provide separate and additional information on the retinal vasculature. Direct funduscopy and indirect funduscopy, while capable of detecting retinal abnormalities, often provide more limited views with less magnification. Direct funduscopy may include the use of a direct ophthalmoscope or the use of a slit lamp with an additional 78D or 90D lens.

Intraocular pressure

The measurement of intraocular pressure (IOP), for the purposes of toxicology assessments, can be adequately made by applanation tonometry. The invasiveness of more accurate measures is usually not warranted. In the rare cases where a more exact estimate of aqueous production is needed, tonography can be performed.

Number of Patients to Test

Common events are easier to identify and characterize than more unusual ones. It is customary to attempt to identify events which occur at a frequency of 1% or higher. Mathematical prin­ ciples of probability dictate that when the true event incidence is 1% or higher, in order to have a 95% change of observing at

least one event, 300 subjects must be monitored. This is often referred to as the rule of 3 (Hanley and Lippman-Hand 1983).

The rule of 3 states that in order to detect events which would occur at X% or more, you need Y patients where 3/Y =  X. Apply­ ing this rule suggests that if an incidence rate of 10% is to be identified, at least 30 patients need to be studied. If an incidence rate of 5% is to be detected, 60 patients must be studied. If an incidence rate of 0.1% is to be detected, 3000 patients must be studied.

Summary

There are many potential ocular toxicity tests. Ocular toxicity tests should be used to investigate potential adverse events that might be either frequent or serious. There should be a justified reason for the selection of each test, and each test should be appropriate for the event in question.

References and Further Reading

Hanley JA, Lippman-Hand A. If nothing goes wrong, is everything all right? Interpreting zero numerators. JAMA 249: 1743–1745, 1983.

Summary

Introduction

Modern structure-based drug design results increasingly in compounds that act very specifically, e.g. as modulators of the function of channels or enzymes that occur not only in the targeted tissue but also in the eye. Increasingly, therefore, ophthalmological symptoms occur in phases I and II of clinical studies without any ophthalmic signs having been reported in preclinical studies. A well-known case is sildenafil (Viagra®), a selective inhibitor of cGMP-specific phosphodiesterase (PDE) 5 in the corpus cavernosum that, with lower selectivity, also affects the retinal PDE6, and elicits luminous phenomena such as blue-tinged objects and phosphenes, and thus reflects an unexpected pharmacological side effect (for a survey see Laties and Zrenner 2002). In such cases, usually long-term multi-center studies are required to exclude irreversible effects such as retinal degeneration and permanent loss of certain visual functions. As more and more drugs are targeted at specific molecular actions, e.g. specific protein structures or receptor subfamilies with particular ligand abilities that may occur in the eye as well, specifically targeted tests and study designs have to be used to differentiate between effects merely reflecting harmless pharmacological actions at an undesired site from those that lead to irreversible, toxic damage. Moreover, it is still unclear which battery of ophthalmic function tests assesses the safety of specific drug actions best and how one sets the threshold for potential clinical concern and the proper endpoint for safety. In addition, given the complexity and multitude of stimulation and recording conditions of tests such as electroretinography, electrooculography and visually evoked cortical potentials (VEPs), it is not easy to assure comparability and quality of test results in multi-center trials, not to mention the large variations in procedures, devices and methods of evaluation.

This chapter therefore outlines some principles that may help to select proper tests on the basis of their specificity for the function of certain retinal cell subgroups. It gives practical advice for selecting those tests that will enable the investigators to assess the specific functions and morphology of the visual system and it also addresses questions of safety and efficacy, based on hypotheses that stem from preclinical data where the psychophysics of the visual system are not available. Moreover, new developments are pointed out that help to improve the quality of multi-center studies by proper standard operating procedures (SOPs) and stringent case report forms (CRFs), based on the development of international standards and recommendations in ophthalmology (for electrophysiology, e.g. www.iscev.org) as well as the development of international networks of certified centers (http://www.europeanvisioninstitute.org/CT_SE/).

The Visual System

Almost half of all neurotoxic chemicals affect some aspect of sensory function (Croft and Sheets 1989), with the visual ­system being most frequently affected. Grant and Schuhman (1993) in their encyclopsoedic Toxicology of the Eye list approximately 3000 substances that produce unwanted side effects in the visual system. Numerous transmitters and mechanisms are involved in processing information in photoreceptors and in the many connected neurons transmitting visual information to perceptual centers whose function can be affected by neurotropic agents, drugs, food and environmental compounds. As shown in Table 4.1, a multitude of transmitters and neuromodulators is found in the various retinal layers, especially in the more than 25 different types of amacrine cells in the inner plexiform layer. Compounds can therefore act specifically on the function and electrophysiological characteristics of these target cells. This specificity often allows the correlation of functional alterations in electrophysiological parameters to the action and adverse reactions of neurotropic compounds. Additionally, there are many non-neuronal cells whose function is important for the integrity of information processing, such as pigment epithelial cells, glial cells and vascular structures, that can be affected by drug action as well. Of further general concern are the factors that determine whether a particular chemical can reach a particular ocular site: concentration and duration of exposure, mode of application and interaction with the various ocular structures as well as integrity of natural barriers. The blood-retinal barrier may be impermeable under normal physiological conditions (Alm 1992). However, in certain areas of the retina, e.g. around the optic disc, the continuous type of capillaries is lacking and hydrophilic molecules may enter the optic nerve head by diffusion from the extra-vascular space.

This chapter aims to convey a basic understanding of toxic mechanisms in the visual system by pointing out cell-specific functional changes and typical symptoms in toxic side effects. In an individual case it is strongly recommended that referenced publications such as Grant and Schuhman (1993) and Fraunfelder (2000) are consulted. These have references on the primary literature concerning the various substances. Additionally, there are very interesting general chapters and books that concern­ ocular toxicology and may be of help (e.g. Chiou 1992; Potts 1996; Ballantyne 1999; Fox and Boyes 2001; Bartlett and Jaanus 2007).

The retinal pigment epithelium

The retinal pigment epithelium (RPE) has four major functions: phagocytosis, vitamin A transport and storage, potassium ­metabolism and protection from light damage (see Fig. 4.1). RPE functions can be altered, e.g. by inhibitors of phagocytosis,

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Table 4.1 – The various transmitters and modulators present in the retinal layers (modified from Vaney 1990 and Kolb et al 2001)

modulation of potassium metabolism, metabolic alterations in the visual cycle and by the action of melanin-binding substances. Additionally, melanin is found in several different locations, such as the pigmented cells of the iris, the ciliary body and the uveal tract. Melanin has a high binding affinity for polycyclic aromatic carbons, calcium and toxic heavy metals such as aluminum, iron, lead and mercury (Meier-Ruge 1972; Potts and Au 1976; Ulshafer et al 1990; Eichenbaum and Zheng 2000). This may result in excessive accumulation and slow release of numerous drugs and chemicals that bind to melanin granula, such as phenothiazines, glycosides and chloroquine (Alkemade 1968, Bernstein 1967).

The outer retina is supplied by the choriocapillaries and capillaries have loose epithelial junctions, multiple fenestrae and are highly permeable to large proteins (Fig. 4.2a). During systemic exposure to chemicals and drugs by inhalation, transdermally or parenterally, certain compounds can be distributed to all parts of the eye via the bloodstream (Fig. 4.2b). For example, chloroquine phosphate binds strongly to the RPE with a half-life of 5 years, 80 times more strongly than to the liver. This drug is not only used for malarial prophylaxis, but also plays an important role in the treatment of rheumatoid diseases (up to 4 mg/kg, or for

Fig. 4.1  The various roles of the retinal pigment epithelial cells.

Photoreceptor (cone)

Fig. 4.2  (a) The RPE, with its tight junctions between the cell membranes, forms a cellular monolayer. On its basal side it is attached to Bruch’s membrane for its metabolic exchange with the choroidal vasculature; on the apical side it is intrinsically connected to the photoreceptors. (b) A standing potential of 6 mV is obtained across Bruch’s membrane, governed by various channels, pumps and exchangers. (Adapted from H. Jaegle, personal communication.)

hydroxychloroquine 6 mg/kg, of body weight per day). At these maximal rates, a critical cumulative dose can be reached within 3 to 6 months (Mavrikakis et al 2003).

Signs of chloroquine phosphate retinopathy (typically following a cumulative dose of 100 to 300 g) are:

•a relative paracentral scotoma, usually an annular perifoveal ring-shaped depression in visual fields

•loss of blue/yellow color discrimination

•RPE depigmentation often observed in a pattern matching the ring-shaped visual field depression (most easily seen during fluorescein angiography as window defects)

•reduced light-evoked amplitude rise in the electrooculogram (EOG)

•reduced b-wave amplitudes and prolonged latencies in the electroretinogram (ERG) due to secondary effects on photoreceptor­ function.

Patients with low body weight and/or poor renal function are particularly susceptible (Ochsendorf et al 1993) therefore care should be taken to avoid over-dosage.

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intestinal peptide, somatostatin, nitric oxide and even angio­ tensin-converting enzyme (Zrenner et al 1989). Altering the metabolism, release or uptake of these substances can affect visual function. The following section discusses several more common examples.

GABA and anti-seizure medications

Anti-epileptic drugs act on the GABA metabolism and carbamazepine (Tegretol®), phenytoin (Phenhydan®) and vigabatrin (Sabril®), for example, quite commonly alter visual function­ (Dyer 1985; Besch et al 2000, 2002; Harding et al 2002). These drugs can cause color vision disturbances. Vigabatrin­ can produce irreversible concentric visual field defects with incidences between 0.1 and 30% (Miller et al 1999; Schmitz et al 2002). Vigabatrin raises GABA levels by irreversibly binding to GABA transaminase, thus preventing the metabolism of GABA. The earliest report of retinal electrophysiological changes in humans associated with vigabatrin treatment is that of Bayer et al (1990). All patients with visual field defects revealed altered oscillatory potential waveforms in the ERG, especially patients with marked visual field defects (Besch et al 2002), which were also visible in multifocal ERGs (mfERGs; Rüther et al 1998). Such changes are usually irreversible (Schmidt et al 2002). In many patients a delayed cone single flash response was found in the Ganzfeld ERG and a reduced Arden ratio in the EOG. Harding et al (2002) developed a special VEP stimulus with a high sensitivity and specificity for identifying visual field defects.

Agents that modulate GABA can be expected to alter the GABAergic functions of horizontal cells and thereby can alter contrast vision and presumably functions of light adaptation as well.

Dopamine

Drugs such as fluphenazine, haloperidol and sulpiride can affect the dopamine metabolism and thereby modify retinal function (Schneider and Zrenner 1991). In the arterially perfused eye all three dopamine antagonists increased the rod b-wave while b-wave latency and implicit time showed no drug-induced changes. On the other hand, the D1 antagonist fluphenazine increased the fast transient ON-component while simultaneously strongly decreasing the OFF-component. In contrast, concentrations of the D2 antagonist sulpiride that had a comparable effect on the fast transient ON-component of the optic nerve response (ONR) did not influence the OFF-component. These findings indicate that D1 and D2 receptors play different roles in the transmission of rod signals at the border of the middle and inner retina. As reported in patch clamp investigations by Guenther et al (1994), application of the receptor antagonists haloperidole, spiperone and SCH23390 reduces calcium influx by between 8 and 77%, while no effect of dopamine itself was observed. The study of Dawis and Niemeyer (1986) in arterially perfused cat eyes suggests that dopamine itself has an inhibitory effect on the rod visual pathway since the rod b-wave amplitude is reduced after dopamine application (for a review see Witkovsky and Dearry 1991; Witkovsky 2004). The action of these drugs may be based on a particular cell population of dopaminergic neurons, the density of which ranges from 10–80 cells/mm2. They surround other amacrine pericaria, probably amacrine cells of the AII type that transmit rod pathway information onto cone bipolar cells.

Other retinal transmitters and modulators

Approximately 20 different transmitter substances and modulators are used by the various types of amacrine cells, as shown in Table 4.1. Consequently, numerous drugs can affect the function

of amacrine cells, and individual wavelets of the oscillatory potentials­ that have their origins in amacrine cells can be affected­ differently. A good example of this is angiotensinergic amacrine cells (Datum and Zrenner 1991; Jurklies et al 1995; Kohler et al 1997; Wheeler-Schilling et al 2001). Angiotensin II antagonists can considerably alter retinal function, as shown in ERGs and ONRs of the arterially perfused cat eye (Dahlheim et al 1988; Zrenner et al 1989) and by electroretinography in cats (Jacobi et al 1994).

Functional changes in retinal ganglion cells

While the parvocellular system is mainly responsible for coding of fine spatial resolution and color vision, the magnocellular system codes primarily movement and contrast. Drugs that ­affect ganglion cell function therefore can show different effects on the parvoand magnocellular systems, e.g. ethambutol.

Ethambutol is widely used for the treatment of tuberculosis. It causes:

•color vision disturbances as early symptoms

•contrast vision changes

•visual field defects, especially central scotomas with loss of visual acuity

•changes in veps.

These alterations can occur within a few weeks after starting the treatment. Also, ethambutol may act at different sites. While initially horizontal cell function and thereby color discrimination are altered as seen in VEPs (Zrenner and Krüger 1981) as well as in fish retina single cell recordings (van Dijk and Spekreijse 1983), advanced forms may lead to loss of ganglion cells and degeneration of the optic nerve. After cessation of drug intake color discrimination improves, as can easily be shown by anomaloscopy (Nasemann et al 1989).

Other substances that typically modify ganglion cell functions are methanol and ethanol, chloramphenicol, quinine, thallium and ergotamine derivatives (for references see Zrenner and Hart 2007).

The optic nerve

Several compounds particularly affect axonal fibres of ganglion cells. For example, exposure to acrylamide produces particular damage to the axons of the parvocellular system, while it spares axons of the magnocellular system, leading to specific visual deficits, such as an increase in the threshold for visual acuity and flicker fusion as well as prolonged latency of pattern-veps.

Carbon disulfide

The changes induced by carbon disulfide (CS2) on the visual function are central scotoma, depressed visual sensitivity in the periphery, optic atrophy, pupillary disturbances, disorders of color perception and blurred vision (for a survey see Beauchamp et al 1983). Vasculopathies including the retina were reported as well but histology in animals points primarily to an optic neuropathy (Eskin et al 1988) and alterations in the VEP are expected in such conditions.

Tamoxifen

Kaiser-Kupfer et al (1981) reported widespread axonal degeneration induced by tamoxifen in the macular and perimacular area, accompanied by retinopathy. Clinical symptoms include a permanent decrease in visual acuity and abnormal visual fields (Ah-Song and Sasco 1997). Following cessation of low dose tamoxifen therapy most of the keratopathy accompanying tamoxifen intake and some of the retinal alterations were seen to be reversible (Noureddin et al 1999).

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Other toxic optic neuropathies

Other more common drugs that can produce alterations of optic nerve function with concomitant VEP changes are ethambutol (see above), isoniazid, and isonicotinic acid hydrazide, as well as streptomycin and chloramphenicol.

Damage to glial cells

The retina has several types of glial cells: Mueller cells, oligo­ dendrocytes and astrocytes. Mueller cells are important for glutamate metabolism (in the uptake of the transmitter released by photoreceptors), as well as for the storage of calcium ions. Damage to glial cells, e.g. by the ammonia toxicity of alcohol syndrome (caused by severe toxic hepatic damage), produces marked changes in the ERG (Eckstein et al 1997). This arises because of damage to Mueller cells, since the b-wave of the ERG is strongly modulated by the function of the retina’s Mueller cells.

CNS damage

As has been outlined for retinal disorders, toxic and pharmacologic disturbances of visual function can also show up in the central nervous system. In addition to neurotropic effects, some drugs can cause an elevation of intracranial pressure, resulting in papillary edema, which is observed, for example, after the treatment with ergotamines and may produce prolonged latency in the pattern-VEP (Heider et al 1986). Since many cortical ­areas are involved in processing visual information, alteration of vision can also be caused by direct drug action on neurons in higher cortical areas and produce visual hallucinations and neurological deficits.

The Role of Non-Invasive

Electrophysiology in Drug Testing

Non-invasive tests allow the recording of bio-electric potentials that arise during the neural processing of visual information by the various elements of the afferent visual pathways.

Changes in the various signals allow conclusions about the locations and kinds of functional disturbances in the afferent neurons of the visual system. Electrophysiology, including the EOG, the ERG and veps have been standardized by the International Society for Clinical Electrophysiology of Vision (ISCEV, http://www. iscev.org/standards/index.html). This site also provides further guidance for the proper application of electrophysiological tests.

Relationship between anatomy and function

To make valid use of electrophysiology it is necessary to know the anatomic origins of the various potentials, the appropriate stimuli to evoke them, and the methods for recording and measuring­ their responses.

Table 4.2 shows a survey (modified after Apfelstedt and Zrenner 2007) arranged according to the various anatomic structures of the visual pathway. Nearly every part of the visual pathway can be studied with at least one electrophysiological method.

Electrooculography

The EOG measures a physiological electrical potential difference between the cornea and the posterior pole of the globe, with the cornea by convention being the positive pole. This so-called ocular resting potential is a physiological, transepithelial electrical potential that lies across the RPE, as shown in Fig. 4.2b. Under steady conditions of illumination, the resting potential maintains a constant value called the baseline potential, but it will vary with changes in illumination. An increase in this potential is a response

Table 4.2 – Origin of the various components of the electrophysiological recordings in relation to morphological sites of the retina (modified after Apfelstedt and Zrenner 2007)

of the RPE to light-induced shifts in ion concentrations in the extracellular fluid of the photoreceptor, induced by the ­activation of the phototransduction process in response to illumination of their outer segments. The light-dependent changes in the ocular resting potential recorded by the EOG require an intact function of the RPE and the photoreceptors.

The EOG is recorded by means of a pair of skin electrodes fixed close to the canthi of the eyelids. The patient is asked to continuously alternate his/her gaze between fixation lights at 1 second intervals for 10 seconds every minute. The fixation lights are separated by a visual angle of 40° from each other and located in a diffusely illuminated Ganzfeld sphere. The gaze-induced change in the eye bulb’s angle alters the relative ­potential difference between the nasal and temporal electrodes. The gaze-induced potential difference is directly related to the size of the ocular resting potential. The gaze-induced modulation of the potential is measured for about 30 minutes.

The EOG procedure is described in the ISCEV Standard by Brown et al (2006).

Dark adaptation of the light-adapted eye results in a decrease in the ocular resting potential, which reaches a minimum value after about 10 minutes, the so-called dark trough (Arden et al 1962).

During subsequent steady illumination of the eye, a continuous rise in the resting potential is evoked, which reaches the so-called ‘light peak’ 8–10 minutes after light onset (lower line in the right part of Fig. 4.5). The essential measure of the EOG is the ratio between light peak amplitude and dark trough amplitude just before light onset, the Arden ratio, which typically amounts to about 1.5–2.5 (right part of Fig. 4.5). Additionally it is possible to evoke a fast oscillation by setting a light on and off every 75 seconds. This potential is shown on the left of Fig. 4.5 and magnified in the inset figure. It stems from the RPE basal membrane.

Widespread damage to the pigment epithelial/photoreceptor complex results in a diminished light/dark ratio up to a complete loss of the light-induced potential increase.

Due to the origin of the standing potential, the EOG is the test of choice in cases where the primary functional alteration is expected in RPE cells, e.g. in chloroquine retinopathy (Johnson and Vine 1987) or phenothazine retinopathy (Henkes 1967). Since the light-induced increase in the standing potential is triggered by photoreceptor excitation, an intact function of the neuro-retina is required to identify a pigment epitheliopathy at an early stage. Electroretinographical recordings must therefore accompany the EOG tests as well as careful ophthalmoscopy of the pigment epithelial layer for biomicroscopic alterations. Biomicroscopy is supported by autofluorescence measurements and fluorescence angiography, methods which allow us to assess the state of the RPE layer more accurately.

Ganzfeld Electroretinography

Illumination of the entire retina by a short flash causes all retinal neurons to respond with a change in their electrical potential, triggered by the phototransduction process that finally modulates

the glutamate release in the photoreceptor synapses. The sum of light-evoked changes in the membrane potential of retinal cells can be recorded by corneal electrodes, such as contact lenses, gold foils or fibre electrodes, resulting in the ERG. The sketch in Fig. 4.6 points out the light-induced increase in extra-cellular potassium (K+) in the outer and inner plexiform layers that is picked up by millions of Müller cells. Each Müller cell directs the potassium current towards the vitreous humor, forming a current loop mainly induced by the activity of the depolarizing ­bipolar cells (blue in Fig. 4.6). While the first event in ERG response to a strong flash is a negative response (the a-wave, shown at the top of Fig. 4.7) that reflects the photoreceptor outer segments at least in the first 10–12 milliseconds, the subsequent positive response (b-wave) is mainly created by the depolarizing bipolar cells, whose excitation is reflected in the Müller cell currents, picked up as b-waves by corneal electrodes.

The various types of amacrine cells contribute to the oscillatory potentials that are visible as little wavelets on the ascending limb of the b-wave and can be made visible by filtering out the slower a- and b-waves electronically or digitally.

While a- and b-waves reflect outer and inner retina layers, respectively, the appropriate choice of the stimulus parameter of the test flash and the ambient light level allows transretinal separation­ of the rod from the cone system. In a completely dark-adapted condition, achieved after 20 minutes in darkness, rods, stimulated with a weak test light, dominate the ERG response. Under light-adapted conditions (approximately 30 cd/m2 of steady white background in a Ganzfeld-bowl), which saturate the rod system, strong test flashes elicit exclusively cone responses. The function of the two systems, rods and cones and the cells in the inner retina connected to the

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Fig. 4.7  This diagram relates the structures of the retina (lower part) to the response components in the ERG (upper part). The a-wave originates mainly in the outer retina (photoreceptor layer), while the b-wave is driven by depolarizing bipolar cells via Müller cell current loops. Amacrine cells contribute to fast wavelets along the ascending limb of the b-wave, which can be isolated by electronic filtering. The c-wave of the ERG (the beginning of which can be seen at the end of the ERG trace on the right) is a very slow and variable potential originating in the RPE cells. It is rarely used clinically because this function can be better assessed by the ERG and is difficult to record. (Sketch modified from H. Jaegle, personal communication.)

3. Oscillatory potentials, obtained from the dark-adapted standard combined flash ERG by passing the response through electronic filters that eliminate frequencies below 75 Hz.

4. 30 Hz flicker ERG elicited under the same conditions as the single flash cone response, but at a higher repetition frequency of the stimulus that cannot be resolved by rods.

5. Single flash cone ERG after adaptation to a white background light of 17–34 cd/m2, presented at the surface of a full field Ganzfeld bowl for at least 10 minutes. This response represents an isolated cone response because the white Ganzfeld light saturates the rod response.

For further references concerning the principles and practice of the electrophysiology of vision see Fishman et al (2001), Heckenlively­ and Arden (2006), and Apfelstedt and Zrenner (2007).

Flash

Fig. 4.8  The five ISCEV standard responses of the electroretinogram. The rod response is elicited by a dim light after 20 minutes of dark adaptation. The strength of the light is the ISCEV-defined standard flash (SF) (in this case 2.4 cds/m2 attenuated by 2.5 log units, resulting

in 0.01 cds/m2). The maximal response is marked by a huge a- and b-wave, representing a mixture of rod and cone responses and is elicited by the ISCEV standard flash (1.5–3 cds/m2). Oscillatory potentials are extracted by electronic filtering of all responses with frequencies of 75 Hz and higher. In the light-adapted state, the cone-driven flicker response is elicited by the standard flash with a repetition rate of

30 Hz. It is also used for the single-flash cone response. This recording reflects the minimum of electroretinographical documentation according to the recommendations of the ISCEV. For clinical trials an extended protocol that is targeted to specific mechanisms is recommended (see below).

Evaluation

In the ERG wave that is elicited by a single flash, typical components of the electrical potentials that correspond to various neuro-anatomic structures can be identified. The two primary components, the a- and b-waves, are particularly clear and easily recognizable in an ERG response that is evoked by a strong flash of light in a fully dark-adapted eye.

•a-wave: A negative component arising at the beginning of the recorded response, produced primarily by the hyperpolarization of the photoreceptor outer segments. This response appears a few milliseconds after a strong light flash stimulus and has its maximum near 16 milliseconds. The a-wave is only the leading edge of the negative potential contributed by the photoreceptors. Its further course is masked by the onset of the b-wave.

•b-wave: The b-wave arises from electrical activity in the inner layers of the retina. On the one hand, the b-wave tests the integrity of the second neuron of the afferent path (the depolarizing bipolar cell) and of Müller cells (see above); on the other hand, it indirectly reflects photoreceptor function, since the activity of the bipolar cells is determined

by the strength of the rod and cone signals. A reduction in the ERG a-wave because of photoreceptor degeneration

Fig. 4.9  Multifocal ERG. (a) While the Ganzfeld ERG does not allow the assessment of the topography of retinal excitability, a special hexagonal pattern, presented in the so-called m-sequence (Sutter and Tran 1992) allows the recording of multiple small ERGs from individual points (64 locations in this example) of the retina.

(b) A three-dimensional plot that shows the excitability of the foveal region in the centre. (c) The amplitude depression near the optic nerve head on the left. (d) In the case of affection of the cone system, the central peak disappears.

therefore also causes a reduction in the b-wave response. Conversely, there are retinal conditions that do not impair photoreceptor function or the a-wave of the ERG, but which selectively depress or delay the b-wave.

In a flash ERG, the following parameters are evaluated:

•the amplitude of the a-wave, measured from the electrical baseline to the negative peak recorded just before the appearance­ of the b-wave

•the amplitude of the b-wave, measured from the trough of the a-wave to the positive peak of the b-wave

•the implicit time, indicating the time elapsed from the flash stimulus to the peak of the corresponding ERG peak (a- or b-wave)

•amplitude and implicit time of the second oscillatory potential

•amplitude and implicit time of the flicker response or its phase.

It should be noted that the 2004 update of the ISCEV recommendation for full field electroretinography additionally recommends a very strong flash (approx. 12 cds/m2) in order to better isolate the a-wave. In particular the slope of the a-wave during the first 10–12 milliseconds is probably the best parameter for isolating alterations in the photoreceptor response.

All these measurements are a minimum requirement and there are many more possibilities to extend the information on the relationship between structure and function, such as OFFresponse, double flash amplitude recovery and others that may be recorded in special situations. These are described in the literature­ (see Heckenlively and Arden 2006).

Multifocal electroretinography

While Ganzfeld electroretinography only allows the assessment of the function of rod and cone pathways as a whole, multifocal electroretinography, as developed by Sutter and Tran (1992), has made it possible to assess the electrical activity of the retina locally from different regions of the posterior retina. The electrical responses are recorded from the eye with corneal electrodes

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just as in conventional ERG recording; however, a special stimulation pattern is presented on a computer monitor that consists of many hexagons, each of which has a 50% chance of being bright or dark (Fig. 4.9a). Although a pattern seems to flicker randomly, each element follows a predetermined sequence that ensures that the overall luminance of the screen over time is stable. The focal ERG signal associated with each element is calculated by correlating the continuous ERG signal with the ON or the OFF phase of each stimulus element. The result is a topographic array of many little ERGs (Fig. 4.9b), which reflects the regional response to the corresponding hexagon in Fig. 4.9a.

The waveform of each response resembles the Ganzfeld ERG in shape and origin, although some peculiarities are evident. The preferred designation for labeling these three negative and positive peaks is N1, P1 and N2, respectively, in order to distinguish them from the Ganzfeld ERG.

As shown in Fig. 4.9c, the amplitude of the local potentials can be plotted in terms of the amplitude/area of each hexagon, resulting in a response density plot (Fig. 4.9c). In this threedimensional representation of the multifocal ERG, the fovea shows the largest response density because of the high cone photoreceptor density in this region. At the left of the peak is a depression in amplitude, indicating the physiologic blind spot. Usually the electrical map of the multifocal ERG represents a retinal area around the foveola with a radius of 25–30°. The amplitude of the pedestal at the borders of the response density array therefore reflects the activities of the retinal eccentricity up to 25–30°. Since multifocal electroretinography is done in a light-adapted state, the responses reflect the cone system’s activity only. Diseases or conditions that affect the fovea result in the picture seen in Fig. 4.9d, where the central peak is lost, while generalized cone affection shows an overall loss of amplitude or response density (from Kretschmann et al 1997). Details can be found in the review by Hood (2000). Pharmacological or toxicological drug action which shows local retinal changes, such as vigabatrin (Harding et al 2002) or hydroxychloroquine (Lai et al 2006; Kellner et al 2006), does show up in multifocal ERGs.

The ISCEV has provided guidelines for basic multifocal electro­ retinography (Marmor et al 2003); a multifocal ERG standard will be finalized by the ISCEV in 2007.

Pattern ERG

The pattern ERG (PERG) is a retinal biopotential that is evoked when a temporary modulated pattern stimuli of constant total luminance, such as a checker-board reversal, is projected onto the retina. While the Ganzfeld ERG does not represent signals from the ganglion cells, the PERG (component P1 vs. N2, also called N95) does reflect the activity of the innermost retina. Its application in dysfunction of the macula is therefore not only strongly correlated to visual acuity (Neveu et al 2006) but also allows the assessment of ganglion cell function (Holder 2001) because its origin is in the ganglion cell level (Baker et al 1988). ISCEV has provided a standard for PERG (Holder et al 2007).

Visually evoked cortical potential

The signals processed in the retina are transmitted to the central nervous system via the axons of the ganglion cells that form the optic nerve, ending in the lateral geniculate body. There the information of both eyes merges and is transmitted to the primary visual cortex at the occipital lobe. The potentials arising in the occipital cortex can be picked up by skin electrodes fixed at the posterior pole, relative to a reference electrode mounted on the cortex. Such VEPs can be elicited by simple flash stimuli;

Fig. 4.10  Visually evoked cortical potentials are usually elicited by a checkerboard pattern. Recorded from the posterior pole of the eye, the response to such pattern reversal results in a strong positive deflection near 100 milliseconds (P100) arising from a negative deflection (N75) followed by another negative deflection (N135). Delays in the response properties indicate changes in the transmission velocity of signals in the optic nerve, e.g. in demyelinization.

Fig. 4.11  Pupillogram. The efferent visual pathway is most easily assessed by measuring eye motility as well as pupilograms. The pupil diameter starts to decrease after a latency time in response to light and reaches its maximum constriction after approximately 1 second. The pupil usually opens again after approximately 3 seconds. Changes in these temporal dynamics point to alterations in the efferent visual pathway and/or the autonomous nervous system. (From B. Wilhelm, personal communication.)

however, a pattern reversal stimulus is the preferred technique for most clinical purposes because the results obtained with pattern reversal stimuli are less variable in wave form and timing. The patient fixates a visual target on the centre of the black and white reversal pattern; it is important that an adequate optical correction is used. The responses at the occipital lobe are very small and are masked by larger potentials not related to visual function. The stimulus must therefore be presented at least 50–100 times in order to average the stimulus-related potential changes and extract them.

The ISCEV has provided a standard for VEPs (Odom et al 2004). A typical VEP is shown in Fig. 4.10 in response to a pattern reversal stimulus. It has a negative (N75), a positive (P100) and another negative component (N135). In clinical routine testing, the P100 amplitude relative to the N75 trough as well as the implicit time of the P100 peak are determined. The value of the P100 implicit time is a good indicator of retino-cortical response latency. Any condition or compound that affects or destroys the myelin sheets of the ganglion

cell axons causes a significant retardation in the velocity of axonal conduction. The prolongation of this component is therefore a good indicator for drug effects on myelin, e.g. in ethambutol.

Pupillography

Pupillography is commonly performed by infrared video systems, providing stable tracking of pupil diameter and automated analysis­ as an important precondition for objective assessment of visual and autonomous function (Fig. 4.11). The systems available have been listed and characterized by Wilhelm and Wilhelm­ (2003). For the majority of purposes the light reflex is the focus of attention when toxicological questions arise. The parameters provided during the dynamic recording of pupil size under certain stimulus conditions give selective information about the pharmacological or toxic effects of compounds in either the sympathetic or parasympathetic afferent or efferent pathway (see Wilhelm et al 1998). The exact site of effect – be it central or peripheral, sympathetic or parasympathetic – can be localized in detail if needed by additional pharmacological blocking (eye drops) of the opponent system in the periphery. Principally the constriction of the pupil to a certain light stimulus is influenced predominantly by the parasympathetic system and depends on the afferent visual pathway, while the redilation part of the light reflex is triggered by the sympathetic system.

An additional pupillographic technique (Wilhelm et al 1998) enables the objective assessment of the central nervous activation level and thus can reveal information about sedative or stimulating effects, for example in antidepressants, antihypertensives or amphetamines. This is possible because a decrease in activation level is accompanied by decreasing inhibition of the parasympathetic Edinger-Westphal nuclei in the oculomotor nerve complex. This provokes spontaneous fluctuations of pupil diameter in the dark that are closely correlated to low-frequency bands in electroencephalography. These oscillations can be recorded and analyzed by an automated software system (www. stz-biomed.de), the pupillographic sleepiness test (PST device by AMTech, Weinheim, Germany).

Visual Electrophysiology

in Drug Development

The visual electrophysiological techniques described here can provide useful information on safety, efficacy and proper dosage of novel chemical entities. When a new compound has been selected, preclinical exploratory tests have to be performed. In vitro toxicity tests are available, e.g. in the isolated perfused eye models (Niemeyer 2001).

At this stage whole cell patch clamp recordings from individual neurons, either in culture or in tissues such as isolated retina or retinal slice, also allow direct evaluation of the function of ligand and voltage-gated ion channels (e.g. Guenther et al 1994). This allows an understanding of the targets of the drug, especially in conjunction with immuno-histochemical studies. In order to monitor drug effects on the visual pathway under more natural physiological conditions, in vivo electrophysiological studies have to be performed (typically in rodents, pigs and dogs). As more animal models of diseases of the visual pathway become available, visual electrophysiology in animals will be crucial for questions of efficacy and safety. Preclinical toxicity studies can also help to identify the most sensitive markers for unwanted side effects or potential toxic effects to be used later in clinical studies. Moreover, functional tests can be related to histopathology in animal eyes and there are many instances where electrophysiological alterations are observed well

Time (min)

Fig. 4.12  Pharmacodynamic effects of sildenafil (Viagra®) in high doses on the ERG b-wave in dogs. The ERG b-wave implicit time becomes considerably prolonged and closely follows the pharmaco­ kinetic profile with a time lag of approximately 30 minutes. The close relationship between the ERG effects of sildenafil and its pharmaco­ kinetics suggests that the transient visual symptoms experienced by patients taking the drug are related to its binding to PDE6. With

long-term exposure to sildenafil the pharmacodynamic effects on the ERG remain constant, with return to pre-exposure baseline following drug wash-out. The absence of any long-term changes in the ERG provides evidence that the effects of sildenafil on PDE are physiological and do not reflect a toxic process. (From Brigell et al 2005.)

ahead of histological changes (see Brigell et al 2005). However, even if an accepted safety margin is present in animal studies, there is no guarantee that this margin will apply across species to humans.

After appropriate preclinical testing, electrophysiological investigations are very important for all phases of the clinical development, as nicely outlined by Brigell et al (2005). In phase I safety studies, usually dose escalation studies, visual electrophysiology can provide variable pharmacodynamic information. It is very important to show reversibility of an effect in order to differentiate a pharmacological drug action from a toxic drug ­action. An example is shown in Fig. 4.12. The effect of sildenafil (Viagra) in very high doses on the dog ERG b-wave is a prolongation of the implicit time that closely follows the pharmacokinetic profile with a time lag of approximately 30 minutes. Sildenafil increases blood flow by binding PDE5 but it also binds with lower affinity to PDE6, an enzyme needed for phototransduction (for a review see Laties and Zrenner 2002). As outlined by Brigell et al (2005), the long-term exposure to sildenafil in the ERG shows the return to pre-exposure baseline following drug washout that is constant, thus reflecting a physiological and not a toxic process. Electrophysiological measures of this kind can help in differentiating toxic from pharmacological effects at this early stage.

In phase II studies, electrophysiology has successfully been applied­ to proof of concept trials. As a marker of an effect on the disease progression ERG or VEP is very useful in phase III studies as evidence for regulatory approval. For example, glaucomatous damage to the nerve fiber of the retina has been well characterized in the pattern ERG (Korth et al 1993). Additionally, methods have been developed to detect very tiny ERG amplitudes in patients with retinal degenerations (Birch and Sandberg 1996). In diabetic retinopathy, changes in the oscillatory potentials of the ERG can monitor the disease process (Bresnik and Palta 1987).

In phase III studies, multi-center randomized studies have to be performed, generally involving large numbers of patients. Safety usually must be demonstrated in at least 100 patients

development drug in electrophysiology Visual

toxicology ocular in psychophysics and electrophysiology of role The  • PART 4