (100 cd/m2) is turned off, but it is not as pronounced as in the 2 patients shown in Figures 7 and 8.
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Ophthalmology Volume 118, Number 2, February 2011 weighted pupil responses would be reduced in patients with RP, whereas the melanopsin-mediated pupil response would remain preserved, because it does not require signaling from the outer photoreceptors to be activated. Our findings largely support this premise. We found that the median pupil responses of the patients, including the melanopsin-mediated response, were significantly reduced compared with the normal subjects, but their range of responses was large. To look for a relationship between the pupil response and the functional photoreceptor deficit, we reexamined the pupil responses after dividing the patients by the degree of their ERG abnormality (nonrecordable, abnormal, or normal in the scotopic and photopic conditions). In other words, the “rod-weighted” pupil response (transient response to a 1 cd/m2 blue light) was compared with the scotopic ERG and the “cone-weighted” pupil response (transient response to a 100 cd/m2 red light) was compared with the photopic ERG. Not surprisingly, patients with a non-recordable ERG tended to have the greatest loss of pupil response, and the median pupil response (rod-weighted and cone-weighted) from this group was significantly lower than in the other 2 groups (patients with an abnormal ERG and patients with a normal ERG). Likewise, the rod and cone-weighted pupil responses tended to be preserved in those patients with normal ERGs, and the difference from normal subjects was not significant. Although these results are highly promising for use of the chromatic pupil light reflex as an alternative, objective parameter of outer and inner retinal function in disease states, the current test protocol still has not been optimized for isolating rod, cone, and melanopsin-mediated pupil responses. Two findings from this study suggest that the stimulus conditions used in this study are not selective enough in activating a single population of photoreceptors. Among the patients with RP who had an abnormal but recordable scotopic ERG, their rod-weighted pupil response was not significantly different (reduced) compared with normal subjects. Yet these patients had clear evidence of field loss and electrophysiologic dysfunction, and we would have expected their pupil response to be similarly abnormal. Admittedly, there might simply not have been enough patients in this group (n⫽8) to reflect any difference in median values. In addition, the spatial summation of pupil response across the retina may be uniquely different than what is measured with a Ganzfeld ERG and what is measured with kinetic perimetry. Yet it may also be that our measure of rod function, defined in this study as the pupil response to a 1 cd/m2 blue light stimulus, is not a selective enough measure of pure rod activity. That cones are simultaneously activated during this light stimulus condition is suggested by the subjective reporting of blue color perception during testing. We currently are developing more selective stimuli using a much lower-intensity blue stimulus under conditions of dark adaptation for the rod-mediated response and bright red stimulus on a rod-suppressing background stimulus for a cone-mediated pupil response. The other unexpected finding of this study was that the intrinsic, melanopsin-mediated pupil response was significantly reduced in the group of patients with non-recordable scotopic or photopic ERG. From a pathophysiologic stand-
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point, this might suggest that RP in its later stages can cause ganglion cell loss or that outer photoreceptors have an enhancing influence on intrinsic phototransduction in ganglion cells that is lost in the advanced stages of disease.13 But it is also possible that our measure of melanopsin function, the pupil response to continuous bright blue light, does not isolate intrinsic melanopsin activity completely but also includes rod and cone input to the pupil light reflex. Even with sustained, steady bright light stimulation, rods may be contributing a constant level of input to the melanopsin ganglion cell signal, as well as cones. In such a situation, a severe loss of rod or cone function might be the reason for the reduced pupil response to 100 cd/m2 blue light seen in our patients with RP with a non-recordable ERG. Further refinement of the stimulus condition and pupil analysis to light onset and offset is being developed for better isolation of the intrinsic melanopsin-mediated pupil response. Our results provide evidence that the sustained pupil contraction after blue light offset compared with red light offset may provide additional diagnostic information correlating to the status of outer photoreceptor disease. In conclusion, this study demonstrated that patients with RP have reduced pupil responses to red and blue light and that the loss of pupil function mediated by outer retinal photoreceptors appears most substantial in those who had a non-recordable ERG. Yet despite the absence of any recordable electrophysiologic signal of rod or cone function, all these patients except 1 had a recordable pupil response. This suggests a potentially important role for chromatic pupillometry, namely, that it may be a clinical test that extends the dynamic range for monitoring outer photoreceptor function. This is a particularly relevant situation in the treatment of patients who have severely constricted visual fields and a flat ERG. Following the pupil responses to red and blue light may be one way to assess functional recovery of the outer photoreceptors to assess new treatments.14 In addition to the static measure of pupil response to a light stimulus, we observed a delay in pupil re-dilation in the eyes of patients with RP after termination of the bright blue light compared with red light offset in the eyes of patients with RP. In a recent article by Kankipati et al,15 the authors described a post-illumination pupil constriction after offset of a bright blue light in normal subjects and ascribed the phenomenon to melanopsin activity. Because our blue stimulus condition did not evoke a post-illumination constriction in any of our normal subjects, we were surprised to see its occurrence in the patients with RP. This suggests that outer photoreceptors have a modifying influence on the intrinsic MGC signal and that assessment of the light OFF period may add diagnostic information for monitoring outer retinal dysfunction. Although the present test protocol does not yet seem to be maximally optimized for selectively activating rods, cones, and melanopsin-mediated pupil responses independently, the results of our study support the potential usefulness of chromatic pupillometry as a noninvasive method for monitoring outer and inner retinal function and also may be a unique means for following patient status when the ERG has become non-recordable.
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References 1. Hattar S, Lucas RJ, Mrosovsky N, et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 2003;424:76 – 81. 2. Güler AD, Ecker JL, Lall GS, et al. Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision. Nature 2008;453:102–5. 3. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science 2002;295: 1070 –3. 4. Jusuf PR, Lee SCS, Hannibal J, Grünert U. Characterization and synaptic connectivity of melanopsin-containing ganglion cells in the primate retina. Eur J Neurosci 2007;26:2906 –21. 5. Wong KY, Dunn FA, Graham DM, Berson DM. Synaptic influences on rat ganglion-cell photoreceptors. J Physiol 2007; 582;279 –96. 6. Schmidt TM, Taniguchi K, Kofuji P. Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development. J Neurophysiol 2008;100:371– 84. 7. Kardon R, Anderson SC, Damarjian TG, et al. Chromatic pupil responses: preferential activation of the melanopsinmediated versus outer photoreceptor-mediated pupil light reflex. Ophthalmology 2009;116:1564 –73.
8. Dacey DM, Liao HW, Peterson BB, et al. Melanopsinexpressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature 2005;433:749 –54. 9. Gamlin PDR, McDougal DH, Pokorny J, et al. Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Res 2007;47:946 –54. 10. McDougal DH, Gamlin PD. The influence of intrinsicallyphotosensitive retinal ganglion cells on the spectral sensitivity and response dynamics of the human pupillary light reflex. Vision Res 2010;50:72– 87. 11. Esterman B. Grid for scoring visual fields. II. Perimeter. Arch Ophthalmol 1968;79:400 – 6. 12. Kwon YH, Kim CS, Zimmerman MB, et al. Rate of visual field loss and long-term visual outcome in primary open-angle glaucoma. Am J Ophthalmol 2001;132:47–56. 13. Wan J, Zheng H, Hu BY, et al. Acute photoreceptor degeneration down-regulates melanopsin expression in adult rat retina. Neurosci Lett 2006;400:48 –52. 14. Maguire AM, High KA, Auricchio A, et al. Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 2009;374: 1597– 605. 15. Kankipati L, Girkin CA, Gamlin PD. Post-illumination pupil response in subjects without ocular disease. Invest Ophthalmol Vis Sci 2010;51:2764 –9.
Footnotes and Financial Disclosures
Financial Disclosure(s): The author(s) have made the following disclosure(s):
Supported by a Merit Review and Rehabilitation Award from the Veterans Administration, Washington, DC (Dr. Kardon, VA Center of Excellence for the Prevention and Treatment of Vision Loss). Dr. Kardon is the recipient of a Lew Wasserman Scholar Award (Research to Prevent Blindness) and the Pomerantz Chair in Ophthalmology. Dr. Kawasaki was supported in part by a grant from the Foundation for Research in Ophthalmology and Loterie Romande Swiss. Unrestricted support was also received from Research to Prevent Blindness (New York, NY). Dr. Stone receives support as an Investigator of the Howard Hughes Medical Institute and from the Carver Family Center and the Center for Macular Degeneration. This article contains additional online-only material. The following figures should appear online-only: Figures 1 and 3 to 6, and Table 1.
Dr. Kawasaki is a paid consultant by Bayer SpA, Milano Italy. None of the authors have a proprietary interest in any of the instrumentation, technology, or products mentioned in this manuscript.
Correspondence: Aki Kawasaki, MD, Hôpital Ophtalmique Jules Gonin, Avenue de France 15, Lausanne-1004, Switzerland. E-mail: [email protected].
Originally received: February 5, 2010. Final revision: June 21, 2010. Accepted: June 25, 2010. Available online: September 23, 2010.
Manuscript no. 2010-194.
1
Department of Ophthalmology and Visual Science, University of Iowa, Iowa City, Iowa.
2
Veterans Administration Hospitals, Iowa City, Iowa.
3
Howard Hughes Medical Institute, Chevy Chase, Maryland.
4
Hôpital Ophtalmique Jules Gonin, University of Lausanne, Switzerland.
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