Doc Ophthalmol DOI 10.1007/s10633-014-9445-y

ORIGINAL RESEARCH ARTICLE

Morphological and electrophysiological outcome in prospective intravitreal bevacizumab treatment of macular edema secondary to central retinal vein occlusion Ivana Gardasˇevic´ Topcˇic´ • Maja Sˇusˇtar Jelka Brecelj • Marko Hawlina • Polona Jaki Mekjavic´



Received: 28 February 2014 / Accepted: 22 May 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose To evaluate intravitreal bevacizumab (IVB) treatment in patients with central retinal vein occlusion (CRVO) by spectral domain optical coherence tomography (OCT) and electroretinography (ERG). Methods Twenty-two CRVO patients were treated with IVB injections and followed for 1 year. Morphological effect of treatment was observed with fluorescent angiography and OCT. Functional effect was followed with best corrected visual acuity (BCVA) and ERG: combined rod-cone response of the standard fullfield ERG (dark adapted 3.0 ERG), photopic negative response (PhNR), and pattern ERG (PERG). Results Best corrected visual acuity (BCVA) improved by 18.2 letters after 6 months (p B 0.001) and additional 4.7 letters by the 12th month (p B 0.001). The central retinal thickness of 829.8 ± 256.7 lm decreased to 398.8 ± 230 lm (p B 0.001) after 6 months and to 303.7 ± 128.9 lm during the following 6 months (p B 0.001). The total macular volume (14.4 ± 4.2 mm3) decreased to 9.6 ±

3.2 mm3 and 8.5 ± 2.0 mm3 after 6 months and 1 year of treatment, respectively (p B 0.001). Electrophysiological measures improved significantly after 6 months and 1 year of treatment: the a-wave implicit time of dark adapted 3.0 ERG from 25.6 ± 2.3 to 24.1 ± 2.1 and 24.1 ± 2.0 ms (p B 0.01); the PhNR from -5.9 ± 6.6 to -9.4 ± 6.1 and -10.4 ± 4.6 lV (p B 0.05); the PERG P50 amplitude from 0.2 ± 0.3 to 0.9 ± 0.6 and 1.1 ± 0.6 lV (p B 0.001); and N95 amplitude from 0.4 ± 0.6 to 1.2 ± 0.9 and 1.6 ± 0.9 lV (p B 0.001). Conclusions Intravitreal bevacizumab (IVB) treatment of macular edema due to CRVO improved standard morphological measures and the electrophysiological function of outer and inner retinal layers, which was most evident in central retina. Keywords Central retinal vein occlusion  Intravitreal bevacizumab  Optical cocherence tomography  Electroretinography  Photopic negative response  Pattern electroretinography

Clinical Trial Registration Number: 57/07/09. I. Gardasˇevic´ Topcˇic´ (&) General Hospital Novo mesto, Sˇmihelska 1, 8000 Novo mesto, Slovenia e-mail: [email protected] M. Sˇusˇtar  J. Brecelj  M. Hawlina  P. Jaki Mekjavic´ Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia

Introduction Retinal vein occlusion is the second most common cause of reduced vision due to retinal vascular disease [1], with the occurrence of central retinal vein occlusion (CRVO) being 2–3 times less common than

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branch retinal vein occlusion [2]. At the onset, visual morbidity in CRVO is mainly a consequence of retinal hemorrhages, macular edema, or macular ischemia; later, of iris rubeosis and neovascular glaucoma, persistent macular edema, retinal pigment epithelial changes, or epiretinal membrane [3]. In CRVO, obstruction of blood flow within the vein generates increased intraluminal pressure, which according to Starling’s law causes transudation of blood products into the retina. In turn, this process results in increased interstitial oncotic pressure, perpetuating tissue edema. The latter will impede capillary perfusion and lead to ischemia resulting in high levels of vascular endothelial growth factor [4, 5]. In eyes affected with either iris or angle neovascularization, therapeutic approaches are limited to panretinal laser photocoagulation in an attempt to prevent neovascular glaucoma. A recent consensus document [6] for management of retinal vein occlusion recommends intravitreal pharmacotherapy with anti-VEGF or glucocorticoids as the first line of treatment for macular edema due to CRVO. Among intravitreal anti-VEGF drugs, off-label anti-VEGF bevacizumab (IVB) is also used in clinical practice [7]. Morphological improvement after treatment of CRVO with IVB has been monitored in different prospective studies on treatment naı¨ve eyes [8–12]. These have demonstrated that central retinal thickness (CRT) [8– 12] and total macular volume (TMV) [12] assessed with optical coherence tomography (OCT), significantly improved after treatment with IVB. Functional effect of treatment was mostly recorded by changes in visual acuity, which primarily reflects foveolar function. To evaluate the severity of impairment, and response to treatment of not only foveola, but also larger macular area, as well as the entire retina, it is essential to establish additional functional examinations. Several studies have demonstrated the usefulness of electrophysiological tests for monitoring the improvement after the treatment of CRVO with IVB [13–16]. While there was no significant change of the full-field ERG [13, 14], there was also no significant worsening of the multifocal ERG (mfERG) responses [13] and some studies described a significant improvement of the mfERG [14, 15]. Pattern electroretinography (PERG) has been previously used in CRVO as an indicator of rubeosis development [17]. Since PERG

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reflects central retinal function, with macular edema being one of the leading causes of visual morbidity in CRVO patients, it might be an appropriate objective method for evaluating the improvement of central retina after treatment with IVB. A sensitive and specific test for identifying CRVO eyes at risk for the development of neovascularization was proved to be a full-field ERG [18, 19] as it provides an evaluation of the entire retina. Combined rod-cone response of the full-field ERG (dark adapted 3.0 ERG) was shown to provide a prognostic value in CRVO [20], but there are many discrepancies among the studies regarding which ERG parameter (a- and b-wave amplitudes, b/a ratio, and a- and b-wave latencies) provides optimal prognosis for later progression to the ischemic type. However, the a-wave and b-wave of the full-field ERG, respectively, reflect the functional integrity of the photoreceptor and inner nuclear layers [21, 22], which in turn depend upon different blood supplies. Accordingly, full-field ERG may be useful in assessing the amount of initial damage and improvement of these layers after treatment. As shown by Chen et al. [23], the amplitude of the photopic negative response (PhNR), which provides information about the general function of the ganglion cell layer, was significantly affected in CRVO. A role of PhNR for predicting visual outcome after IVB therapy in patients with macular edema secondary to CRVO was investigated by Moon et al. [16]. They concluded that the relative amplitude of PhNR can be a useful prognostic factor for visual outcome after IVB therapy. Therefore, the PhNR amplitude could have a potential role in assessing inner retinal damage and also evaluating the effect of treatment. Until recently, the effect of CRVO treatment with IVB was monitored by different methods. OCT currently represents a valuable diagnostic test for macular edema and the assessment of CRT and TMV by OCT, which provides information of the morphological outcome. For functional evaluation of the treatment, only the visual acuity has been assessed in most reports. Taking into account the location and extent of retinal pathology in patients with CRVO, additional functional tests might provide more information. Therefore, the aim of this study was to evaluate IVB treatment in patients with CRVO by using morphological and objective electroretinographic functional tests.

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Patients and methods Examination procedure The current study was designed as a prospective noncomparative cohort study. The protocol of the study was approved by National Committee for Medical Ethics at the Ministry of Health (Republic of Slovenia) and conformed to the guidelines of the Declaration of Helsinki. All patients treated with IVB due to CRVO at the Eye Hospital, University Medical Centre Ljubljana, Slovenia from September 2009 to September 2013, and complying with the study inclusion criteria, were informed of the study and were requested to consider participating in the study. They received detailed information regarding the study, particularly the off-label character of the IVB injections, the potential risk of endophthalmitis, and retinal detachment, as well as the required repetition of the treatment received, and gave their written informed consent for participation in the study. All patients consenting to participate in the study were included. The diagnosis of CRVO was based on clinical examination: retinal hemorrhages and dilated venous system in all four quadrants and decreased VA due to macular edema. The inclusion criteria included as follows: best corrected visual acuity (BCVA) of less than 0.4 according to Snellen, evident macular edema on OCT, and fluorescent angiography (FA) with symptoms lasting for up to 3 months. The exclusion criteria were other retinal diseases, previous vitreoretinal surgery, intravitreal injections, or laser treatment; all eyes were naı¨ve to treatment of macular edema. Eyes with neovascularization at baseline were not included. Patients underwent a complete ophthalmological examination, including BCVA, using the Early Treatment in Diabetic Retinopathy Study Chart (ETDRS; 4-m distance), intraocular pressure (IOP) determination, slit lamp biomicroscopy, fundus examination, and OCT at the beginning and every 4–6 weeks thereafter. Patients underwent FA and all 3 ERG examinations: at the inclusion in the study, half a year, and 1 year after beginning of the treatment. Morphological effect of treatment was observed with OCT and FA [8–11]. Its functional effect was followed with BCVA and ERG. ERG tests were chosen to selectively evaluate the function of various retinal layers. With combined rod-cone response of the standard full-field ERG (dark adapted 3.0 ERG), the function of the

photoreceptor layer (a-wave) and inner nuclear layer (b-wave) was evaluated [18–20, 24–27]; other responses of the standard full-field ERG were not recorded, in order to shorten the testing time. The PhNR, a relatively new full-field ERG test, has been shown to assess general function of the ganglion cell layer [28, 29]. PERG was chosen as an objective test of the macular function and the function of the ganglion cells of the central retina [30–32] . Fluorescein angiography (FA) Fluorescein angiograms (ImageNet 2000; Topcon, Tokyo, Japan and OCT-SLO Spectralis Heidelberg Retina Angiograph 2, Heidelberg Engineering, Dossenheim, Germany) were obtained by injecting 2 ml of 25 % sodium fluorescein solution. Two retina specialists were assigned to interpret the FA images. CRVO was considered ischemic, if FA revealed retinal non-perfusion of 10 or more disk areas (DA) according to the definition of the CRVO Study Group [33]. In eyes with a large amount of intraretinal hemorrhage, the area of non-perfusion was determined by comparing, when available, the angiogram with the color photograph when available. Morphologic imaging using OCT On SD-OCT (3D OCT-1000; Topcon), central retinal thickness (CRT, lm) and total macular volume (TMV, mm3) were measured and automatically computed by the SD-OCT software afterward. CRT is defined as the mean thickness of the neurosensory retina in a central 1-mm-diameter area, and TMV as the total volume of the scanned neurosensory retina covering a 6-mm (horizontal) 9 6-mm (vertical) 9 1.7-mm (axial) block of the macular region, centered on the fovea. Since the error of the instrument consisted of the inexact identification of the borders between retinal layers, the manual repositioning of the incorrectly placed points was performed. OCT has been provided by two independent investigators that have been supervising the quality of the scans as well as the results of the OCT imaging. Functional testing with electroretinography The recording electrodes were HK loops (HK electrodes: Agencija Avanta, Slovenia) [34] that were

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placed in the fornices of the lower eyelids. The silver chloride reference electrodes (Skin reference electrodes: Carefusion, Germany) were placed on the ipsilateral temples and the ground electrode on the forehead. The skin was prepared by cleaning with abrasive gel, and conductive paste was used to ensure good electrical connection [35]. PERG was recorded without dilatation, while prior to recording the fullfield ERG and PhNR, pupils were dilated with 1 % tropicamide (MydriacylÒ). Combined rod-cone response of the standard fullfield ERG (dark adapted 3.0 ERG) was recorded following the standards of International Society of Clinical Electrophysiology of Vision (ISCEV) [36]. Patients were subjected to adaptation to darkness for 20 min before recording. ERG was elicited. Ganzfeld stimulator of the RETI port unit (Roland Consult, Wiesbaden, Germany). Eight responses to single white light flash stimuli were averaged for each trace, and two recordings were repeated. The band filter was set between 1 and 300 Hz. The amplitude of the a-wave was measured from the baseline to the wave trough, and the a-wave implicit time was measured from the stimulus onset to a-wave trough. The amplitude of the b-wave was measured from the a-wave trough to the wave peak, and the b-wave implicit time from the stimulus onset to b-wave peak. The b/a ratio was calculated from the b- and a-wave amplitude values. Pattern ERG (PERG) was recorded according to the ISCEV standard [37] by the use of RETIscan System (Roland Consult, Wiesbaden Germany). Before testing, the correct distance refraction was determined and used. The visual stimulus was a black-and-white reversing check board pattern (mean luminance 80 cd/m2, contrast 92 %) that reversed four times per second with analysis time of 180 s. The stimulus size subtended a visual angle of 12° 9 16° at viewing distance of 140 cm. The check size was set to 0.8°. Two recordings of 150 sweeps were obtained. The N35/P50 and P50/N95 amplitudes were measured on the average traces. The P50 was measured from the trough of N35, while the N95 amplitude was measured from the peak of P50. Photopic negative response was elicited with Ganzfeld Espion ColorDome stimulator (Diagnosys LLC, Littleton, MA, USA). Monochromatic red (635 nm) stimuli of 2.5 cd s m-2 luminance were delivered on the blue (470 nm) 10 cd m-2 background after 5 min of light adaptation. Twenty

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responses to flash stimuli were averaged for each trace, and the recordings were repeated three times. The amplitude of PhNR was measured from baseline to the negative trough following the b-wave [38]. Treatment procedure Intravitreal bevacizumab (IVB) 1.25 mg in 0.05 ml was injected via the pars plana under sterile conditions in accordance with the recommendations of Aiello et al. [39]. All of the patients received the initial loading dose of four bevacizumab injections separated by 4–6 weeks. Thereafter, patients were followed at monthly intervals and an injection was repeated in the case of persistent or recurrent macular edema with CRT more than 350 lm. Statistical analysis Statistical analysis of the results of those patients who completed 1-year follow-up was performed using SPSS 20.0. The analysis was done in three steps. Repeated measures ANOVA was used to determine differences in mean values of our results before treatment, after the 6th month of treatment, and after 1 year of treatment. Patients were then assigned into two separate subgroups according to whether they had ischemic or non-ischemic CRVO at the end of the study (after 1 year). Repeated measures ANOVA was further done separately for each of the subgroups. Additionally, mixed ANOVA was used to determine whether there is an interaction between measured dependent variables and the fact that someone was ischemic or non-ischemic at the end of the study. Statistical power analysis was done using GPower 3.1 statistical tool.

Results Thirty eyes of 30 patients were originally assigned to IVB. Three patients discontinued the study during the first 6 months for the following reasons: Two patients missed a control visit after 6 months; one patient suffered from subdural hematoma after head injury. During the subsequent 6 months, another five patients discontinued the study; one patient was given intravitreal triamcinolon because of persistent macular

Doc Ophthalmol Table 1 Results for 22 cases of CRVO Baseline

After 6 months

After 1 year

BCVA—ETDRS

38.5 ± 23.5

56.7 ± 21.4***

61.4 ± 17.8***

OCT—CRT (lm)

829.8 ± 256.7

398.8 ± 230.0***

303.7 ± 128.9***

OCT—TMV (mm3) Max.res. a-wave ampl. (lV)

14.4 ± 4.2 93.9 ± 24.3

9.6 ± 3.2*** 100.5 ± 19.6

Max.res. a-wave impl.t. (ms)

25.6 ± 2.3

Max.res. b-wave ampl. (lV)

164.9 ± 60.7

185.5 ± 57.5

186.9 ± 67.7

Max.res. b-wave impl.t. (ms)

49.6 ± 4.7

48.4 ± 3.8

48.6 ± 4.4

1.8 ± 0.4

1.8 ± 0.4

-5.9 ± 6.6

-9.4 ± 6.1

b/a-wave ratio PhNR ampl. (lV)

24.1 ± 2.1**

8.5 ± 2.0 *** 94.6 ± 30.2 24.1 ± 2.0**

2.0 ± 0.3** -10.4 ± 4.6*

PERG—P50 ampl. (lV)

0.2 ± 0.3

0.9 ± 0.6***

1.1 ± 0.6***

PERG—N95 ampl. (lV)

0.4 ± 0.6

1.2 ± 0.9***

1.6 ± 0.9***

Includes morphological and functional results before treatment, and after 6 months, and 1 year of treatment Data are mean ± SD *, **, *** Significance level of pairwise comparison to baseline results (p B 0.05, p B 0.01, and p B 0.001, respectively, according to repeated measures ANOVA. Post hoc multiple comparisons were made with Bonferroni adjustment) BCVA best corrected visual acuity, ETDRS early treatment diabetic retinopathy study, OCT optical coherence tomography, CRT central retinal thickness, TMV total macular volume, Max. res. a-wave ampl. maximal response a-wave amplitude, Max.res. a-wave impl.t. maximal response a-wave implicit time, Max.res. b-wave ampl. maximal response b-wave amplitude, Max.res. b-wave impl.t. maximal response b-wave implicit time, PhNR ampl. photopic negative response amplitude, PERG—P50 ampl. pattern electroretinography P50 wave amplitude, and PERG—N95 ampl. pattern electroretinography N95 wave amplitude

edema; one patient received laser treatment, and three patients had difficulties in returning to control visits. Accordingly, 22 patients were followed for 1 year. Eight CRVO patients were female, and 14 were male. The average age was 63.5 ± 12.8 years. The duration of macular edema due to CRVO before the initial IVB ranged from 6 weeks to 3 months. In 1 year, patients received a mean of 8.18 IVB. All patients included in the study needed more than an initial loading dose of three injections. There were no events of endophthalmitis, retinal tear, or detachment. No serious non-ocular adverse events were reported either. Fluorescein angiography On the basis of retinal vascular perfusion status assessed with FA, eyes were defined as non-ischemic or ischemic. At baseline, there were five eyes with ischemic CRVO and 17 with non-ischemic, additional two progressed from non-ischemic to ischemic after the first 6 months of treatment. After the following 6 months, no one progressed to ischemic. Out of 22 patients who entered the study, 15 were non-ischemic and seven were ischemic after 1 year.

Visual acuity Changes in BCVA as a function measure of central macula were assessed. BCVA improved by 18.2 letters after 6 months, and a further gain of 4.7 letters was seen by the 12th month (Table 1; Fig. 1a). After separating the ischemic from non-ischemic eyes and comparing the BCVA gain, as seen in Table 2, it was significant in both groups after 6 months and 1 year, respectively. Morphologic outcomes Morphologic improvement was monitored with two OCT measures; CRT and TMV. The former reflecting changes in thickness of the central macula, and the latter changes in the volume of the entire macula. The mean pretreatment CRT decreased by more than half after 6 months. Significant improvement was also observed after 1 year of treatment (Table 1; Fig. 1b). The improvement in TMV was statistically significant after 6 months and 1 year of treatment (Table 1; Fig. 1c). After separating ischemic from non-ischemic eyes, we observed a significant improvement of both measures in both groups after 6 months and 1 year (Table 2).

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Fig. 1 BCVA (a), CRT (b), and TMV(c) before, 6 months and 1 year after treatment of CRVO with IVB. *, **, ***. Significance level of pairwise comparison to baseline results (p B 0.05, p B 0.01, and p B 0.001, respectively, according to repeated measures ANOVA. Post hoc multiple comparisons were made with Bonferroni adjustment)

of the dark adapted 3.0 ERG were observed. In our patient population, only the a-wave implicit time improved after 6 months and 1 year of treatment, respectively, and b/a ratio (a ratio between b-wave and a-wave amplitudes) improved after 1 year of treatment, while no significant changes were observed of either b-wave implicit time, nor a- or b-wave amplitudes (Table 1; Fig. 2a–c). When patients were assigned to the ischemic and non-ischemic groups, we noted the same improvement in the above parameters in the nonischemic, but not in ischemic group (Table 2). The PhNR was recorded to investigate the function of the inner retinal layer. As seen in Fig. 3a and Table 1, there was a significant improvement of PhNR amplitude after 1 year of treatment. After separating the eyes according to perfusion status, the significance of PhNR amplitude improvement was not detected in any of the groups (Table 2). Central retinal function at the level of the ganglion cells was measured by the amplitudes of P50 and N95 waves of the pattern PERG. This was seen as an increase of the amplitude after 6 months and 1 year of treatment, respectively (Table 1; Fig. 3b, c), while the implicit time remained unchanged. After separating ischemic from non-ischemic eyes, there was a significant improvement of PERG amplitudes in both groups after 1 year. In non-ischemic group, the improvement was significant already after 6 months (Table 2). Except for the PhNR amplitude, the non-ischemic group showed an identical course of treatment effect, as previously seen for the whole group (Tables 1, 2). However, the results were less favorable in the ischemic group, with a significant improvement of only those measures referring to the central retina (Table 2). Results of mixed ANOVA showed no interaction between measured dependent variables (BCVA, CRT, TMV, a-wave implicit time or amplitude, b-wave implicit time or amplitude, b/a ratio and PhNR, P50, and N95 amplitudes) and the perfusion status of the retina at the end of the study. This indicates that the rate of improvement (in measured dependent variables) between the ischemic and non-ischemic group did not significantly differ.

ERG outcomes

Discussion

In order to investigate the functional integrity of photoreceptors and inner nuclear layers, a- and b-waves

The results of this prospective clinical trial on treatment naı¨ve eyes showed that IVB treatment in

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9.2 ± 1.5***

1.9 ± 0.3

1.6 ± 0.8***

1.1 ± 0.5***

-10.28 ± 5.6

68.6 ± 15.8**

1.7 ± 0.9***

1.1 ± 0.6***

-11.5 ± 4.1

2.0 ± 0.3*

49.5 ± 4.2

201.1 ± 68.5

24.3 ± 2.2**

101.5 ± 32.1

8.3 ± 1.8 ***

296.3 ± 117.5***

20.9 ± 16.99

0.1 ± 0.3

0.1 ± 0.3

-2.97 ± 6.5

1.8 ± 0.5

48.4 ± 5.1

154.0 ± 61.2

25.3 ± 2.1

86.1 ± 17.3

15.2 ± 4.5

876.0 ± 250.7

0.5 ± 0.4

0.5 ± 0.6

-7.5 ± 7.2

1.6 ± 0.5

47.1 ± 2.1

154.7 ± 59.3

23.3 ± 2.3

94.7 ± 18.3

10.4 ± 5.5*

393.0 ± 331.5**

37.9 ± 17.8**

After 6 months

1.2 ± 0.7**

1.0 ± 0.6*

-7.9 ± 4.8

1.9 ± 0.4

46.7 ± 4.3

156.6 ± 59.4

23.7 ± 1.6

79.7 ± 20.6

8.8 ± 2.4**

319.6 ± 159.5*

46.00 ± 10.8**

After 1 year

BCVA best corrected visual acuity, ETDRS early treatment diabetic retinopathy study, OCT optical coherence tomography, CRT central retinal thickness, TMV total macular volume, Max. res. a-wave ampl. maximal response a-wave amplitude, Max.res. a-wave impl.t. maximal response a-wave implicit time, Max.res. b-wave ampl. maximal response b-wave amplitude, Max.res. b-wave impl.t. maximal response b-wave implicit time, PhNR ampl. photopic negative response amplitude, PERG—P50 ampl. pattern electroretinography P50 wave amplitude, and PERG—N95 ampl. pattern electroretinography N95 wave amplitude

*, **, *** Significance level of pairwise comparison to baseline results (p B 0.05, p B 0.01, and p B 0.001, respectively, according to repeated measures ANOVA. Post hoc multiple comparisons were made with Bonferroni adjustment)

Data are mean ± SD

Includes morphological and functional results before treatment, after 6 months, and 1 year of treatment

0.3 ± 0.3 0.5 ± 0.6

PERG—P50 ampl. (lV)

PERG—N95 ampl. (lV)

-7.3 ± 6.4

1.7 ± 0.4

PhNR ampl. (lV)

b/a-wave ratio

48.9 ± 4.3

50.1 ± 4.7

Max.res. b-wave impl.t. (ms)

24.4 ± 2.1*** 199.9 ± 52.5

25.7 ± 2.5 170.0 ± 62.0

103.2 ± 20.2

Max.res. a-wave impl.t. (ms)

Max.res. a-wave ampl. (lV)

65.5 ± 17.0** 401.5 ± 179.5***

Max.res. b-wave ampl. (lV)

14.0 ± 4.1 97.6 ± 26.6

OCT—TMV (mm3)

46.7 ± 21.9 808.3 ± 265.2

BCVA—ETDRS

OCT—CRT (lm)

Baseline

After 1 year

Baseline

After 6 months

Ischemic CRVO

Non-ischemic CRVO

Table 2 Results for 22 cases of CRVO for ischemic and non-ischemic eyes separately

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Fig. 2 Combined rod-cone response of the full-field ERG: a-wave amplitude (a), a-wave implicit time (b), and b-wave amplitude (c) before, 6 months and 1 year after treatment of CRVO with IVB. *, **, ***. Significance level of pairwise comparison to baseline results (p B 0.05, p B 0.01, and p B 0.001, respectively, according to repeated measures ANOVA. Post hoc multiple comparisons were made with Bonferroni adjustment)

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Fig. 3 PhNR wave amplitude (a), P50 wave amplitude (b), and N95 wave amplitude (c) before and 6 months (center) and 1 year (right) after treatment of CRVO with IVB. *, **, ***. Significance level of pairwise comparison to baseline results (p B 0.05, p B 0.01, and p B 0.001, respectively, according to repeated measures ANOVA. Post hoc multiple comparisons were made with Bonferroni adjustment)

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patients with macular edema secondary to CRVO was associated with significant improvement, where not only a good morphological, but also a favorable functional effect of treatment was observed. In the total study population, there was a marked reduction of CRT and TMV, corresponding with a resolution of macular edema. Simultaneously, while a significant improvement of the macular function was seen in BCVA and P50 and N95 amplitudes of PERG, an improvement of outer and inner retinal layers was also detected with a-wave implicit time of combined rodcone response, b/a ratio, and PhNR amplitude (Table 1). To date, treatment success with IVB on treatment naı¨ve eyes with CRVO has mostly been monitored with BCVA, CRT, and TMV improvement [8–11, 16]. Our results compare favorably with these studies. In comparison with the prospective study of Algvere et al. [8], there was a similar gain in BCVA after 1 year even though in the present study visual acuity improved gradually during the whole year, while such gain was achieved already at month 6 and remained stable after 1 year in their study [8]. Similar to our results, Epstein et al. [9] also reported a gradual improvement of VA, but the final gain after 1 year was smaller than in our study, which may be due to the older age of the population and longer duration of symptoms before treatment in their study. Previously, a decrease of visual acuity was noted by a meta-analysis of the natural history of visual acuity in untreated CRVO, conducted by McIntosh et al. [40]. They showed a mean decrease in visual acuity of ten letters from baseline to 6 months and three letters from baseline to 12 months or beyond for non-ischemic CRVO. For ischemic CRVO, a mean decrease of visual acuity was even greater, 15 letters from baseline after 6 months and 35 letters from baseline after 1 year or beyond. Accordingly, there was no sham group in the present study, and all the included patients received IVB treatment. Regarding morphological parameters after 6 months, the improvement in CRT was comparable with that observed in previous prospective studies [8, 9], whereas the decrease in TMV was smaller compared with the results of Costa et al. [12]. After 1 year, we noted a greater improvement in CRT and TMV compared with previous studies [8, 9]. However, the studies differed in time before treatment, number of eyes treated, perfusion status, and inclusion criteria.

Beside morphological improvement and improvement of the visual function by means of VA, this study demonstrates an objective functional evaluation of the treatment effects through several parameters of different electroretinographic tests. The electrophysiological improvement could be most clearly recognized at the level of the central retina, as identified through the PERG parameters, correlating well with morphologically measured remission of macular edema. PERG abnormalities were previously reported in several diseases causing macular edema, such as inflammatory ocular diseases, central serous retinopathy, and retinal vascular diseases [41–43]. In PERG, P50 wave has been shown to be partially derived from ganglion cells with significant contributions from retinal neurons distal to ganglion cells [30], therefore, depending on a functional integrity of the macular photoreceptors [31, 32]. N95 wave is generated in relation to retinal ganglion cells function of central part of retina [44]. Because PERG provides an objective measure of central retinal function through several retinal layers, we chose this method for electrophysiological evaluation of macular function in CRVO patients. Analysis of PERG data demonstrated that after 6 months and 1 year after IVB treatment, the amplitudes of both P50 and N95 waves significantly improved. The improvement of the former is comparable with the improvement of mfERG measures in two prospective studies monitoring the short-term functional and structural effects at the macula following single IVB for macular edema [14, 15], while the third did not observe any worsening of the mfERG measures [13]. This functional improvement correlates well with OCT data, which also showed a significant morphological improvement. Finally, the possibility that the improvement in outer retinal function leads to a downstream improvement in inner retinal responses cannot be excluded. The combined rod-cone response of the full-field ERG was recorded to investigate the effects of treatment on the outer retinal layers, and it showed a significant improvement of a-wave implicit time after 6 months and 1 year after IVB treatment and a significant improvement of b/a ratio after 1 year (Table 1). It has previously been shown that retinal circulatory disturbances in CRVO preferentially affect the inner retinal layers, seen as significant b-wave amplitude or b/a ratio reductions being most significantly associated with later development of iris

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rubeosis [18, 45, 46]. In the present study, a significant improvement of b/a ratio was observed after 1 year of treatment (Table 1). According to this, it could be assumed that treatment resulted in improvement of perfusion of inner retinal layers. Furthermore, according to Johnson et al. [20], not only the function of the inner retina, but also photoreceptor function might be affected in patients with CRVO. Johnson et al. [20] hypothesized that this could be the result of a change in retinal O2 gradient due to reduced inner retina perfusion, which results in lack of oxygen for the photoreceptors in hypoxic eyes. In the present study, a-wave latency improved significantly already after 6 months (Table 1). Additionally, significant delays of the a-wave implicit time were previously shown in a study conducted by Moschos et al. [27], which compared the affected CRVO eyes and the corresponding normal fellow eyes. The prolongation was more marked in eyes prone to develop NV [27]. The results of a present study may imply that treatment influences also the improvement of photoreceptor function despite their huge demands for oxygen and metabolic substrates are provided by choriocapillaries. To additionally investigate the effect of IVB treatment on the inner retina, PhNR was recorded. This slow negative potential, which follows the b-wave of the photopic ERG, most likely originates from spiking activity of the retinal ganglion cells [28, 29]. Previously, Chen et al. [23] demonstrated that the amplitude of the PhNR was significantly smaller in CRVO and BRVO eyes, than those in the unaffected fellow or control eyes. Hence, they suggested that PhNR could have a potential role in assessing inner retinal damage and evaluating the effect of treatment. In our study, PhNR amplitudes showed a significant improvement after 1 year of treatment (Table 1; Fig 3a). On the contrary in a recent study by Moon et al. [16], the improvement of PhNR amplitude after three injection of IVB in 6 weeks intervals was not significant. However, the comparison of outcome between two studies is difficult because their treatment was different to the one employed here [16]. Since there was improvement of electroretinographic measures reflecting outer, as well as inner retinal function, it can be hypothesized that early CRVO treatment with IVB might help stabilize retinal circulation. Furthermore, it might also promote reperfusion of non-perfused areas of inner layers of the

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retina and consequently improve the bipolar and ganglion cell function. The latter might stop the oxygen shift from outer to inner retinal layers and improve the function of outer retinal layers. Two previous studies [25, 47] suggested that retinal impairment due to transient ischemia can be reversible when either the period of ischemia is sufficiently short and/or the degree of ischemia is moderate. After a retrospective analysis of prospectively collected data from BRAVO and CRUISE trials (a 6-month phase III, multicenter, randomized, injection-controlled studies, designed to evaluate efficacy and safety of intraocular injections of ranibizumab in patients with macular edema following RVO), Campochiaro et al. [47] suggested that high levels of VEGF contribute to the progression of retinal non-perfusion, with vascular occlusion as an inciting event and retinal ischemia due to high levels of VEGF as consequence. High levels of VEGF in turn become an important contributor to the disease by worsening retinal ischemia, promoting retinal non-perfusion and suppressing reperfusion of non-perfused retina. It was shown for the central part of the retina that non-perfused retina can become perfused, if treated with ranibizumab [47]. In the present study, only the central part of the retina showed morphological and electrophysiological improvement in both groups; non-ischemic as well as ischemic, but in the ischemic group, the improvement was evident later, after 1 year of treatment. In contrast to the whole study population, in the ischemic group, however, there was seen no improvement of the electrophysiological measures reflecting the function of the peripheral retina (Table 2). Further studies with a greater number of patients will be needed for clarification if improvement in ischemic eyes should be based only on CRT, TMV, and BCVA changes. The expectation that in the long term, ischemic patients would gradually require fewer injections is yet to be confirmed. They may actually require aggressive and prolonged treatment with periodic monitoring of perfusion status, all with the aim to promote long-term stability. In conclusion, although the present study has its limitation in the relatively small sample size, an attempt was made to compare several different morphologic and functional parameters used to evaluate IVB treatment success of central and peripheral retina in eyes with CRVO. In the current setting, it has been demonstrated that there was not only improvement of

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standard measures most often being used for monitoring of treatment success, but also in the objective electroretinographic measures depicting function of the outer and inner retina. The results of the present study suggest benefit of early intervention with antiVEGF for macular edema due to CRVO, when there is still a chance for reperfusion to occur, and for photoreceptor function to be restored which might lead to improvement of not only BCVA but also function of the entire macular region. Acknowledgments The authors wish to thank Mrs. Anja Milenkovic´ Kramer, MSc for all the statistical support, Mrs. Marija Jesensˇek, Mrs. Ana Jersˇin, and Miss Helena Lindicˇ for PERG and PhNR recordings. Mrs. Barbara Klemenc, Miss Anja Hudoklin, Mr. Darko Perovsˇek, MSc, Mr. Matjazˇ Miljan and Mr. Mitja Pipan for performing FA and OCT. This study was financially supported by the Slovene Research Agency Grant No. P3-0333 and No. 57/07/09.

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Conflict of interest The authors declare no actual or potential conflicts of interest relevant to this subject matter.

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Morphological and electrophysiological outcome in prospective intravitreal bevacizumab treatment of macular edema secondary to central retinal vein occlusion.

To evaluate intravitreal bevacizumab (IVB) treatment in patients with central retinal vein occlusion (CRVO) by spectral domain optical coherence tomog...
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