Michael A. Sandberg
INTRODUCTION
This chapter describes the methodology application, and normal variation of some objective measures of retinal function that are used to establish diagnoses in known or suspected hereditary retinal diseases. Objective assessment of retinal function is important for at least three reasons. First, it provides evidence about the site of localization of visual loss (e.g., whether the site of visual loss is within the eye or visual pathways or whether the abnormality is restricted to the macula or involves the entire retina). Second, in most cases it permits a quantitative assessment of the degree of malfunction that can be followed up over time for the purpose of projecting long-term prognosis or evaluating a prospective treatment. Third, outcomes from these measures may be shown to the patient as variations in fundus reflectance or as waveforms on photographs or paper so that the patient can appreciate the type and magnitude of his or her visual malfunction and thereby actively participate with the ophthalmologist in the initial and follow-up examinations.
The techniques addressed include fundus reflectometry, fundus autofluorescence, optical coherence tomography (OCT), early receptor potential (ERP) recording, electrooculography, full-field and focal flash electroretinography, pattern-reversal electroretinography, and pattern-reversal visual evoked response (VER) recording. The first three methods, though not measures of function per se, are always presented in the context of measures of function in interpreting hereditary retinal disease. (OCT is considered in greater detail elsewhere in this publication.) Although VER recording does not reflect retinal function directly, it is a necessary test when measures of retinal function fail to disclose the site of abnormality. It is therefore discussed briefly at the end of the chapter. Guidelines are presented for obtaining reliable and reproducible results and, in some cases, for interpreting variations within a single session and between visits in a given patient as well as differences that may exist among normal subjects.
FUNDUS REFLECTOMETRY
Fundus reflectometry (also known as retinal densitometry) is a method for estimating the mass optical density, absorption spectra, and regeneration kinetics of the photolabile pigments within photoreceptors. Although still primarily a research tool, it may be used clinically to help identify stationary forms of nyctalopia that have normal rhodopsin densities but a defect in transmission between rod photoreceptors and more proximal retinal cells[1]; diseases of dark adaptation involving slowed pigment regeneration, such as fundus albipunctatus (Fig. 124.1)[2]; diseases of dark adaptation with normal pigment regeneration, such as Oguchi's disease[3]; and photoreceptor mosaicism in carriers of X-linked retinitis pigmentosa.[4,5] It is also useful in subtyping patients with dominant retinitis pigmentosa based on generalized rod loss versus regionalized rod and cone loss[6] and in monitoring the course of diseases affecting the uvea and retinal pigment epithelium (RPE).[7]
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FIGURE 124.1 Visual pigment regeneration for two brothers (LD and ED) with fundus albipunctatus. Recovery times to 50% of maximum were ?1h for rhodopsin in the peripheral retina and 20 min for cone pigments in the fovea, increases of approximately 20-fold and 16-fold, respectively, compared with normal times with this test system. |
Two types of reflectometer are in use at present, normally in a clinical research setting. The first, and original, type involves collecting and comparing the amount of light reflected by 1-2° of the fundus, initially from the dark-adapted eye and then again after exposure to a light that bleaches most of the available visual pigment.[8-10] The light from a brief test flash, which in itself bleaches little of the visual pigment, is reflected from the fundus and focused on the head of a photomultiplier tube. The difference in the reflected light before and after the bleaching episode provides a measure of the amount of visual pigment. The second, and more recent, type of reflectometer involves imaging the fundus over a visual angle of at least 10° and capturing the reflected light either photographically or on videotape. Density differences are then quantified by comparing unbleached with bleached areas captured in a single image[11,12] or between successive images.[13,14]
Both systems may be properly used only in patients with clear media and stable fixation. The pupil is maximally dilated and the eye undergoes dark adaptation for at least 30 min, during which time an impression of the patient's bite is made with dental wax. With the patient's head stabilized by the wax impression and the fellow eye fixating on a red lamp or light-emitting diode, the examiner aligns the eye to be tested in dim red or infrared light that causes negligible bleaching. For assessing rhodopsin density, the mid-peripheral fundus, in which cone photoreceptors are scarce, is generally chosen. For assessing cone pigment density, the fovea, in which rods are fewest, is chosen. A test flash of narrow-band wavelength near the peak of the absorption spectrum (i.e., ?500nm for rods and ?560nm for cones) is then presented to quantify reflectance of the dark-adapted eye. After reaffirming alignment in red light, a bright white light is usually presented for many seconds to bleach at least 95% of the visual pigment. The test flash is presented again to quantify reflectance without significant absorption by visual pigment. The logarithmic difference between the two measurements represents the pigment density. This is a 'double-density' difference in that the test beam has passed through the photoreceptor layer twice, the second time by reflection from the pigment epithelium and choroid. Additional test flashes may be presented either at other wavelengths in rapid succession to both dark-adapted and bleached eyes to determine the absorption spectrum of the visual pigment or at known intervals after the bleaching episode to assess the time course of pigment regeneration. Figure 124.2 illustrates results from an imaging densitometer. In this case, a mid-peripheral region of retina was initially bleached for 10s over only the right half of the image, the left half remaining adapted to the dark. The figure shows the image photographed by a 500-nm test flash and captured on videotape. The bleached half appears brighter than the unbleached half, indicating less remaining visual pigment in the former.
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FIGURE 124.2 Midperipheral regions of a normal human fundus photographed on videotape with 502-nm light immediately after a greater than 95% rhodopsin bleach over the right half performed with an imaging reflectometer. Squares at the bottom were copied from areas designated with letters to facilitate comparison of double-density gray values, which differ by ?0.2 log unit. |
Normal values for rhodopsin double density range from 0.08 to 0.15 log-unit in different peripheral regions for young adult subjects[15] and from 0.06 to 0.14 log-unit between regions of a single subject.[16] For cone pigment, the range between subjects is 0.14-0.30 log-unit within the central 2°, which falls to 0.05-0.11 log-unit at an average eccentricity of 2.5° degrees.[14] Foveal cone pigment density has been reported to decline linearly with age.[17]
FUNDUS AUTOFLUORESCENCE
Delori and associates in 1995 used noninvasive fundus spectrophotometry to demonstrate that lipofuscin in the human RPE exhibits a red fluorescence when stimulated by a shorter-wavelength light.[18]Emission peaked between 620 and 640nm, and excitation peaked at 510nm. The shape of the emission spectrum changed for excitation wavelengths >470nm, indicating a second fluorophore. The authors also showed that fluorescence was minimal at the fovea, apparently due to excitation absorbance by macular pigment and by the increased melanin in this region, and increased markedly with increasing age for excitation >470nm (Fig. 124.3, left).
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FIGURE 124.3 (Left) Variation of RPE fluorescence with retinal location and age. Fluorescence at 620 nm was excited by a wavelength of 510 nm. Spatial resolution = 2°. (Right) Fluorescence of the RPE at 620 nm to an excitation of 510 nm for one or both eyes of patients with Stargardt disease/fundus flavimaculatus (triangles) and for healthy controls (circles, n = 45). Error bars designate ±1 standard deviation. The solid and dashed lines represent the best-fitting regression line and its 99.99% confidence limits. |
Later in the same year, Delori et al showed that patients with autosomal recessive juvenile macular degeneration (Stargardt disease/fundus flavimaculatus) and a dark choroid on fluorescein angiography had increased autofluorescence of the RPE after taking into account age (Fig. 124.3, right), indicating abnormally high accumulation of lipofuscin in these patients.[19] Since then, the adaptation of a confocal scanning laser ophthalmoscope in London has led to a series of papers describing the imaging of fluorescence of the RPE in patients with different retinal diseases. These authors reported increased autofluorescence in patients with macular dystrophy in general (i.e., even in the absence of a dark choroid on fluorescein angiography),[20] although some patients with Stargardt disease had reportedly normal or reduced autofluorescence.[21] Measurements with this instrument have also been performed in retinitis pigmentosa, where rings of increased autofluorescence in the macula and areas of decreased fluorescence in the periphery have been observed.[22,23] Conversely, some patients with Leber congenital amaurosis, an early-onset severe form of retinitis pigmentosa, have been found to have minimally, if at all, disturbed autofluorescence, which the authors interpreted as indicating morphologically preserved photoreceptors and RPE in those regions.[23] However, since the confocal scanning laser ophthalmoscope excites autofluorescence with a lower wavelength (488nm) and measures autofluorescence over a broader spectrum (with a 50% cut-on at 510nm) than the results of Delori and colleagues would recommend, it is possible that differences in lenticular absorption, in macular pigment density, in uveal pigment density, and in the relative concentrations of multiple retinal and RPE fluorophores may, in part, confound interpretation when this instrument is applied to different types and stages of hereditary retinal degeneration.
OPTICAL COHERENCE TOMOGRAPHY
OCT is a new rapid noninvasive method for obtaining cross-sectional images of the retina based on differential near-infrared light reflection at optical interfaces. Although its most obvious application has been to help monitor the surgical treatment of macular holes, epiretinal membranes, and retinal tears, OCT can also be used to help assess hereditary retinal disease. Figure 124.4 illustrates representative tomograms from a normal control subject and two patients with retinitis pigmentosa. The tomograms were obtained with a commercial third-generation instrument (OCT3). The upper tomograms from the normal control show the low-reflectance outer nuclear layer peaking in thickness in the center beneath the foveal depression and visible from edge to edge. A third high-reflectance band can be distinguished just above a thicker high-reflectance RPE/choriocapillaris complex; the third high-reflectance band is slightly convex in the center, possibly reflecting longer cone outer segments in this region. The middle tomograms from a patient with retinitis pigmentosa and normal visual acuities have a normal appearance in the center (with an intact third high-reflectance band) but show loss of the outer nuclear layer more peripherally. The lower tomograms from a patient with retinitis pigmentosa and poor visual acuities show widespread loss of the outer nuclear layer with apparent thinning of the inner retina. By examining patients with retinitis pigmentosa and no cystic changes in the macula, these investigators showed that both retinal thinning (due to cell loss) and retinal thickening (due to presumed edema) were associated with lower visual acuity (Fig. 124.5).[24] This study also found that an increase or decrease in retinal thickness of more than 17 ?m at fixation at follow-up can be considered a significant (p < 0.01) change in patients with retinitis pigmentosa.
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FIGURE 124.4 Tomograms recorded with a Zeiss Stratus High-Resolution Optical Coherence Tomographer from a 39-year-old female normal control with Snellen visual acuities of 20/20 OD and 20/20 OS (upper images), a 34-year-old male with retinitis pigmentosa (RP) and visual acuities of 20/20 OD and 20/20 OS (middle images), and a 33-year-old male with RP and visual acuities of 20/200 OD and 20/80 OS (lower images). Each tomogram subtended 6 mm centered on the fovea. The horizontal arrows (upper right) designate the low-reflective outer nuclear layer (ONL), the third high-reflectance band possibly designating the photoreceptor inner segment/outer segment junction (third HRB), and the high-reflective RPE/choriocapillaris (RPE). |
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FIGURE 124.5 Regression of ETDRS visual acuity on OCT retinal thickness at fixation based on data from 288 eyes of 162 patients with retinitis pigmentosa without macular cysts. The straight line with large dashes is the best-fitting linear function. The curve with small dashes is the best-fitting log function. The solid curve is the best-fitting second-order polynomial and best describes the data. The different symbols designate the definition of the third high-reflectance band (third HRB) which is thought to represent the inner segment/outer segment junction. |
OCT has also been used to detect cystoid macular edema (CME) in patients with retinitis pigmentosa. One of these studies found that the area of the cystoid spaces was positively correlated with the grade of the fluorescein angiogram and the visual acuity,[25] and two studies have reported that some eyes with cystoid lesions showed no angiographic evidence of dye leakage or pooling.[25,26] Figure 124.6 illustrates tomograms from a patient with retinitis pigmentosa, before and after treatment with acetazolamide for CME; the patient showed no evidence of CME on fundus examination or fluorescein angiography prior to treatment.[26] Several studies have also used OCT to visualize cystic lesions in patients with hereditary retinoschisis.[27-30]
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FIGURE 124.6 OCT images from the right eye of a 44-year-old male with retinitis pigmentosa before a and 3 weeks after b beginning treatment with acetazolamide, at which time the macular cysts had decreased in size. |
EARLY RECEPTOR POTENTIAL RECORDING
The ERP is a very short latency, biphasic response from the eye elicited by a very bright flash of light.[31] It consists of a cornea-positive component (R1) followed by a cornea-negative component (R2), which is then followed by the cornea-negative a-wave of the electroretinogram (ERG) (Fig. 124.7). It derives almost entirely from photoreceptors[32]- R1 reflects the conversion of lumirhodopsin to metarhodopsin I,[33] whereas R2 reflects the conversion of metarhodopsin I to metarhodopsin II.[34] As the ERP amplitude is proportional to the amount of pigment bleached by the flash[35] and depends on the orientation of visual pigment molecules within the outer segment,[36] it may be used as a measure of mass outer segment optical density over most of the retina. In humans, 60-70% of the R2 response is generated by cones and the remainder by rods,[37,38] so that it may not be possible to specify whether one or both receptor systems are involved if the amplitude reduction is less than 40%. The ERP has been used to demonstrate photoreceptor impairment in a variety of retinal diseases, including retinitis pigmentosa.[31,39,40]
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FIGURE 124.7 Normal human ERP followed by an a-wave of ERG (left tracing) and normal ERP with high sweep speed and amplification (right tracing). Both cornea-positive peak (R1) and later cornea-negative peak (R2) of ERP are designated. Stimulus onset is at the beginning of each trace. Calibration symbol = 2 ms horizontally and 100 ?V vertically for the left tracing; 0.5 ms horizontally and 50 ?V vertically for the right tracing. |
The ERP must be elicited with a light flash ?10 million times brighter than that required to elicit the b-wave of the ERG (Fig. 124.8).[41] This can be achieved, for example, with a flashgun focused in the plane of the pupil in Maxwellian view. Care must be taken to avoid a photovoltaic artifact superimposed on R1. Investigators have developed custom monopolar contact lenses with either nonmetallic electrodes or metallic electrodes shielded from incident light[42-44] to record an ERP uncontaminated by a photovoltaic artifact. Since the light flash must bleach a considerable amount of visual pigment to generate a detectable response, time for pigment regeneration must be allowed before presenting successive flashes to illustrate reproducibility. Intraindividual and interindividual variations in ERP amplitude may be as much as 3:1.[45]
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FIGURE 124.8 Amplitudes of the b-wave and a-wave of the ERG and the R2 component of the ERP for the dark-adapted albino rat as a function of the log of the flash energy. Responses were obtained with 0.7-ms full-field white flashes. Log flash energy of zero corresponds to one quantum absorbed/rod. Amplitudes were measured from baseline. |
ELECTROOCULOGRAPHY
The light-rise of the electrooculogram (EOG) is a measure of the integrity of the RPE and overlying photoreceptors. It arises from a depolarization of the basal (i.e., choroidal side) membrane of the RPE.[46]The EOG is a measure of the slowly changing voltage difference between the front (positive) and the rear (negative) surfaces of the globe recorded over time under different conditions of illumination (Fig. 124.9). The light-rise represents the largest difference measured in illumination divided by the smallest difference measured in darkness.[47] In clinical practice, its important use is to help diagnose Best's vitelliform macular degeneration, a dominantly inherited condition. In this disease, the EOG light-rise is reduced or absent.[48-51] Asymptomatic carriers of Best's disease may also have abnormal EOGs.[50]The EOG also differentiates Best's disease from pseudovitelliform macular degeneration, in which there is generally a normal ratio.[51] In other retinal diseases, EOG testing does not add to whatever diagnosis may have been made with the ERG alone.[52] Fast oscillations of the EOG have also been recorded[53] and have been found to be dissociated from the light-rise of the EOG in some maculopathies. For example, in one report the fast oscillations were found to be normal or nearly normal, while the light-rise was abnormal, in Best's disease (Fig. 124.10).[54]
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FIGURE 124.9 EOG recorded from a normal subject. Eye movements were made twice each minute by alternately viewing fixation points separated by 30° in a Ganzfeld dome, differentially amplified at a gain of 200 (-3 dB at 0.1 and 100 Hz), digitized, and peak-to-peak amplitudes for each saccade quantified by computer. Vertical dotted lines are separated by 2.5-min intervals; horizontal dotted lines are separated by 200 ?V. Arrow designates onset of background illumination. |
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FIGURE 124.10 EOG from a normal subject (A) and from three patients with Best disease (B-D). Event marker line below indicates 'on' or 'off' of the 20 ftL background illumination. Fast oscillation peaks, indicated on normal EOG by closed arrows, are normal for the patients. Peaks of the light-rise are indicated by open arrows, and are prolonged and/or subnormal for the patients. |
The EOG may be measured in cooperative patients with stable fixation who have a visual-field diameter of at least 60°. After exposure to ambient room illumination for 30 min or longer, during which time the pupils are dilated, cup electrodes filled with electrode cream are attached with tape just lateral to the inner and outer canthus of each eye (i.e., two electrodes per eye) and to the forehead as ground. On verbal instruction from the examiner, the subject alternately fixates red light-emitting diodes in a Ganzfeld dome placed at 0° and 30° with respect to forward gaze, first in the dark for ?12 min and then in the presence of a full-field 10ftL white background for ?12 min to monitor the light-rise. A Ganzfeld dome, rather than an X-ray viewing box (or equivalent), should be used so that the entire retina will be illuminated as evenly as possible. Otherwise the EOG will reflect primarily only posterior pole function.[55] Intervals of ?12 min of darkness and 12 min of illumination are said to reduce the normal variation.[56]The maximal voltage in the light is compared with the minimal voltage in the dark to derive the light-rise to dark-trough ratio, which is normally greater than 1.8 in patients younger than 50 years. Care should be taken that the onset of illumination is not too abrupt or else the tested eye might begin to tear, which could alter electrode resistance. Fast oscillations may be elicited with light-dark periods of 2.5 min and can be immediately followed by monitoring the dark phase of the conventional EOG.[54] Responses from each eye should be differentially amplified at a gain of ?200 (0.1-100 Hz) and displayed on an x-yplotter or digitized and displayed by computer. Repeat recordings on a given patient should be done at approximately the same time of day because of the presence of an underlying circadian rhythm (see further on).
Intraindividual variability in the light-rise to dark-trough ratio of the EOG generally does not exceed 60%.[57-59] The normal range for the light-rise to dark-trough ratio has been placed at 1.9-2.8,[60] although values of 1.5-3.4 have been reported.[61] It should be noted that the ratio increases with luminance.[62] The ratio also appears to decline with age, at least among women.[60,61,63] Significant differences in the ratio between sexes have been reported, with larger values for females.[59,61] Between 20% and 50% of the variation in the EOG light-rise may be due to circadian rhythmicity.[64] Recordings done at 2-h intervals for six normal subjects showed a sinusoidal temporal pattern in which the ratio was highest in the early morning and late afternoon and lowest around midday.
FULL-FIELD ELECTRORETINOGRAPHY
The normal human ERG elicited by a moderate-intensity white flash from the dark-adapted eye consists of a cornea-negative deflection, called the a-wave, followed by a cornea-positive deflection, called the b-wave (Fig. 124.11). The a-wave is known to reflect photoreceptor function,[65,66] and the b-wave is generated by Müller's cells reflecting activity in the inner nuclear layer of the retina.[67,68] In actuality, the recorded b-wave is a summation of photoreceptor and more proximal retinal function, since the photoreceptor component continues beyond the onset of the b-wave.[66] Several wavelets, known as oscillatory potentials (Fig. 124.11), may normally be seen superimposed on the ascending portion of the b-wave. These wavelets reflect bipolar cell responses generated by feedback from amacrine cells.[69,70] It is also possible to record a positive response - the d-wave - to light offset, most typically for square-wave light modulation but, recently, also to sawtooth-modulated stimuli.[71,72] If the conventional brief strobe- or photo-flash is used to elicit the ERG, it is thought that the response lacks the positive d-wave that otherwise occurs at light-offset.[73]
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FIGURE 124.11 Dark-adapted ERGs recorded from a normal subject in response to full-field white flashes of varying integrated luminance. Traces begin at flash onset. Cornea-negative a-wave and cornea-positive b-wave are designated by letters; oscillatory potentials are designated by asterisks. Arrow points to inflection in the a-wave, representing a combination of cone and rod components. |
Separation of the ERG into a-wave, b-wave, and oscillatory potentials has important application for objectively diagnosing, classifying, and staging retinal diseases. For example, in response to a moderate-intensity white light presented to the dark-adapted eye, loss of the a-wave with a slowing of the b-wave in patients with clear media may signify a photoreceptor degeneration in which photoreceptors have lost optical density. This follows from the fact that in normal eyes reducing stimulus intensity affects a-wave amplitude before b-wave amplitude (see Fig. 124.11). Conversely, loss of the b-wave with preservation of the a-wave may signify retinoschisis[74] or congenital nyctalopia with myopia[1] (Fig. 124.12). In both of these conditions, synaptic transmission from photoreceptors to more proximal elements is disturbed. When 'on' versus 'off' responses to sawtooth stimuli were compared in patients with retinoschisis, the investigators found that the patients with retinoschisis had a normal a-wave but a b-wave to light onset that was relatively reduced when compared to their positive d-wave to light offset (Fig. 124.13).[71] The authors concluded that the patients had a greater impairment of their depolarizing bipolar system than their hyperpolarizing bipolar system. Selective loss of oscillatory potentials, in contrast, is usually interpreted as specifically reflecting inner retinal ischemia, as occurs in diabetic retinopathy[75]and central retinal vein occlusion (Fig. 124.14).[76]
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FIGURE 124.12 Dark-adapted ERGs recorded from a normal subject, a patient with juvenile X-linked retinoschisis, and a patient with congenital nyctalopia with myopia in response to a full-field white flash. Calibration - 100 ?V vertically and 50 ms horizontally. |
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FIGURE 124.13 ERG waveforms of patients with X-linked retinoschisis (XLRS) (panels 1 and 3) and control subjects (panels 2 and 4) in response to 8-Hz rapid-on (panels 1 and 2) and rapid-off (panels 3 and 4) sawtooth flicker. Dashed lines: time of stimulus onset; stimulus waveform is illustrated on the x-axis. The waveforms are arranged in order of increasing b-wave amplitude. Numbers next to the waveforms in the left panel refer to patient designations. |
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FIGURE 124.14 Dark-adapted ERGs recorded from a normal subject, a patient with central retinal vein occlusion (CRVO), and a patient with diabetic retinopathy in response to a full-field bright white flash. |
Careful inspection of the a-wave reveals an inflection (see Fig. 124.11, arrow) that reflects a summation of cone and rod components of differing time course. Although not apparent, the same is true of the b-wave. The fact that the normal dark-adapted ERG is a summation of rod and cone components may be demonstrated in two ways. First, placing in turn a blue filter or a red filter scotopically matched to the blue filter (i.e., matched in brightness for the rods) in front of the eye and then flashing a white light results in different waveforms (Fig. 124.15). The waveform with the blue filter in front of the eye consists of a late-onset, bell-shaped b-wave, whereas that with the red filter in front of the eye consists of an early-onset a-wave and b-wave (with oscillations) followed by the same late-onset b-wave. The early components observed for red, but not blue, light represent cone activity, whereas the late b-wave represents rod activity. When the cone and rod components are well separated in time, they appear to summate linearly.[77-79] However, when the two b-waves occur at the same time with near-maximal amplitudes, as for the conventional white flash, the summation appears to be nonlinear, as may be seen by using a method of digital subtraction.[80] Linear subtraction of a cone-isolated response to red light from a mixed cone and rod response to a bright blue light photopically matched to the red light yields a rod-isolated waveform in which the b-wave is 'scalloped' (Fig. 124.16). This implies that the b-wave to the bright blue light represents a sublinear summation of rod and cone components. Conversely, even for these bright lights, the two a-waves appear to summate linearly. The presence of a steady white background that desensitizes rods and eliminates their contribution to the ERG reveals a 'photopic' a-wave and b-wave from the cone system (Fig. 124.17).[81] In addition to background illumination, a flash rate of 30Hz may be used to isolate cone function to white light (see Fig. 124.15).[78] This is true because the rod system is normally incapable of responding to these rates of flicker. Lights of different wavelength and background adaptation have been used to demonstrate both a rod and a cone contribution to oscillatory potentials.[82]
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FIGURE 124.15 Full-field ERGs to scotopically matched red (column 1) and blue (column 2) flashes, to photopically matched orange (column 3) and blue-green (column 4) flashes in the presence of 5-10 ftL of background light and to 30-Hz white flashes (column 5) are shown successively from top to bottom for a patient with Nougaret-type night blindness, a normal subject, and a patient with advanced cone degeneration. Two or three responses to the same stimulus are superimposed. Calibration - 60 ms horizontally and 50 ?V vertically for columns 1 and 2; 30 ms horizontally and 50 ?V vertically for columns 3 and 4; 60 ms horizontally and 100 ?V vertically for column 5. Corneal positivity is an upward deflection. Stimulus onset, vertical hatched line for columns 1-4; shock artifacts for column 5. |
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FIGURE 124.16 Computer-averaged dark-adapted full-field ERGs from a normal subject to photopically matched blue (top) and red (middle) flashes and the result of subtracting the second response from the first to derive a rod ERG in isolation (bottom). The rod component elicited by the red flash had already been eliminated by subtracting the response to a scotopically matched blue flash. In the bottom waveform, the lower solid line represents the result of subtraction and the hatched area illustrates the suggested rod b-wave correction for nonlinear summation of the cone and rod components to blue light. |
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FIGURE 124.17 Analysis of ERG in a dark-adapted human eye as the resultant of photopic (dashed line) and scotopic (dotted line) components. The a-wave is composed of photopic (ap) and scotopic (as) components, and the b-wave is similarly composed of photopic (bp) and scotopic (bs) components. The solid line shows the algebraic sum of the lower waveforms. |
Isolation of rod and cone contributions to the ERG, like comparative analysis of the a-wave and b-wave components, is also important for classifying retinal diseases. For example, selective loss of cone function may signify congenital rod monochromatism or advanced cone degeneration, whereas loss of a rod contribution may signify an early stage of dominant retinitis pigmentosa. In the cone-isolated response to flicker, the high rate of presentation and sinusoidal nature of the waveform in cases of advanced retinitis pigmentosa make it possible to resolve amplitudes as small as 0.05 ?V with signal averaging and electronic filtering (see further on), which can be used to follow up the course of this condition.[83]
The full-field ERG may also be used to quantify the amount of remaining function for each of the three cone mechanisms. The middle-wavelength or green-cone system and the long-wavelength or red-cone system may be evaluated by comparing cone ERGs elicited by a short-wavelength flash or a photopically matched long-wavelength flash superimposed on a photopic background (see Fig. 124.15)[78] or flickering at 30Hz.[84] If the responses are equivalent in amplitude, the patient is considered to have comparable numbers of cones of each type, as has been observed in carriers of blue-cone monochromatism.[85] However, if the two responses are unequal in amplitude or differ by a factor of two or greater, then one of the two cone systems is considered to be reduced in number relative to the other or absent, respectively.[84] Use of a short-wavelength flash superimposed on a bright white background has been shown to isolate in time the short-wavelength or blue-cone system from the other systems (Fig. 124.18).[86]
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FIGURE 124.18 Full-field ERGs elicited by red and blue flashes at different levels of background illumination indicated by the photopic troland values on the left above each trace. L, M, and S signify the responses of, respectively, the long-wavelength-, the middle-wavelength-, and the short-wavelength-sensitive cone systems. Calibration - vertically 4 ?V for the upper four and 40 ?V for the lower four traces and 9 ms horizontally. |
Interest has developed in clinical recording of b-wave amplitude versus flash intensity functions under conditions of dark adaptation in patients with retinal disease. The intent of this approach is to distinguish changes in maximal amplitude (Vmax), which are thought to reflect cell number and response gain, from changes in sensitivity (k), which are thought to reflect outer segment optical density and media clarity. Some studies have used white flashes to elicit these functions,[87-89] which complicates interpretation because of the variable summation of cone and rod contributions in diseases that may affect these two systems unequally. One study used digital subtraction to isolate rod function in patients with retinitis pigmentosa or cone-rod degeneration and showed that reductions in sensitivity, irrespective of changes in maximal amplitude, may be used to infer losses of rod photoreceptor optical density.[90] Another study showed that patients with an apparently rare form of retinal degeneration could have increased maximal rod b-wave amplitude with reduced sensitivity, as well as implicit times that were more delayed for dim flashes than for bright flashes (Fig. 124.19), a combination that could better be explained by an elevation of retinal cyclic guanosine monophosphate than in terms of cell and optical density loss.[80]
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FIGURE 124.19 Rod b-wave amplitude and implicit time (i.e., time to peak) versus retinal illuminance functions for 17 normal subjects (mean ± SD) and three patients with an elevated cyclic guanosine monophosphate-type retinal degeneration. Rod ERGs were elicited with full-field blue flashes at low retinal illuminances and by a method of digital subtraction involving photopically matched blue and red flashes at high retinal illuminances. |
A corresponding approach has been used by a few laboratories to evaluate the a-wave and, by inference, the photoreceptor response. a-Waves were elicited by a series of flash intensities that included intensities far brighter than those needed to saturate the b-wave. A computational model was then applied to the leading edge of the a-wave to estimate parameters of the phototransduction cascade. Representative cone a-waves obtained in the presence of a rod-saturating background and rod a-waves derived from subtracting the cone a-waves from dark-adapted responses of a normal observer and the fitted functions are illustrated in Fig. 124.20.[91] With such fitted functions it is possible to estimate the maximal amplitude, sensitivity, and delay of the respective photoresponse. Normative values are presented in the afore-mentioned paper.[91] When this methodology was applied to patients with retinitis pigmentosa, the authors found that cone and rod a-waves were quantifiable in 30-33% of cases and, in those cases, the maximum amplitude and sensitivity parameters were below normal in all subgroups.[92] In this cross-sectional study there was no evidence that phototransduction efficiency decreased with increasing age.
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FIGURE 124.20 Representative a-waves from a 65-year-old control subject. a Responses in dark to intensities ranging from 3.2 to 4.4 log scotopic troland-seconds (log sc td-s). b Same 4 intensities presented against a 3.2-log td background. c Rod-isolated responses and fitted functions (dashed lines). d Cone responses and model fits (dashed lines. |
Full-field ERGs, like EOGs, are conventionally elicited with a Ganzfeld dome (Fig. 124.21) that provides a nearly homogeneous distribution of light over the central 120° of the retina.[93] Although retinal illuminance falls as a consequence of decreasing apparent pupillary area for retinal eccentricities greater than 60°, this is compensated in large part by the curvature of the retina and by reduced light absorption in the ocular media with eccentricity.[94] With virtually uniform retinal illumination, the faster cone and slower rod components of the ERG across the retina respond with a minimal variation in latency and therefore may be separated in time and quantified. In addition, this retinal light distribution is altered little by small changes in eye position, which fosters reproducibility between successive responses.[93,95]
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FIGURE 124.21 Ganzfeld stimulator. Flashlamp enclosed in case and attached to the top of the diffusing sphere illuminates the inner white surface of this dome (40 cm in diameter), providing a full-field stimulus. Lights are recessed in the top of the dome so that the patient can be tested in the presence of steady full-field background light. |
The eye(s) to be tested should be initially dilated and adapted to the dark for at least 45 min. Dilatation maximizes amplitudes and generally minimizes implicit times.[96,97] Complete dark adaptation, which may require 45 min or longer depending on the level of prior exposure, also maximizes amplitudes (Fig. 124.22)[98] while also tending to maximize implicit times. Recordings are best done with a bipolar contact lens electrode, with the positive electrode being a ring around the contact lens and the negative or reference electrode a conductive coating on a lid speculum (Fig. 124.23). The bipolar configuration, in which the lid versus ground response is subtracted from the cornea versus ground response, localizes the response to the eye and provides the best elimination of surrounding 60-Hz noise and any photovoltaic artifact that may be generated by the flash lamp. The lid speculum also prevents the upper and lower eyelids from partially covering the cornea and thereby obstructing the passage of light into the eye. A reduction in retinal illuminance could artifactually reduce ERG amplitudes and increase implicit times. Some facilities use alternative electrodes for recording ERGs. In this country, these include the disposable Jet electrode, the Arden gold foil electrode, and the Dawson-Trick-Litzkow (DTL) fiber electrode. All three are monopolar electrodes that will give larger voltages than the bipolar Burian-Allen electrode,[99] but also be more subject to artifact contamination.
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FIGURE 124.22 Computer-averaged full-field rod ERG percent amplitudes to a 0.5-Hz 10-?s blue flash (max 440 nm) of 16 ftL recorded over time in the dark from a normal observer after a 10-min full-field white light bleach of 10 or 500 ftL presented to the dilated pupil. Amplitudes for 60 min of dark adaptation were arbitrarily designated 100%. All the data appear to reflect rod function, except for the low-amplitude 5-min value after the stronger bleach, which probably represents residual cone function. Each set of data represents a single run. |
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FIGURE 124.23 Double-electrode (Burian-Allen) contact lenses used to obtain ERG responses. |
With the patient fixating on a red light-emitting diode, 0.5-Hz dim, short-wavelength flashes may be given first to isolate the rod ERG. Next, 0.5-Hz scotopically matched long-wavelength flashes can be given to obtain a mixed response consisting of a faster cone component and a slower rod component of amplitude comparable with that elicited by the short-wavelength flash. After that, 0.5-Hz dim white flashes may be given to elicit an a-wave and maximal b-wave consisting of a summation of both cone and rod components. Finally, 30-Hz dim white flashes or 0.5-Hz dim white flashes superimposed on a background to isolate a cone response may be given.[95] For cases of generalized cone degeneration or when abnormal color vision is found, the spectral lights described earlier may be presented to assess the different cone systems across the retina. When isolating the cone response with flicker or background illumination, the retina may need to be illuminated for several minutes before a maximal response is obtained.[100-102]
Patients with small dilated pupils or those whose pupils cannot be dilated or, in some cases, those who have media opacities that obscure visualization of the fundus may have ERGs that are smaller in amplitude than expected or nondetectable with single flashes because of reduced stimulus retinal illuminance. An electronic photoflash, with ?1000 times the energy of the conventional full-field flash when illuminating a Ganzfeld dome, can be used to elicit larger responses from such eyes.[103] In order to separate the optical density effect of the media obstruction from any change that may be due to a retinal abnormality, responses to a series of stimulus intensities should be compared with those obtained with conventional full-field flashes in normal eyes (e.g., see Fig 124.11). If a-wave and b-wave amplitudes and implicit times to the brighter flashes in eyes with opaque media can be matched to those obtained to the dimmer flashes in normal eyes with clear media, large areas of the retina can be considered to be functioning normally.[104] In eyes with large pupils and relatively clear media, these bright flashes may be used to elicit a maximal a-wave with oscillatory potentials superimposed on the ascending limb of the b-wave.[103] Bright flash stimulation should be presented to the dark-adapted eye (i.e., before flicker or background illumination), with each flash separated from the next by an interval of ?1 min to minimize light adaptation from the preceding flash.
Full-field ERG responses may be photographed in real time from an oscilloscope or digitized and stored for subsequent analysis and hard copy. Digitization may be used to eliminate baseline variation and to isolate oscillatory potentials, whose frequency content (50-180Hz) exceeds that of the a-wave (<50Hz) and b-wave (<25Hz).[103,105-107] Oscillatory potentials may then be quantified either in conventional time domain or, by Fourier analysis, in frequency domain (Fig. 124.24). Consecutive digitized responses may also be summed and averaged and, for 30-Hz flicker, smoothed with narrow band-pass filtering to resolve very small amplitudes (Fig. 124.25).[83,108] However, for this purpose, recordings should be performed with a bipolar contact lens electrode to minimize contamination of the waveform with electrical or photovoltaic artifacts. Figure 124.26 shows responses to 30-Hz xenon flashes recorded without and with narrow band-pass filtering from different electrodes placed in saline solution. The monopolar gold foil electrode generates a large artifact with constant phase for both types of filtering. The monopolar Jet electrode generates smaller responses reflecting an electrical artifact alone. In contrast, the two bipolar electrodes (the Burian-Allen and GoldLens) show minimal noise.[109] With a well-conditioned Burian-Allen contact lens electrode, test-retest reproducibility for sub-?V full-field cone ERGs to 30-Hz flashes revealed highly correlated implicit times and amplitudes.[109] Signal averaging may also be used to reduce large voltages due to eye movements, as, for example, in cases of nystagmus.
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FIGURE 124.24 Quantification of mean oscillatory potential amplitude in the time domain between the a-wave and the b-wave peaks after 62-Hz high-pass filtering for a normal subject in response to a bright full-field flash white flash. Vertical lines represent some of the amplitudes whose absolute values with respect to baseline were summed before calculation of the mean (left). Quantification of mid- and high-frequency amplitudes for oscillatory potentials by the magnitude fast Fourier transform for a normal subject in response to the same flash (right). |
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FIGURE 124.25 ERGs elicited with full-field white 30-Hz flashes from a normal subject and four patients with different genetic types of RP at ages spanning intervals of 11-15 years. Responses were recorded without (left column) or with (right column) computer averaging and band-pass filtering. Stimulus onset, vertical markers. Calibration (left column, lower right) - 100 ?V vertically for the normal subject and the top three patients; 40 ?V vertically for the bottom patient; 50 ms horizontally for all traces. Calibration (right column, lower right) - 2 ?V vertically for the dominant, X-linked, and isolate patients; 0.3 ?V for the recessive patient; 20 ms horizontally for all traces. The b-wave implicit times are designated with arrows. |
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FIGURE 124.26 Responses to full-field 30-Hz xenon flashes of 1.3 log td-s without (left) and with (right) narrow-band filtering from four different electrodes placed in saline solution. |
The variation in full-field ERG amplitude for a given stimulus condition across normal subjects is as much as 2.5:1.[110,111] Age, sex, refraction, ocular pigmentation, and time of day may all contribute to this variation: with increasing age among adults, a-wave amplitude declines,[110] b-wave amplitude declines,[110,112-114] oscillatory potential amplitude declines (Fig. 124.27), and b-wave implicit time increases.[110,114] The same trends have been observed in the b-wave component isolated from the short-wavelength-sensitive cones in response to a blue flash on a bright background.[115] These decreases in ERG amplitudes and increases in ERG implicit times with increasing age probably reflect decreasing retinal illuminance with increasing age caused by reduced light transmissivity by the lens[116] and, possibly, to a smaller dilated pupillary diameter with increasing age. Similarly, increased uveal pigmentation lowers amplitude, and decreased uveal pigmentation raises it.[117,118] Increased uveal pigmentation related to race appears to reduce the maximal amplitude by an average of 17%, but does not affect the sensitivity or the implicit time of the b-wave as measured in response to blue flashes of varying retinal illuminance.[119] The ERG b-wave is also larger in hyperopic eyes than in myopic eyes (Fig. 124.28)[112,120,121] and is slightly larger in women than in men.[110,112,120,122,123]
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FIGURE 124.27 A-wave, b-wave, and oscillatory potential amplitude versus age in normal subjects. Responses were elicited from dark-adapted eyes with full-field dim white flashes for the upper and middle graphs or bright white flashes for the lower graph. Oscillatory potential amplitudes are calculated as described for Figure 124.24. |
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FIGURE 124.28 Linear regression of b-wave amplitude on refraction based on average of regression equations for men and women from measurements on 86 normal subjects. Maximal b-waves were elicited from the semi-dark-adapted eye with white light flashes derived from a lamp at different distances from the subject and presented through the dilated pupil. |
Intrasubject variation in amplitude in normal subjects across days can be as much as ±25% for large responses. Normal subjects, unentrained to a cyclical pattern of illumination, have shown no significant variations in rod ERG amplitude over daylight hours.[124] However, in normal subjects entrained to cyclical illumination for at least 3 days, rod ERG amplitudes follow a regular pattern, being on average 10-15% smaller 1.5 h after light onset than at other times of day.[124] The physiologic basis for this ERG diurnal rhythm appears to be an alteration in photoreceptor function associated with rod outer segment disk shedding, since recordings in normal light-entrained rats have demonstrated an ERG variation similar to that seen in humans and that is significantly correlated with the number of phagosomes appearing in the RPE.[125]
FOCAL CONE ELECTRORETINOGRAPHY
Since only some 7% of cone photoreceptors are in the central macula, patients with macular degeneration typically retain normal full-field ERGs and must be assessed with a focal stimulus in order to reveal and quantify malfunction.[52] Foveal cone ERGs have been used by various centers since the late 1960s to detect macular malfunction.[126-133] Abnormal foveal cone responses have been found in patients with Stargardt's disease,[129,134] in which amplitude varied directly with visual acuity, and in those with age-related macular degeneration (Fig. 124.29).[130,135] Patients with age-related macular degeneration may also show delays in foveal cone ERG implicit time that are associated with abnormal choroidal perfusion.[136] In patients with macular holes, foveal amplitude varied inversely with hole diameter, and subnormal responses appeared to predict impending holes in the fellow eye.[137] Foveal ERGs may be abnormal in symptomatic patients even when no diagnostic abnormalities can be seen by ophthalmoscopy and fluorescein angiography,[138,139] and thus they provide an aid in early detection of macular malfunction (i.e., the so-called 'occult maculopathy'). Conversely, foveal cone ERGs are normal in patients with strabismic amblyopia[140] or optic neuropathy,[140-142] indicating that the test provides a differential diagnosis for diseases of the macula versus diseases of the optic nerve or visual cortex (Fig. 124.30). Focal cone ERGs should, in general, be used in conjunction with full-field recording in order to rule out generalized cone degeneration, which may not have visible signs of peripheral retinal disease.
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FIGURE 124.29 Representative foveal cone ERGs from a normal subject and four patients with AMD. Responses were recorded with a stimulator ophthalmoscope. Each 100-ms trace contains 4 responses to the 42-Hz stimulus. Two consecutive traces (each the average of 200 sweeps) are shown for each patient. Arrows indicate b-wave implicit time (time between light flash and response). |
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FIGURE 124.30 Foveal and parafoveal cone ERGs recorded with a stimulator ophthalmoscope from a normal subject and four patients with visual acuity 6/60. Two or more consecutive computer summations (n = 128) are shown. Vertical lines denote stimulus onset; arrows denote b-wave implicit time to corresponding response peak. Calibration symbol in lower right corner denotes 20 ms horizontally and 0.25 ?V vertically. |
Focal cone ERGs may be elicited with a commercially available hand-held stimulator ophthalmoscope, the Maculoscope. With this instrument, the examiner can visualize the fundus through a short-stalk Burian-Allen bipolar contact lens electrode over the dilated pupil. A 42-Hz, white, 4° diameter stimulus is placed on the fovea and centered within a steady white 12° surround. This stimulus/surround configuration allows the examiner to visually track an area to be tested even in patients with eccentric or variable fixation. The high rate of flicker not only isolates cone function but also renders the response sinusoidal so that amplitudes less than 1 ?V, which occur normally, can be resolved with the help of electronic filtering, computer averaging, and Fourier analysis.[129] White flashes, rather than red, are used to avoid eliciting subnormal amplitudes from patients with congenital protanopia. Focal cone ERGs may be performed in an unshielded room in dim ambient room illumination. Since foveal cone ERG amplitudes are very small and tend to increase during continued light adaptation,[143] consecutive response averages should be recorded over a period of a few minutes to prove reproducibility.
Foveal cone ERG amplitude has been reported to be inversely correlated with age for 100 normal eyes of subjects between the ages of 5 and 75 years.[130] A fourfold range for normal foveal cone ERG amplitude has been reported.[134] Although the reason for this large normal range is unclear, it can be effectively reduced by 30% on average by adopting a foveal to parafoveal amplitude ratio.[144] This approach should be used with caution, as some regional variation in the parafoveal cone ERG has been reported.[145] The foveal cone ERG is also smaller in amplitude in eyes with brown irides than in eyes with lighter-pigmented irides, presumably because of the corresponding differences in uveal pigmentation.[146] Normal intervisit variability in foveal cone ERG amplitude has not been reported.
Another commercial instrument (the VERIS System) exists for recording focal cone ERGs simultaneously over an interval of 16 min from up to 241 locations within the central 50°, called the 'multifocal ERG'.[147] Although this approach assumes phase (implicit time) constancy between adjacent 5° locations and relies on the voluntary fixation of the patient, the output is a detailed three-dimensional plot of response amplitude by location. These a-wave and b-wave responses appear to be the same as those obtained by conventional recording and, thereby, appear to be of value in identifying which cells are contributing to the retinal malfunction.[148] This approach ought to be most useful for characterizing maculopathies[149-151] by revealing a relative foveal amplitude depression (e.g., Fig. 124.31),[150] but the diagnosis of maculopathy rests on the assumption that the patients maintained stable central fixation throughout testing.
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FIGURE 124.31 Multifocal ERGs and the corresponding three-dimensional plot for a normal control subject (left) and a patient with occult macular degeneration (right). |
The multifocal ERG may be most appropriate for generating an objective map of the central visual field of patients with generalized retinal disease who retain central fixation. Several studies, for example, have performed multifocal ERG testing on patients with early stages of retinitis pigmentosa. Based on a sample of 111 patients with retinitis pigmentosa, 24% who were anticipated to have sufficiently large signals for testing were found to have a detectable multifocal ERG, consisting of a central peak of variable diameter, with implicit times that tended to increase towards the periphery.[152] A study on a small group of patients with retinitis pigmentosa tried to correlate multifocal cone ERGs with multifocal rod ERGs and multifocal ERG values with visual field sensitivities by location. The authors reported a significant correlation between multifocal cone ERG amplitude and cone-mediated visual field sensitivity, but there were many instances of reduced amplitude with normal sensitivity. The correlations between rod ERG amplitude and rod-mediated visual field sensitivity and between cone ERG amplitude and rod ERG amplitude were very low, perhaps due to methodological problems in isolating multifocal rod ERGs.[153]
Although full-field ERG recording has been shown to detect retinal malfunction in 90-96% of obligate carriers of X-linked retinitis pigmentosa,[154,155] complementary tests of localized function could potentially enhance this sensitivity by revealing an underlying mosaicism as predicted by random X-chromosome inactivation. Multifocal ERG recording in five carriers from three families revealed local amplitude losses and implicit time delays in two patients, local amplitude losses alone in one patient, local implicit time delays alone in one patient, and no abnormality in one patient. In the two patients with both types of defect there was poor correspondence between the locations showing amplitude losses and the locations showing implicit time delays.[156] Although based on only a small number of patients, these results suggest that multifocal ERG recording may not be a useful additional test to detect carriers of X-linked retinitis pigmentosa.
Multifocal ERG recording has also been used to reveal the distribution of retinal malfunction in patients with X-linked juvenile retinoschisis.[157,158] These patients present with a foveal schisis in all cases and a peripheral schisis in 50% of cases. Multifocal ERG recording has demonstrated a greater amplitude loss centrally than peripherally, and amplitudes have been found to be normal even in areas with visible peripheral schisis. The multifocal ERG waveform also generally does not show the negativity characteristic of the full-field ERG, perhaps because of constraints in the software algorithm that derives the multifocal ERG waveform. Interestingly, multifocal ERG implicit times have been found to be delayed at all, or nearly, all tested locations, indicating involvement of areas without visible schisis and areas with normal amplitudes.
FOCAL ROD ELECTRORETINOGRAPHY
Since rod photoreceptors are especially sensitive to reflected light, it has been impossible, until recently, to record rod ERGs to bright flashes of light from localized areas. In addition to allowing one to characterize transduction among homogeneous populations of rod photoreceptors,[159] these techniques allow the assessment of retinal function in areas outside of the macula that are not easily assessed by focal cone ERGs because cone photoreceptors are normally scarce. For example, Figure 124.32 shows dark-adapted focal rod ERGs from a patient with the multiple evanescent white dot syndrome and an enlarged blind spot in her left eye.[160] In comparison with the large, symmetric focal responses recorded from the nasal and temporal retina of a normal subject, the response from the nasal retina (within the scotoma) was nondetectable whereas the response from the temporal retina was normal in the patient. These recordings prove that her visual loss was of retinal origin.
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FIGURE 124.32 (a) Kinetic visual field for the left eye (visual acuity = 20/25) of a patient with multiple evanescent white dot syndrome (MEWDS) and an enlarged blind spot. The visual field was to a V4e white test light presented with a Goldmann perimeter on a background luminance of 31.5 asb. The blind spot normally subtends ?5° between 10° and 20° eccentricity in the temporal field. b Focal rod ERGs to a 30°-diameter blue flash of 2.1 log scot. td.-s. along the horizontal meridian at an eccentricity of 25° in the temporal or nasal field of the eye of a normal volunteer and the affected eye of the patient. Responses were computer averaged (n = 4). Focal rod ERGs for another normal volunteer (not illustrated) had b-wave amplitudes that were 44 ?V temporally and 43 ?V nasally. |
One approach involving three steps allows one to record the focal rod a-wave.[159] In step 1, ERGs are recorded from the dark-adapted eye in response to a 40°-diameter stimulus. In step 2, ERGs are recorded in response to the same stimulus 1 s after a white conditioning flash of the same diameter. The conditioning flash is used to desensitize rods directly illuminated by the flash without significantly affecting the a-wave generated by rods and cones illuminated by reflected light. Subtracting the a-wave obtained in step 2 from that obtained in step 1 derives the a-wave generated by rods directly illuminated by the flash. The validity of this method rests on the assumption that the rods and cones outside the stimulus are not light-adapted by the conditioning flash.
A second technique allows recording both a-waves and b-waves from the dark-adapted eye in response to stimuli as small as 10°.[160] This technique is illustrated in Figure 124.33. The response to a 30°-diameter focal blue flash (a) consists of a faster, smaller 'focal' component and a slower, larger 'stray light' component. The response to some dimmer full-field blue flash (b) consists of a negative deflection (a-wave) followed by a positive deflection (b-wave) that closely matches the stray light b-wave of the first response. Subtraction of the second from the first response (a-b) results in isolation of a local response (c) with an a-wave and b-wave that are smaller than, but otherwise similar to, those of the full-field, rod-isolated response to a blue flash of the same retinal illuminance (d). The focal response appeared to be generated entirely by rods because the ERG to a 30°-diameter red flash that would elicit the same cone response as the blue flash was nondetectable for these recording conditions (e). When using a 10°-diameter stimulus, the stray light component may not overlap the focal component that can then be quantified without resorting to a subtractive technique.
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FIGURE 124.33 ERGs from a normal volunteer. a ERG to a 30°-diameter blue flash of 2.1 log scot. td.-s. at a temporal retinal eccentricity of 30°. b Full-field ERG to a blue flash of 0.4 log scot. td.-s. c Subtraction of b from a. d ERG to a full-field blue flash of 2.1 log scot. td.-s. after subtraction of the response to a photopically matched full-field red flash. eERG to a 30°-diameter red flash at a temporal retinal eccentricity of 30°s and photopically matched to the blue flash used to elicit a. Responses were computer averaged (n = 4). A second normal volunteer gave similar ERGs (not illustrated). |
PATTERN ELECTRORETINOGRAPHY
Pattern electroretinography uses a spatiotemporal exchange of stripes or checks in which mean luminance is held fixed to elicit a focal ERG.[161] The theory is that local, time-varying changes in luminance responses generated by photoreceptors cancel at the cornea (or at least make a minimal contribution to the response), whereas cells with center-surround receptive fields (i.e., retinal ganglion cells) are strongly stimulated. One defense of this idea is that the pattern electroretinogram (PERG) shows spatial tuning under some situations. That is, amplitude is larger for stimulus elements of medium size and smaller for stimulus elements of smaller or larger sizes (Fig. 124.34).[162-170] In other instances, however, a low-pass relationship occurs in which amplitude increases monotonically with the size of the stimulus element.[168] In these cases, primarily resulting from high-luminance, high-contrast targets, a significant luminance component is thought to contaminate the responses to large stimulus elements.[162,167] Luminance components presumably arise from nonlinear summation of opposite sign responses to on and off stimulation.[169,170]
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FIGURE 124.34 Pattern ERG amplitude and phase versus spatial frequency for a normal subject. Responses were elicited with a 1.6 × 1.7°, 100% contrast sinusoidal grating reversing sinusoidally at 8 Hz. Responses were recorded with a gold-foil electrode. |
Unlike the focal luminance-evoked ERG (usually referred to as the focal ERG or FERG), the PERG has been found to be predominantly abnormal in patients with glaucoma,[171-174] optic neuritis,[175,176] or optic atrophy (Fig. 124.35),[177,178] which further supports the idea that it derives from ganglion-cell activity. It has been reported to be both more sensitive[179] and less sensitive[180] than full-field oscillatory potentials in detecting inner retinal malfunction in patients with early stages of diabetic retinopathy. It remains controversial whether the PERG tends to be abnormal in ocular hypertension, as some studies have found many abnormal responses,[181,182] whereas others have found mostly normal responses.[183] It also remains controversial whether the PERG is abnormal in amblyopia, as some studies have found mainly abnormal responses,[184-186] whereas others have found normal responses.[165,187]
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FIGURE 124.35 Pattern-reversal ERGs (PERRs) a and VERs b from the right and left eyes of a patient with a traumatic unilateral (right) optic nerve section. Responses were elicited by a matrix of 40 min of arc dots, as illustrated at upper right. Arrows designate major negative and positive components, respectively. The pattern ERGs were recorded simultaneously from both eyes with DTL electrodes. The pattern VERs were recorded sequentially with the positive electrode 2 cm above the inion referenced to an earlobe. |
Based on the preceding normative and clinical studies, studies in mammals involving section of the optic nerve,[188,189] and current source density analysis,[190] it is now fairly certain that the PERG derives, at least in part, from the ganglion cells. However, the PERG should not be used alone to diagnose the site of retinal malfunction, since it necessarily depends on photoreceptor function and has been shown to be abnormal in macular disease involving the photoreceptors.[191] If the PERG is abnormal, it would also be advisable to obtain a focal flash ERG to evaluate photoreceptor function before assuming that the inner retina is abnormal.
PERGs are usually elicited by phase-reversing square-wave stripes or checks presented on a television monitor or modulated by a moving mirror within an optical system. The temporal modulation may be at a low frequency (i.e., <8Hz) to elicit transient responses (see Fig. 124.35), or at a high frequency (i.e., ?8Hz) to elicit a steady-state, sinusoidal response. The transient response consists of a cornea-negative deflection followed by a cornea-positive deflection (see Fig. 124.35), much like the luminance ERG. The temporal modulation is usually square wave but may be sinusoidal. Contrast (i.e., the luminance difference between light and dark elements relative to the mean luminance) may be 100% or less. Field size is typically 20° or less, but the pattern must contain an even number of spatial elements in order to minimize any luminance component, particularly if low spatial frequencies are used.
The patient's pupil may or may not be dilated. In either case, care must be taken that the patient is properly refracted and can focus on the pattern, which is usually placed between 0.5 and 1 m distant. A pattern that is not focused will have reduced retinal contrast and result in a reduction in amplitude.[162,165] Two diopters of blur may reduce the amplitude by 50%.[165] The probability that patterns will be out of focus for patients with reduced acuity must be considered when interpreting responses, an issue that has been raised with respect to evaluating strabismic amblyopia.[165]
Signals may be monitored with a silver cup skin electrode on the lower lid,[192] a corneal gold foil or DTL electrode placed in the lower conjunctival sac,[192-194] or a Burian-Allen bipolar contact lens electrode.[195] With the gold foil electrode, but apparently not with the DTL electrode,[196] pattern stimulation of one eye can by volume conduction lead to an artifactual response from the fellow eye.[197-199] With the DTL electrode, the ratio of signal to noise was said to be best if the reference electrode was placed by the outer canthus of the stimulated eye.[196] If a contact lens is used, the eye must be refracted with it in place. PERG signals are typically less than 5 ?V in amplitude and must be averaged, preferably with an artifact reject buffer. The skin electrode is reported to be little affected by blinking but yields smaller amplitudes than does a corneal electrode.[192] The contact lens electrode alone prevents reduction of stimulus luminance resulting from lid closure.
Although the stimulus fields for eliciting PERGs are usually large relative to those used for eliciting FERGs, fixation near the center of the pattern is still important because the major contribution to the response for a mid-frequency check size comes from the fovea. It has been shown that PERG amplitude can fall by 50% for an eccentricity of 4°[165] and by 63% for an eccentricity of 12°.[200] Eccentric fixation may also help to generate a luminance component, which may be conveniently checked by quantifying the fundamental component of the response by Fourier analysis.[166]
Interocular differences in PERG amplitude among normal subjects have been estimated to be as much as 100%.[165] Test-retest correlations were 0.58 comparing two measurements made on the same day but only 0.01 comparing measurements made 1 week apart.[201] The PERG does not appear to be significantly different between males and females,[195] but a small decline in amplitude[195,202] and an increase in latency[195] with increasing age have been reported. In a comparison of responses for a group of normal subjects of mean age 21 years with a group of normal subjects of mean age 72 years, the latter had an average 40% reduction in amplitude.[203]
VISUAL EVOKED RESPONSE TESTING
Pattern-reversal VERs are now preferred over flash VERs for the evaluation of the visual pathways, owing in large part to their smaller range of variation in normal subjects and, undoubtedly related to this, their enhanced sensitivity in detecting axonal conduction defects. The pattern VER is a multiphasic response that, when detectable, contains an inion-positive component with a latency of ?100 ms, called P100 (see Fig. 124.35). The primary use of the pattern-reversal VER is to identify visual loss secondary to diseases of the optic nerve and anterior visual pathways versus those of psychogenic origin. For example, demyelination and compressive lesions of the optic nerve produce reliable slowing of the pattern response (Fig. 124.36), whereas flash responses in these cases often have normal latencies.[204] As many as 96% of patients with multiple sclerosis might be expected to have delayed pattern VERs.[205] Although P100 remains delayed, its amplitude varies monotonically with visual acuity during recovery from optic neuritis.[206] Determining contrast thresholds for a range of spatial frequencies permits the objective assessment of visual acuity (at 100% contrast),[207] which can be used to detect malingering.
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FIGURE 124.36 (Left) Pattern-reversal VERs recorded from a midline occipital electrode from the left and right eyes of a normal subject (a) and two patients who were recovering from acute attacks of optic neuritis in the right eye with onset 4 weeks (b) and 3 weeks (c) previously. Pattern reversal is at 20 ms; positivity downward. (Right) Distribution of the peak latencies of the major positive component of the pattern-reversal VER from the affected eye of 18 patients with optic neuritis (upper histogram), from the unaffected eye of 19 patients with optic neuritis (middle histogram), and from the right eye of 17 normal subjects (lower histogram). |
The stimulus for the pattern VER may be essentially the same as for the PERG, allowing for both measures to be recorded simultaneously, as is becoming increasingly the custom.[163,184,195,200,202] It is essential that the checkerboard pattern be in proper focus, since lack of focus leads to decreases in VER amplitude[208,209] and increases in VER latency.[210] Recordings are generally made from the midline, using either a bipolar configuration with the positive electrode above the inion and the negative electrode on the vertex or monopolar configurations with positive electrodes above the inion and over the parietal lobe. Earlobes may serve as reference and ground. The electrodes may be conventional electroencephalographic cup electrodes and should be filled with electrode cream and applied to the scalp after reducing scalp resistance with an abrasive. The intent is to obtain a resistance less than 5000?. The electrodes may be held in place with colloidin. Responses must be averaged by computer because amplitudes are generally less than 10 ?V.
The use of on-off modulated gratings (in which a grating alternates with a blank screen of the same mean luminance) has been recommended as superior to pattern-reversal stimulation for eliciting the steady-state VER.[211] The on-off grating stimulus has been reported to evoke a bell-shaped spatial-frequency tuned response more consistently across normal subjects than the pattern-reversal stimulus. In addition, the spatial-tuning characteristics obtained by psychophysical testing and by the VER are more similar for the on-off grating stimulus than for the pattern-reversal stimulus (Fig. 124.37).
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FIGURE 124.37 Contrast-sensitivity function (CSF) as obtained through the visual-evoked potential (VEP) regression technique (filled symbols) and psychophysically (open symbols). (Left) Pattern reversal (Rev.). (Right) On-off modulation. |
Unlike retinal responses, scalp responses to pattern reversal show marked interindividual variation in waveform among normal subjects.[212] Nevertheless, remarkable agreement exists for the normal latency for the P100 component across studies. Upper limits for latency differences between left and right eyes of normal subjects have been estimated at 8-10 ms.[213] Repeat testing of the same subjects on separate days showed a substantial variation in latency with values that still mostly fell within normal limits obtained on the same patients at a single session.[213] P100 latency increases with age in normal subjects, at a rate of ?2 ms/decade depending on spatial frequency (Fig. 124.38).[214] In addition, a 50-year increase among adults was associated with a 50% reduction in VER amplitude for temporal modulation below 6Hz. For faster modulation, age had no effect on amplitude.[203] VER acuity (derived by extrapolating VER amplitude as a function of increasing spatial frequency to zero amplitude) increased 300% in infants for increasing age up to the adult level reached at age 30 weeks.[215] P100 latency is also greater for small pupils than for large pupils.[216] Some (but not all) of the increase with age may be due to age-related miosis.
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FIGURE 124.38 P100 (P1) latency for 48 min (closed circles) and 12 min (open circles) checks as a function of age. Each data point is the mean latency of the right and left eyes of each subject. For 48-min checks, y = 0.14 x + 101.8; for 12-min checks, y = 0.26 x + 105.6. For ease of comparison, the regression line for 48-min checks has also been plotted as a dashed line on the 12-min graph. |
CHOOSING A TEST
When a patient presents with reduced visual acuity, blur, or metamorphopsia or with difficulty seeing at night, increased sensitivity to bright lights, or an impairment in discriminating colors (or any combination of these symptoms), the ophthalmologist must decide whether objective tests of visual function are required in addition to a routine examination. It has been our preference to first rule out distal retinal malfunction by performing full-field and focal flash electroretinography. Tests based on differential fundus reflectance - especially OCT - can be used to corroborate this impression. If results from these tests are abnormal, no additional measurements of function are necessary, and the patient can be advised that the problem is within the eye in the outer retina. If results from flash electroretinography are normal, it would be appropriate to consider the PERG to rule out ganglion cell and optic nerve dysfunction, and then the pattern VER to rule out visual pathway and cortical dysfunction.
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Key Features |
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