Introduction

Age-related macular degeneration (AMD) is a chronic, debilitating, and progressive degenerative disease affecting central vision of patients aged 55 years or older in industrialized countries [1]. AMD includes a wide spectrum of clinical presentations, from early to advanced forms, with an increasing degree of visual acuity (VA) and central visual field impairment [2, 3]. Patients affected by early AMD usually show normal VA, but ophthalmoscopic signs, such as central drusen, pigmentary abnormalities of retinal pigment epithelium (RPE), and extrafoveal geographic atrophy (GA), are already visible. Drusen correspond to the focal depositions of extracellular material, principally lipid-derived, between basal lamina of RPE and the inner collagenous layer of Bruch’s membrane. Intermediate AMD is characterized by at least one large drusen (size ≥ 125 µm) or multiple soft intermediate drusen (≥ 63 µm, ≤ 125 µm) and the presence of extrafoveal GA, and corresponds to AREDS 3 grade classification [2].

In clinical daily practice, macular involvement due to different stages of AMD can be functionally and structurally evaluated using electrophysiological tests (i.e., focal or multifocal electroretinogram, mfERG) and optical coherence tomography (OCT), respectively.

MfERG is an electrophysiological method that allows one to detect selectively the bioelectrical responses originating from photoreceptors and bipolar cells (pre-ganglionic elements) in localized central retina areas [4,5,6,7,8]. Abnormal mfERG responses have been recorded in the early or intermediate stages of AMD, thus suggesting early dysfunction of the macular pre-ganglionic elements [5,6,7,8].

OCT evaluation is a routine structural test for quantitative and qualitative assessment of retina and choroid [9,10,11,12,13,14,15,16,17,18]. In early AMD, thinning of all retinal layers (from ganglion cell to RPE) associated with outer retinal complex changes has been observed [9,10,11,12]. On spectral domain-OCT (SD-OCT), drusen appear as hyperreflective dome-shaped mound deposits under the RPE, possibly associated with additional retinal abnormalities, such as reticular pseudodrusen (RPD) [13,14,15]. The degenerative process extends also to the choriocapillary. Indeed, choroidal dropout and thinning have been observed in early stages of AMD, and the number and density of subretinal drusenoid deposits are linearly related to the disease progression [16,17,18].

Considering increasing life expectancy and to ameliorate quality of life in the elderly, an important goal for ophthalmologists is to offset AMD progression by adopting adequate therapeutic strategies. In this context, several studies reported that regular intake of lutein and zeaxanthin (constituents of the macular pigment) can reduce the risk of developing advanced AMD and their supplementation may induce a stabilization/increase of VA and of the contrast sensitivity with consequent improvement of the quality of life in patients with AMD [19, 20].

MfERG is an interesting tool for assessing functional changes (increase of the function of the pre-ganglionic elements) after supplementation with zeaxanthin and lutein in AMD patients [5]. In contrast, there is a lack of evidence about possible changes of the macular chorio-retinal structure in patients treated with the aforementioned molecules proven to induce functional benefit in pre-ganglionic macular elements (see above).

Therefore, the aim of our study was to assess whether oral supplementation with carotenoids and antioxidants, including lutein and zeaxanthin, as well as rutin, N-acetylcysteine, bromelain, alpha-lipoic acid, vitamin C, and vitamin D3 would induce functional (evaluated by mfERG) and/or morphological (assessed by SD-OCT) changes of the macular region in patients affected by intermediate AMD.

Methods

Patients

Seventy patients (35 male and 35 female, mean age 70.34 ± 9.82 years) affected by AMD were screened for enrollment in the study.

Clinical diagnosis of AMD was based on slit-lamp and indirect ophthalmoscopic examination using + 90D or + 78D non-contact lens (Volk Optical, Mentor, OH) after pupillary dilatation with tropicamide 1% drops. Stereoscopic color fundus photographs (30° centered on the fovea) were also taken, and independently analyzed and graded by two masked observers (MT and MV) in accordance with the AREDS classification [2]. Macular features included drusen number, size and confluence, focal hyperpigmentation, or hypopigmentation of RPE.

Only eyes with AREDS category 3 features (intermediate AMD) were selected for this study. Inclusion criteria for the selected eyes were as follows: VA ≥ 20/32 [0.2 logarithm of the minimum angle of resolution (logMAR)], 74 letters of the Early Treatment Diabetic Retinopathy Study (ETDRS) charts], extensive (as measured by drusen area) intermediate (≥ 63 µm, < 125 µm) drusen, at least one large (≥ 125 µm) drusen or GA not involving the center of the macula. In addition, AMD patients were never supplemented with carotenoids or antioxidants.

Exclusion criteria, based on the fact that several pathologies may influence the bioelectrical responses derived from the macular region [21], were moderate to dense lens opacity, implanted intraocular lens, corneal opacities, previous history of refractive surgery, glaucoma or ocular hypertension, previous history of intraocular inflammation such as anterior or posterior uveitis, previous history of retinal detachment or laser treatment for peripheral retinal diseases, diabetes or systemic hypertension under medical treatment, previous history of ocular trauma, usage of systemic treatments with known toxic effects on the macula (e.g., chloroquine, oxazepam), neurological diseases, or presence of any signs of advanced AMD (choroidal neovascularization or central GA) in the study eye.

When both eyes fulfilled the inclusion criteria, the eye with the best VA was selected; when both eyes had the same VA, the right eye was chosen for analysis.

As a result, 30 eyes with intermediate AMD from 30 patients (9 male and 21 female, mean age 69.32 ± 9.21 years) were enrolled in the study.

Study Design

This study has been designed as a monocentric, randomized, prospective, and double blind (placebo vs active treatment). The research followed the tenets of the Helsinki Declaration (1964 and further revisions) and the study was approved by the local ethics committee (Comitato Etico Centrale IRCCS Lazio, Sezione IFO/Fondazione Bietti, Rome, Italy). The present study was registered at ClinicalTrials.gov (NCT03919019). Upon recruitment, executed from February to July 2017, at the IRCCS Fondazione Bietti, each patient signed an informed consent.

Baseline

All enrolled AMD patients were randomly divided into two age-similar groups, each made up of 15 patients:

  • AMD-M group: 15 enrolled patients providing 15 eyes; mean age 68.50 ± 8.79 years

  • AMD-P group: 15 enrolled patients providing 15 eyes; mean age 70.14 ± 9.87 years

The random separation of AMD patients (screened by MT and MP) was performed by an electronically generated randomization system on the basis of age, gender, and mfERG ring 1 response amplitude density (RAD) (see below).

Months 0–6

Throughout a 6-month period, AMD-M patients received two tablets per day on an empty stomach, before meals of a dietary complementary supplement containing in total lutein (20 mg), zeaxanthin (4 mg), N-acetylcysteine (140 mg), bromelain 2500GDU (80 mg), vitamin B12 (18 mg), vitamin D3 (800 IU), alpha-lipoic acid (140 mg), rutin (157 mg), vitamin C (160 mg), zinc oxide (16 mg), Vaccinium myrtillus 36% anthocyanosides (90 mg), Ganoderma lucidum (600 mg) (Macuprev®, Farmaplus Italia s.r.l., Italy). AMD-P patients received a daily dietary complementary supplement of placebo (two tablets per day on an empty stomach, before meals) containing microcrystalline cellulose (885 mg), talcum (28 mg), calcium phosphate tribasic (688 mg), vegetable magnesium stearate (14 mg), and calcium carbonate (344 mg).

During this period, one eye belonging to the AMD-M group was excluded for lack of compliance and therefore 14 AMD-M eyes out of 15 eyes completed the study. One eye belonging to the AMD-P group was excluded on the basis of IOP increase (> 21 mmHg and < 24 mmHg) and therefore 14 AMD-P eyes out of 15 eyes completed the study.

Following a criterion previously used in other published works [5], in order to evaluate mfERG and OCT data, independently from the clinical conditions and patients’ assignment group, all examinations were performed at baseline and after 6 months of follow-up in the presence of four operators (DM, PG, LZ, and VVF; see Acknowledgements), who were masked for each patient evaluation.

At month 6, compliance to administration (active or placebo treatment) was assessed by evaluating the ratio between the number of tablets delivered at baseline and the number of tablets returned from each AMD patient after 6 months. “Good compliance” was considered as a ratio > 90%. This value was reached by all AMD enrolled patients but one (from the AMD-M group, see above) that was excluded from the analysis at follow-up.

The key was opened to all the investigators at the end of the follow-up.

Electrophysiological Examination (mfERG Recordings)

In AMD-M and AMD-P eyes, mfERG was recorded according to the standard ISCEV [22], and to our previously published method [5, 6, 21].

In the analysis of mfERG responses, we analyzed the average RAD (measured in nV/degree2) between the first negative peak, N1, and the first positive peak, P1, obtained in five concentric annular retinal regions (rings) centered on the fovea. Therefore, we analyzed the N1–P1 RADs derived from 0° to 2.5° (ring 1, R1), from 2.5° to 5° (ring 2, R2), from 5° to 10° (ring 3, R3), from 10° to 15° (ring 4, R4), and from 15° to 20° (ring 5, R5).

MfERGs were performed three times on three different days in each AMD patient. The recording with the highest R1–R5 N1–P1 RADs was considered in the statistical analysis (see below).

Morphological Evaluation (SD-OCT Assessment)

All patients underwent a structural SD-OCT scan using Heidelberg Spectralis (version 1.10.4.0, Heidelberg Engineering, Heidelberg, Germany). The SD-OCT imaging protocol, after pupil dilation, consisted of at least 20° × 15° volume scans of the macula area with 19 B-scans. Furthermore, the enhanced depth imaging (EDI)-OCT scans were acquired to better visualize the choroid.

All B-scan images were checked for errors in automatic segmentation and manual adjustments were used if uncorrected automatic segmentation occurred. Central macular thickness (CMT), inner retinal layer (IRL) and outer retinal layer (ORL) thickness and volume were automatically measured in the macular map centered on the fovea, through inbuilt software of Heidelberg Spectralis (version 1.10.2.0). The IRL included the sum of inner plexiform and nuclear layers thickness, whereas the ORL thickness corresponded to the sum of outer plexiform and nuclear layers thickness.

Two experienced operators (EC and DM) manually measured the choroidal thickness (CT) in EDI-OCT scans, as the distance between hyperreflective inferior limit of RPE and the hyperreflective sclera-choroidal junction, using a caliper integrated in the device. Interobserver agreement was calculated (Cohen’s kappa 0.98 and 0.92), and measurements from the first operator (EC) were used for the study analysis. The CT was measured at the fovea (SCT), 1000 µm nasally (NCT) and 1000 µm temporally (TCT) to the fovea.

In the analysis on SD-OCT we considered the following parameters: CRT (micron), central macular volume (CMV, mm3), central inner retinal thickness and volume (C-IRT, micron and C-IRV, mm3, respectively), central outer retinal thickness and volume (C-ORT, micron and C-ORV, mm3, respectively), subfoveal choroidal thickness (SCT, micron), temporal choroidal thickness (TCT, micron), and nasal choroidal thickness (NCT, micron).

Morpho-Functional Correlations

In order to evaluate whether the possible electrofunctional changes induced by the active treatment were dependent or not on the morphological changes, we correlated mfERG and OCT data derived from the same central macular areas. Therefore, in these correlations we compared exclusively the R1 and R2 mfERG results with corresponding SD-OCT data.

Statistics

Sample size estimates were obtained from pilot evaluations of mfERG recordings performed in 10 non-advanced AMD patients other than those included in the current study (unpublished results), using R1 RAD as the main outcome measure. With a power of 90% at an alpha of 0.01, to detect an expected difference of 35% in mfERG RAD, a sample size of 10 subjects was obtained.

A dropout rate of 40% was estimated; thus, the number of subjects required was 14 for both groups.

Test–retest data of mfERG results were expressed as the mean difference between two recordings obtained in separate sessions ± the standard deviation (SD) of this difference. The Anderson–Darling and Kolmogorov–Smirnov tests were applied to verify that the data were normally distributed. Indeed 95% confidence limits of test–retest variability in patients were established assuming a normal distribution. In AMD patients, test–retest data were calculated considering the entire cohort of enrolled patients that completed the study (28 AMD eyes).

At baseline, the differences of mfERG and SD-OCT values detected in the groups (AMD-P and AMD-M eyes) were evaluated by one-way analysis of variance (ANOVA).

At the end of follow-up, individual changes (values detected at 6 months minus those detected at baseline) of mfERG data detected in AMD-P and AMD-M were calculated by performing a logarithmic transformation.

Mean values of absolute changes in mfERG and SD-OCT data observed in AMD-P and AMD-M eyes after 6 months were compared to baseline values by one-way ANOVA.

The linear correlations between the individual differences (difference of logarithmic transformation of values at 6 months minus logarithmic transformation of values at baseline) of mfERG and SD-OCT data were assessed by Pearson’s test. All statistical analyses were performed by using MedCalc V.13.0.4.0 (MedCalc, Mariakerke, Belgium), and a p value less than 0.05 was considered as statistically significant.

Results

Electrophysiological (mfERG) Data

Figure 1 shows examples of a tri-dimensional mfERG plot recorded in three different AMD-P and AMD-M eyes at baseline conditions and after 6 months.

Fig. 1
figure 1

Examples of multifocal electroretinogram (mfERG) tri-dimensional plots, recorded from six eyes with intermediate age-related macular degeneration (AMD) at baseline and after 6-month follow-up. AMD patients were treated with placebo (AMD-P eyes) or with Macuprev® (AMD-M eyes). At 6 months, with respect to baseline, the tri-dimensional plot in AMD-P eyes showed unmodified or worsened central localized amplitudes, whereas AMD-M eyes showed improved central localized amplitudes

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At baseline, AMD-P and AMD-M eyes showed non-significantly (p > 0.05) different R1–R5 RAD values. Mean RAD values and relative statistical analysis are reported in Table 1 (baseline).

Table 1 MfERG data at baseline and during follow-up
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At 6-month follow-up, a great percentage of AMD-M eyes (> 85%) showed increased R1 and R2 RAD values, whereas an increase of RADs was found in R3–R5 in a percentage ranging from 42.86% to 57.14%. In AMD-P eyes, reduced RAD values were detected in R1 in a great percentage of eyes (57.14%), whereas in the other rings (R2–R4) RAD values were unmodified in a percentage of eyes ranging from 42.86% to 78.57%. The individual changes observed at 6 months in AMD-P and AMD-M eyes with respect to baseline are reported in Table 2 and Fig. 2.

Table 2 MfERG changes during follow-up
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Fig. 2
figure 2

Individual changes after 6-month follow-up with respect to baseline of multifocal electroretinogram (mfERG) R1–R5 N1–P1 response amplitude density (RAD) values observed in AMD eyes treated with placebo (AMD-P eyes) or treated with Macuprev® (AMD-M eyes). R1–R5 refers to localized mfERG responses averaged in five eccentricity areas between the fovea and mid-periphery: 0–2.5° (R1), 2.5–5° (R2), 5–10° (R3), 10–15° (R4), and 15–20° (R5). The percentage of unmodified eyes (within the 95% confidence test–retest limit—values within solid and dashed line), eyes with improvement (values over the 95% confidence test–retest limit—solid line), and eyes with worsening (values under the 95% confidence test–retest limit—dashed line) are reported in Table 2

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On average, at 6-month follow-up in comparison with baseline, AMD-P eyes showed non-significant (p > 0.05) changes in R1–R5 RAD values. In AMD-M eyes, a significant (p < 0.05) increase of RAD values was found in R1 and R2, whereas non-significant (p > 0.05) RAD changes were observed in R3–R5. Mean data and relative statistical analyses of mfERG responses are shown in Table 1 (6 months).

SD-OCT Data

At baseline, AMD-P and AMD-M eyes showed non-significantly (p > 0.05) different values for each SD-OCT parameter. Mean values and relative statistical analysis are reported in Table 3.

Table 3 SD-OCT data at baseline and during follow-up
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At 6-month follow-up, on average, all SD-OCT values were not significantly (p > 0.05) modified in comparison with those observed at baseline in both AMD groups.

Correlations Between Electrophysiological and Morphological Data

In AMD-M, the logarithmic differences (6 months minus baseline) of mfERG parameters (R1 and R2 RADs) were not significantly correlated (p > 0.05) to the corresponding differences of morphological (SD-OCT) parameters. The values of these correlations are shown in Table 4.

Table 4 Correlations between functional and morphological changes at 6-month follow-up
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Discussion

The aim of our study was to assess whether the oral supplementation with Macuprev® can induce an increase in the function of pre-ganglionic retinal elements, associated or not with morphological changes in macular chorio-retinal ultra-structure in patients with intermediate AMD.

This monocentric, randomized, prospective, and double-blind (placebo vs active treatment) study was conducted to assess retinal functional changes by mfERG recordings and macular structure by SD-OCT evaluation.

After 6 months of supplementation with Macuprev®, eyes with intermediate AMD showed increased mfERG R1 and R2 RADs, suggesting a functional improvement of the pre-ganglionic retinal elements located in the 0–5 central retinal degrees. Since in the more external retinal areas (5–20°, rings 3, 4, and 5), non-significant functional changes were observed, the effects induced by the oral intake can be consider as limited to the central macular areas. Despite these functional changes, no differences of the macular chorio-retinal structural parameters were detected at the end of follow-up, as demonstrated by the absence of any significant differences in OCT values of the AMD-M group. As a consequence, the increased function was not significantly correlated with the structural changes from the same tested retinal areas. It is worth noting that in eyes with intermediate AMD treated with placebo, no functional or morphological differences were seen at the end of follow-up.

Since the oral supplement contains different compounds (lutein, zeaxanthin, N-acetylcysteine, bromelain 2500GDU, vitamin B12, vitamin D3, alpha-lipoic acid, rutin, vitamin C, zinc oxide, Vaccinium myrtillus 36% anthocyanosides, Ganoderma lucidum), we are not able to define the specific effects of each of them.

Nevertheless, the observed increase of macular function (improved R1 and R2 mfERG RADs) in active-treated AMD eyes requires an explanation based on the available information on the mechanisms of action of the principal compounds of the supplement. The main and well-studied components are lutein and zeaxanthin, which are known to increase retinal function in healthy subjects [23] and in human pathological conditions [24,25,26]. Indeed, our results are in agreement with our previous study [5], in which similar mfERG changes, despite the lower concentration of lutein (10 mg) and zeaxanthin (1 mg), were found.

To explain why mfERG improvement in our active-treated AMD eyes was found exclusively in the most central retinal area, it is relevant that the highest concentration of lutein and zeaxanthin can be found in the fovea, as studied in cone-rich animal models [27, 28]. This is corroborated by the finding that these functional changes were not observed in areas external to the fovea (5–20°). Therefore, considering also the photoreceptor distribution and topography in human retina [29, 30], it is unlikely that the supplementation with lutein and zeaxanthin would induce an over-normal function in the extra-foveal areas.

Supplementation of AMD patients with lutein and zeaxanthin increases macular pigments and consequently prevents light-induced damage and aging oxidative injury [31]. These protective mechanisms, widely described in a review by Stahl and Sies [32], can explain analogous mfERG findings in AMD patients supplemented with similar content of lutein and zeaxanthin [33].

The relevance of dietary intake of lutein and zeaxanthin has been highlighted by a clinical epidemiological study [34] and by a study from AMD donor eyes [35], showing that food rich of these macular pigments, as well as high plasma levels of these two compounds, was associated with lower risk of AMD. The importance of dietary intake is supported by opposite data showing that drusen develop at the RPE level in monkeys fed with a xanthophyll-depleted diet [36] and by the finding that the concentration of zeaxanthin was inversely and significantly related to the number of light-induced apoptotic photoreceptors in quail retina [37]. In these animal models, lutein and zeaxanthin were considered to have significant antioxidant effects, preventing or delaying photoreceptor dysfunction or loss [36, 37].

Among the other components used in the current study, N-acetylcysteine, rutin, alpha-lipoic acid, and vitamin C may also play a significant role by reducing the oxidative injury [38,39,40,41] and decreasing photoreceptor impairment in AMD. Also bromelain, a cysteine protease enzyme extracted from the stems of pineapple, with its anti-inflammatory activities observed in several therapeutic fields [42], is likely to play a role in the reduction of the AMD photoreceptor dysfunction, possibly contributing to reduce the inflammation involved in the AMD degenerative process [43].

In the present study, we also evaluated the possible changes induced by oral supplementation on the chorio-retinal structures of AMD eyes. Non-significant changes between baseline and the end of supplementation were found for each SD-OCT parameter, as described by the absence of differences in the inner and outer retinal layers’ thickness and volume and by the absence of changes in choroidal thickness during the follow-up. This also led us to believe that in our patients with intermediate AMD, the oral supplementation with the aforementioned components is not able to modify the chorio-retinal morphology, and no other findings available in the literature can confirm or contrast our results at this moment.

Conclusions

In intermediate AMD patients, supplementation with combined components of Macuprev® (lutein, zeaxanthin, N-acetylcysteine, bromelain 2500GDU, vitamin B12, vitamin D3, alpha-lipoic acid, rutin, vitamin C, zinc oxide, Vaccinium myrtillus 36% anthocyanosides, Ganoderma lucidum) increases the function of pre-ganglionic elements located in the most central macular areas, confirming functional improvement [5, 33] that is not associated with morphological changes.

Nevertheless, the present study presents the following limitations: our results, observed after a 6-month period of treatment with Macuprev®, need to be confirmed by long-term supplementation and, since combined compounds were administered, the specific role of each of them in improving macular function needs to be identified. However, our findings could constitute a rationale for further clinical studies, assessing whether the improvement of macular pre-ganglionic function obtained with Macuprev® can be associated or not with the already know changes in psychophysical (i.e., visual acuity, contrast sensitivity [20], and macular microperimetry [33]) measurements in AMD.