Inter-ocular and inter-session reliability of the electroretinogram photopic negative response (PhNR) in non-human primates

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Abstract

Purpose. To assess the inter-ocular and inter-session reliability for a range of parameters derived from the photopic electroretinogram (ERG) in a group of normal non-human primates.

Methods. Inter-ocular differences for photopic ERGs were assessed in a group of normal anesthetized adult rhesus monkeys (Macaca mulatta, n=29); inter-session reliability was assessed for 23 eyes of 23 animals tested 3 months later. Signals were acquired using Burian-Allen contact lens electrodes, whereby the contralateral cornea served as a reference. Photopic ERGs were elicited using red Ganzfeld flashes (−0·5–0·67 log photopic cd.s m−2) on a rod suppressing blue-background (30 scotopic cd m−2). Measurement reliability was established for a-wave, b-wave, photopic negative response (PhNR) and oscillatory potential (OP) amplitudes, as well as for their implicit times, by calculation of the 95% limits-of-agreement (LOA) and the coefficient-of-variation (COV) for each parameter.

Results. OP and a-wave amplitudes increased with intensity up to 0·67 log photopic cd.s m−2, following a typical saturating function, whereas b-wave and PhNR amplitudes both declined above 0·42 log photopic cd.s m−2. Inter-session variability was greater than inter-ocular variability. The inter-session COVs for PhNR amplitude (10–20%) were similar to the other photopic ERG components (a-wave: 12–17%, b-wave: 12–17%, OPs: 13–19%). Inter-session LOAs were also similar across components, but on average, were smallest for responses to moderate intensities (0·0–0·42 log photopic cd.s m−2).

Conclusion. In non-human primates, the 95% LOA for inter-session measurements of the photopic ERG a-wave, b-wave, OPs and PhNR are all similar. Inner-retinal damage may best be measured using the PhNR amplitude for moderately bright stimulus intensities. B-wave and PhNR amplitudes for brighter flashes are smaller and more variable. The ratio of PhNR:b-wave amplitudes manifests smaller variability and may therefore be useful for detection of selective PhNR loss.

Introduction

The flash electroretinogram (ERG) is frequently used for assessment of retinal function in both clinical and laboratory settings. As with any such test, its inter-session reliability determines the confidence with which the effects of a clinical disease, disease model, or therapeutic intervention can be distinguished from inherent measurement variability. To date, few studies of full-field ERG test–retest reliability appear in the literature; and to our knowledge, none for non-human primates. Given the extensive use of non-human primates in ophthalmic research, the inter-session reliability of the full-field ERG should be quantified.

Although well established as a measure of photoreceptor and bipolar cell activity, the full-field flash ERG was thought to contain little, if any contribution from ganglion cells. Thus, its application to basic and clinical studies of glaucoma, or other diseases of the optic nerve, has been relatively limited (Vaegan et al., 1991, Korth, 1997, Graham and Klistorner, 1998, Holopigian et al., 2000, Bach, 2001). More recently, studies by Frishman and colleagues have demonstrated that slow negative components in the flash ERG response, measured under both dark-adapted (scotopic), and light-adapted (photopic) conditions are dependent on ganglion cell activity and are reduced in experimental and human glaucoma (Frishman et al., 1996a, Frishman et al., 1996b, Viswanathan et al., 1999, Frishman et al., 2000, Viswanathan et al., 2000). This has led to renewed interest in the use of the full-field ERG for detection of abnormal function in human glaucoma and experimental models of glaucoma (Frishman et al., 1996a, Frishman et al., 1996b, Colotto et al., 2000, Cursiefen et al., 2001, Drasdo et al., 2001, Viswanathan et al., 2001).

The slow negative potential recorded in the scotopic flash response is relatively small and only observed near ERG threshold (Sieving et al., 1986, Naarendorp et al., 2001). Thus named the scotopic threshold response (STR), its measurement requires sufficient dark-adaptation and extensive signal averaging. Furthermore, some controversy exists regarding the extent to which the STR is affected by ganglion cell loss in humans (Sieving, 1991, Korth et al., 1994). In contrast, an analogous slow negative potential that follows the b-wave of the photopic ERG (called the photopic negative response or ‘PhNR’) is substantially larger and easier to record. Perhaps not surprisingly then, the PhNR has been used by several other investigators in studies of human glaucoma (Colotto et al., 2000, Cursiefen et al., 2001, Drasdo et al., 2001).

Given that the PhNR may be of use in human and experimental glaucoma, it is important to establish its reliability, especially for longitudinal studies of disease progression and/or therapeutic intervention. The purpose of this study was to compare inter-ocular and inter-session reliability of the PhNR with other components of the photopic ERG in a group of normal macaque monkeys. In addition, changes in the PhNR were compared with changes in other photopic ERG parameters following pharmacologic suppression of inner-retinal light responses.

Section snippets

Experimental animals

All experimental methods and animal care procedures adhered to the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local Institutional Animal Care and Use Committee (IACUC). Inter-ocular variability was assessed in 29 adult female rhesus monkeys (Macaca mulatta) ranging in age from 9 to 14 years, whereas inter-session reproducibility was considered for 23 eyes of 23 animals (all left eyes)

Results

Examples of photopic ERG responses to four stimulus intensities are presented in Fig. 1. For all flash intensities the photopic ERG response of the monkey recorded under these conditions had the characteristic a-wave and b-wave complex. OPs were found superimposed on the rising slope of the positive b-wave, while the late, negative PhNR followed the b-wave. Note that waveform morphology changed between 0·42 and 0·67 such that the b-wave became smaller and the PhNR implicit time abruptly shifted

Discussion

The photopic ERG waveforms measured in this study were similar to those previously reported for primates using short-duration, red full-field flashes across a range of stimulus intensities (Viswanathan et al., 1999). The results of this study show that while OP and a-wave amplitudes increased along a typical saturating function with stimulus intensity, the amplitude of the PhNR actually began to decline above 0·42 log cd.s m−2 much like that observed for the b-wave. This observation suggests

Acknowledgements

The authors wish to thank Karin Novitsky for her assistance with data collection. This study was supported in part by grants from the National Institutes of Health EY05231 (G.A.C.), M.J. Murdock Charitable Trust, Vancouver, WA, USA, and Allergan, Inc., Irvine, CA, USA.

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