Send Orders of Reprints at Reprints@benthamscience.net Correlation from Undiluted Vitreous Cytokines of Untreated Central Retinal Vein Occlusion with Spectral Domain Optical Coherence Tomography

Purpose: To correlate inflammatory and proangiogenic key cytokines from undiluted vitreous of treatment-naïve central retinal vein occlusion (CRVO) patients with SD-OCT parameters. Methods: Thirty-five patients (age 71.1 years, 24 phakic, 30 nonischemic) underwent intravitreal combination therapy, including a single-site 23-gauge core vitrectomy. Twenty-eight samples from patients with idiopathic, non-uveitis floaterectomy served as controls. Interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF-A) levels were correlated with the visual acuity (logMar), category of CRVO (ischemic or nonischemic) and morphologic parameters, such as central macular thickness-CMT, thickness of neurosensory retina-TNeuro, extent of serous retinal detachment-SRT and disintegrity of the IS/OS and others. All cytokines correlated highly with one another (correlation coefficient r=0.82 for IL-6 and MCP-1; r=0.68 for Il-6 and VEGF-A; r=0.64 for MCP-1 and VEGF-A). IL-6 correlated significantly with CMT, TRT, SRT, dIS/OS, and dELM. MCP-1 correlated significantly with SRT, dIS/OS, and dELM. VEGF-A correlated not with changes in SD-OCT, while it had a trend to be higher in the ischemic versus the nonischemic CRVO group (P=0.09). Conclusions: The inflammatory cytokines were more often correlated with morphologic changes assessed by SD-OCT, whereas VEGF-A did not correlate with CRVO-associated changes in SD-OCT. VEGF inhibition alone may not be sufficient in decreasing the inflammatory response in CRVO therapy.


INTRODUCTION
Today, we can assess CRVO patients not only for changes in central macular thickness (CMT), but we can also conduct a detailed analysis of the neurosensory retina layers. Prognosis on visual acuity can be based upon the integrity of the IS/OS (photoreceptor inner and outer segments) and the external limiting membrane (ELM), which are important landmarks for good visual acuity rehabilitation. The development of subfoveal serous detachment seems thereby to be a potential negative clinical indicator [1][2][3][4]. In terms of *Address correspondence to this author at the Department of Ophthalmology, Hospital of the Goethe University, Frankfurt am Main, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany; Tel: +49 -69 -6301 5649; Fax: +49 -69 -6301 5621; E-mail: Michael.Koss@me.com reducing the frequent intravitreal reinjections, careful SD-OCT analysis allows for flexible anti-VEGF treatment, which has demonstrated significant functional and anatomic changes [5].
Macular edema secondary to central retinal vein occlusion (CRVO) occurs after multifactorial pathophysiologic changes, which affects intraocular cytokine levels [6][7][8]. Cytokines mediate between endothelial cells (EC) and inflammatory cells, which themselves interact with cytokine expression [9,10]. The prolonged contact of ECs to proinflammatory cytokines might promote more thrombosis on top of the initial venous occlusion. Upregulated vascular endothelial growth factor (VEGF) is thereby a known chemoattractant cytokine for macrophages and leukocytes and thus plays an important role in the pathophysiologic dysbalance of CRVO [11]. Funk et al. recently demonstrated that anti-VEGF monotherapy has impact on the expression of VEGF and inflammatory markers, including interleukin 6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1) [6]. IL-6 thereby is a major promoter of acute-phase proteins. Secondly it mediates by VEGF the change from acute to chronic inflammation, as it thus combines the inflammatory process with angiogenesis [12]. It could be demonstrated that the severity of macular edema (ME) is correlated with cytokine dysbalance [13].
Our group has previously described the rationale and the clinical outcome of a combination therapy including a core vitrectomy with the application of anti-VEGF agents and steroids and we have described the differences of intravitreal cytokines in different RVO categories [14,15]. Thereby we were able to acquire undiluted vitreous from CRVO patients before the drug injection and correlate the load of cytokines with detailed intraretinal layer changes assessed with SD-OCT.

METHODS
This study was conducted after local institutional review board (IRB) approval. Following the sixth revision of the Declaration of Helsinki each participating patient consented to the study.
Patients were included with a CRVO associated significant macular edema (CSME) involving the fovea, but a macular thickness of not more than 1000 μm, which led to a visual acuity of not worse than 2.0 LogMAR. Not included were patients with CRVO associated complications, like iris rubeosis or neovascularization. Excluded were additionally patients with a history of previous intravitreal drug injections or surgery.
Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) was assessed at 5 m with stopping at three out of five optotypes and presented as the logarithm of the minimum angle of resolution (LogMAR).
A SD-OCT (SD-OCT; 3D OCT-2000; Topcon, Tokyo, Japan) scan depth of 2.3 mm with a horizontal resolution of 20μm, and a longitudinal resolution of 5-6 μm was acquired at an A-scan speed of 27.000 A scans/second. Consecutive sections and vertical and horizontal scans within the macular region were obtained by a well-trained OCT-certified technician (certification by the reading center, Vienna, Austria). Using OCT images, a standardized reading protocol was performed on OCT scans with a quality score over 16 dB, including six measurements ( Fig. 1):

1.
Central macular thickness (CMT) was calculated as the distance of the inner limiting membrane (ILM) to the basal membrane (BM) of the retinal pigment epithelium (RPE) including all compartments in between.

2.
Total retinal thickness (TRT) was defined as the biggest distance of the ILM to the BM of the RPE within the 3D scan field (127 A-scans), including 3. The Thickness of the neurosensory retina (TNeuro) and 4. The Subfoveal serous retinal thickness (SRT) and

5.
The disintegrity of the inner and outer photoreceptor segments (dIS/OS) 6.
The disintegrity of the external limiting membrane (dELM), both at the foveal region.
These measurements were reviewed with a caliper that was built into the software of the OCT machine by two retina specialists (M.P./F.K.) who were blinded to the visual acuity results and the results of the cytokine evaluation. Intergrader reliability ( ) was assessed with a value of 0.88 to 0.95. Additionally, the A-scans were evaluated for the occurrence of intraretinal cysts or hyperreflective spots. To exclude false positive occurrence of disrupted/ disintegrated IS/OS or ELM sections due to overlying cystic edema or intraretinal bleeding a-scans were scrolled through the macular region. Ischemic retinopathy was declared with >ten disc areas of nonperfusion with fluorescein angiography using the OIS WinStation (11K™, CCS Pawlowski GmbH, Jena, Germany). In case of early, fresh CRVO with extensive hemorrhages blocking retinal parts, FA was delayed until bleeding cleared up.
The surgical technique that yielded the sample collection was described earlier [16]. Briefly, 0.6 to 0.8ml of undiluted vitreous fluid from the mid-to posterior vitreous cavity were extracted by an assistant under the guidance of the surgeon, who controlled the vitrector with a headset and a magnifying 28-diopter lens.
Samples from idiopathic, non-uveitic floaterectomy served as controls. All samples were saved prior to drug application and frozen at -80°C.
The cytometric bead array system with flex sets (BD™, Heidelberg, Germany) was used to determine IL-6, MCP-1 and VEGF-A according to the manufacturer´s instruction manual including the measurement on a FACSArray™ Bioanalyzer (BD™, Heidelberg, Germany) with FCAP array software (BD™, Heidelberg, Germany). The data were saved in EXCEL (Microsoft Office 2010, Redmond, USA) and statistically analyzed with Bias software (Version 8.3.8, Epsilon, Darmstadt, Germany). The data had a nonparametrical distribution, which was checked with the David's test (error level of 5%), thus the Wilcoxon-Mann-Whitney test could be applied with a P value of <0.05.
Spearman rank correlation coefficient was used to examine the relationship between the influences of the cytokines on other parameters, like or changes in SD-OCT.

Patients
The CRVO group (14 men and 21 women) was aged 71.1 ± 11.7 years (mean ± standard deviation -SD), and the control group (12 men and 16 women) was aged 66.2 ± 7.9 years (P=0.89 and 0.17, respectively, Table 1). The visual acuity was 1.28 ± 0.59 LogMAR in the CRVO group and 0.51 ± 0.22 in the control group (P<0.001). Twenty-four out of 35 in the CRVO group and 8 out 28 in the control group were phakic (P<0.005). Among the 35 CRVO patients, 30 were nonischemic. The duration of the CRVO was 7.4 ± 3.5 months for all CRVO patients and all were treatment naïve before study start. The patients were distributed in a subgroup of fresh CRVO with a duration of 5.1 ± 2.0 months (n=22) and a subgroup of old CRVO (n=13) with a duration of 11.2 ± 1.6 months after onset of the disease (P<0.001).

Vitreal Cytokine Levels
The mean IL-6 vitreal, MCP-1 and VEGF-A levels can be found in Table 2 and Fig. (2).

Correlation Between Cytokines
In the CRVO group, all cytokines were positively correlated with one other. IL-6 was positively correlated ( Table 3)

Visual Acuity
Visual acuity was analyzed with LogMAR values and correlated to objective SD-OCT parameters and the vitreal cytokine values ( Table 3). There was no significant correlation between any of the parameters and visual acuity.

DISCUSSION
We could demonstrate, that primarily the inflammatory cytokines were more often correlated with morphologic changes assessed by SD-OCT, whereas VEGF-A did not correlate with CRVO-associated changes in SD-OCT.
Recent studies with SD-OCT have shown that macular edema secondary to retinal vein occlusion is often characterized by numerous cystoid spaces and marked retinal swelling, especially in the outer retinal layers. We are not aware, that there is a correlation of SD-OCT parameters with intraocular cytokine levels published up to date. In our study, IL-6 correlated more often with SD-OCT parameters than MCP-1, whereas VEGF-A did not correlate with CRVOassociated changes in SD-OCT ( Table 3). CRVO macular edema is often accompanied by serous retinal detachment, which might occur as pointed or dome-shaped and might be associated with a poorer visual acuity prognosis [1]. The most suitable predictor of visual acuity, however, seems to be the integrity of the photoreceptor layer and the integrity of the external limiting membrane [3,4]. Subretinal thickness, dIS/OS, and dELM were significantly correlated with the inflammatory marker IL-6 and not with VEGF-A. The visual acuity at the time of combination therapy was not correlated with objective SD-OCT parameters or the cytokines. Cystoid spaces themselves are often accompanied by hyperreflective spots, which might indicate chronicity [17]. Cystoid spaces and hyperreflective spots were correlated with central macular thickness and IL-6, which might underline chronicity. MCP-1, however, was not correlated with cystoid space or with hyperreflective spots. Generally, the role of MCP-1 in the pathophysiology of RVO is until today not well perceived. It has strong eosinophilic chemotactic properties and is crucial in monocyte recruitment at the endocytes, which supports the role of eosinophils in tissue remodeling [18].
It is noteworthy that in our study all cytokines are positively correlated with each other ( Table 3), which was highly statistically significant. Because of one limitation of our study, the relatively small number of ischemic CRVO patients (n=5), we can only speculate about the implication of the results in terms of the comparison between ischemic versus nonischemic patients. While Noma et al. were able to demonstrate elevated IL-6 and VEGF levels in 18 ischemic CRVOs (versus 9 nonischemic CRVO) [7], we could not confirm this observation (Fig. 3). There was a trend towards elevated VEGF-A levels in the ischemic group (p=0.09), but the correlation among the cytokines was insignificant, while there was a positive correlation between the cytokines in the  nonischemic group. Based on routine clinical practice [10,19] and previous preclinical publications [20,21], it is likely that in CRVO with retinal ischemia, VEGF and IL-6 are both higher than in nonischemic CRVO. Cataract extraction after ischemic CRVO may imply a clinical risk of developing rubeosis iridis, but generally the implication of lens status (phakic or pseudophakic) is not well understood. We did not observe a significant difference between the two subgroups. The limitations of our study are a small sample size, which may have to oversee especially in the ischemic CRVO group significant differences.
In conclusion, we demonstrated significantly higher values of inflammatory and proangiogenic cytokines in eyes with "fresh", treatment-naïve CRVO eyes as compared with control eyes. Although there was no correlation between morphologic SD-OCT parameters and visual acuity seven months after CRVO onset, inflammatory cytokines, IL-6 and MCP-1 were more often correlated with predictive morphologic changes (SRT, IS/OS, ELM), which are clinically important in visual acuity prognosis, than VEGF-A. VEGF-A had a tendency to be higher in the ischemic than in the nonischemic CRVO group. Fig. (2). Bar plots with the values of cytokines (statistical extremes are marked with dots) for all CRVO patients (left bar plots; n=35) versus the control group (right bar plot; n=28); significant differences are noted with stars. Fig. (3). Bar plots with the values of cytokines (statistical extremes are marked with dots) for ischemic CRVO patients (left bar plots; n=15) versus non-ischemic CRVO patients (right bar plot; n=30); p=0.09 is marked with a star in brackets.