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Alterations of Photopic Negative Response of Multifocal Electroretinogram in Patients with Glaucoma a

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Muneyoshi Kaneko , Shigeki Machida , Yuya Hoshi & Daijiro Kurosaka

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Department of Ophthalmology, Iwate Medical University School of Medicine, Uchimaru, Morioka, Iwate, Japan Published online: 25 Jul 2015.

Click for updates To cite this article: Muneyoshi Kaneko, Shigeki Machida, Yuya Hoshi & Daijiro Kurosaka (2015) Alterations of Photopic Negative Response of Multifocal Electroretinogram in Patients with Glaucoma, Current Eye Research, 40:1, 77-86 To link to this article: http://dx.doi.org/10.3109/02713683.2014.915575

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Current Eye Research, 2015; 40(1): 77–86 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.915575

ORIGINAL ARTICLE

Alterations of Photopic Negative Response of Multifocal Electroretinogram in Patients with Glaucoma Muneyoshi Kaneko, Shigeki Machida, Yuya Hoshi and Daijiro Kurosaka

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Department of Ophthalmology, Iwate Medical University School of Medicine, Uchimaru, Morioka, Iwate, Japan

ABSTRACT Purpose: To determine the effect of glaucoma on the multifocal electroretinograms (ERGs) (mfERGs) elicited by low-frequency stimuli. Methods: Forty-four patients with open-angle glaucoma and 15 normal subjects were studied. The stimulus frequency was 6.25 Hz, and the stimulus was a circle with a 6.8 radius that was centered on the fovea (center). MfERGs were also elicited by a quarter of an annulus placed around the macula (superior/temporal; inferior/ temporal; superior/nasal; and inferior/nasal quadrants). The radius of the inner border of the annulus was 6.8 and that of the outer border was 20 . The actual sensitivity was determined by standard automated perimetry. The thickness of the ganglion cell complex (GCC) was measured by optical coherence tomography. Results: The mfERGs consisted of a negative wave (N1) followed by a positive wave (P1), and followed by a slow negative wave (N2). There were no significant differences in the response densities of N1 and P1 between the normal control and glaucomatous eyes in any areas. The N2 response density was significantly reduced with the severity of glaucoma in the center. There was a significant reduction even at an early stage of glaucoma compared to control values. In the center, the N2 response density was significantly correlated with the GCC thickness and mean sensitivity. However, in other stimulus areas, there was no significant reduction of any components of the mfERGs. Conclusions: These results suggest that the N2 component of the slow-sequence mfERGs is affected by glaucoma in the central retinal area. Regional variations in the contribution of the retinal ganglion cell activity to the N2 should be considered when examining the mfERGs in glaucoma patients. Keywords: Glaucoma, multifocal ERG, multifocal PhNR, photopic negative response, retinal ganglion cells

detected in glaucomatous eyes.2,3 Therefore, a new measure of the functional activity of RGCs needs to be developed to detect early stage glaucoma. One possible candidate for this might be the photopic negative response (PhNR), which is a negative-going wave that occurs after the b-wave of the full-field photopic electroretinogram (ERG).4 Animal studies have shown that the PhNR originates from the neural activities of RGCs.4,5 Accumulating evidence from clinical cases has demonstrated the PhNR can be used to evaluate RGC function in patients with retinal

INTRODUCTION Open-angle glaucoma (OAG) is a prevalent disease especially in individuals over 40 years of age.1 Standard automated perimetry (SAP) has been widely used to evaluate the visual function in patients with OAG. However, it has been shown that the anatomic changes of retinal ganglion cells (RGCs) can precede the development of visual field defects determined by SAP. Thus, a 20–50% loss of the RGCs is necessary before visual field defects can be

Received 30 December 2013; revised 9 March 2014; accepted 8 April 2014; published online 14 May 2014 Correspondence: Shigeki Machida, MD, PhD, Department of Ophthalmology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan. Tel: 81-19-651-5111 (ex 6902 or 8691). Fax: 81-19-653-2864. E-mail: [email protected]

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78 M. Kaneko et al. and optic nerve diseases affecting the RGCs.6–16 In glaucoma patients, clinical studies have shown that the amplitude of the PhNR was correlated with the visual sensitivity, retinal nerve fiber layer (RNFL) thickness and optic disc topography.12 However, the results also show that the diagnostic sensitivity of the full-field PhNR was not high for eyes with early-stage glaucoma.12,17 The PhNR of the full-field ERGs originates from the neural activity of the RGCs over a wide area of the retina. We have demonstrated that the amplitude of the focal PhNR was significantly correlated with the reduction of the SAP-determined visual sensitivity,13 with the RNFL thickness, and with the alterations of the rim area of the optic nerve head in eyes with glaucoma.18 We have also shown that analyzing the PhNR of the focal ERGs from multiple retinal loci increased the sensitivity of detecting early functional loss to nearly 90%.14 These findings suggested that the PhNRs recorded from focal retinal areas could be used to analyze RGC function in focal areas of the retina. Another technique that can be used to examine the function of focal retinal areas is the multifocal ERGs (mfERGs).19,20 The mfERGs allow clinicians to record focal ERGs from numerous retinal locations in a short time. The mfERGs are typically recorded by a fastsequence stimulation with a frame rate of 75 Hz with an inter-stimulus interval of 13.3 ms. Therefore, the mfERG waveform is somewhat different from that of the full-field photopic ERG waveform. However, mfERGs that resemble the conventional full-field photopic ERGs are obtained by reducing the stimulus frequency, called slow-sequence mfERGs.21,22 The mfERGs elicited by slow-sequence stimulation have components that correspond to the a- and b-waves of the full-field ERGs. In addition, there is a slow negative wave following the b-wave, which resembles the PhNR. The purpose of this study was to determine the effect of glaucoma on the different components of the mfERGs elicited by slow-sequence stimuli.

METHODS Patients Forty-four eyes of 44 patients with OAG were studied. The patients were being treated in the Glaucoma Unit of the Iwate Medical University Hospital, and their ages ranged from 34 to 87 years with a mean ± standard deviation of 63.2 ± 14.9 years. The diagnosis of OAG was based on the presence of a glaucomatous optic disc associated with visual field defects determined by SAP. The glaucomatous optic disc was assessed according to the guideline provided by Japanese Glaucoma Society (www.nichigan.or.jp/ member/guideline/glaucoma3.jsp). All patients

underwent gonioscopy to confirm that the anterior chamber angles were open. A visual field defect was determined to be glaucomatous when it met one of three criteria:23 (1) the pattern deviation plot showed a cluster of three or more non-edge points that had lower sensitivities than that in 5% of the normal population (p50.05) and one of the points had a sensitivity that was lower than 1% of population (p50.01); (2) the value of the corrected pattern standard deviation was lower than that of 5% of the normal visual field (p50.05); and (3) the Glaucoma Hemifield test indicated that the field was outside the normal limits. In all glaucomatous eyes, the intraocular pressure was controlled under 21 mmHg by anti-glaucoma eye drops at the time of the mfERG recordings. Patients who underwent filtering surgery were excluded from the study. Fifteen eyes of 15 agematched normal volunteers, ranging in age from 40 to 75 years with a mean of 58.6 ± 10.1 years, were studied under the same conditions. This research was conducted in accordance with the Institutional Guidelines of the Iwate Medical University, and the procedures conformed to the tenets of the Declaration of Helsinki. An informed consent was obtained from all subjects after a full explanation of the nature of the experiments.

Visual Field Analyses The Humphrey Visual Field Analyzer (Model 750, Humphrey Instruments, San Leandro, CA) was used for SAP. The SITA Standard strategy was applied to program 30-2, and the measurements of the visual sensitivity were made after at least three minutes of adaptation to the background lights. The mean deviation (MD) was defined as the mean of the differences between the measured sensitivity and normal values of age-matched controls. Thus, the MDs represented the depression of sensitivity over the whole visual field. From the MD, we classified the patients with glaucomatous visual fields into three groups; early (MD4 6 dB, n = 17), intermediate ( 6 dB MD  12 dB, n = 13) and advanced (MD5 12 dB, n = 14) glaucoma. The MDs ranged from 5.99 to 0.94, from 11.99 to 6.09 and from 31.07 to 12.77 dB for the early, intermediate and advanced groups, respectively. The average of MDs was 3.32 ± 1.80, 8.73 ± 1.90 and 20.5 ± 6.04 dB for the early, intermediate and advanced groups, respectively. The actual sensitivity of the most central 4 points and fovea was taken to be the visual sensitivity in the central retinal area. Because the decibel (dB) value is 10  log (1/Lambert), we converted the actual sensitivity of each measured point (dB, log unit) to 1/ Lambert (linear unit), and then averaged it to obtain the mean sensitivity. We then determined whether the Current Eye Research

Multifocal PhNR in glaucoma means of the sensitivities were significantly correlated with the N2 response density in the central area.

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mfERG Recordings The pupils were dilated to approximately 8 mm in diameter by a mixture of 0.5% tropicamide and 0.5% phenylephrine HCL. The refractive error was measured with an autorefractometer (ARK-503 A, Nidek Co. Ltd., Gamagori, Aichi, Japan), and a lens with the spherical equivalent of the refractive error plus a + 3.0 diopter lens was placed in front of the examined eyes. The other eye was covered with an eye patch. Stimuli were generated on a CRT monitor (VERISTM 7, Electro-Diagnostic Imaging, San Mateo, CA) and consisted of five stimulus elements with a dart pattern (Figure 1A). The mfERGs were elicited by a circular stimulus with a 6.8 radius centered on the fovea (center) and by a quarter of an annulus placed around the macula (superior/temporal, inferior/temporal, superior/nasal and inferior/nasal). The radius of the inner border of the annulus was 6.8 and that of the outer border was 20 . White (200 cd/m2) or black (4 cd/m2) elements were presented in a pseudorandom binary-m sequence at a frequency of 6.25 Hz. A steady background of 100 cd/m2 surrounded the stimulus field. After corneal anesthesia with 4% lidocaine HCL and 0.4% oxybuprocaine HCL, a Burian-Allen bipolar contact lens electrode (Hansen Ophthalmic Laboratories, Iowa City, IA) was placed on the cornea. A chlorided silver electrode was placed on the left ear lobe as the ground electrode. The responses were digitally band pass filtered from 3 to 30 Hz. The mfERGs were analyzed with the VERIS software (VERIS Science 4.1.1, Mayo, Nagoya, Japan), and the all-trace waveforms of the first-order kernels (A)

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were obtained by averaging the local retinal responses from the five different retinal loci. The N1 amplitude was measured from the baseline to the trough of the first negative response, and the P1 from the baseline to the peak of the following positive wave and the N2 from the baseline to the following trough (Figure 1B). The amplitudes of the mfERG were expressed as response density (nV/deg2) representing the amplitude as a function of the stimulus area.

Optical Coherence Tomography The ganglion cell complex (GCC) is composed of the RNFL, the ganglion cell layer and the inner plexiform layer. It has been used to evaluate the thickness of the inner retina in glaucoma patients.24 The GCC thickness was measured at 512  128 points in the posterior pole of the eye with a radius of 15 corresponding to 4.5 mm by spectral-domain optical coherence tomography (OCT) (SD-OCT, RS-3000 Advance, Nidek Co. Ltd.). SD-OCT was used to obtain GCC thickness maps, and the values were compared to the embedded age-matched data base (Figure 2). The mean GCC thickness was obtained for the central circular area with a radius of 2.25 mm corresponding to 7.5 except for the foveal area within 0.75 mm, and these values were correlated with the N2 response density in the central area. The tracking system of the SD-OCT allowed us to average images at exactly the same measuring points.

Statistical Analyses One-way ANOVA was used to determine the statistical significance between control subjects and glaucoma patients at different stages. In addition,

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FIGURE 2 Responses from representative early and advanced glaucomatous eyes. Visual fields obtained by SAP, GCC maps obtained by SD-OCT and mfERGs elicited from the five retinal loci are shown. SAP: standard automated perimetry, GCC: ganglion cell complex, SD-OCT: spectral-domain optical coherence tomography and mfERG: multifocal electoretinogram.

Bonferroni’s multiple comparison tests were performed after the ANOVA as post-hoc tests. Pearson’s coefficients of correlation were calculated to determine the strength of correlation between the ERG parameters and the GCC thickness or mean sensitivity. These analyses were performed using Prism 5.1 (GraphPad Software Inc., San Diego, CA). The level of statistical significance was set at p50.05.

RESULTS

with localized visual field loss with an MD of 5.99 dB. Although the N1 and P1 response densities were not significantly different from those of the control, the N2 response density was reduced in the center while it remained normal in other retinal areas. In the advanced glaucoma case, the GCC thickness was diffusely reduced resulting in a diffuse reduction of visual sensitivity in the SAP with an MD of 26.13 dB. In the advanced case, the N2 response density was reduced only in the center with relatively good preservation of the N2 response density in other retinal areas.

Representative Waveforms of Normal and Glaucomatous Eyes Representative mfERGs recorded from a normal subject are shown in Figure 1(B). The waveforms consisted of the N1 and P1 components immediately followed by the slow negative-going N2. The waveforms were similar in all stimulus areas. This waveform is similar that of the focal ERGs that consist of an a-wave and b-wave and the PhNR.13 Because the high-cut filter was set at 30 Hz, waves with highfrequency components, such as oscillatory potentials, were not recorded. The N2 recorded from the center appeared to be proportionally larger than that in the other areas. The mfERGs recorded from representative cases of early and advanced glaucoma are shown in Figure 2. In the early glaucoma case, the GCC map showed an accurate-shaped loss in the lower fundus associated

Averaged Response Densities in Normal Subjects The response density of all mfERG components was lower in the surround areas than in the center. The regional variations of the averaged N2 response density are shown in Figure 3(A). The averaged ratios of the response densities of N2 to P1 (N2/P1 ratio) were significantly different in the different regions (Figure 3B, p50.0001). The N2/P1 ratio was significantly smaller in the superior/temporal (p50.05), superior/nasal and inferior/nasal (p50.0005) quadrants than that in the center. These findings indicate that N2 contributes more to the mfERGs in the center than in the other areas. Current Eye Research

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FIGURE 4 Averaged response density of the N1 of the mfERG recorded from normal subjects and patients with early, intermediate and advanced glaucoma in the center (A), superior/temporal (B), inferior/temporal (C), superior/nasal (D), inferior/nasal (E) and all retinal areas (F). White: normal subjects; light gray: early; gray: intermediate; and dark gray bars: advanced glaucoma patients. The boxes represent the 25%–75% interquartile ranges. The horizontal line represents the mean values, and the bars represent the 5% and 95% confidence intervals. mfERG: multifocal electroretinogram.

Comparison of Averaged Response Densities between Normal Subjects and Glaucoma Patients The averaged response densities of the N1, P1 and N2 and the response density ratio of N2/P1 for normal subjects and glaucoma patients with early, !

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intermediate or advanced stage are shown in Figures 4–7. There was no significant difference of the N1 and P1 between the control and glaucomatous eyes at any stages and in any areas (Figures 4 and 5). The N2 response density in the center was significantly reduced in eyes with the severity of glaucoma (p50.0001, Figure 6A), but no significant changes

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FIGURE 5 Averaged response density of the P1 of the mfERG recorded from normal subjects and patients with early, intermediate and advanced glaucoma in the center (A), superior/temporal (B), inferior/temporal (C), superior/nasal (D), inferior/nasal (E) and all retinal areas (F). White: normal subjects; light gray: early; gray: intermediate; and dark gray bars: advanced glaucoma patients. The boxes represent the 25%–75% interquartile ranges. The horizontal line represents the mean values, and the bars represent the 5% and 95% confidence intervals. mfERG: multifocal electroretinogram.

were observed in the all-trace waveform and in the other areas (Figure 6B–F). The N2/P1 response density ratio was significantly decreased with the severity of glaucoma in the center (Figure 7A, p50.0001) and in the inferior/temporal quadrant (Figure 7C, p50.05), and in the all-trace waveform (Figure 7F, p50.005). Multiple comparison tests revealed significant differences in the N2 response density and N2/P1 response density ratio recorded from the center between the control and all stages of glaucoma (Figures 6A and 7A, early: p50.01, intermediate and advanced: p50.00001).

Correlation between N2 Response Density and GCC Thickness and Mean Sensitivity Because the N2 response density in the center was solely associated with the severity of glaucoma, we plotted the center N2 response density against the GCC thickness (Figure 8A) and also against the mean sensitivity of the central retinal areas (Figure 8B) for the control subjects and glaucoma patients. The mean sensitivity was converted to linear values and represented by 1/Lambert. The N2 response density was significantly correlated with the GCC thickness (R2 = 0.330, p50.0001) and mean sensitivity (R2 = 0.358, p50.0001).

DISCUSSION Our results demonstrated that the N2 component of the slow-sequence mfERGs elicited from the central visual field was most susceptible to the glaucomatous damage compared to those elicited from other retinal areas including the superior/temporal, superior/ nasal, inferior/temporal and inferior/nasal quadrants. Even at the early stage of glaucoma, the N2 response density was significantly reduced in the center, while it remained not significantly changed in the other retinal areas even in eyes with advanced glaucoma. In the central retinal area, the N2 response density was significantly correlated with the GCC thickness and SAP-determined sensitivity.

N2 in Central Area Corresponds to PhNR of Focal Macular ERGs In the central area, the N2 response density was selectively attenuated and the degree of attenuation was correlated with the severity of glaucoma and also with the loss of the retinal sensitivity and GCC thickness. These changes are similar to those obtained for the PhNR of the focal macular ERGs.13,14,17,25,26 This indicates that the origin of the N2 of the slowsequence mfERG is the same as that of the PhNR of Current Eye Research

Multifocal PhNR in glaucoma Center

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FIGURE 6 Averaged response density of the N2 of the mfERG recorded from normal subjects and patients with early, intermediate and advanced glaucoma in the center (A), superior/temporal (B), inferior/temporal (C), superior/nasal (D), inferior/nasal (E) and all retinal areas (F). White: normal subjects; light gray: early; gray: intermediate; and dark gray bars: advanced glaucoma patients. The boxes represent the 25%–75% interquartile ranges. The horizontal line represents the mean values, and the bars represent the 5% and 95% confidence intervals. mfERG: multifocal electroretinogram. Asterisks indicate statistical significance after Bonferroni’s multiple comparison test. *p50.01, **p50.00001.

the focal macular ERG. Based on evidence obtained from experimental and clinical researches, the PhNR of the focal macular ERG originates from the neural activity of RGCs in the macular area.13,14,17,25–27 Thus, the N2 component of the mfERGs recorded from the central area represents the RGC activity of the corresponding retinal area.

Regional Variations of N2 in Normal Subjects As expected, the response density of all mfERG components was smaller in the surround areas than in the central area because the cone density is highest in the fovea and decreases with increasing eccentricities from the fovea. If the reduction of the cone photoreceptor density is the only factor that determines the regional variations of the mfERGs, the response density of all ERG components should decrease proportionately with increasing eccentricity. However, the N2/P1 response density ratio was small in the surrounding areas especially the retinal area nasal to the macula, compared to the central area. This indicates that the N2 response density predominately decreases with eccentricity from the fovea. !

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It has been reported that RGC density decreases and the receptive field size increases with eccentricity from the fovea.28,29 These changes could explain the weaker contribution of RGC activity to the N2 of the mfERGs in the surrounding areas. Alternatively, a balance of the ON- and OFFresponses may be another possible explanation. Kondo et al. demonstrated differences in the regional distribution of ON- and OFF-responses using mfERGs,30 in which the contribution of the OFFresponses became larger with increasing eccentricity from the fovea. In the cone ERGs elicited by short stimuli, the OFF-response shapes the i-wave, a small positive hump following the b-wave.31 The i-wave counteracts the PhNR because it has a peak time similar to the PhNR, although some parts of the i-wave may be filtered out by the 30 Hz high cut filter. In this study, the slow-sequence stimuli can produce waveforms resembling the full-field cone ERGs. Therefore in the surrounding areas, the OFF-response could counteract the PhNR and result in smaller N2 amplitudes. There is a possibility that the glial mediator of the PhNR is more influential in the central than area in the surrounding area. Although the PhNR originates from the RGCs, it is likely mediated by glial cells.

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FIGURE 7 Averaged N2/P1 response density ratio of the mfERG recorded from normal subjects and patients with early, intermediate and advanced glaucoma in the central (A), superior/temporal (B), inferior/temporal (C), superior/nasal (D), inferior/nasal (E) and all retinal areas (F). White: normal subjects; light gray: early; gray: intermediate; and dark gray bars: advanced glaucoma patients. The boxes represent the 25%–75% interquartile ranges. The horizontal line represents the mean values, and the bars represent the 5% and 95% confidence intervals. mfERG: multifocal electoretinogram. Asterisks indicate statistic significance after Bonferroni’s multiple comparison test. *p50.01, **p50.005 and ***p50.00001.

In animal models with inherited photoreceptor degeneration, we have shown that the PhNR is enhanced despite the progressive photoreceptor degeneration.32 Intravitreal injections of antibodies against inward rectifying potassium (Kir) channels reduced this enhancement.33 Because the Kir2.1 and Kir4.1 channels are expressed on Mu¨ller cells,34 the potassium flux through the Kir channels may partly contribute to generating the PhNR. It has been reported that glial cell density is much higher in the central than in the peripheral retina,35 which could explain the larger N2 in the central areas than in the surrounding areas.

Ganglion Cell Component of N2 in Surrounding Area Even at an advanced stage of glaucoma, the N2 response density remained normal in the surrounding regions despite a diffuse loss of RGCs as demonstrated by visual field defects and GCC maps. This can be interpreted by the following explanations. First, the N2/P1 response density ratio was smaller in the surrounding areas than in the central area in normal controls indicating that the N2 response density is proportionally small in the surrounding

area even in healthy eyes. In addition, changes of the N2 response density with the severity of glaucoma were small in the surrounding areas. As a result, individual variations of the N2 response density likely masked the functional loss in the surrounding areas. Second, the N2 may not represent the neural activity of only the RGCs. In rodents, the PhNR also receives contribution from the neural activity of amacrine cells.31,32,36 The electrical response driven by amacrine cells could partially shape the PhNR even in humans because the PhNR of the full-field cone ERGs does not completely disappear in patients with complete visual loss caused by traumatic optic neuropathy.7 This suggests that other neural elements contribute partially to the amplitude of the PhNR especially in the surrounding areas.

Future Clinical Use of mfERGs for Glaucoma Our results suggest that the N2 component recorded from the central area is most susceptible to glaucomatous damage. On the other hand, the N2 from the surrounding areas are resistant to glaucomatous damage despite apparent loss of SAP-determined sensitivity and GCC thickness. These findings suggest Current Eye Research

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that functional analysis using the mfERG should be applied to the central retina rather than the surrounding areas when the mfERG is used for examining glaucoma patients. Detailed analysis in the central retina using smaller elements would provide useful information regarding functional loss in glaucoma patients.

CONTRIBUTIONS OF AUTHORS Design of the study (M. K., S. M. and D. K.), conduct of the study (M. K. and Y. H.) and data analysis (M. K. and S. M.).

REFERENCES CONCLUSIONS The results suggest that the N2 component of the slow-sequence mfERGs is affected by glaucoma in the central retinal area. This response probably corresponds to the PhNR of the focal macular ERG representing the RGC function in the central area. Regional variations in the contribution of the RGC activity to the N2 should be considered when applying the mfERG to glaucoma patients.

ACKNOWLEDGEMENTS We thank Dr. Duco Hamasaki for editing the manuscript.

DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by a Grant-in-Aid for Scientific Research C from Ministry of Education, Science and Culture in Japan No. 24592677 (MK) and 24592677 (SM). !

2015 Informa Healthcare USA, Inc.

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Current Eye Research

Alterations of photopic negative response of multifocal electroretinogram in patients with glaucoma.

To determine the effect of glaucoma on the multifocal electroretinograms (ERGs) (mfERGs) elicited by low-frequency stimuli...
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