Additional psychophysical tests

Detailed psychophysical tests may be used to investigate rod and cone function, such as dark adapted perimetry, dark adapted spectral sensitivities and cone critical flicker fusion tests. These tests are not used routinely and tend to be reserved for the research setting. They were used in the LRAT subjects in this study and will be explained further in chapter 4.6.

1.3.6 Electrophysiology

The objective measure of visual pathway function is achieved through visual electrophysiological testing, and such testing is fundamental to the diagnosis of all retinal dystrophies. It allows the assessment of the nature and severity of visual dysfunction, which may or may not be evident through clinical ocular examination. It is particularly useful in children, in whom subjective measures of visual dysfunction may not be accurate or possible. The main test armamentarium includes: the electro-oculogram (EOG), which measures retinal pigment epithelium (RPE) function, and its relationship with the rod photoreceptor; the full-field electro-retinogram (ERG), which measures the massed retinal response to light, and is a measure of

photoreceptor and inner nuclear layer function; the pattern electro-retinogram (PERG), which arises from retinal ganglion cell function and can give a measure of the macular function; and the visual evoked potential (VEP), which measures the function of the intracranial visual pathways. The International Society for Clinical Electrophysiology of Vision (ISCEV) regularly publishes minimum standards (and updates) to establish standardised worldwide protocols for electrophysiological examinations (http://www.iscev.org/standards/).

1.3.6.1 The ISCEV standard Electro-Oculogram

In this test, changes in the electrical potential across the RPE are measured during successive periods of dark and light adaptation [59]. The difference in electrical potential between the cornea and the posterior pole of the eye is known as the standing potential. This potential is generated by the RPE and it changes in response to the background retinal illumination. When switching to darkness, the potential continues to decrease for 8-10 minutes; there is then an initial fall in the standing potential over 60-75 seconds when the retina is subsequently illuminated (the fast oscillation), and then a slower but larger rise over 7-14 minutes (the light response).

These changes are generated by changes in permeability of ion channels across the RPE basal membrane, which are (in part) encoded by the Bestrophin-1 (BEST1) gene.

When measuring the EOG, the amplitude of the standing potential is measured in the dark, and then again at its maximum amplitude in the light, with electrodes placed at the medial and lateral canthi. This is done by the patient making fixed 30^o lateral eye movements during a period of 20 minutes dark adaptation, and then again during a 15

minute period of light adaptation. Eye movements are made every 1-2 seconds for 10 seconds every minute. The ratio of the maximum (peak) amplitude in the light to the minimum (trough) amplitude in the dark is expressed as the ‘Arden’ ratio (EOG light rise or light/dark ratio) and a normal EOG light rise is greater than 175%. This test is relatively difficult to perform and may not be possible in children below the age of 10 years. The principal use for the EOG in clinical practice is in the diagnosis of Bestrophin related conditions. It may also be used in inflammatory conditions of the choroid and retina.

1.3.6.2 The ISCEV standard Electro-Retinogram

The full field ERG (ffERG) represents the combined electrical activity of different cells of the retina to uniform illumination. This is measured using corneal electrodes and an integrating sphere, the Ganzfeld bowl, to deliver stimuli with whole field illumination. The ISCEV standard specifies 5 responses: (1) dark adapted 0.01 ERG (rod response); (2) dark adapted 3.0 ERG (combined rod-cone response); (3) dark adapted 3.0 oscillatory potentials; (4) light adapted 3.0 ERG (cone response); and (5) light adapted 3.0 flicker (30Hz flicker) [60]. An additional ‘maximal’ ERG is also undertaken in the dark adapted state. Measurements are taken with pupils pharmacologically dilated, and dark adapted ERGs are measured after 20 minutes dark adaptation, and light adapted ERGs after 10 minutes light adaptation. For the

‘maximal’ ERG, an 11 cd.s.m^-2 flash is presented in the dark adapted state. The stimuli presented to attain the other responses range from a dim white flash of 0.01 cd.s.m^-2 (dark adapted 0.01 ERG) to a bright white flash of 3.0 cd.s.m^-2 on a dark adapted background (dark adapted 3.0 ERG) or 30 cd.s.m^-2 background luminance

(light adapted 3.0ERG). In the 3.0 flicker ERG the same 3.0 cd.s.m^-2 stimulus is presented at a rate of 30 stimuli per second (30Hz), on an illuminated background.

The maximal ERG has an (negative) a-wave, the initial 8-10ms of which predominantly reflects rod photoreceptor hyperpolarisation. The subsequent (positive) b-wave is generated post-receptorally, secondary to depolarisation of the ON-bipolar cells. The oscillatory potentials are small oscillations on the ascending limb of the b-wave and are thought to arise from the amacrine cells. Rod system dysfunction will be seen as a reduction in the rod specific ERG b-wave, but this is generated in the inner nuclear layer so does not localise disease to the photoreceptors themselves. The maximal response a-wave does allow localisation as it is predominantly driven by rod photoreceptor function. If the rod response is poor, it could reflect either an ON-bipolar abnormality or poor rod photoreceptor function, so the bright flash maximal response in the dark adapted state needs to be measured. If it is a bipolar problem then the a-wave will be normal or near normal (an ‘electronegative’ ERG); if it is a rod photoreceptor problem then both the a and b waves will be reduced.

A cone-specific waveform, generated at the inner retinal level, is recorded when the 30Hz flicker is presented on a rod-suppressing background (in the light adapted state).

The single flash cone response allows better localisation within the retina, with the photopic a-wave reflecting cone photoreceptor and OFF-bipolar cell hyperpolarisation, and photopic b-wave reflecting post-phototransduction activity. As the ERG is a massed retinal response, disease isolated to one part of the retina, eg the macula, will have a normal ERG.

1.3.6.3 Other electrodiagnostic tests

ISCEV have published standards on a number of other electrodiagnostic tests [61-63].

The Pattern ERG (PERG) assesses the retinal response to a contrast-reversing stimulus such as a black and white checkerboard, when the eyes are fixated centrally, and provides information regarding macular and retinal ganglion cell function [61].

The PERG waveform consists of a small initial negative waveform of 35 ms peak time (N35), followed at 45-60 ms by a large positive waveform (P50) and then a large negative component at 90-100 ms (N95). This is a ‘transient’ response that is complete before the next contrast reversal. The amplitudes of the PERG are measured from the peaks to the troughs of the waveform. The N95 component arises in relation to the retinal ganglion cells; the P50 component reflects macular function. In contrast to the ff-ERG, the PERG is a local response to the area that is covered by the stimulus image, and thus can sensitively indicate macular dysfunction. It can also be used in conjunction with the visual evoked potential in order to differentiate a central retinal abnormality from optic pathway dysfunction, when the VEP is abnormal.

The visual evoked potentials (VEP) provide diagnostic information regarding the functional integrity of the visual system. They are the visually evoked electrophysiological signals obtained from electroencephalographic activity of the visual cortex that are recorded from the scalp overlying this area [62]. They are used to assess the intracranial visual pathways, in particular the optic nerves and chiasm.

ISCEV have published a range of stimuli and recording conditions including: pattern reversal VEPs and pattern onset/offset VEPs, both elicited by checkerboard stimuli with large 1^o (60 minutes of arc) and small 0.25^o (15 minute) check sizes; and flash

VEPs, elicited by a brief luminance increment, a flash, which subtends a visual field of at least 20^o. Pattern reversal is used for most clinical situations, pattern onset/offset is useful to assess for malingering and in patients with nystagmus, and flash VEPs are used if there is poor cooperation, very poor vision levels or poor optics that make the use of pattern stimuli difficult.

1.3.6.4 Electrodiagnostic testing in children

Electrodiagnostic testing can be essential in the visual assessment and diagnosis of children with visual dysfunction, and can provide an indication of vision levels in non- or pre-verbal children. In very young or premature children it may not be possible to apply adult protocols, and as such they may require non-standard protocols. Most paediatric patients can be tested without anaesthesia or sedation, which is the preferred situation as anaesthesia carries a small risk and also may alter the VEP measurements. Although corneal electrodes may be applied, most young children (particularly between age 3 months to 5 years) will not tolerate them. In these situations, infra-orbital skin electrodes, close to the rim of the lower eyelid, may be used, but consideration must be taken into the variety of physical and physiological factors that can interfere with readings obtained in this manner [64]. Some laboratories advocate the recording of both the ERG and VEP concurrently, using skin electrodes [65]. In this situation, an estimate of gross retinal function (from the ERG) and macular pathway function (from the VEP) in a single recording session lasting 30-40 minutes can be achieved. In LCA, 56% show an un-recordable ERG or VEP; and in 44% no recordable ERG activity is detected but a small, degraded flash VEP is detectable, suggesting some residual retinal and visual pathway function. [66].