Diosmin alleviates retinal edema by protecting the blood-retinal barrier and reducing retinal vascular permeability during ischemia/reperfusion injury

Abstract

Background and purpose: Retinal swelling, leading to irreversible visual impairment, is an important early complication in retinal ischemia/reperfusion (I/R) injury. Diosmin, a naturally occurring flavonoid glycoside, has been shown to have antioxidative and anti-inflammatory effects against I/R injury. The present study was performed to evaluate the retinal microvascular protective effect of diosmin in a model of I/R injury.

Methods: Unilateral retinal I/R was induced by increasing intraocular pressure to 110 mm Hg for 60 min followed by reperfusion. Diosmin (100 mg/kg) or vehicle solution was administered intragastrically 30 min before the onset of ischemia and then daily after I/R injury until the animals were sacrificed. Rats were evaluated for retinal functional injury by electroretinogram (ERG) just before sacrifice. Retinas were harvested for HE staining, immunohistochemistry assay, ELISA, and western blotting analysis. Evans blue (EB) extravasation was determined to assess blood-retinal barrier (BRB) disruption and the structure of tight junctions (TJ) was examined by transmission electron microscopy.

Results: Diosmin significantly ameliorated the reduction of b-wave, a-wave, and b/a ratio in ERG, alleviated retinal edema, protected the TJ structure, and reduced EB extravasation. All of these effects of diosmin were associated with increased zonular occluden-1 (ZO-1) and occludin protein expression and decreased VEGF/PEDF ratio.

Conclusions: Maintenance of TJ integrity and reduced permeability of capillaries as well as improvements in retinal edema were observed with diosmin treatment, which may contribute to preservation of retinal function. This protective effect of diosmin may be at least partly attributed to its ability to regulate the VEGF/PEDF ratio.

Figure 1. Effect of diosmin on the scotopic a- and b-wave amplitudes as assessed by ERG.

(A) Typical ERG records for the three groups 24 hours and 7 days after ischemia. (B) The ERG was recorded for quantitative analysis. The a- and b-wave amplitudes were measured, and the b/a ratio was determined. Data are expressed as the mean±SD (n = 8). ***P<0.001.

Figure 2. Histological examination after retinal ischemia.

The retinal I/R injury induced retinal edema and retinal atrophy 24 h and 7 d after ischemia, respectively. These insults were reversed by diosmin, as shown in the HE analysis. (A) Representative photographs of rat retinas from the three groups 24 h and 7 d after ischemia. The thickness of the retinas was analyzed quantitatively (B) 24 h and (C) 7 d after ischemia. (D) Changes of the whole retinal thickness 24 h after retinal ischemia for three groups. RGC, retinal ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium; IRT, inner retinal thickness; ORT, outer retinal thickness, OT, overall thickness, ONH, optic nerve head. Data are expressed as the mean±SD (n = 6). *P<0.05, **P<0.01, ***P<0.001. Scale bar, 50 µm.

Figure 3. The ultrastructure of tight junctions (TJ) and the expression of TJ associated protein, ZO-1 and occludin.

In control retinas (A, detail in D), the arrowhead shows “closed” tight junctions, which reflect the integrity of the TJ structure. Retinal ischemia induced “opened” tight junctions, as shown by the arrow in the vehicle groups (B, detail in E), indicating damage to the integrity of the TJ structure. Preserved “closed” tight junctions shown by the arrowhead after diosmin treatment (C, detail in F) indicated its protective effect on maintaining the integrity of the TJ structure after ischemic insult. A-C, magnification × 45,000; D-F, magnification × 150,000. (G) Representative Western blot showing the expression of ZO-1 and occludin. (H) Quantitative analysis of the Western blot. RBC, red blood cells; RCEC, retinal capillary endothelial cells. Data are expressed as the mean±SD (n = 6). *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Visualization of retinal blood vessel leakage after intravascular perfusion with Evans Blue.

(A) Low levels of background fluorescence and vasculature sharply outlined by the dye are seen in control retinas. (B) Twenty-four hours after ischemia, focal sites of leakage and diffusely distributed dye are found in the vehicle retinas. (C) Diosmin treatment markedly reduced the vascular permeability. (D) Quantitative analysis of the Evans Blue leakage. The photographs are representative of four eyes per group and the data are expressed as the mean±SD (n = 6). ***P<0.001. Scale bar = 100 µm.

Figure 5. ELISA analysis of VEGF and PEDF protein expression.

(A) Twenty-four hours after ischemia, there was a significant increase in VEGF expression and a slight increase in PEDF expression in the vehicle group compared with the control group; diosmin treatment reversed this trend. (B) Seven days after ischemia, the VEGF and PEDF protein expression returned to normal in both the vehicle and diosmin groups. (C) The VEGF/PEDF ratio for the three groups 24 h and 7 d after ischemia. Data are expressed as the mean±SD (n = 8). **P<0.01, ***P<0.001.

Figure 6. The Immunoreactivity for VEGF and PEDF in retina.

No significant immunoreactivity for VEGF and PEDF was found in control retina. I/R injury induced a stronger immunoreactivity for VEGF in retina and diosmin administration relieved this trend. The PEDF immunoreactivity was nearly similar to the control group after 24 h retinal ischemia, while diosmin administration immensely increased this immunoreactivity. Scale bar, 100 µm.