Aqueous cytokine levels are associated with reduced macular thickness after intravitreal ranibizumab for diabetic macular edema

Abstract

Purpose: It is controversial whether the administration of anti-vascular endothelial growth factor drugs for diabetic macular edema (DME) affects intraocular inflammatory cytokines. In this study, we measured cytokine concentration in aqueous humor before and after intravitreal injection of ranibizumab (IVR). The aim was to determine changes in cytokine concentration and their effects on DME reduction.

Methods: Twelve patients (13 eyes) with DME received two IVR (0.5 mg) with a 1 month interval, and a total of 26 aqueous humor samples were obtained. Macular thickness was measured with an optical coherence tomography (OCT) using thickness-map mode with an Early Treatment Diabetic Retinopathy Study (ETDRS) 9-zone grid that was divided into two zones: a central circle with a diameter of 1 mm (zone1); and an outer circle with a diameter of 6 mm (zone2).

Results: The concentration of eotaxin-1 in aqueous humor samples decreased significantly after IVR. Baseline cytokine concentration was associated with IVR-induced DME reduction. In zone1, higher baseline concentration of interferon-induced protein (IP)-10, and in zone 2, higher baseline concentration of granulocyte-macrophage colony-stimulating factor, IP-10, and tumor necrosis factor (TNF) α; and lower baseline concentration of eotaxin-1, interleukin (IL)-5, and IL-8 were associated with improved DME. Cytokine changes were associated with IVR-induced DME reduction. In zone1, lower concentration of IP-10 compared to baseline or higher concentration of macrophage inflammatory protein (MIP) -α, and in zone 2, lower concentration of IL-5 compared to baseline, IL-8, and IP-10 or higher concentration of eotaxin-1 and MIP-1β were associated with improved DME.

Conclusions: These findings suggest that ranibizumab affects the concentration of cytokines in aqueous humor. Various cytokines contribute to a decrease in retinal thickness, both in the center of the macula and in a larger area of the retina.

Fig 1. Measurement and evaluation of DME at baseline and post-IVR.

OCT findings at baseline and 1 month post-IVR. (A-D) Macular thickness was measured on OCT images using thickness-map mode with an ETDRS 9-zone grid. (B,D) Thickness-map showing the average retinal thickness (μm) in each grid, which projects to the fundus of the eye.

Fig 2. Classification of DME.

DME was categorized according to extent and form. The ETDRS grid was divided into two zones: the central circle with a diameter of 1 mm (zone 1); and the outer circle with a diameter of 6 mm (zone 2). Zone 2 allowed the evaluation of larger areas of macular edema. In addition, macular edema was also defined as diffuse if retinal thickness was at least ±30% beyond the reference thickness of zone 1; otherwise, it was defined as non-diffuse.

Fig 3. Measurement method of cytokine concentration (the example of eotaxin-1).

After plotting the standard curve of eotaxin-1, the fluorescence intensity (FI) of each sample was measured, and the value read from the standard curve was defined as the concentration. S1—S6 are standard samples. S1[9298.11 (concentration), 25760.9 (FI)], S6[3.37 (concentration), 21.9 (FI)]. The detection limit of eotaxin-1 was 3.367 pg/mL. LLOQ = lower limit of quantification ULOQ = upper limit of quantification.

Fig 4. Changes in aqueous humor cytokine concentrations post-IVR.

Changes in aqueous humor cytokine concentrations in the 13 eyes between baseline and 1 month post-IVR are shown (IL-6 review of 12 eyes). Eotaxin-1 concentration decreased significantly in aqueous humor samples after IVR (n = 13; P = 0.047). IL-6 concentration also tended to decrease after IVR (n = 12; P = 0.075).