Protocol measures cell damage


Chromatic pupillometry dissects function of different light-sensitive retinal cell populations

An optimized chromatic pupillometry protocol for assessing retinal cell function in patients with RPE65 deficiency has been found to be more sensitive in quantifying cone sensitivity loss than the original protocol.

"This pupillometry protocol could be useful as an objective method of evaluating inner and outer retinal damage in the absence of measurable electroretinography recordings, which is typical in patients with RPE65 deficiency," said Dr Birgit Lorenz, PhD, at the annual meeting of the Association for Research in Vision and Ophthalmology.

The protocol could also be valuable in analysing the effect of experimental treatment protocols for early onset severe retinal dystrophy or Leber's congenital amaurosis, she noted.

Both protocols, developed by the same group of investigators, gather functional information on three types of cell input: rods, cones and intrinsic photosensitive retinal ganglion cells (ipRGC), each of which has different wavelength dependencies and different relative sensitivities. When blue (462 nm) and red (640 nm) light stimuli are used, the sensitivities can be compared at different wavelengths. Depending on the absolute and relative differences, investigators can determine whether rod, cone, or ipRGC cells triggered the response.

Dr Lorenz and colleagues at the Department of Ophthalmology, Justus-Liebig University Giessen, Giessen, Germany, evaluated the two protocols, which vary in light intensities, stimulus duration and background, and then correlated the data with the clinical phenotype.

The first protocol, published by Randy Kardon et al. in 2009, consisted of red and blue stimuli of increasing intensity; the response to the strongest red stimulus corresponded with cone-mediated pupillary response, the transient response to the weakest blue stimulus was considered to be rod-mediated, and the sustained response after 13 seconds to the strongest blue stimulus was associated with the ipRGCs.

The modified protocol, published by Jason C. Park et al. in 2011, has a one-second stimulus time and a different sequence of stimuli: the weak blue stimulus for analysing rod responses is followed by a strong blue stimulus for analysing ipRGC responses. To eliminate intrusion by rodmediated responses, a weak blue background is switched on and a strong red stimulus is applied corresponding to the transient cone-mediated response, then a blue stimulus is used to eliminate the small ipRGC contribution.

Dr Lorenz and colleagues measured responses with a custom-made binocular chromatic pupil­lometer (Bino 1, AMTech) connected to a stimulator (Color-Dome Ganzfeld, Diagnosys LLC). The study group consisted of 11 patients with RPE65 mutations and a control population of 18 healthy probands and two subjects with achromatopsia. Investigators also performed visual field examinations and full-field stimulus testing (FST) and correlated these with the pupillometry responses.

After testing the patients with RPE65 deficiency using both protocols, the investigators found that the modified Park protocol was superior for assessing change in rod, cone, and ipRGC responses to stimuli, Dr Lorenz explained.

In the two patients with achromatopsia, the modified Park protocol produced much better separation of cone- and rod-mediated responses, as expected, because eyes with achromatopsia do not typically have a cone-mediated response.

Separately, investigators compared rod-mediated response in pupillometry with the results of Goldmann visual fields and FST in six patients. In one patient with the least severe phenotype and 20/20 visual acuity, all three tests detected some pupillary response. However, in the Park protocol, four patients had no pupillary response to the weak blue stimulus. This suggests that it might be necessary to use a stronger stimulus to increase the dynamic range of response when evaluating the effect of treatment, Dr Lorenz said.

She also observed that the post-illumination pupil response was prolonged in RPE65deficient eyes, as measured by both protocols, but not in the eyes of normal subjects; this was evaluated by looking at pupil constriction 30 seconds after the last blue stimulus. Several theories may explain this, such as missing input from rods or cones.

"While either protocol measured absence of rod function and reduced ipRGC function, the optimized protocol was more sensitive in measuring cone sensitivity losses," Dr Lorenz said. This demonstrates that, especially in the absence of any other electrophysiological data, chromatic pupillometry is both highly sensitive and objective as a testing method and should be added to the panel of examination tools not only for analysing the natural history of retinal degenerative diseases but also for quantifying treatment results with experimental therapeutic strategies.

Related manuscript

†B. Lorenz et al., Invest. Ophthalmol. Vis. Sci., 2012;53(9):5641–5652.

Related Videos
J. Morgan Micheletti, MD, speaks at the 2024 ASCRS meeting
Dr William Wiley of Cleveland Eye Clinic, Northeast Ohio
© 2024 MJH Life Sciences

All rights reserved.