Neuroprotective effect of bilberry extract in a murine model of photo-stressed retina

<div><p>Excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration and retinitis pigmentosa. However, the underlying mechanisms of photo-induced retinal degeneration are not fully understood, and a generalizable preventive intervention has not been proposed. Bilberry extract is an antioxidant-rich supplement that ameliorates ocular symptoms. However, its effects on photo-stressed retinas have not been clarified. In this study, we examined the neuroprotective effects of bilberry extract against photo-stress in murine retinas. Light-induced visual function impairment recorded by scotopic and phototopic electroretinograms showing respective rod and cone photoreceptor function was attenuated by oral administration of bilberry extract through a stomach tube in Balb/c mice (750 mg/kg body weight). Bilberry extract also suppressed photo-induced apoptosis in the photoreceptor cell layer and shortening of the outer segments of rod and cone photoreceptors. Levels of photo-induced reactive oxygen species (ROS), oxidative and endoplasmic reticulum (ER) stress markers, as measured by real-time reverse transcriptase polymerase chain reaction, were reduced by bilberry extract treatment. Reduction of ROS by N-acetyl-L-cysteine, a well-known antioxidant also suppressed ER stress. Immunohistochemical analysis of activating transcription factor 4 expression showed the presence of ER stress in the retina, and at least in part, in Müller glial cells. The photo-induced disruption of tight junctions in the retinal pigment epithelium was also attenuated by bilberry extract, repressing an oxidative stress marker, although ER stress markers were not repressed. Our results suggest that bilberry extract attenuates photo-induced apoptosis and visual dysfunction most likely, and at least in part, through ROS reduction, and subsequent ER stress attenuation in the retina. This study can help understand the mechanisms of photo-stress and contribute to developing a new, potentially useful therapeutic approach using bilberry extract for preventing retinal photo-damage.</p></div

Suppression of photo-induced histological changes in the retina by bilberry extract.

<p>(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.</p

Suppression of photo-induced tight junction disruption in the RPE by bilberry extract.

<p>(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.</p

Suppression of photo-induced retinal oxidative and ER stress by both bilberry extract or NAC.

<p>(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.</p

A proposed model of rapamycin’s neuroprotective effect during retinal inflammation.

<p>The basal level of activated mTOR promoted NF-κB activation during inflammation, which induced the retinal expression of the inflammatory cytokines, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Activated NF-κB elevated the activated and phosphorylated mTOR (pmTOR) level during inflammation, which may have exacerbated the pathogenesis. The subsequent retinal activation of signal transducer and activator of transcription 3 (STAT3), at least partly, reduced the rhodopsin protein expression in a post-transcriptional manner and impaired rod photoreceptor function. Rapamycin also attenuated inflammatory leukocyte adhesion to the vessel walls, and increased potentially neuroprotective autophagy in the retina during inflammation.</p

Suppression of photo-induced visual function impairment by bilberry extract.

<p>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.</p

Suppression of nuclei ATF4 in Müller glial cells by bilberry extract.

<p>(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.</p

The interaction between mTOR and NF-κB in the retina during inflammation.

<p>Immunoblot analyses (A-H). The protein level of phosphorylated and activated mTOR (pmTOR) was not increased in the retina 3 hours after the induction of inflammation by an intraperitoneal lipopolysaccharide (LPS) injection, although the level of pmTOR was suppressed by the rapamycin treatment (A, B). At 12 hours after the LPS injection, the pmTOR expression was increased in the retina, which was suppressed by the rapamycin treatment (C, D). The pmTOR level in the retina at 12 hours was also suppressed by the NF-κB inhibitor, DHMEQ (E, F). The ratio of the LC3II to LC3I protein levels (an autophagy marker) in the retina was not changed after LPS injection but was increased by rapamycin at 12 hours after LPS injection (G, H); n = 5. *p < 0.05, **p < 0.01.</p

Rapamycin suppressed inflammatory molecules in the retina during inflammation.

<p>Immunoblot analysis showed that phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65 (pNF-κBp65) was increased in the retina 3 hours after the induction of inflammation by an intraperitoneal injection of lipopolysaccharide (LPS), and that this was attenuated by the rapamycin treatment (A, B). ELISA assays showed that the protein levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were increased in the retina 12 hours after the induction of inflammation, and that these changes were attenuated by the rapamycin treatment (C, D). Real-time PCR showed that the mRNA levels of both <i>IL-6</i> and <i>MCP-1</i> were increased 6 hours after the induction of inflammation, and that the mRNA level of <i>IL-6</i> (E), but not that of <i>MCP-1</i> (F), was attenuated by the rapamycin treatment. Immunoblot analyses showed that the expression level of phosphorylated and activated signal transducer and activator of transcription 3 (pSTAT3) was increased at 12 hours after inflammation, and that this increase was attenuated by the rapamycin treatment (G, H); n = 5. *p < 0.05, **p < 0.01.</p

Inhibition of photo-induced ROS accumulation in the retina by bilberry extract.

<p>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.</p