Protective Effect of Vaccinium myrtillus Extract on X-Ray Irradiation-Induced Retinal Toxicity via eNOS and 8-OHdG expression

Abstract

Every year, hundreds of thousands of cancer patients receive radiotherapy treatment. Oxidative stress is observed in healthy tissues due to irradiation exposure. The present study is the first to address the effects of Vaccinium myrtillus (whortleberry, WB) against the effects of X-ray irradiation on retinal tissue. Twenty-four Sprague-Dawley rats were randomly allocated into 4 groups: (1) control group: rats without any treatment, (2) X-ray irradiation group: 8 Gray (Gy) RT for 2 days, (3) 100 mg WB extract + X-ray irradiation group: 8 Gy irradiation for 2 days and followed by intraperitoneal (IP) WB extract (100 mg/kg) supplementation for 10 days, (4) 200 mg WB extract + X-ray irradiation group: 8 Gy irradiation for 2 days and followed by IP WB extract (200 mg/kg) supplementation for 10 days. Eyes were enucleated on the 10th day after RT for histopathological, immunohistochemical (8-hydroxy-2'-deoxyguanosine [8-OHdG], endothelial nitric oxide synthase [eNOS]), and biochemical analyses (glutathione peroxidase [GSH], and malondialdehyde [MDA]). The GSH levels significantly decreased and MDA levels and 8-OHdG staining increased after X-ray irradiation compared to the control group. Combined X-ray irradiation + WB treatment significantly increased GSH levels and significantly decreased MDA production and 8-OHdG staining. However, eNOS staining was not affected in any of the groups. Besides, X-ray irradiation significantly increased cell losses and edematous areas. The WB significantly reversed the cellular damage in ganglion cells, inner nuclear, and outer nuclear layers in quantitative analyses. The X-ray irradiation caused significant retinal impairment, and additional WB therapy provided protective effects against radiation-induced retinopathy. These results may suggest WB extract as an adjuvant therapy to reverse retinal impairments after X-ray irradiation.

Keywords: Endothelial nitric oxide synthase; Oxidative stress; Radiotherapy; Retinopathy; Vaccinium myrtillus.

Fig. 1.

Quantitative analysis of retinal thickness with the DP2 (Olympus Inc., Japan) software computer program arbitrary probe.

Fig. 2.

Retinal thickness measurements of the study groups. a: p = 0.001: vs. the control group, b: p = 0.04: vs. irradiation, c: p <0.001: vs. irradiation.

Fig. 3.

Histologic sections of retinal tissues stained with Harris Hematoxylin and Eosin G. a Control group: The retinal layer is observed to be in normal structure in the sections of the control group eye tissue (the retinal layer thickness: 93.41 ± 7.93 μm), ×60. b Irradiated group: In the RT group sections, loss and accompanying edematous areas are observed mostly in the ganglion cell layer (blue arrow) and the cells in the INL (star). Also, edematous areas are observed in the ONL due to cell loss in some areas. Furthermore, there is a decrease in the thickness of the retina layer (retina layer thickness: 73.53 ± 9.87 µm). ×60. c The Irradiated + WB 100 mg group: In the sections belonging to WB 100 mg treatment group, it is observed that cell losses and edematous areas decreased in the GCL, INL (retina layer thickness: 86.97 ± 6.70 μm), ×60. d The Irradiated + WB 200 mg group: In the sections belonging to the WB 200 mg treatment group, it is observed that losses in GCL, INL, ONL layers, and areas of edematous decreased (retina layer thickness: 96.54 ± 7.11 µm), ×60. PL, photoreceptor layer; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; ILM, inner limiting membrane.

Fig. 4.

Semiquantitative analysis results. ^ap = 0.024: vs. the control group, ^bp = 0.001: vs. the irradiation.

Fig. 5.

Light microscopic representations of retinal tissue sections incubated with the eNOS primary antibody. a Control group: The retinal layer was observed to be normal in the sections of the eye tissue of the control group (eNOS positivity score median value: 0.00 [0–1]), ×100. b Irradiation group: No positive positivity was observed in the cells of the retinal layer in the RT group eye tissue sections (eNOS positivity score median value: 0.5 [0–1]), ×100. c, d Irradiation + WB 100 mg group (eNOS positivity score median value: 0 [0–0]) and Irradiation + WB 200 mg group (eNOS positivity score median value: 0 [0–0]) in the cells of the retinal layer in sections of the eye tissue no positivity was observed, ×100.

Fig. 6.

Light microscopic representation of retinal tissue sections incubated with 8-OHdG primary antibody. a Control group: The retinal layer was observed to be normal in the control group (8-OHdG positivity score median value: 0 [0–0]), ×100. b Irradiation group: Irradiation group 8-OHdG positivity was observed in the retinal layers, especially the GCL and INL layers of the retina (8-OHdG positivity score median value: 1 [1–1]), ×100. c, d Irradiation + WB 100 mg (8-OHdG positivity score median value: 0 [0–0]) and Irradiation + WB 200 mg groups (8-OHdG positivity score median value: 0 [0–0]) No positivity was observed in the retinal layer cells, ×100.