Dark-adapted red flash ERGs in healthy adults

Dark-adapted red flash ERGs in healthy adults Doc Ophthalmol (2018) 137:1–8 https://doi.org/10.1007/s10633-018-9642-1(0123456789().,-volV)(0123456789().,-volV) ORIGINAL RESEARCH ARTICLE Dark-adapted red flash ERGs in healthy adults R. Hamilton K. Graham Received: 22 February 2018 / Accepted: 22 May 2018 / Published online: 1 June 2018 The Author(s) 2018 Abstract Conclusions This small study suggests that x-wave Purpose The x-wave of the dark-adapted (DA) ERG visibility in healthy subjects after 20 min dark adap- to a red flash reflects DA cone function. This tation is improved by using flashes weaker than around -2 -2 exploratory study of healthy adults aimed to investi- 0.6 cd s m ; for flash strengths of 1.5 cd s m , gate changes in the DA red ERG with flash strength x-wave visibility is enhanced by recording after only and during dark adaptation to optimise visualisation around 5 min of dark adaptation. No evidence was and therefore quantification of the x-wave. found that interim red flash ERGs affect the dark- Methods The effect of altering red flash strength was adapted state of the normal retina. investigated in four subjects by recording ERGs after 20 min dark adaptation to red flashes Keywords ISCEV standard  Dark-adapted red flash -2 (0.2–2.0 cd s m ) using skin electrodes and natural ERG  x-wave  Dark-adapted pupils. The effect of dark adaptation duration was investigated in 16 subjects during 20 min in the dark, by recording DA 1.5 red ERGs at 1, 2, 3, 4, 5, 10, 15 and 20 min. Introduction Results For a dark adaption period of 20 min, the x-wave was more clearly visualised to weaker The dark-adapted (DA) ERG to a red flash has an -2 (\ 0.6 cd s m ) red flash strengths: to stronger initial positive peak called the x-wave [1, 2] which is flashes it became obscured by the b-wave. For red seen only in species with cone-rich retinae [3]. X-wave -2 flashes of 1.5 cd s m , the x-wave was most promi- amplitude is largest to wavelengths around 630 nm nent in ERGs recorded after 1–5 min of dark adapta- [2–4], and increases during dark adaptation, peaking tion: with longer dark adaptation, it was subsumed into within a few minutes [4–6]. Its peak time, unlike the the b-wave’s rising edge. b-wave, changes little with wavelength [7]. Visibility of the x-wave can be enhanced by using a dim background to suppress rods [4, 8]. R. Hamilton (&)  K. Graham No x-wave is evident in protanomalous subjects Department of Clinical Physics and Bio-Engineering, NHS Greater Glasgow and Clyde, Glasgow, UK [4, 7, 9] nor in subjects with achromatopsia [7, 9]. e-mail: ruth.hamilton@glasgow.ac.uk Conversely, the x-wave is preserved but the b-wave is attenuated or absent in RDH5 retinopathy (fundus R. Hamilton  K. Graham albipunctatus) [10, 11] and in vitamin A deficiency College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 123 2 Doc Ophthalmol (2018) 137:1–8 [12]. The DA ERG to a red flash is present—albeit artificially lit. The first investigation explored the with impaired kinetics [13]—in bradyopsia (RGS9/ effect of altering red flash strength on four subjects (all R9AP mutation), indicating preserved cone function, female, aged 22–53). After 20 min dark adaptation, whereas the light-adapted white flash ERG is absent, a ERGs were recorded as described below from one combination reported to be pathognomonic for the random eye to red flashes of 0.2 or 0.3–2.0 phot -2 -2 condition [14]. The x-wave is, therefore, interpreted as cd s m in 0.1 steps. A white 0.01 phot cd s m a measure of dark-adapted cone function. Although a ERG (DA 0.01 ERG) was also recorded at each step DA red ERG is not part of the ISCEV ERG standard for comparison and to check for any evidence, e.g. [15], it is used ‘sometimes’ or ‘often’ in around half of reducing amplitude, that the retina was becoming light visual electrophysiology clinics [16]. Red flash adapted. -2 strengths of 0.05–2.5 cd s m [8, 10, 17–21] have The second investigation explored the effect of been used, but some studies report clinical use of the altering duration of dark adaptation on the red flash red flash ERG without giving flash strength or spectral ERGs, and was conducted as part of the concomitant characteristics, instead describing flash strength ‘‘such study [23]. In the baseline phase of the experiment that in a normal subject the amplitude of the rod (Fig. 1), subjects were dark adapted for 20 min, at the -2 component to the red flash is equivalent to that of the end of which a DA 1.5 cd s m red flash ERG rod-specific response to a dim white flash (dark- (followed by ISCEV standard DA 0.01 and DA 3.0 -2 adapted 0.01 cd s m )’’ [11, 12, 14, 22]. ERGs) was recorded. Subjects were then light adapted The x-wave can be swamped by the later, larger rod to the artificial room lighting for 10 min before b-wave, appearing as only a shoulder which hampers beginning the experimental phase of the protocol. In quantification [20, 21]. Clearer visualisation and the experimental phase, dark adaptation was recom- hence quantification of the x-wave might be achieved menced and interim DA 1.5 red ERGs were recorded by a suitable combination of red flash strength and during this second 20-min dark adaptation period at 1, dark adaptation duration. This exploratory study 2, 3, 4, 5, 10, 15 and 20 min. The baseline phase was aimed to investigate changes in the DA red ERG with included in order to compare ERGs recorded after flash strength and during dark adaptation. 20 min uninterrupted dark adaptation with those recorded after 20 min dark adaptation punctuated with multiple, interim ERGs, in order to test whether Methods the experimental design itself affected the ERGs. The study was approved by the Ethics Committee of ERGs the College of Medical, Veterinary and Life Sciences, University of Glasgow. Subjects gave informed, ERGs were recorded from both eyes using adhesive, written consent. disposable skin electrodes placed on the lower lid, referenced to skin electrodes at the ipsilateral temporal Subjects orbital rim. A ground electrode was placed on a mastoid. Skin was prepared to ensure low (\5kX) Sixteen adult subjects (20–58 years old) without self- and matched impedances; amplifier bandpass was reported neurological or ocular conditions were 0.3–300 Hz (IIR digital, 2 pole Bessel emulations), recruited without incentive. Inclusion criteria were with a sampling frequency of 1000 Hz. In the interests refractive errors of \ 3 dioptres, and a normal Ishihara of investigating protocols with greater patient test colour test result. The sample size was selected to acceptability, no dilating drops were used, contrary to power a concomitant study of shorter dark adaptation the stipulation of the ISCEV standard [15]. Pupil sizes for ISCEV standard DA ERGs [23]. were measured towards the end of the baseline period of dark adaptation using a half-moon rule with 0.5 mm Study design precision and an infrared camera. Diameters ranged from 7 to 10 mm (median 8.5 mm), very similar to Subjects were restricted to interior lighting for at least those from an earlier study on similar subjects 1 h prior to any recordings. The test room was 123 Doc Ophthalmol (2018) 137:1–8 3 Fig. 1 Timeline illustration of recording protocol. Black and white bands indicate dark and light adaptation, respectively. Boxes below the timeline indicate ERG recordings, labelled by duration of dark adaptation after which they were made Results (7–9 mm) with mydriasis [24]. A dim red fixation mark aided eye stability during recordings. ERG changes with red flash strength Stimulation and acquisition were driven by a visual electrophysiology system (Espion, Diagnosys LLC, The red flash ERG grew in amplitude as flash strength Lowell, MA, USA). Ten ERGs were averaged with an inter-stimulus interval of 1 s to ensure adequately high increased for all four subjects tested (Fig. 2). The SNR since skin electrodes were used. Flashes were a-wave was present, typically at around 17–20 ms. generated by LEDs within a ganzfeld (ColorDome, Two further troughs were evident, typically shallower, Diagnosys LLC, Lowell, MA, USA) with stated peak at around 25–28 ms and at around 30–37 ms, depend- wavelength k = 635 nm and CIE coordinates ing on flash strength. The x-wave was also present, x = 0.702, y = 0.298. Annual manufacturer’s calibra- typically at around 45–50 ms, and was more clearly seen at lower than at higher flash strengths: as flash tion before and after the investigation showed no changes; values were confirmed for white flashes strength increased, it became larger, but was increas- ingly obscured by the b-wave, appearing as a shoulder using a photometer (ILT1700, International Light Technologies, MA, USA) in integrating mode. Values on the b-wave rising edge. At lower flash strengths, the b-wave was also clearly visualised at around 100 ms, given here are manufacturer’s nominal values. For the second investigation, a red flash strength of 1.5 with a similar form to the b-wave of the dim white -2 (photopic) cd s m was chosen somewhat arbitrarily, flash ERG. As red flash strength increased, the b-wave as it fell within the range of flash strengths described shortened and merged with the x-wave peak. elsewhere for use with a preceding 20 min period of The white flash DA 0.01 ERGs recorded at each -2 dark adaptation (0.05–2.5 cd s m [8, 10, 17–21]); step showed no evidence of reducing in amplitude additionally, it generated a b-wave of a similar over the 10 ERGs used for the average in each step, nor over the whole investigation for any subject, despite a amplitude to that produced by the DA 0.01 ERG white flashes (Fig. 2). 1-s inter-stimulus interval, rather than the 2 s stipu- lated in the ERG standard [15]. Similarly, the red flash Amplitudes of a-waves were measured from base- line, and x- and b-waves were measured from a-wave ERGs showed no evidence of reducing in amplitude over the 10 ERGs used for the average in each step, troughs. No systematic inter-ocular ERG differences existed, so parameters from eyes of each subject were suggesting that, even for relatively strong red flashes, a averaged [25]. Data were treated nonparametrically 1-s inter-stimulus interval was adequate to maintain because of the small sample size and some skew. the dark-adapted state of the retina. 123 4 Doc Ophthalmol (2018) 137:1–8 Fig. 2 Illustrative red flash ERGs (red traces) and ISCEV strength on subject #16’s peak times (b); x and b-wave standard DA 0.01 ERGs (grey traces) from subject #16, recorded amplitudes (c) and a-wave amplitudes (d). Circles: a-waves, after 20 min dark adaptation (a). The numbers to the right of diamonds: x-waves, squares: b-waves. Open symbols close to each pair of traces indicate the strength of the red flash in y-axes represent mean (sd) values for DA 0.01 ERGs for -2 photopic cd s m . Right panels show effect of red flash comparison 123 Doc Ophthalmol (2018) 137:1–8 5 ERG changes with duration of dark adaptation Comparing ERGs recorded at the end of the baseline phase with those recorded at the end of the experi- mental phase revealed no statistically significant differences in ERG parameters (Mann–Whitney U tests, p values all C 0.50), establishing that interim ERGs recorded during the experimental phase did not affect the measured parameters of ERGs recorded at the end of the dark adaptation period (Fig. 3). This also implies that delivering red flashes with 1-s inter- stimulus intervals does not affect the adaptation state of the retina, notwithstanding the additional white flashes delivered during the experimental phase [23]. The morphology of the DA 1.5 red ERG changes during dark adaptation (Fig. 4). A series of oscillations at the a-wave and rising edge of the b-wave are evident, with the largest positive peak—the x-wave— being most prominent at around 40 ms in ERGs recorded after 1–5 min of dark adaptation. As the presumably rod-driven, later b-wave gains amplitude during dark adaptation, this 40-ms x-wave peak becomes subsumed into the b-wave’s rising edge, often being no longer apparent, or present as only a Fig. 4 Illustrative DA 1.5 red ERGs from a typical subject (#8). shoulder with no following trough. A later oscillation, The numbers to the right of each trace indicate the duration of dark adaptation (minutes) before each ERG. Vertical grey lines usually [ 50 ms, is more clearly visualised as the mark the two positive peaks which exchange dominance (largest x-wave than the 40-ms peak in ERGs recorded after amplitude) as dark adaptation proceeds. The largest peak is 10–20 min of dark adaptation. measured as the x-wave (continuous line). Note the triple trough The development of the DA red 1.5 ERG during which forms the a-wave, with troughs typically at around 17, 24 and 31 ms dark adaptation was quantified by normalising indi- vidual subject’s ERG parameters to those of their Fig. 3 Upper panels: scatterplots of ERG parameters recorded indicate the median difference between experimental and at the end of baseline phase versus end of experimental phase for baseline recordings, and grey dashed lines indicate the 5th and all 16 subjects. Grey diagonal lines indicate equality. Lower 95th percentiles of the difference panels: corresponding difference plots. Grey horizontal lines 123 6 Doc Ophthalmol (2018) 137:1–8 Fig. 5 DA red 1.5 ERG changes during dark adaptation. Left: amplitudes. Right: peak times. Circles: individual subject’s normalised data points; triangles: median values; dashed lines: 95% prediction intervals of growth curves fitted to median data; solid horizontal line highlights the 100% level. Data are normalised relative to values after 20 min dark adaptation, hence the lack of variability at 20 min. Note change of scale for a-wave amplitudes 20 min DA ERG (Fig. 5). The red a-wave amplitude lengthening dark adaptation as might be expected for a reduced a little over the first 3 min of dark adaptation, rod system b-wave. while peak times did not change at all over the whole Summarised reference data are presented in 20 min. The x-wave amplitude increased, and peak Table 1 for the DA 1.5 red flash ERG after 5, 10 and time lengthened between five and 10 min of dark 20 min of dark adaptation: data are presented as adaptation, primarily due to the later peak at * 50 ms ranges as the sample size is inadequate for percentiles becoming dominant as the growing b-wave obscured with confidence intervals [26]. These data do not the earlier x-wave peak at * 40 ms. The b-wave change substantially with duration of dark adaptation, amplitude increased, and peak time shortened with except for the x-wave peak time which more tightly 123 Doc Ophthalmol (2018) 137:1–8 7 Table 1 Summarised reference data (ranges) for DA 1.5 red flash ERGs, skin electrodes, undilated pupils, N = 16 subjects Duration of dark adaptation (min) a-wave x-wave b-wave Amplitude (lV) PT (ms) Amplitude (lV) PT (ms) Amplitude (lV) PT (ms) 5 5.2–24 15–20 31–100 41–50 21–97 62–89 10 6.3–25 15–19 38–110 41–58 40–128 66–92 20 6.6–22 15–20 45–114 41–56 50–129 62–89 PT peak time defined after 5 min in the dark than after longer dark feasible that the ISCEV standard dark-adapted ERG adaptation. protocol could incorporate a dim red flash ERG delivered at some point during the currently stipulated 20 min of dark adaptation, adding no further burden of Discussion time to the patient or tester. These data also suggest that additional flashes, even delivered once per In this small, exploratory study of healthy adult second, are unlikely to affect the dark-adapted state subjects, we found that after 20 min of dark adapta- of the normal retina, although this may not be the case tion, the x-wave was more clearly visualised with for patients with retinal dysfunction. -2 weaker (about 0.6 cd s m or less) than with stronger Compliance with ethical standards flashes, as described elsewhere [18]. We also found -2 that that with a relatively strong 1.5 cd s m red Conflict of interest The authors declare that they have no flash, the x-wave was more clearly visualised after conflicts of interest. shorter (about 5 min or less) than longer dark adap- Ethical approval All procedures performed in studies tation, also confirming findings elsewhere [6]. involving human participants were in accordance with the eth- The subjects in this study were mostly in their ical standards of the institutional and/or national research second or third decades, so findings cannot be committee and with the 1964 Helsinki declaration and its later generalised to all age groups. As an exploratory study, amendments or comparable ethical standards. we chose a minimally invasive protocol with non- Statement of human rights All procedures performed in corneal electrodes and no mydriasis: this will have studies involving human participants were in accordance with resulted in higher signal-to-noise ratios than usually the ethical standards of the institutional and/or national research found with corneal electrodes. This is unlikely to committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. affect conclusions since mostly relative outcome measures were used, but signal noise increases uncer- Statement on the welfare of animals This article does not tainty of peak labelling in some instances. contain any studies with animals performed by any of the The DA red flash ERG is quite widely used, and the authors. International Society for Clinical Electrophysiology Informed consent Informed consent was obtained from all of Vision has recently prepared a new Extended individual participants included in the study. Protocol to inform current and potential users. Its utility lies in an extant x-wave revealing the presence Open Access This article is distributed under the terms of the of functioning, dark-adapted cones. Thus, the visibil- Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unre- ity of the x-wave is of primary importance for the stricted use, distribution, and reproduction in any medium, optimisation of protocols. The current findings suggest provided you give appropriate credit to the original that x-wave visibility in normal subjects is impaired by author(s) and the source, provide a link to the Creative Com- -2 flashes stronger than around 0.6 cd s m (for 20 min mons license, and indicate if changes were made. dark adaptation), or by dark adaptation longer than -2 around 5 min (for flash strengths of 1.5 cd s m ). Other combinations remain uninvestigated. It seems 123 8 Doc Ophthalmol (2018) 137:1–8 References 14. Vincent A, Robson AG, Holder GE (2013) Pathognomonic (diagnostic) ERGs: a review and update. Retina 33:5–12. https://doi.org/10.1097/iae.0b013e31827e2306 1. Motokawa K, Mita T (1942) Uber eine einfachere Unter- 15. McCulloch DL, Marmor MF, Brigell MG, Hamilton R, suchungsmethode und Eigenschaften der Aktionsstro¨me der Holder GE, Tzekov R, Bach M (2015) ISCEV Standard for Netzhaut des Menschen. Tohoku J Exp Med 42:114–133. full-field clinical electroretinography (2015 update). 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Hamilton R, Graham K (2016) Effect of shorter dark Moore AT, Robson AG, Holder GE, Webster AR (2011) adaptation on ISCEV standard DA 0.01 and DA 3 skin Phenotypic variability in RDH5 retinopathy (fundus ERGs in healthy adults. Doc Ophthalmol 133:11–19. albipunctatus). Ophthalmology 118:1661–1670. https://doi. https://doi.org/10.1007/s10633-016-9554-x org/10.1016/j.ophtha.2010.12.031 24. Abdlseaed A Al (2014) Pupil dilation, light source, gender 12. McBain VA, Egan CA, Pieris SJ, Supramaniam G, Webster and pigmentation effects on normal adult human ERGs, and AR, Bird AC, Holder GE (2007) Functional observations in an exploration of short latency VEPs. PhD thesis, Glasgow vitamin A deficiency: diagnosis and time course of recovery. Caledonian University Eye 21:367–376. https://doi.org/10.1038/sj.eye.6702212 25. Armstrong RA (2013) Statistical guidelines for the analysis 13. Nishiguchi KM, Sandberg MA, Kooijman AC, Marte- of data obtained from one or both eyes. Ophthalmic Physiol myanov KA, Pott JWR, Hagstrom SA, Arshavsky VY, Opt 33:7–14. https://doi.org/10.1111/opo.12009 Berson EL, Dryja TP (2004) Defects in RGS9 or its anchor 26. Horowitz GL (2010) EP28-A3C defining, establishing, and protein R9AP in patients with slow photoreceptor deacti- verifying reference intervals in the clinical laboratory; vation. Nature 427:75–78. https://doi.org/10.1038/ approved guideline, 3rd edn. Clinical and Laboratory nature02170 Standards Institute, San Diego http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Documenta Ophthalmologica Springer Journals

Dark-adapted red flash ERGs in healthy adults

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Doc Ophthalmol (2018) 137:1–8 https://doi.org/10.1007/s10633-018-9642-1(0123456789().,-volV)(0123456789().,-volV) ORIGINAL RESEARCH ARTICLE Dark-adapted red flash ERGs in healthy adults R. Hamilton K. Graham Received: 22 February 2018 / Accepted: 22 May 2018 / Published online: 1 June 2018 The Author(s) 2018 Abstract Conclusions This small study suggests that x-wave Purpose The x-wave of the dark-adapted (DA) ERG visibility in healthy subjects after 20 min dark adap- to a red flash reflects DA cone function. This tation is improved by using flashes weaker than around -2 -2 exploratory study of healthy adults aimed to investi- 0.6 cd s m ; for flash strengths of 1.5 cd s m , gate changes in the DA red ERG with flash strength x-wave visibility is enhanced by recording after only and during dark adaptation to optimise visualisation around 5 min of dark adaptation. No evidence was and therefore quantification of the x-wave. found that interim red flash ERGs affect the dark- Methods The effect of altering red flash strength was adapted state of the normal retina. investigated in four subjects by recording ERGs after 20 min dark adaptation to red flashes Keywords ISCEV standard  Dark-adapted red flash -2 (0.2–2.0 cd s m ) using skin electrodes and natural ERG  x-wave  Dark-adapted pupils. The effect of dark adaptation duration was investigated in 16 subjects during 20 min in the dark, by recording DA 1.5 red ERGs at 1, 2, 3, 4, 5, 10, 15 and 20 min. Introduction Results For a dark adaption period of 20 min, the x-wave was more clearly visualised to weaker The dark-adapted (DA) ERG to a red flash has an -2 (\ 0.6 cd s m ) red flash strengths: to stronger initial positive peak called the x-wave [1, 2] which is flashes it became obscured by the b-wave. For red seen only in species with cone-rich retinae [3]. X-wave -2 flashes of 1.5 cd s m , the x-wave was most promi- amplitude is largest to wavelengths around 630 nm nent in ERGs recorded after 1–5 min of dark adapta- [2–4], and increases during dark adaptation, peaking tion: with longer dark adaptation, it was subsumed into within a few minutes [4–6]. Its peak time, unlike the the b-wave’s rising edge. b-wave, changes little with wavelength [7]. Visibility of the x-wave can be enhanced by using a dim background to suppress rods [4, 8]. R. Hamilton (&)  K. Graham No x-wave is evident in protanomalous subjects Department of Clinical Physics and Bio-Engineering, NHS Greater Glasgow and Clyde, Glasgow, UK [4, 7, 9] nor in subjects with achromatopsia [7, 9]. e-mail: ruth.hamilton@glasgow.ac.uk Conversely, the x-wave is preserved but the b-wave is attenuated or absent in RDH5 retinopathy (fundus R. Hamilton  K. Graham albipunctatus) [10, 11] and in vitamin A deficiency College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 123 2 Doc Ophthalmol (2018) 137:1–8 [12]. The DA ERG to a red flash is present—albeit artificially lit. The first investigation explored the with impaired kinetics [13]—in bradyopsia (RGS9/ effect of altering red flash strength on four subjects (all R9AP mutation), indicating preserved cone function, female, aged 22–53). After 20 min dark adaptation, whereas the light-adapted white flash ERG is absent, a ERGs were recorded as described below from one combination reported to be pathognomonic for the random eye to red flashes of 0.2 or 0.3–2.0 phot -2 -2 condition [14]. The x-wave is, therefore, interpreted as cd s m in 0.1 steps. A white 0.01 phot cd s m a measure of dark-adapted cone function. Although a ERG (DA 0.01 ERG) was also recorded at each step DA red ERG is not part of the ISCEV ERG standard for comparison and to check for any evidence, e.g. [15], it is used ‘sometimes’ or ‘often’ in around half of reducing amplitude, that the retina was becoming light visual electrophysiology clinics [16]. Red flash adapted. -2 strengths of 0.05–2.5 cd s m [8, 10, 17–21] have The second investigation explored the effect of been used, but some studies report clinical use of the altering duration of dark adaptation on the red flash red flash ERG without giving flash strength or spectral ERGs, and was conducted as part of the concomitant characteristics, instead describing flash strength ‘‘such study [23]. In the baseline phase of the experiment that in a normal subject the amplitude of the rod (Fig. 1), subjects were dark adapted for 20 min, at the -2 component to the red flash is equivalent to that of the end of which a DA 1.5 cd s m red flash ERG rod-specific response to a dim white flash (dark- (followed by ISCEV standard DA 0.01 and DA 3.0 -2 adapted 0.01 cd s m )’’ [11, 12, 14, 22]. ERGs) was recorded. Subjects were then light adapted The x-wave can be swamped by the later, larger rod to the artificial room lighting for 10 min before b-wave, appearing as only a shoulder which hampers beginning the experimental phase of the protocol. In quantification [20, 21]. Clearer visualisation and the experimental phase, dark adaptation was recom- hence quantification of the x-wave might be achieved menced and interim DA 1.5 red ERGs were recorded by a suitable combination of red flash strength and during this second 20-min dark adaptation period at 1, dark adaptation duration. This exploratory study 2, 3, 4, 5, 10, 15 and 20 min. The baseline phase was aimed to investigate changes in the DA red ERG with included in order to compare ERGs recorded after flash strength and during dark adaptation. 20 min uninterrupted dark adaptation with those recorded after 20 min dark adaptation punctuated with multiple, interim ERGs, in order to test whether Methods the experimental design itself affected the ERGs. The study was approved by the Ethics Committee of ERGs the College of Medical, Veterinary and Life Sciences, University of Glasgow. Subjects gave informed, ERGs were recorded from both eyes using adhesive, written consent. disposable skin electrodes placed on the lower lid, referenced to skin electrodes at the ipsilateral temporal Subjects orbital rim. A ground electrode was placed on a mastoid. Skin was prepared to ensure low (\5kX) Sixteen adult subjects (20–58 years old) without self- and matched impedances; amplifier bandpass was reported neurological or ocular conditions were 0.3–300 Hz (IIR digital, 2 pole Bessel emulations), recruited without incentive. Inclusion criteria were with a sampling frequency of 1000 Hz. In the interests refractive errors of \ 3 dioptres, and a normal Ishihara of investigating protocols with greater patient test colour test result. The sample size was selected to acceptability, no dilating drops were used, contrary to power a concomitant study of shorter dark adaptation the stipulation of the ISCEV standard [15]. Pupil sizes for ISCEV standard DA ERGs [23]. were measured towards the end of the baseline period of dark adaptation using a half-moon rule with 0.5 mm Study design precision and an infrared camera. Diameters ranged from 7 to 10 mm (median 8.5 mm), very similar to Subjects were restricted to interior lighting for at least those from an earlier study on similar subjects 1 h prior to any recordings. The test room was 123 Doc Ophthalmol (2018) 137:1–8 3 Fig. 1 Timeline illustration of recording protocol. Black and white bands indicate dark and light adaptation, respectively. Boxes below the timeline indicate ERG recordings, labelled by duration of dark adaptation after which they were made Results (7–9 mm) with mydriasis [24]. A dim red fixation mark aided eye stability during recordings. ERG changes with red flash strength Stimulation and acquisition were driven by a visual electrophysiology system (Espion, Diagnosys LLC, The red flash ERG grew in amplitude as flash strength Lowell, MA, USA). Ten ERGs were averaged with an inter-stimulus interval of 1 s to ensure adequately high increased for all four subjects tested (Fig. 2). The SNR since skin electrodes were used. Flashes were a-wave was present, typically at around 17–20 ms. generated by LEDs within a ganzfeld (ColorDome, Two further troughs were evident, typically shallower, Diagnosys LLC, Lowell, MA, USA) with stated peak at around 25–28 ms and at around 30–37 ms, depend- wavelength k = 635 nm and CIE coordinates ing on flash strength. The x-wave was also present, x = 0.702, y = 0.298. Annual manufacturer’s calibra- typically at around 45–50 ms, and was more clearly seen at lower than at higher flash strengths: as flash tion before and after the investigation showed no changes; values were confirmed for white flashes strength increased, it became larger, but was increas- ingly obscured by the b-wave, appearing as a shoulder using a photometer (ILT1700, International Light Technologies, MA, USA) in integrating mode. Values on the b-wave rising edge. At lower flash strengths, the b-wave was also clearly visualised at around 100 ms, given here are manufacturer’s nominal values. For the second investigation, a red flash strength of 1.5 with a similar form to the b-wave of the dim white -2 (photopic) cd s m was chosen somewhat arbitrarily, flash ERG. As red flash strength increased, the b-wave as it fell within the range of flash strengths described shortened and merged with the x-wave peak. elsewhere for use with a preceding 20 min period of The white flash DA 0.01 ERGs recorded at each -2 dark adaptation (0.05–2.5 cd s m [8, 10, 17–21]); step showed no evidence of reducing in amplitude additionally, it generated a b-wave of a similar over the 10 ERGs used for the average in each step, nor over the whole investigation for any subject, despite a amplitude to that produced by the DA 0.01 ERG white flashes (Fig. 2). 1-s inter-stimulus interval, rather than the 2 s stipu- lated in the ERG standard [15]. Similarly, the red flash Amplitudes of a-waves were measured from base- line, and x- and b-waves were measured from a-wave ERGs showed no evidence of reducing in amplitude over the 10 ERGs used for the average in each step, troughs. No systematic inter-ocular ERG differences existed, so parameters from eyes of each subject were suggesting that, even for relatively strong red flashes, a averaged [25]. Data were treated nonparametrically 1-s inter-stimulus interval was adequate to maintain because of the small sample size and some skew. the dark-adapted state of the retina. 123 4 Doc Ophthalmol (2018) 137:1–8 Fig. 2 Illustrative red flash ERGs (red traces) and ISCEV strength on subject #16’s peak times (b); x and b-wave standard DA 0.01 ERGs (grey traces) from subject #16, recorded amplitudes (c) and a-wave amplitudes (d). Circles: a-waves, after 20 min dark adaptation (a). The numbers to the right of diamonds: x-waves, squares: b-waves. Open symbols close to each pair of traces indicate the strength of the red flash in y-axes represent mean (sd) values for DA 0.01 ERGs for -2 photopic cd s m . Right panels show effect of red flash comparison 123 Doc Ophthalmol (2018) 137:1–8 5 ERG changes with duration of dark adaptation Comparing ERGs recorded at the end of the baseline phase with those recorded at the end of the experi- mental phase revealed no statistically significant differences in ERG parameters (Mann–Whitney U tests, p values all C 0.50), establishing that interim ERGs recorded during the experimental phase did not affect the measured parameters of ERGs recorded at the end of the dark adaptation period (Fig. 3). This also implies that delivering red flashes with 1-s inter- stimulus intervals does not affect the adaptation state of the retina, notwithstanding the additional white flashes delivered during the experimental phase [23]. The morphology of the DA 1.5 red ERG changes during dark adaptation (Fig. 4). A series of oscillations at the a-wave and rising edge of the b-wave are evident, with the largest positive peak—the x-wave— being most prominent at around 40 ms in ERGs recorded after 1–5 min of dark adaptation. As the presumably rod-driven, later b-wave gains amplitude during dark adaptation, this 40-ms x-wave peak becomes subsumed into the b-wave’s rising edge, often being no longer apparent, or present as only a Fig. 4 Illustrative DA 1.5 red ERGs from a typical subject (#8). shoulder with no following trough. A later oscillation, The numbers to the right of each trace indicate the duration of dark adaptation (minutes) before each ERG. Vertical grey lines usually [ 50 ms, is more clearly visualised as the mark the two positive peaks which exchange dominance (largest x-wave than the 40-ms peak in ERGs recorded after amplitude) as dark adaptation proceeds. The largest peak is 10–20 min of dark adaptation. measured as the x-wave (continuous line). Note the triple trough The development of the DA red 1.5 ERG during which forms the a-wave, with troughs typically at around 17, 24 and 31 ms dark adaptation was quantified by normalising indi- vidual subject’s ERG parameters to those of their Fig. 3 Upper panels: scatterplots of ERG parameters recorded indicate the median difference between experimental and at the end of baseline phase versus end of experimental phase for baseline recordings, and grey dashed lines indicate the 5th and all 16 subjects. Grey diagonal lines indicate equality. Lower 95th percentiles of the difference panels: corresponding difference plots. Grey horizontal lines 123 6 Doc Ophthalmol (2018) 137:1–8 Fig. 5 DA red 1.5 ERG changes during dark adaptation. Left: amplitudes. Right: peak times. Circles: individual subject’s normalised data points; triangles: median values; dashed lines: 95% prediction intervals of growth curves fitted to median data; solid horizontal line highlights the 100% level. Data are normalised relative to values after 20 min dark adaptation, hence the lack of variability at 20 min. Note change of scale for a-wave amplitudes 20 min DA ERG (Fig. 5). The red a-wave amplitude lengthening dark adaptation as might be expected for a reduced a little over the first 3 min of dark adaptation, rod system b-wave. while peak times did not change at all over the whole Summarised reference data are presented in 20 min. The x-wave amplitude increased, and peak Table 1 for the DA 1.5 red flash ERG after 5, 10 and time lengthened between five and 10 min of dark 20 min of dark adaptation: data are presented as adaptation, primarily due to the later peak at * 50 ms ranges as the sample size is inadequate for percentiles becoming dominant as the growing b-wave obscured with confidence intervals [26]. These data do not the earlier x-wave peak at * 40 ms. The b-wave change substantially with duration of dark adaptation, amplitude increased, and peak time shortened with except for the x-wave peak time which more tightly 123 Doc Ophthalmol (2018) 137:1–8 7 Table 1 Summarised reference data (ranges) for DA 1.5 red flash ERGs, skin electrodes, undilated pupils, N = 16 subjects Duration of dark adaptation (min) a-wave x-wave b-wave Amplitude (lV) PT (ms) Amplitude (lV) PT (ms) Amplitude (lV) PT (ms) 5 5.2–24 15–20 31–100 41–50 21–97 62–89 10 6.3–25 15–19 38–110 41–58 40–128 66–92 20 6.6–22 15–20 45–114 41–56 50–129 62–89 PT peak time defined after 5 min in the dark than after longer dark feasible that the ISCEV standard dark-adapted ERG adaptation. protocol could incorporate a dim red flash ERG delivered at some point during the currently stipulated 20 min of dark adaptation, adding no further burden of Discussion time to the patient or tester. These data also suggest that additional flashes, even delivered once per In this small, exploratory study of healthy adult second, are unlikely to affect the dark-adapted state subjects, we found that after 20 min of dark adapta- of the normal retina, although this may not be the case tion, the x-wave was more clearly visualised with for patients with retinal dysfunction. -2 weaker (about 0.6 cd s m or less) than with stronger Compliance with ethical standards flashes, as described elsewhere [18]. We also found -2 that that with a relatively strong 1.5 cd s m red Conflict of interest The authors declare that they have no flash, the x-wave was more clearly visualised after conflicts of interest. shorter (about 5 min or less) than longer dark adap- Ethical approval All procedures performed in studies tation, also confirming findings elsewhere [6]. involving human participants were in accordance with the eth- The subjects in this study were mostly in their ical standards of the institutional and/or national research second or third decades, so findings cannot be committee and with the 1964 Helsinki declaration and its later generalised to all age groups. As an exploratory study, amendments or comparable ethical standards. we chose a minimally invasive protocol with non- Statement of human rights All procedures performed in corneal electrodes and no mydriasis: this will have studies involving human participants were in accordance with resulted in higher signal-to-noise ratios than usually the ethical standards of the institutional and/or national research found with corneal electrodes. This is unlikely to committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. affect conclusions since mostly relative outcome measures were used, but signal noise increases uncer- Statement on the welfare of animals This article does not tainty of peak labelling in some instances. contain any studies with animals performed by any of the The DA red flash ERG is quite widely used, and the authors. International Society for Clinical Electrophysiology Informed consent Informed consent was obtained from all of Vision has recently prepared a new Extended individual participants included in the study. Protocol to inform current and potential users. Its utility lies in an extant x-wave revealing the presence Open Access This article is distributed under the terms of the of functioning, dark-adapted cones. Thus, the visibil- Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unre- ity of the x-wave is of primary importance for the stricted use, distribution, and reproduction in any medium, optimisation of protocols. 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Documenta OphthalmologicaSpringer Journals

Published: Jun 1, 2018

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