Focal Macular Electroretinograms after Intravitreal Injections of Bevacizumab for Age-Related Macular Degeneration

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1 Retina Focal Macular Electroretinograms after Intravitreal s of Bevacizumab for Age-Related Macular Degeneration Eiji Iwata, Shinji Ueno, Kohei Ishikawa, Yasuki Ito, Ruka Uetani, Chang-Hua Piao, Mineo Kondo, and Hiroko Terasaki PURPOSE. To evaluate the changes in the best-corrected visual acuity (BCVA), macular thickness, and focal macular electroretinograms (FMERGs) after three intravitreal injections of bevacizumab for a choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD). METHODS. The medical records of 18 eyes of 18 patients who had received three consecutive monthly intravitreal injections of bevacizumab were retrospectively studied. The BCVA, macular thickness determined by optical coherence tomography (OCT), and FMERGs were measured before the first injection, and 10 days after each of the intravitreal bevacizumab injections. RESULTS. The number of eyes with improvement in BCVA after the first injection was one (6%), after the second injection was four (22%), and after the third injection was five (28%). The number of eyes with reduction in macular thickness was 4 (33%), 8 (44%), and 10 (56%) after each of the three injections. The number of eyes with increase in b-wave amplitude of the FMERGs was 7 (38%), 6 (33%), and 10 (56%) after each of the three each injections. The mean macular thickness was significantly thinner after the first injection, and the mean BCVA was significantly improved after the second injection. The mean amplitude and implicit time of the b-wave of the FMERGs were significantly improved only after the third injection (P < 0.05). CONCLUSIONS. All parameters improved but the best was after the third injection, indicating that three monthly intravitreous injections with bevacizumab may be an effective treatment regimen for AMD. (Invest Ophthalmol Vis Sci. 2012;53: ) DOI: /iovs Several studies have shown that anti-vegfs, for example, bevacizumab, 1,2 pegaptanib, 3 and ranibizumab, 4,5 are effective in suppressing the choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD). 1 5 At present, these anti-vegf agents are the main treatment for the CNVs associated with AMD. For optimal effectiveness, pegaptanib and ranibizumab are recommended to be given every month indefinitely. There is no official regimen for the bevacizumab treatment but the standard practice is to begin with three monthly injections and to continue with additional injections on a pro re nata basis for better results with the additional injections. 6,7 Changes in the best-corrected visual acuity (BCVA), macular retinal thickness, ophthalmoscopic appearance of the fundus, and fluorescein angiograms have been mainly used to assess the effectiveness of the different anti-vegf agents. In addition to these tests, we hypothesized that it would be better to assess the macular functions objectively. Our laboratory has used focal macular electroretinograms (FMERGs) to assess the function of the macular area of AMD patients before and after the surgical removal of a CNV, 8 macular translocation surgery with 3608 retinotomy, 9 transpupillary thermotherapy, 10 and photodynamic therapy (PDT). 11 FMERGs have been used to evaluate not only the positive effects but also the adverse effects of the different types of therapy. It has been reported that anti-vegf agents can affect the choroid circulation 12 by reducing the endothelial cell fenestrations in the choriocapillaris in animal experiments. 13 Other studies have shown that intravitreal ranibizumab can induce retinal arteriolar vasoconstriction in patients with neovascular AMD. 14 In addition, it has been reported that chronic inhibition of VEGF-A function can lead to a significant reduction in the number of retinal ganglion cells. 15 Thus, the purpose of this study was to evaluate objectively the therapeutic and adverse effects of intravitreal injections of bevacizumab in eyes with a CNV associated with AMD. To accomplish this, we analyzed the BCVA and optical coherence tomographic (OCT) images of the retina before the first injection and 10 days after each of the three injections of intravitreal bevacizumab. FMERGs were recorded to assess the function of the macular area of the retina. From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan. Supported by Grants-in Aid (YI), (MK), (HT), and (HT) from the Ministry of Education, Science, Sports, and Culture, Japan. Submitted for publication December 16, 2011; revised April 11 and May 11, 2012; accepted May 14, Disclosure: E. Iwata, None; S. Ueno, None; K. Ishikawa, None; Y. Ito, None;R. Uetani, None;C.-H. Piao, None;M. Kondo, None; H. Terasaki, None Corresponding author: Hiroko Terasaki, Department of Ophthalmology, Nagoya University Graduate School of Medicine, 65 Tsurumacho, Showa-ku, Nagoya , Japan; terasaki@med.nagoya-u.ac.jp. METHODS Patients The medical records of 18 eyes of 18 patients (13 men, 5 women, mean age years, mean 6 SD), who had received three consecutive monthly intravitreal injections of 1.25 mg of bevacizumab at the Nagoya University Hospital from November 2006 through January 2008, were studied. All of the patients signed an informed consent for the injections and agreed to the recording of the FMERGs as a functional follow-up. These studies were performed before the Japanese government approved pegaptanib and ranibizumab. All of Investigative Ophthalmology & Visual Science, June 2012, Vol. 53, No. 7 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 4185

2 4186 Iwata et al. IOVS, June 2012, Vol. 53, No. 7 FIGURE 1. Mean BCVA before the first injection and 10 days after each intravitreal bevacizumab injection. * P < 0.05 versus baseline and error bars are the SEMs. the patients had a subfoveal CNV associated with the AMD or with polypoidal choroidal vasculopathy (PCV). Patients with a CNV lesion caused by other retinal diseases were excluded. Of the 18 eyes, 15 eyes (83%) had AMD and 3 eyes (16%) had PCV. Patients were excluded if they had been treated by laser photocoagulation, radiation, transpupillary thermotherapy, surgery, or photodynamic therapy. The procedures used conformed to the tenets of the World Medical Association s Declaration of Helsinki. The intravitreal injections of bevacizumab were performed after obtaining approval of the Nagoya University Hospital Ethics Review Board and a written informed consent from each patient. The Nagoya University Hospital Ethics Review Board approved this retrospective analysis of the patients data. Visual Acuity A standard Japanese visual acuity chart was used to measure the visual acuity in decimal units, and the results were converted to the logarithm of minimal angle of resolution (logmar) units for statistical analyses. Macular Thickness Measured by OCT The macular thickness was determined by OCT (Stratus OCT; Carl Zeiss Meditec, Dublin, CA) before the first injection, and 10 days after the first, second, and third intravitreal bevacizumab injections. Six radial scans of 6-mm length were made with the Fast Macular Thickness Map (FMTM) protocol. Because the FMTM protocol often misreads the exact borders of the retina in the presence of a CNV, the thickness of the retina was determined by a program developed in our laboratory. 16 With this program, the user is able to set 8 or more cursors above and below the area of interest manually. Our examiner set the upper cursors on the internal limiting membrane and the lower cursors on the RPE side of the retina. Another set of cursors was set at the fovea of the OCT images. When a CNV extended above the RPE, the lower cursors were set on the retinal side of the CNV contour. Then, the program automatically calculated the average macular thickness within a 3-mm circle centered on the fovea. To measure the retinal thickness accurately, one experienced masked examiner performed the measurements based on the protocol we developed. 16 FMERGs FMERGs were recorded before the first injection, and 10 days after the first, second, and third intravitreal bevacizumab injections. Our system for eliciting and recording FMERGs has been described in detail. 17,18 Briefly, an infrared fundus camera equipped with a stimulus light, background illumination, and fixation target was used. The image from the camera was fed to a television monitor, and the examiner used the image on the monitor to maintain the stimulus centered on the fovea. The size of the stimulus spot was 158, and the background light from the fundus camera illuminated nearly the entire visual field. The luminance of the white stimulus light was cd/m 2 and that of the background light was 2.89 cd/m 2. These luminances were measured at the corneal surface and then converted to that at the retinal surface. A Burian-Allen bipolar contact lens electrode was used to pick up the FMERGs. This contact lens electrode system had not only low electrical noise but also permitted a clear view of the fundus by the camera during the recordings. After the patients pupils were fully dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride, FMERGs were elicited by 5 Hz square-wave modulated central stimulus spot (100-ms light on and 100- ms light off). A total of 512 responses were averaged by a signal processor. A time constant of 0.03 seconds with a 100-Hz high-cut filter on the amplifier was used to record the a- and b-waves. The amplitude of the a-wave was measured from the baseline to the first negative trough, and the amplitude of the b-wave was measured from the trough of the a-wave to the positive peak of the b-wave. The noise level of our recording system was less than 0.4 lv. Treatments A 0.12-mL aliquot of commercially available bevacizumab (25 mg/ml) was prepared for each patient and placed in a 1.0-mL syringe with a 30- gauge needle using aseptic techniques in the pharmaceutical department of our university hospital. After the eye had been prepared in a standard fashion and a paracentesis made, 0.05 ml containing 1.25 mg of bevacizumab was injected through the pars plana into the vitreous cavity 3.0 to 3.5 mm posterior to the limbus. After the injection, the patients were instructed to apply topical antibiotics for 3 days. Statistical Analyses The BCVA, macular thickness, and amplitudes and implicit times of the FMERGs before the first injection and 10 days after each of the intravitreal bevacizumab injections were compared by nonparametric Wilcoxon signed-rank tests. The Mann-Whitney U test was used to compare two groups. The data were analyzed with the StatView version 5 (SAS Institute, Cary, NC) computer software. A P less than 0.05 was considered to be statistically significant. RESULTS BCVA The mean BCVA was 20/48 before the intravitreal bevacizumab injections, and it improved to 20/43 10 days after the first injection, to 20/37 after the second injection, and to 20/36 after the third injection. The mean BCVA after the second and third injection were significantly better than the pre-injection mean BCVA (P ¼ after second injection; P ¼ after third injection, Wilcoxon signed-rank test, Fig. 1). An increase of more than 0.3 logmar units was defined as a significant improvement in the BCVA, and a decrease of more than 0.3 logmar units was defined as a significant worsening. Ten days after the third intravitreal bevacizumab injection, 5 (28%) eyes had a significant improvement in the BCVA, 12 (67%) eyes were unchanged, and 1 (6%) eye had a significant decrease in the BCVA. The mean BCVA before the bevacizumab injections was negatively correlated with the improvement in the BCVA 10 days after the first, second, and third injections (r ¼ 0.559, P ¼ ; r ¼ 0.511, P ¼ ; r ¼ 0.576, P ¼ , respectively). Thus, patients with better baseline visual acuity improved less after the treatments. This was because the visual acuity of patients with good baseline acuity had only a small range of increase before it attained a plateau visual acuity of 1.0, a ceiling effect, but patients with poor baseline visual acuity had a greater range to improve their visual acuity.

3 IOVS, June 2012, Vol. 53, No. 7 Focal Macular ERG after Bevacizumab for AMD 4187 FIGURE 2. Examples of the changes of the OCT images and FMERGs in seven patients before the first injection and 10 days after third intravitreal bevacizumab injection. The pre-injection (left side) and after third injection (right side) images are shown. (A) Examples of the OCT images of seven patients. Exudative changes such as subretinal fluid, pigment epithelium detachment, and retinal edema were eliminated in all cases except Cases 4, 5, and 7. (B) FMERGs recorded from seven patients. An increase in the a-wave can be seen in Cases 1 and 4 and in the b-wave in Cases 1 through 4 after the third injection. In Cases 5 and 6, the a- and b-waves were unchanged. In Case 7, a decrease in the a- and b-waves can be seen. Macular Thickness The OCT images of seven patients before the first injection (left column) and 10 days after the third injection (right column) are shown in Figure 2A. The exudative changes, for example subretinal fluid, retinal edema, and detachment of the retinal pigment epithelium (RPE), were not present postinjection in 4 cases but remained in three cases (Cases 4, 5, and 7). The mean macular thicknesses within a 3-mm-diameter circle centered on the fovea before and after treatment are plotted in Figure 3. The mean macular thickness was lm before the first injection, lm 10 days after the first injection, lm after the second injection, and lm after the third injection. The mean macular thickness was significantly thinner than that of the pretreatment thickness at all times even after the first injection (P ¼ , P ¼ , P ¼ , respectively; Wilcoxon signed-rank test). FMERGs FMERGs were recorded from seven patients before the first injection (left column) and after the third injection (right column; Fig. 2B). A significant increase in the a-wave amplitude in Cases 1 and 4 and in the b-wave amplitudes in Cases 1, 2, 3, and 4 can be seen after the third injection. The a- and b-wave amplitudes were unchanged in Cases 5 and 6. In Case 7, the a- and b-wave amplitudes were decreased.

4 4188 Iwata et al. IOVS, June 2012, Vol. 53, No. 7 FIGURE 3. Mean macular thickness before the first injection and 10 days after each intravitreal bevacizumab injection. * P < 0.05 versus baseline, ** P < 0.01 versus baseline. Error bars indicate the SEM. The mean amplitudes and implicit times of the a- and b- waves of the FMERGs before the first injection and 10 days after the first, second, and third injections are shown in the Table, along with the data from 112 healthy subjects. The mean amplitude of the a-waves in all 18 eyes was lv (mean 6 SEM) before the first injection, lv after the first injection, lv after the second injection, and lv after the third injection. None of these differences was significant. The mean amplitude of the b-waves in all 18 eyes was lv before the first injection, lv after the first injection, lv after the second injection, and lv after the third injection. The mean b-wave amplitude was significantly increased over the baseline amplitude only after the third injection (P ¼ ; Wilcoxon signed rank test, the Table). The b-wave after third injections increased in 13 of the 18 eyes (Fig. 4). There were no significant changes in the mean amplitude of a-wave, the b/a ratio, and the mean implicit times of the a- waves 10 days after each injection (the Table). However, the mean implicit time of the b-wave was significantly shortened 10 days after the third injection (P ¼ ; Wilcoxon signed rank test, the Table). We then investigated whether the macular thickness was correlated with the parameters of the FMERGs and BCVA. The amplitude of the b-waves of FMERGs had increased by greater than or equal to 30% in 7 of 10 eyes in which the macular thickness had decreased by greater than or equal to 10% FIGURE 4. Amplitude of the b-waves before the first injection (abscissa) and 10 days after third intravitreal bevacizumab injections (ordinate). (Fig. 5). But we found that the correlations between the macular thickness and the FMERGs, and between the macular thickness and the BCVA were not significant. The distribution of the changes in the BCVA, macular thickness, and the amplitudes and implicit times of the b-waves of FMERGs at the baseline and at 10 days after the first, second, and third injections are shown in Figure 6. We categorized the patients into those who had an improvement, had not changed, or had decreased for each test. The number of eyes whose BCVA had improved more than three lines after the first, second, and third injections was one (6%), four (22%) and five (28%), respectively. The number of eyes whose macular thickness was reduced by greater than or equal to 10% of the pre-injection thickness was 4 (33%), 8 (44%) and 10 (56%) after each of the three injections. The number of eyes whose implicit times of the b-waves of the FMERGs shortened by more than 3 ms was 6 (22%), 10 (56%), and 10 (56%) after each of the three injections. The number of eyes whose amplitude of the b-waves increased by greater than or equal to 30% of the pre-injection amplitude was 7 (38%), 6 (33%), and 10 (56%) after each of the three injections. The number of eyes whose BCVA, macular thickness, amplitude and implicit time of the b- waves of FMERGs worsened was one (6%), one (6%), one (6%), and four (22%) 10 days after the third injection in spite of the three consecutive monthly intravitreal injections of bevacizumab. TABLE. FMERGs before and 10 Days after the First, Second, and Third s Before After First After Second After Third Healthy Subject (n ¼ 112) Amplitide, lv a-wave P * b-wave P * b/a ratio P * Implicit time, ms a-wave P * b-wave P * * Wilcoxon signed-rank test (compared with before injection).

5 IOVS, June 2012, Vol. 53, No. 7 Focal Macular ERG after Bevacizumab for AMD 4189 FIGURE 5. The percentage increase of the macular thickness (abscissa) and the percentage increase of the amplitude of the b- waves (ordinate). The amplitude of the b-waves of FMERGs has increased by 30% or more in the 7 of 10 eyes in which the macular thickness has recovered by 10% or more. Complications None of the cases had an increase of intraocular pressure higher than 21 mm Hg, and none of the cases required cataract surgery within 3 months. No other complications were observed. DISCUSSION Our results showed that the mean macular thickness was significantly thinner 10 days after the first injection, the mean BCVA was significantly better after the second injection, and the mean amplitudes and implicit times of b-wave of the FMERGs were significantly improved after the third injection (P < 0.05). The distribution of the changes in the BCVA and macular thickness after the bevacizumab injections was different from those of the FMERG parameters (Fig. 6). The number of eyes with an improvement of the BCVA and a reduction in macular thickness gradually increased after each injection, but the number of eyes with an improvement of the FMERG parameters did not increase. We do not know the reason for these differences; however, the number of improved patients were maximum after the third injection for all of these clinical parameters. These results indicate that the three monthly intravitreous injections with bevacizumab may be an effective treatment regimen for AMD. In eyes with AMD, a preliminary study reported a significant increase in the P1 amplitude density of the multifocal ERGs recorded from the fovea and parafovea 1 month after an intravitreal bevacizumab. 19 In addition, the P1 amplitude density of multifocal ERGs from a small area in the lesionassociated zone 20 or the central zone 21 was reported to be significantly correlated with the improvement in the BCVA. Intravitreal ranibizumab has also been reported to improve the P1 amplitude density of the multifocal ERGs in the central ring at 4 weeks, and the improvement was positively correlated with the improvement in the BCVA and negatively with the macular thickness. 22 Multifocal ERGs are elicited from small areas of the retina, and the site analyzed can be selected to correspond to the area of interest. Thus, by selecting the appropriate site, significant correlations between the P1 amplitude density and the BCVA can be obtained. On the other hand, our results showed that the correlation between the FMERG improvements and the changes in the visual acuity was not significant. This is not too surprising because the FIGURE 6. Distribution of the (A) changes in BCVA, (B) macular thickness, and the (C) amplitudes and the (D) implicit times of the b-waves of the FMERGs between baseline and 10 days after the first, second, and third injections. All of the parameters had maximum improvement after the third injection.

6 4190 Iwata et al. IOVS, June 2012, Vol. 53, No. 7 FMERGs represent the sum of the neural activity from a 158 area of the macula, whereas the visual acuity is determined by a small foveal area. However, the different parameters (e.g., the a- and b-waves) of the FMERGs can be evaluated more effectively with FMERGs. For example, the mean b-wave amplitude but not the a-wave had increased significantly after the third bevacizumab injection. We further analyzed the relationship between the recovery of the FMERGs and the decrease in macular thickness at an early stage. Seven of the 10 eyes with decreased macular thickness (greater than or equal to 10%) had increased amplitude of the b-waves of the FMERGs (greater than or equal to 30%) (Fig. 5); however, the correlation between the increase in the amplitude of the b-waves and the decrease in the macular thickness was not statistically significant. This discrepancy is because these tests also do not test the same spatial areas. Another reason is that other factors, such as the time required for the CNV to contract and the hemorrhages to resolve, may affect the recovery process. Thus, if the duration of disease was long or there were subretinal hemorrhages, the photoreceptor function could be severely damaged. In these cases, if the macular thickness decreased after the intravitreal bevacizumab, the a-wave and the b-wave of FMERGs might not recover. We compared the FMERG parameters after three monthly intravitreal injections of bevacizumab with the data collected after PDT by our laboratory. 11 Although the conditions of the AMD before the treatment were not necessarily equal, the b- waves improved by 21% after bevacizumab, whereas the b- wave decreased by 7% after PDT. Three monthly intravitreal injections of bevacizumab seemed to be better therapy than PDT from the results of our FMERG data. The limitations of this study were its retrospective nature without a control group and the small number of the eyes. In addition, the follow-up period was short, although we had planned to study the changes induced by intravitreal bevacizumab during the early stages. In conclusion, the time course of the recovery was different for the BCVA, macular thickness, and FMERGs after the three intravitreal bevacizumab injections for AMD. These results indicate that electrophysiological tests may be a useful method to follow these patients. All parameters had improved the most after the third injection, and these results indicated three monthly intravitreous injections with bevacizumab may be an effective treatment regimen for AMD. References 1. Avery RL, Pieramici DJ, Rabena MD, Castellarin AA, Nasir MA, Giust MJ. Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology. 2006;113: , e Spaide RF, Laud K, Fine HF, et al. Intravitreal bevacizumab treatment of choroidal neovascularization secondary to agerelated macular degeneration. 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