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Cite this article: Pescosolido N, Rocca D, Rusciano D (2015) The Value of Flicker Erg B-Wave Implicit Time in Macular Degeneration. JSM Ophthalmol 3(2): 1029.

*Corresponding authorDario Rusciano, Scientific Department, Sooft Italia SpA, Via Salvatore Quasimodo 136, 00144 Roma, Italy, Email:

Submitted: 12 February 2015

Accepted: 09 March 2015

Published: 11 March 2015

ISSN: 2333-6447

Copyright© 2015 Rusciano et al.

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Research Article

The Value of Flicker Erg B-Wave Implicit Time in Macular DegenerationNicola Pescosolido1, Daniela Rocca2 and Dario Rusciano3*1Department of Cardiovascular, Respiratory, Nephrologic, Anesthesiologic and Geriatric Science, Faculty of Medicine and Dentistry, Sapienza University of Rome, Italy2Department of Ophthalmology, Vittorio Emanuele University Hospital, Via Santa Sofia, Catania, Italy3Scientific Department, Sooft Italia SpA, Rome Italy

INTRODUCTIONFlash electroretinography (ERG) is an electro-functional test

able to evaluate the massive bioelectric response of the retina to a non-structured luminous stimulus (flash). ERG is thus an extremely complex analysis in as much as it reflects the activity of different retinal cell types, from photo-receptors (cones and rods) to bipolar cells, to Mueller’s cells.

In 1989 the International Society for Clinical Electrophysiology of Vision (ISCEV) standardized a basic protocol so that ERG could be evaluated and compared worldwide [1]; from then, thanks to a better understanding of electrophysiology, this protocol was updated in 1999 [2], in 2003

[3] in 2008 [4] and finally in 2015 [5].

The protocol includes six tests that represent the basic evaluations for a correct clinical diagnosis based on electrophysiology: 1) dark-adapted 0.01 ERG; 2) dark-adapted 3.0 ERG; 3) dark-adapted 3.0 oscillatory potentials; 4) light-adapted 3.0 ERG; 5) light-adapted 3.0 flicker ERG; 6) light-adapted 3.0 ERG with rapidly repeated stimulus (30 Hz flicker).

Among these basic tests, photopic 30 Hz flicker ERG has a very interesting clinical potential. The flicker test, in fact, accurately studies the system of cones; this is obtained with repeated stimuli of a single flash, under the same light-adapted conditions

used by ERG for the cones with a single flash (3cd x s x m-2 and maximum duration of 5 m sec). The flashes occur at a speed of about 30 stimuli per second, thus obtaining a series of generally sinusoidal waves of which the voltage and the phase retardation with respect to the stimulus will be evaluated later. Moreover, increasing the frequency of stimulation, the amplitude of the wave decreases until the trace is completely flat (critical fusion frequency). Experimental studies of ocular electrophysiology on monkeys have shown that the response of the flicker ERG is 80% dependent on the post-receptive component [6].These studies were clinically confirmed in humans by Verma and colleagues in 2009 [7].

The evaluation of the flicker ERG is based on two parameters inherent in the b-wave: amplitude and implicit time (time from the beginning of stimulation and the peak amplitude response) [8]. Both parameters are altered in pathologies that affect photoreceptors [9], but the implicit time is an index that discriminates better between normal and pathologic subjects. In fact, in our previous study [10] we showed how, comparing healthy patients with patients affected by various typologies of retinopathy, the implicit time was found to be a more reliable index with respect to the amplitude, so much so as to be considered a good parameter for the diagnosis and probably the follow-up of various types of retinopathies. This finding was

Keywords• Flicker ERG• Implicit time• Macular degeneration

Abstract

The B-wave of photopic 30 Hz flicker ERG can be used to study the altered response of the photoreceptor system in pathologic retinas. We had previously shown that its implicit time is a more reliable parameter than its amplitude to discriminate between normal and pathologic retinas. In this pilot study we confirm the observation that implicit time differs between healthy normal eyes (31.71 ±1.41 ms) and retinopathic eyes (38.16 ±3.97). Moreover, we show that implicit time also discriminates between eyes of patients affected by different forms of macular degeneration, such as atrophic AMD (34.54 ± 2.17) or neovascular MD (either age-related or myopic: 40.61 ± 3.13). Finally, we show that the contralateral eye of patients in which the fellow eye was affected by a neovascular retinal disease also shows an abnormal implicit time, suggesting that the physiological dysfunction may precede the morphologic, anatomic damage.

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confirmed by us in a further study [11] in which both the implicit time of the flicker ERG and the optical coherence tomography (OCT) examination were used for the evaluation of results.

Our current study evaluates the implicit time of the flicker ERG in a group of patients affected by macular degeneration (bilateral neovascular, unilateral neovascular or bilateral atrophic) with respect to a group of healthy patients. We also examine these parameters comparing them with the contralateral eye in the forms of unilateral neovascular maculopathy.

MATERIALS AND METHODSAll patients were of Caucasian ethnicity. We enrolled in the

study 62 eyes of 33 patients sub divided into two groups: the first, the control reference group was composed of young adults without ophthalmic or systemic pathologies, and included 34 eyes of 17 patients (Table 1); the second group included 28 eyes of 16 patients affected by various types of macular degeneration. Six patients (8 eyes) had neovascular age-related macular degeneration (AMD); one patient was single-eye and 3 eyes of 3 patients that presented with the atrophic form were not considered (Table 2). Five patients (10 eyes) were affected by the bilateral atrophic form (Table 3), and 5 patients (10 eyes) by the unilateral neovascular form.

All patients had a complete ophthalmologic examination with evaluation of the ocular fundus by means of a binocular indirect ophthalmoscope after dilatation of the pupil with Visumidriatic (tropicamide 1%, Visupharma, Italy) and by OCT examination and photopic 30 HZ flicker ERG. The OCT examination was carried out with an RS 3000 tomograph (Nidek Co. Ltd Japan) with radial acquisition. The electrophysiological evaluation of the retina was carried out using a Retimax Advanced Plus (CSO ophthalmological instruments, Florence, Italy) following the standard ISCEV protocols for the 3.0 flicker ERG with photopic

adaptation. The instrument used included a particular software application that evaluates the retinal response of the patient to the stimulation flicker. The response was detected by HK electrodes positioned inside the inferior conjunctival fornices, after instillation of anesthetic eye drops. Amplitude and implicit time of the b-wave of the flicker 30 HZ were evaluated in the diverse conditions of macular degeneration: bilateral wet or atrophic, unilateral neovascular versus the contralateral eye.

To evaluate the different responses of the two groups a non-parametric statistical test (Mann Whitney) was used with a confidence interval of 99%.

RESULTSAll the individual values reported in the tables are averaged

and the respective SD is reported. For sake of simplicity, the text reports the average value between right and left eye. Visual acuity is measured on a decimal scale. Table 1 shows the implicit times of the 17 healthy patients (7 males, 10 females; average age 58.65 ± 13.01 years), without retinal pathologies who show a range of physiologic values between 28.86ms and 33.39ms (mean: 31.71 ±1.41 ms). Figure 1 shows a normal macular OCT with a profile characterized by physiological foveal depression, perfectly conserved retinal thickness and a 30 HZ flicker with standard deviations within normal limits and with the typical shape of a roughly sinusoidal wave.

The next tables (II to IV) report the data of patients with different types of macular degeneration. Fourteen patients, all affected by AMD, were over 63 years of age (mean: 78.6 ± 6.8), and two patients affected by neovascular myopic MD were under 50 years. None of the patients was affected by other ocular pathologies, so that no influence on the flicker response was expected. Table 2 shows the data of the 6 patients (4 females, 2 males, mean age 81.83 ± 4.79 years) affected by bilateral

Sex Age Implicit Time (ms) VisusRE LE RE LE

F 32 33.07 33.07 1 1M 40 29.83 29.83 1 1F 46 32.1 33.07 0.8 1F 48 33.39 33.39 0.9 1F 50 28.86 29.83 1 1F 55 32.42 32.42 1 1F 55 30.48 30.15 1 1M 56 31.45 31.45 1 1M 57 33.39 34.04 1 1F 63 30.48 33.07 1 1M 66 31.77 31.45 1 1F 67 30.8 30.15 1 1M 68 30.8 30.8 0.9 0.9F 68 33.39 33.07 1 0.8F 70 32.42 32.42 1 1M 72 32.1 33.07 1 1M 84 30.15 29.83 1 1

MEAN 58.65 31.58 31.83SD 13.01 1.37 1.47

Table 1: Implicit time values and BCVA of normal subjects.

Abbrevations: RE: Right Eye; LE: Left Eye

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Figure 1 Top: OCT (left eye) and Bottom: photopic flicker ERG 30 Hz of a normal control patient.

Neovascular Disease

Sex AgeImplicit Time (Ms) Visus

RE LE RE LE

F 76 36.96 0.1

F 83 34.69 DRUSEN 0.05 0.4

M 80 43.12 43.45 0.1 0.5

F 85 41.5 DRUSEN 0.05 0.5

F 78 DRUSEN 40.85 0.2 FINGERCOUNT

M 89 41.82 42.47 FINGERCOUNT 0.05

MEAN 81.83 39.62 42.26

SD 4.79 3.61 1.31

Table 2: Implicit time values and BCVA of wet AMD patients.

Abbrevations: RE: Right Eye; LE: Left Eye

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neovascular macular degeneration. One female patient had only one eye and the other three female patients had NVMD in one eye and drusen in the contralateral eye. Only the implicit time of the eye affected by NVMD was considered in the analysis to avoid the confounding effect of a different affection. The values of the implicit time range between 34.69 and 43.45ms (40.61 ± 3.13ms), and differ in a statistically significative way with respect to the subjects without retinopathy. Figure 2 illustrates the parameters observed in patients of this group. The profile of the flicker ERG is clearly altered in both eyes. OCT revealed the presence, in front of the pigment epithelium, of a neovascular membrane that raised the neuroretina.

Table 3 and Figure 3 refer to the group of 5 patients (all females, mean age 76 ± 8.86 years) with bilateral atrophic macular degeneration. Table 3 shows values of implicit time between 31.12 and 36.96 ms (mean 34.54 ± 2.17ms) that were significatively increased with respect to those of the control group (p<0.001). The OCT examination typically reveals an altered foveal profile and a reduction of the thickness of the retina with an increase of the reflectivity of the pigment epithelium due to atrophy.

Table 4 and Figure 4 summarize the data of the 5 patients (3 females, 2 males) affected by unilateral wet macular degeneration. The two youngest patients (a male of 48 and a female of 40) were affected by myopic choroidal neovascularization, while the other

3 (1 male and 2 females, average age 78.6 ± 6.02 years) had AMD. Given the same nature of the damage (choroidal neovascular disease), although due to different etiologies, their data were analyzed together. All five patients showed the confirmed pathology in only one eye, while the contralateral eye appeared normal at the OCT examination. However, the implicit time appeared pathologic in both eyes with variable values between 40.85 and 42.15 ms in the eye with pathology (mean 41.50± 0.46ms), and between 37.29 and 39.88 ms (mean 38.84±1.11ms) in the apparently normal eye. The OCT examination of one of the myopic patients showed a picture of neovascular membrane that raised the neuroretina in the right eye, and a normal picture in the fellow eye (Figure 4).

Figure 5 shows an interpolation of all the data, confirming how the values of the implicit time of the normal subjects do not overlap those of the patients affected by macular degeneration. It appears clear that also the implicit time values of the apparently healthy contralateral eye of the patients with the unilateral neovascular form have altered values (even if less altered with respect to the contralateral pathologic eye), intermediate between those registered for the neovascular form and the atrophic form.

DISCUSSIONIn this pilot study we verified how the values of implicit time

Atrophic Disease

Sex AgeImplicit Time (ms) Visus

RE LE RE LE

F 86 36.96 37.71 0.2 MOTOMANU

F 73 33.72 36.64 0.3 0.05

F 63 31.45 31.12 0.5 0.1

F 76 34.69 34.69 0.1 0.05

F 82 34.04 34.37 0.05 0.15

MEAN 76 34.17 34.91

SD 8.86 1.98 2.53

Table 3: Implicit time values and BCVA of dry AMD patients.

Abbrevations: RE: Right Eye; LE: Left Eye

Unilateral Neovascular Disease

SEX AgeImplicit Time (ms) Visus

NVD “normal” NVD “normal”

F 85 41.5 39.88 FINGERCOUNT 0.7

F 78 40.85 39.88 FINGERCOUNT 0.3

M 48 41.5 37.29 0.2 1

M 73 41.5 38.91 0.5 1

F 40 42.15 38.26 0.05 0.3

MEAN 64.80 41.50 38.84

SD 19.66 0.46 1.11

Table 4: Implicit time values and BCVA of young myopic or AMD patients.

Abbrevaions: RE: Right Eye; LE: Left Eye; NVD: Neovascular Disease

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Figure 2 Top: OCT (left and right eye) and Bottom: photopic flicker ERG 30 Hz of eyes with bilateral neovascular AMD.

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Figure 3 Top: OCT (left and right eye) and Bottom: photopic flicker ERG 30 Hz of eyes with bilateral atrophic AMD.

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Figure 4 Top: OCT (left and right eye) and Bottom: photopic flicker ERG 30 Hz of the one eye with neovascular myopic maculopathy (B), and the contralateral eye morphologically normal (A).

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28

30

32

34

36

38

40

42

44

46

NormalControl

NVDbilateral

Drusenbilateral

NVDmonolateral

contralateral

Figure 5 Box plot of the implicit time values of all eyes plotted for category: normal control eyes and eyes presenting with bilateral disease were evaluated without distinction between left and right; the eyes of patients with unilateral neovascular disease were considered separately.

of the photopic 30 Hz flicker ERG differ between the eyes with maculopathy with respect to normal ones. Moreover, we have shown how this parameter also differs between the diverse maculopathies (atrophic vs. neovascular). Finally, we have shown how the implicit time of the flicker is pathological also in the apparently normal fellow eye of patients affected by unilateral wet degeneration, suggesting that functional alterations could precede morphological ones.

AMD is a heterogeneous, complex pathology whose pathogenesis is linked to both genetic and environmental factors [12-14]. Historically, the use of the electroretinogram in this pathology has been under estimated both because macular degeneration has been considered a pathology involving only a small area of the retina, that is the macula, and because the macular cones are only a part of the retinal cones. Over the last few years, instead, there have been numerous studies that have shown how in macular degeneration retinal functionality, measured by the electro retinogram, has been generally compromised [15-18]. In particular, interesting possibilities have been provided by the evaluation of the implicit time of the photopic 30 Hz flicker ERG. This is considered as one of the best predictive parameters of neo vascularization in cases of central retinal vein occlusion [19-21]. Larsson [22] showed that the implicit time is a predictive index also for the development of secondary glaucoma in central retinal vein occlusion. Kjeka [23] noted that all the patients with central retinal vein occlusion and implicit time of 35 ms or more developed ocular neo vascularization, showing, moreover, how the values of implicit time in the contralateral eye are significatively greater than those of the group without neo vascularization, even if they were within normal limits. Other authors [24,25] , however,

have shown pathologic ERG also in the fellow eye. This finding could be explained by an altered retinal circulation that surely also involves the contralateral eye and by systemic risk factors of the subject [16,26]. Other authors [27] used the implicit time of the flicker 30 Hz also in the evaluation of type II diabetes. In these subjects without diagnosis of retinopathy altered flicker was also present; this was in line with our observation that functional alterations could precede the morphological ones. In one review of 2012 [28] Bhutto and colleagues showed how macular degeneration is a pathology characterized by the loss of the mutualistic relationship between the diverse structures of the Photoreceptor/Retinal Pigment Epithelium (RPE)/Bruch’s Membrane (BrMb)/Choriocapillaris (CC) complex. Bhutto also showed how, in the atrophic form, the primum movens is the alteration and the loss of the RPE (hypopigmentation following drusen formation) and in the neovascular form, instead, it is the loss and the alteration of the choroidal vascularization that lead to the degeneration of the photoreceptors due to lack of nutrients. The loss of this delicate equilibrium, whatever the initial alteration was, leads to the dysfunction of all the elements of the complex. We believe, however, that the increase of implicit time of the photopic 30 Hz flicker ERG in the patients with macular pathologies could derive from the variation of the normal parameters of choroidal circulation correlated to the degree of severity of the neo vascularization [29]. The alterations of the choroidal dynamics, characteristic of the neovascular form, cause massive and early photoreceptor damage, while the atrophic form of this degeneration is later and less massive, possibly explaining the more altered implicit times in the neovascular form with respect to the atrophic form. Moreover, the formation of drusen

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or the thickening of Bruch’s membrane increase the distance between the choriocapillaris and the retina, reducing the flow of oxygen to the photoreceptors and thus inducing hypoxia and cell damage [28].

Therefore, it could be hypothesized that these alterations of the choroidal dynamics – that in part seem to be due to genetic factors [12,30] – can influence the photo receptor response (measurable with flicker ERG) preceding the morphologic damage visible using ophthalmoscopy and OCT. Similarly, another possible mechanism that contributes to the increase of the implicit time could be the phlogosis due to the activation, above all in the neovascular form, of the alternative routes of the complement both in the vitreous body [31] and at the systemic level [32]. These events could explain the pathological profile of the implicit time in the morphologically normal contralateral eye of patients with unilateral neo vascular maculopathy.

It is well known how the presence of exudative macular degeneration in one eye is one of the major risk factors for the development of neovascularization in the fellow eye [33-]. It is thus particularly useful to have diagnostic tests that can reveal early photoreceptor alterations. Therefore, the routine use of the 30 Hz flicker in all patients with unilateral macular degeneration could be recommended for an early diagnosis of new retinal alterations.

A limit of this pilot study could be that the mean age of the normal healthy population taken as control group is significantly lower than the mean age of pathologic subjects; therefore the two groups are not age-matched. It is, however, difficult to find a group of individuals above 65 years of age devoid of any ophthalmic pathology, also in the light of the results that we have reported here, showing that also eyes that are morphologically normal may indeed hide some incipient defect, resulting in alterations of the flicker ERG. Therefore, between two possible biases, we decided to go for the lesser, and establish the parameters of a normal flicker ERG response on a young adult population.

Further studies are obviously needed to confirm the predictive role of the analysis illustrated in this preliminary study.

ACKNOWLEDGEMENTSWe would like to thank Dr Antony Bridgewood for his English

proofreading of the manuscript.

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Pescosolido N, Rocca D, Rusciano D (2015) The Value of Flicker Erg B-Wave Implicit Time in Macular Degeneration. JSM Ophthalmol 3(2): 1029.

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