The Dynamic Focal Cone Electroretinogram

6.8.1 Introduction

Over the last fifty years many studies have evaluated outer retinal function by exposing the eye to an intense, bleaching light source and measuring the time taken for visual function to return to a pre-bleach level (Balliart, 1954; Severin et al, 1963; Glaser et al, 1977; Collins &

Brown, 1989; Sandberg et al, 1998). Recovery of visual function after exposure to an intense

source is primarily dependent on the speed with which photopigment can regenerate. Therefore, any disease compromising the integrity of, or limiting the supply of metabolites to the photoreceptor / RPE complex is likely to prolong recovery time. This is exemplified by the observation of extended recovery times in age-related macular degeneration. There are limitations however, to the basic clinical ‘photostress test’.

One limitation relates to the bleach itself. The percentage o f photopigment bleached is affected by factors such as pupil size, media opacification and, for short bleaches of low intensity, modest timing errors (Margrain & Thompson, 2002). Another limitation relates to the belief that the test can differentiate outer retinal from inner retinal disorders (Glaser et al,

1977). More recent studies have reported prolonged photostress recovery times (PSRT) in

diseases such as primary open angle glaucoma {Sherman & Henkind, 1988; Parisi & Bucci,

Chapter 6 Electroretinogram Technique Development

These findings suggest that recovery o f visual function obtained during conventional photostress testing may be influenced by inner and post retinal activity.

The greatest limitation o f this clinical test is its subjective nature. Visual acuity measurement is prone to intersubject variability inherent in the nature of the task. Inter-individual differences in the speed o f reading letters, inclination to guess at uncertain letters and maximum acuity are all factors which may increase variability in the recovery times recorded in normal subjects (Amos, 1991). The effect o f learning and increased confidence in the task involved may also mean a variable result from a single subject over several visits.

In order to overcome this limitation a number o f studies have monitored the recovery of visual function objectively using the visual evoked potential (Lovasik, 1983; Franchi et al, 1987;

Parisi & Bucci, 1992; Parisi et al, 1994; Parisi et al, 1995; Parisi et al, 1998; Parisi et al, 2002). Although this technique provides a means of obtaining an objective assay of the

recovery o f visual function, it does not give a direct measure of outer retinal activity. Hence, its value in the detection o f conditions which exclusively disrupt outer retinal function, such as early age-related macular degeneration, may be limited. In this study, therefore, a protocol was developed to enable the objective monitoring o f the recovery of outer retinal function following a photopigment bleach.

A pilot study was conducted to determine whether the recovery o f visual function following exposure to a bleaching light could be evaluated using the 41 Hz focal cone electroretinogram in a group of healthy adults. The 41 Hz focal cone ERG appeared to be particularly well suited to the task for two reasons. Firstly because the signal, or more specifically the first harmonic of the signal, is dominated by receptoral activity (Falsini et al, 2000), and secondly because this high frequency, steady state signal would facilitate the substantial averaging necessary to improve the post-bleach signal-to-noise ratio.

6.8.2 Method

• Ten healthy adults, aged 27-54 years (mean age 30.4 years, ± 9.2 SD), were recruited to

the study. All subjects had a corrected visual acuity of 6/6 or better, clear ocular media, a normal retinal appearance, no history o f retinal or systemic disease and, none were taking medication which could influence the results o f the study. Each subject gave their informed consent after being provided with a full description of the procedures involved.

Chapter 6 Electroretinogram Technique Development

• Each subject was dilated with 1.0% tropicamide, and prepared for ERG recording in the manner described in section 6.1.

• A 20° diameter, amber (595 nm) stimulus, with a luminance of 30 cd.m'2.sec _1, flickering at a temporal frequency o f 41 Hz, was presented in the centre of a ganzfeld bowl. Subjects were required to fixate the centre o f the stimulus, which was surrounded by a white (30 cd.m*2) adaptational background.

• A pre-bleach 41 Hz focal cone ERG was recorded as a baseline measure. One hundred ERG responses were averaged on a 50 ms timebase, and bandpass filtered from 1-100 Hz. • The test eye was subsequently light-adapted to a bright white background (5.6 log

trolands) for a period o f 2 minutes, resulting in an 86% bleach of cone visual pigment

(Hollins & Alpern, 1973). This bleaching light was presented within the ganzfeld bowl.

• The 41 Hz focal cone ERG was then recorded at 20 second intervals for a period of approximately 4 minutes.

6,8.3 Data Analysis

In order to objectify amplitude measurements, individual traces were modelled using a cosine function (1), which was fitted to the data on a least squares basis.

Amp(t) = (m.t + c) + ([Amp/2] x cos [k/t]) (1)

Where t = time in ms, m = constant allowing for drift, c = constant allowing for bias, Amp = amplitude in pv, and k = 543.2 (constant to convert ms to radians).

This technique was particularly valuable at early times post-bleach, when the signal-to-noise ratio was relatively low. The recovery o f outer retinal function was evaluated by plotting these amplitude measurements as a function o f time post-bleach.

Traditionally, it has been assumed that photopigment regeneration, which underlies the recovery of outer retinal function, proceeds as a first order chemical reaction and therefore it may be appropriate to summarise the amplitude recovery data using an exponential function in the form:

Amp(t) = a.(l -exp(-0.693(t-x) /r) (2)

Where a is the pre-bleach amplitude, t is a time constant, x is the time at which the recovery

Chapter 6 Electroretinogram Technique Development

exponential time constant varies with bleaching level (Paupoo et al, 2000). This finding is inconsistent with a ‘first order’ process and it has been suggested that the recovery is instead ‘rate-limited’. An alternative model based on Michaelis Menton kinetics has therefore been proposed:

Amp(t) = 1- Km.W{B/Km exp(B/Km) exp-r(l+Km)/Km} (3)

where Km is the Michaelis constant, W is the ‘Lambert W function’, B is the initial bleach, and r is the maximum rate o f recovery (Mahroo et al., 2003; Paupoo et al. 2000; Lamb, 2003

personal communication).

In order to determine if the amplitude recovery data agreed with a ‘first order’ or ‘rate limited’ reaction the recovery data was modelled using both equations (2) and (3).

6.8.4 Results

Figure 6.23 shows a typical set o f recovery data.

10 pV

Time After Bleach ( secs) 10 30 50 70 90 110 130 150 170 190 210 230 5 ms

Figure 6.23. Typical set of traces recorded from subject FJ after 2 minutes adaptation to a bright adapting light. The amplitude o f the 41 Hz focal cone ERG is negligible 10 seconds after the bleach but returns there after.

Chapter 6 Electroretinogram Technique Development

It may be seen that the first trace obtained 10 seconds after exposure to the bleaching light was very nearly a flat line. Thereafter, the amplitude of the 41 Hz focal ERG gradually increased and in all cases had reached pre-bleach levels within the 4 minute recording period. The cosine fitting procedure is exemplified in Figure 6.24, which shows the how the model (equation 1) was fitted to the data obtained 30 seconds after the bleach (see the bold trace in Figure 6.23). This procedure allowed standardisation of the assessment of the amplitude of the fundamental component of the signal thereby minimising the effects of noise and higher harmonics. 1.5 1 0.5 0 0.5 1 1.5 0 10 20 30 40 50 Tim e (ms)

Figure 6.24. Demonstrates how an objective estimate of the amplitude of the first harmonic of each trace was obtained i.e. by fitting a cosine function. The trace used in this example is the one obtained 30 seconds after the bleach for subject FJ (see bold trace in Figure 6.23). The resulting peak to trough amplitude data was plotted as a function of time after bleach. Figure 6.25 shows the results obtained with the Dynamic Focal Cone ERG for each of the ten subjects. Both the ‘first order’ and ‘rate limited’ models provided a good fit of the data. The mean values for x and t for the ‘first order’ model were -0.06 min (SD 0.17) and 0.87 min (SD 0.48) respectively. The mean values for Km, r and B in the ‘rate limited’ model were 0.18 (SD 0.05), 0.68 (SD 0.21) and 0.95 (SD 0.05) respectively. A visual inspection of the data showed that there was little to choose between the two models. Evaluation of the residual standard deviation (RSD), which provides a measure of the goodness of fit suggests that the ‘rate limited’ model (i.e. the one based on Michaelis Menton kinetics) is better able to describe our data (‘rate limited’ RSD = 0.32pV; ‘first order’ RSD = 0.35pV). However, the ‘rate limited’ model includes more free parameters and a superior fit may be expected on this basis alone.

Chapter 6 Electroretinogram Technique Development 5 4 3 2 1 0 0 2 3 4 DA 5 4 3 2 1 0 0 2 4 RP 5 4 3 2 1 0 0 2 3 4 BC 5 4 3 2 1 0 0 2 3 4 AN 5 4 3 2 1 0 2 4 KM 5 4 3 2 1 0 5 4 3 2 1 0 0 2 3 4 'P 5 3 4 3 2 1 0 a. 2 0 1 3 4 HT ^ c > 5 r t r 4 2 0 3 4

Time post-bleach (mins)

51 4 3 2 11 0 TM 1 2 3 4

Time post-bleach (mins)

Figure 6.25. Recovery data obtained using the Dynamic Focal Cone ERG for each of our subjects. The dashed line is best fitting ‘first order’ model (equation 1) and the solid line the best fitting ‘rate limited’ model (equation 2).

Chapter 6 Electroretinogram Technique Development

The Akaike criterion is a statistic that may be used to determine if the inclusion of additional parameters is warranted on the basis o f their ability to improve the description of the data

(Carson et al, 1983). The most appropriate model has the lowest Akaike criterion. The mean

Akaike criterion values determined for the ‘first order’ and ‘rate limited’ models were 17.2 and 17.3 respectively. On the basis o f this analysis there was no significant improvement from the addition o f an extra parameter.

6.8.5 Discussion

The results o f this study suggest that the Dynamic Focal Cone ERG may be used to evaluate the recovery o f outer retinal function following exposure o f the eye to intense illumination. The electrophysiological data was objective, thus overcoming problems associated with visual acuity measurement in the traditional photostress test (Amos, 1991). Interpretation of the data was objectified by the use o f a cosine model, thus providing an estimate of the amplitude of the fundamental frequency o f the signal.

An evaluation of the Akaike criterion suggests that there is no difference in goodness of fit between the ‘rate limited’ and ‘first order’ models o f macular recovery and thus the first order model has been chosen for the analysis o f subsequent data, as the model has fewer variables. The values obtained for the free parameters in the model will be used to compare the results of subjects in the subsequent study on people with AMD.

6.9 Conclusion

A series of pilot studies has led to the development of a battery o f electrophysiological techniques that may be o f value in the early detection o f ARM and the assessment of visual function in people who suffer from the condition.

The focal rod ERG protocol was developed in response to reports that parafoveal rod photoreceptor loss occurs at an early stage in ARM, and appears to precede cone loss (Curcio

et al, 1996; 2000; Medeiros et al, 2001). Preliminary investigation indicated that the

2.1 log scot tds.sec*1 stimulus employed by Sandberg et al (1996) in the recording o f the focal rod ERG might elicit a signal contaminated by cone response. An intensity-response series was therefore recorded, and it was determined that the maximum intensity of blue flash which would not result in a response from the cones was 1.7 log scot tds sec'1. The stimulus was 20°

Chapter 6 Electroretinogram Technique Development

in diameter, thus effectively targeting the area most vulnerable to ARM related changes

(Curcio et al, 1996, 2000). Results o f a pilot study comparing two techniques for recording

the focal rod ERG indicated that a background adaptation technique would be most suitable for this study, reliably eliciting both a- and b-waves.

The focal cone ERG was included in this study as a means of assessing central retinal cone function. The response recorded to a 5 Hz stimulus in a preliminary study showed distinct a- and b-waves, and bandpass filtering from 100-1000 Hz provided good isolation of the macular OPs. In addition to this technique, a 41 Hz flickering stimulus was included to produce a steady state focal cone response. The first harmonic of this signal has been hypothesised to strongly reflect outer retinal function (Porciatti et al, 1989; Remulla et al,

1995; Falsini et al, 2000). It therefore has the potential to provide a direct assay of the retinal

layers affected at an early stage by ARM. Both o f these stimuli were 20° in diameter, again targeting the entire macular region shown to be affected in early ARM.

A preliminary assessment was made o f a C-wave recording technique described by Moschos et al (1994). It was hoped that this could provide a means of investigating the integrity of the RPE in subjects with ARM. However the C-wave protocol has been excluded from this study on the basis o f the variability o f results recorded from control subjects in the preliminary investigation, and in published literature (Hamasaki et al, 1997; Hock & Marmor, 1983). The literature suggests the occurrence o f selective dysfunction of S-cone function in early ARM (e.g. Eisner et al, 1987; Haegerstrom- Portnoy et al, 1988; Sunness et al, 1989; Eisner

et al 1991; 1992; Cheng & Vingrys, 1993; Collins & Brown, 1989). A protocol was therefore

included in this study to allow electrophysiological assessment o f the integrity of the short- wavelength sensitive pathway in ARM. The technique chosen was an established full-field silent substitution technique (Drasdo et al 2001; Chiti et al, 2003; Drasdo et al 2002). Attempts to elicit a focal response to this stimulus resulted in a signal too small to be reliably recorded and measured even in young control subjects.

The final protocol described in this chapter was the dynamic ERG - a technique designed to objectively assess the recovery o f cone function following a photopigment bleach. There is much evidence to suggest that photostress recovery is abnormal in subjects with ARM, but this has previously been assessed by psychophysical techniques (Balliart, 1954; Severin et al,

Chapter 6 Electroretinogram Technique Development

developed for this study employs the amplitude recovery o f the 41 Hz ERG to more directly assess outer retinal activity.

The following chapters describe the study in which these techniques were applied to a group of subjects with ARM, and a group o f age matched controls. Further psychophysical tests were also included to allow a full assay o f retinal function in these subjects.