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Vitreous inflammatory factors and macular oedema
  1. Hideharu Funatsu1,
  2. Hidetaka Noma1,
  3. Tatsuya Mimura2,
  4. Shuichiro Eguchi3
  1. 1Department of Ophthalmology, Yachiyo Medical Center, Tokyo Women's Medical University, Yachiyo, Japan
  2. 2Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan
  3. 3Department of Ophthalmology, Eguchi Eye Hospital, Hakodate, Japan
  1. Correspondence to Dr Hideharu Funatsu, Department of Ophthalmology, Yachiyo Medical Center, Tokyo Women's Medical University, 477-96 Owada-Shinden, Yachiyo, Chiba 276-8524, Japan; hfunatsu{at}tymc.twmu.ac.jp

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Introduction

Diabetic retinopathy (DR), branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) are frequently complicated by macular oedema (MO), a condition that arises as a direct consequence of blood–retinal barrier (BRB) breakdown and the subsequent increase in vascular permeability. Accumulating evidence suggests that the upregulation of inflammatory factors or the downregulation of anti-inflammatory factors and a subsequent increase in leucocyte–endothelial interactions contribute to disruption of tight junctions and BRB breakdown.1 2

Recently, clinical treatment has become available to reduce the ocular expression of inflammatory cytokines. Intravitreal injection of triamcinolone acetonide (TA) is effective for reducing macular thickness in patients with diabetic macular oedema (DMO) and those with MO due to BRVO or CRVO.3 4 In addition, the intravitreal injection of antivascular endothelial growth factor (VEGF) antibody has been reported to be effective for reducing MO.5 6 At the usual concentration, intravitreal injection of TA is more effective for reducing DMO and improving the visual acuity than anti-VEGF antibody, suggesting that the pathogenesis of DMO is attributable not only to VEGF, but also to other mechanisms that are suppressed by the corticosteroid.7 Furthermore, the therapeutic effect of TA for MO due to BRVO and CRVO shows similarities.8 9 It has been unclear whether there is any difference in therapeutic effect and which inflammatory cytokines are increased or decreased in the clinical setting.

In this study, we measured the vitreous levels of VEGF and interleukin-6 (IL-6) in patients who had MO associated with DMO or MO due to BRVO and CRVO. The vitreous levels of VEGF and IL-6 were compared among patients with DMO, MO due to BRVO and MO due to CRVO in order to clarify the influence of these molecules on the response to treatment.

Patients and methods

Samples of undiluted vitreous fluid (0.3 to 1.0 ml) were harvested at the start of vitrectomy after informed consent was obtained from each subject following an explanation of the purpose and potential adverse effects of the procedure. This study was performed in accordance with the Helsinki Declaration of 1975 (1983 revision) and the institutional review boards of Eguchi Eye Hospital, Hiroshima University, and Tokyo Women's Medical University approved the protocol for collection of vitreous fluid and blood samples. Vitreous fluid samples were obtained from 36 patients with clinically significant DMO, 25 patients with MO due to BRVO and 27 patients with MO due to CRVO (table 1). The indications for pars plana vitrectomy were MO with a best-corrected visual acuity of less than 20/40 and foveal thickness more than 400 μm. Exclusion criteria for the present study were: (1) ocular disease apart from non-proliferative DR, BRVO and CRVO, (2) previous ocular surgery, (3) previous intravitreal injection of TA and/or anti-VEGF agents within 6 months or laser photocoagulation within 3 months before entry into the study and (4) vitreous haemorrhage.

Table 1

Characteristics of patients

The DMO group (20 men and 16 women) was aged 63.2±7.2 years (mean±SD). The mean duration of diabetes was 18.8±5.1 years, and the haemoglobin A1c (HbA1c) was 7.3±0.7%. The mean duration of MO was 6.0±2.3 months (range: 3–12 months). The BRVO group (10 men and 15 women) was aged 64.3±8.4 years, and the mean duration of BRVO was 4.8±3.0 months (range: 3–10 months). The CRVO group (16 men and 11 women) was aged 65.1±6.5 years, and the mean duration of CRVO was 3.6±1.6 months (range: 3–7 months). Nine eyes were perfused, and 18 eyes were non-perfused. Those eyes did not have new vessels elsewhere or new vessels on the disc.

All of the patients underwent careful biomicroscopic examination using a fundus contact lens, and patients who had clinically significant DMO, BRVO and CRVO were enrolled in this study. Fundus findings were confirmed preoperatively by standardised fundus colour photography and fluorescein angiography (FA), which was performed with a Topcon TRC-50IA fundus camera (Topcon, Tokyo, Japan), an image-net system (Tokyo Optical Co., Tokyo, Japan), and a preset lens with a slit-lamp.

The severity of MO was graded from the retinal thickness at the central fovea, which was measured in each subject within 1 week before vitrectomy by time-domain optical coherence tomography (Zeiss-Humphry Instruments, San Leandro, California). The retinal thickness at the central fovea was calculated from the optical coherence tomography images and was defined as the distance between the inner retinal surface and the retinal pigment epithelium. The mean thickness calculated from four scans of the fovea was used for analysis. The retinal morphologies were assessed using cross-sectional OCT images indicating the reflectivities of retinal structures, and these were classified into four patterns (diffuse retinal thickening, cystoid macular oedema, serous retinal detaching and vitreomacular interface abnormalities).10

Samples of vitreous fluid were collected into sterile tubes at the time of vitrectomy and were rapidly frozen at −80°C. Blood samples were also collected and were immediately placed on ice for centrifugation at 3000 g for 10 min at 4°C, after which the plasma was rapidly frozen and stored at −80°C until assay.

VEGF and IL-6 levels were measured in vitreous fluid and plasma samples by ELISA using kits for human VEGF and IL-6 (R&D System, Minneapolis, Minnesota).11 Each assay was performed according to the manufacturer's instructions. The levels of both factors in the vitreous fluid were within the detection ranges of the relevant assays, with the minimum detectable concentration being 15.6 pg/ml for VEGF (the intra-assay coefficient of variation (CV) was 5.6%, and the interassay CV was 7.5%) and 0.156 pg/ml for IL-6 (intra-assay CV of 4.4% and interassay CV of 7.0%).

All statistical analyses were performed with SAS System 9.1 software (SAS Institute, Cary, North Carolina). Results are presented as frequencies or as the mean±SD. Data with a skewed distribution were transformed to a logarithmic scale, and the geometric mean was calculated together with the 1-SD range on either side of the mean. The Turkey–Kramer test was used to compare patients normally distributed continuous variables among the patients with DMO, BRVO and CRVO, while the Mann–Whitney U test was used for other variables. The χ2 test was employed to compare nominal variables. To evaluate the relations among variables, Pearson correlation coefficients were calculated. Because most variables had a skewed distribution, analysis was performed after logarithmic transformation. Two-tailed p values of less than 0.05 were considered to indicate a statistically significant difference.

Results

The vitreous fluid concentration of VEGF did not differ significantly among the patients with DMO (1080.1 pg/ml (15.6–3042.0)), BRVO (1263.0 pg/ml (15.6–4710.0)) or CRVO (1618.0 pg/ml (15.6–9040.0)) (*p=0.7662, **p=0.3029, and ***p=0.5222, respectively) (figure 1A). In contrast, the vitreous fluid concentration of IL-6 was significantly higher in patients with DMO (389.1 pg/ml (21.0–918.0)) than in patients with BRVO (41.3 pg/ml (0.945–192.0)) or patients with CRVO (94.1 pg/ml (6.01–383.0)) (*p<0.0001, **p<0.0001, respectively). It was also significantly higher in the patients with CRVO than in those with BRVO (***p=0.0265) (figure 1B). There was a significant correlation between the vitreous concentration of VEGF and that of IL-6 in the patients with DMO (r=0.3914, p=0.0036), BRVO (r=0.6086, p=0.0012) and CRVO (r=0.5862, p=0.0029).

Figure 1

Variation in vascular endothelial growth factor (VEGF) among patients with diabetic macular oedema (DMO), branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). In (A), the vitreous fluid concentration of VEGF was not significantly different among patients with DMO, BRVO and CRVO (*p=0.7662, **p=0.3029, ***p=0.5222, respectively). In (B), the vitreous fluid concentration of IL-6 was significantly higher in patients with DMO than those with BRVO *p<0.0001 or CRVO (**p<0.0001), as well as in patients with CRVO than those with BRVO (***p=0.0265).

Vitreous levels of VEGF and IL-6 were independently correlated with the severity of DMO (p<0.0001 and p=0.0282, respectively), as well as with the severity of BRVO (p=0.0008 and p=0.0191, respectively), and that of CRVO (p=0.0014 and p=0.0047, respectively). In contrast, the plasma levels of VEGF and IL-6 were not correlated with the severity of DMO (p=0.7641, p=0.6147, respectively), BRVO (p=0.9197, p=0.5781, respectively) or CRVO (p=0.8573, and p=0.9820, respectively). The ratios of VEGF in the vitreous and plasma for DMO, BRVO and CRVO were 22.0, 52.5 and 39.3, respectively. Furthermore, the ratios of IL-6 in the vitreous and plasma for DMO, BRVO and CRVO were 11.5, 122.0 and 111.2, respectively.

Discussion

Previously, we reported that the vitreous levels of inflammatory cytokines, VEGF and IL-6, were related to the severity of DMO and MO caused by RVO.11 12 In this study, we investigated whether there were any differences in cytokine levels in the vitreous fluid between DMO and MO due to BRVO and CRVO. Our results showed that the vitreous fluid level of VEGF did not differ significantly among these three groups. In contrast, the vitreous fluid level of IL-6 was significantly higher in DMO patients than in those with BRVO or CRVO. On the other hand, it has been reported that aqueous humour levels of VEGF were significantly higher in DMO patients than in those with CRVO, while the VEGF level was also significantly higher in CRVO than in BRVO.13 Their data also suggested that VEGF is the major contributor to MO associated with BRVO and CRVO. Furthermore, there was no difference in the aqueous levels of IL-6, IL-1β and tumour necrosis factor-α between patients who had residual oedema and patients with resolution of oedema, suggesting that none of these cytokines have a greater role than VEGF in the development of MO secondary to BRVO or CRVO. Aqueous and vitreous samples have been obtained during surgery for measurement of various proteins in other studies, revealing a strong correlation between aqueous and vitreous levels of VEGF and IL-6.14 15 Although we also think that measurement of aqueous levels of factors could be useful for diagnosis and for investigating the role of molecules in various disease processes, further studies will be needed to fully elucidate the relation of various inflammatory cytokines to DMO and MO secondary to BRVO or CRVO.

Recently, there have been reports that intravitreal injection of triamcinolone acetonide is effective for reducing macular thickness in DMO.3 More recently, intravitreal injection of bevacizumab (a full-length humanised monoclonal anti-VEGF antibody) has also been reported to be effective against DMO.5 Comparative studies have shown that the reduction in macular thickness achieved by bevacizumab was smaller and of shorter duration than that due to triamcinolone acetonide.7 16 With regard to the improvement of DMO, therefore, anti-VEGF therapy (bevacizumab) is less established than anti-inflammatory therapy (triamcinolone acetonide). Although the pathogenesis of DMO is not fully understood, corticosteroids can modulate vascular permeability by suppressing the expression of VEGF and its receptor, and IL-6 and intercellular adhesion molecule-1 (ICAM-1), as well as by reducing the activity of inflammatory cells that release cytokines, stabilising cell membranes and tight junctions, and upstream of pigment epithelium-derived factor (PEDF) expression.17 Thus, triamcinolone acetonide is a drug with multiple actions that may promote the regression of DMO compared with bevacizumab, which only reduces the amount of free VEGF in the eye. DMO is related not only to VEGF, but also to many other molecules. Because steroids seem to be more effective than anti-VEGF antibody for DMO, it seems that abnormalities of various molecules other than VEGF also contribute to enhancement of vascular permeability in DMO.

On the other hand, the changes of visual acuity and foveal thickness did not show any significant differences between TA and bevacizumab in patients with MO secondary to BRVO and CRVO.8 9 TA may have the same beneficial effects on vision and macular remodelling as bevacizumab for management of MO associated with RVO. In other words, there was no difference of the therapeutic effect for MO due to RVO between inhibiting plural inflammatory cytokines and inhibiting only VEGF. Corticosteroids affect a number of different cytokines including VEGF and IL-6, so it may be necessary to reduce more than one cytokine to make an effective reduction in MO. The mechanism of action of corticosteroids on MO secondary to BRVO and CRVO is still under investigation, but previous reports and our results suggest that VEGF may have a more important role in MO due to RVO than DMO. Further investigations will be needed to fully elucidate the relationship between various inflammatory cytokines and anti-VEGF agents or anti-inflammatory cytokine therapies.

In the present study, we found that different inflammatory molecules may be important for MO in patients with DMO, BRVO or CRVO. We should consider both the treatment modality and levels of inflammatory molecules when managing MO.

Acknowledgments

We thank K Shimada (Department of Biostatistics, STATZ Institute Co.) for his assistance in conducting the statistical analysis.

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Footnotes

  • Funding Supported by a Health Science Research Grant (#10060101 to HF) from the Japanese Ministry of Health, Labour and Welfare (Tokyo, Japan).

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Ethics approval was provided by the Tokyo Women's University, Hiroshima University, Eguchi Eye Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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