Fig 1.
Suppression of photo-induced visual function impairment by bilberry extract.
Analysis of full-field scotopic (a-e) and photopic (f-h) ERGs following light exposure. Representative waveforms of scotopic (a) and photopic (f) ERG from individual mice treated with vehicle or bilberry extract in response to one flash 4 days after light exposure. The amplitude of a- and b-waves in scotopic ERG (b and c) and b-wave in photopic ERG (g) was decreased by light exposure, and these changes were attenuated by treatment with bilberry extract. No differences were observed in a- or b-wave implicit times in both scotopic and photopic ERG (d, e and h). n = 6/ group. ERG, electroretinogram. **P < 0.01 and *P < 0.05.
Fig 2.
Suppression of photo-induced histological changes in the retina by bilberry extract.
(a) TUNEL assay performed 2 days after light exposure. TUNEL-positive cells (red) appeared only in the ONL following light exposure. The number of apoptotic cells was significantly reduced by bilberry extract. DAPI staining of the control is shown as a guide for retinal layers. (b) H&E staining of retinal sections 4 days after light exposure. ONL thickness was lower in vehicle-treated, light-exposed mice than in vehicle-treated, non-light-exposed mice. Photo-induced ONL thinning was significantly attenuated by bilberry extract treatment. (c and d) Rhodopsin (c) and blue opsin (d) immunostaining 4 days after light exposure. The OS lengths of both rod (c) and cone (d) photoreceptors were lower in vehicle-treated light-exposed mice than in vehicle-treated non-light exposed mice. Photo-induced OS shortening was significantly attenuated by bilberry extract treatment (n = 4/ group). Scale bar, 25 μm. ONL, outer nuclear layer; DAPI, 4',6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; IS, inner segment; OS, outer segment. **P < 0.01.
Fig 3.
Inhibition of photo-induced ROS accumulation in the retina by bilberry extract.
Retinal ROS levels were evaluated by DCFH-DA fluorescence. Photo-induced increases in retinal ROS levels were suppressed by bilberry extract and NAC treatment 6 h after light exposure; n = 4/ group. ROS, reactive oxygen species; DCFH-DA, 2’,7’-dichlorofluorescein-diacetate; NAC, N-acetyl-l-cysteine. *P < 0.05.
Fig 4.
Suppression of photo-induced retinal oxidative and ER stress by both bilberry extract or NAC.
(a–f) mRNA expression of markers of oxidative and ER stress was measured using real-time RT-PCR. Expression of (a) ho-1, (b) bip, (c) chop, (d) aft4, (e) xbp1s, and (f) sec24d mRNA was upregulated at 12 h in photo-stressed retinas; however, treatment with bilberry extract attenuated these increases in mRNA expression. n = 5/ group. ER, endoplasmic reticulum. **P < 0.01 and *P < 0.05.
Fig 5.
Suppression of nuclei ATF4 in Müller glial cells by bilberry extract.
(a-d) Co-immunostaining for ATF4 (green) and glutamine synthetase (red), a marker for Müller glial cell, 12 h after light exposure. ATF4 staining was observed in the nuclei (DAPI, blue) of Müller glial cells after light exposure; however, this translocation was suppressed by bilberry extract (c) or NAC (d). n = 5/ group. Scale bar, 10 μm.
Fig 6.
Suppression of photo-induced tight junction disruption in the RPE by bilberry extract.
(a) Immunostaining for ZO-1 in flat mount samples 24 h after light exposure showed a decrease in RPE cells. (b) The number of RPE cells with an intact ZO-1 pattern at all cell edges per total RPE cells is graphically represented. (c) ho-1 mRNA expression was measured using real-time RT-PCR. RPE, retinal pigment epithelium; n = 6/ group. **p < 0.01. Scale bar, 20 μm.